Genetic approaches to molecular motors.

Annu. Rev. Cell BioI.

1992. 8:29-{56 1992 by Annual Reviews Inc.

Copyright ©

Further

ANNUAL REVIEWS All

Quick links to online content

rights reserved

GENETIC APPROACHES TO

Annu. Rev. Cell. Biol. 1992.8:29-66. Downloaded from www.annualreviews.org Access provided by University of Florida - Smathers Library on 11/12/15. For personal use only.

MOLECULAR MOTORS Sharyn A. Endow Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710

Margaret A. Titus Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

KEY WORDS; motor protein mutants, genetic interactions, myosin, kinesin, dynein

CONTENTS MOLECULAR MOTORS ............................................

.

MyOSINS ........................................................

.

MyosinIl '" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myosin I .... . . .. . . ... . ................... . . ... . ... . ....... . . ... .

KINESIN

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

DYNEINS ........................................................

.

Axonemal Dynein . . . . . . . . . . . . . . . . . .. . . . . . . . ... . . . . . . . . . . . . . . .. . . . . Cytoplasmic Dynein . . . . . . . . .. . . . .. . . . ... . . .. . . . ... . ..... . .... . ... .

DISCOVERY OF NEW FAMILY MEMBERS ..............................

';!fnO::�� : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

i Dyneins .. . . . .... . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . .

MUTANT PHENOTYPES AND MOTOR PROTEIN INTERACTIONS ..........

.

';!fnO::i�� : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : Dyneins . . . . . . ... . . . . . . . . ... . ... . ..... . . . . . . . . . . . . . . . . . . ... . . . .

.

GENETIC EVIDENCE FOR MOTOR PROTEIN INTERACTIONS ............. FUTURE PROSPECTS Roles in the Cell.

.

.

30 31 31 32 33 34 34 34 35 35 39 44 45 45 48 51 53

.

57 57 58 59

CONCLUDING REMARKS .......................................... .

60

AFTERWORD .....................................................

60

.

.

.

.

.

.

.

.

... .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.. .

.

.

.

. ... . . .... ... . .. . . . Mechanisms of Motor Protein Action ............. .. . . . .. Regulation of Motor Protein Activity . . . ... . . .. . . . . . . . . .. . . .... . . . . . . . .

..

..

.

.

.

29

0743-4634/92/1115-0029$02.00

30

ENDOW &

TITUS


MOLECULAR MOTORS The field of molecular motors has expanded greatly with the discovery of multiple new myosins and kinesins and the recent evidence that the dyneins comprise a multigene family. The application of genetic and molecular genetic analysis to the study of the new proteins has revealed their involvement in diverse systems of cellular motility. A common feature of molecular motor proteins is their ability to undergo directional movement upon hydrolysis of ATP. These proteins, therefore, constitute force-generating components of the cell with essential roles in many basic cellular processes. The molecular motors fall into three families of proteins: the myosins, kinesins, and dyneins. The myosins utilize actin filaments as "tracks" for movement, while the kinesins and dyneins move along tracks of microtubules. A potential fourth class of molecular motors is represented by the protein dynamin. Dynamin was originally reported as a microtubule-associated protein that required a soluble activating fraction and ATP to support sliding of microtu buIes against one another in vitro (Shpetner & Vallee 1989). Molecular cloning and DNA sequence analysis have revealed homology between dynamin and GTP-binding proteins (Obar et al 1990), and dynamin has been demonstrated to be a microtubule-stimulated GTPase in vitro (Shpetner & Vallee 1992). These observations suggest that GTP is the physiological substrate for dynamin, in contrast to the myosins, kinesin, and dyneins, which utilize ATP in vivo. However, evidence has also been reported recently (Toyoshima et al 1991) that dynamin can function as a microtubule-stimu lated ATPase which supports microtubule translocation in vitro, in apparent contradiction to its molecular similarity to GTPase. Further studies are needed to define the nucleotide requirements and motor properties of dynamin and its role in the cell. These studies are expected to progress rapidly, however, since the gene has been cloned not only from rat (Obar et al 1990), but also from yeast and Drosophila (Yeh et al 1991; van der Bliek & Meyerowitz 1991; Chen et al 1991). The application of genetics to the analysis of function should aid in determining the role of dynamin in the cell. Because the role of dynamin as a motor protein requires further investigation, this review considers only the myosins, kinesins, and dyneins. We first briefly describe founding members of each motor protein family, and then discuss the discovery of newly identified related proteins, with emphasis on the genetic and molecular genetic approaches that have led to their discovery, as well as on the genetic methods that are being applied to analysis of function. The information thus far obtained regarding the primary structures of the proteins, the phenotypes of mutants, and interactions of proteins with one another suggests means of identifying genes that encode

MOLECULAR MOTORS

31

further new family members . It seems likely that some of the newly identified proteins will be involved in presently unrecognized roles within the cell .


MYOSINS The myosins form a diverse class of related proteins that includes the filament-forming myosin II and nonfilament-forming myosin I proteins. Classical biochemistry and cell biology were used in the identification and characterization of the best known members of the myosin family , muscle and nonmuscle myosin II, and the Acanthamoeba and brush border myosin I proteins.

Myosin II Native myosin II proteins consist of two heavy chains of �200K associated with two regulatory and two essential light chains. Critical information about the various domains of the myosins was provided ' by analysis of the Caenorhabditis elegans unc-54 gene. unc-54 mutants are viable but paralyzed, exhibiting an extreme uncoordinated phenotype, and have disorganized muscle tissue containing reduced numbers of thick filaments. Comparison of proteins in wildtype and mutant strains revealed that the unc-54 gene encodes a myosin heavy chain abundant in body wall muscle (MacLeod et al 1 977b). One mutant allele of unc-54, E675 has a small internal deletion of � 1 00 amino acids in the unc-54 myosin heavy chain (MacLeod et al 1 977a,b). This deletion was used in the molecular cloning of the gene by taking advantage of the reduced Mr of in v itro-translated E675 myosin to identify sucrose gradient fractions containing E675 RNA. This allowed the partial purification of E675 RNA and the subsequent recovery of unc-54 cDNA and genomic DNA sequences (MacLeod et al 1 981). DNA sequence analysis of the cloned gene resulted in the first complete amino acid sequence of a motor protein (Karn et al 1 983) . Comparison of the deduced unc-54 amino acid sequence with partial sequence for a second C. elegans myosin heavy chain revealed a region of 82 % identity. Alignment of the C. elegans myosin sequences with peptide sequences and known proteolytic cleavage sites for rabbit skeletal myosin showed that the region of identity corresponded to the globular enzymatic portion of rabbit myosin. This region had been implicated in nucleotide binding in rabbit myosin by its ability to bind photoaffinity analogues of ATP. The conserved region contained

MUSCLE MYOSINS Mr

,

�

lIn order to distinguish between the gene and the protein, names of genes are italicized, but those of proteins are not. Thus

unc·54

refers to the gene, while une-54 denotes the protein. Names of

genes or proteins refer to wildtype, and mutants are given by allele designation (e.g. £675). Names of wildtype S. cerevisiae genes are in upper case. mutant names are in lower case, and protein names are capitalized.


32

ENDOW & TITUS

an amino acid sequence motif found in ATP-binding proteins (Walker et al 1 982). Other invariant sequences in the globular region of the proteins were identified (Karn et al 1 983) that have been implicated in actin binding (Sutoh 1 983). The ATP-binding sequence motif, together with the actin-binding region, comprise the mechanochemical or motor domain of the proteins, which is a conserved structural feature of the myosins and has been used in the identification of new myosins. The remainder of the proteins showed greater sequence variability. This early comparison of myosin sequences established a theme now recognized as common to the motor protein families. Proteins within each family show a high degree of amino acid sequence similarity in the motor domain, but the remainder of the molecules are divergent in sequence. This is true even for proteins such as the muscle myosins that are thought to carry out similar cellular functions . CYTOPLASMIC MYOSIN The nonmuscle or cytoplasmic myosins resemble their muscle counterparts in overall structure and interaction with actin, yet their assembly , regulation , and cellular function differ significantly from that of the muscle myosins (reviewed in Kom & Hammer 1 988). Evidence implicating cytoplasmic myosin in cytokinesis was obtained by localization of the protein to the cleavage furrow of HeLa cells (Fujiwara & Pollard 1978) and by analysis of the effect of antibodies on cell division in starfish embryos (Mabuchi & Okuno 1977; Kiehart et al 1 982). Injection of anti-myosin antibodies into starfish blastomeres prevented cell division by inhibiting cleavage furrow formation while blastomeres injected with buffer continued normal division. The complete amino acid sequences of several cytoplasmic myosins have been obtained through molecular cloning and DNA sequence analysis (for review , see Warrick & Spudich 1987). Comparison with skeletal myosin sequences shows a high degree of sequence similarity in the motor domain. The remainder of the protein sequences diverge from those of skeletal muscle myosins, but retain sequence elements characteristic of ex-helical coiled coil formation.

Myosin I Myosin I was first identified in a search for cytoplasmic myosins in Acanthamoeba castellanii (Pollard & Kom 1 973) as a protein with high + ATPase activity in the presence of EDTA and K , as is typical of muscle myosins . However, the Mr of - 1 40K of the protein was strikingly low compared with known myosin heavy chains. In addition to its unexpectedly low relative mass, the Acanthamoeba protein failed to form thick filaments as was characteristic of known myosins . However, the protein was classified as a myosin because of its actin-stimulated Mg-ATPase activity and ability


MOLECULAR MOTORS

33

to crosslink actin filaments (Kom 1 99 1 ) . Further studies have established the Acanthamoeba protein as a myosin based on its ability to generate ATP-de pendent movement along actin filaments in vitro (Albanesi et al 1 985) . An ATP-extractable actin-binding protein of Mr 1 10K was subsequently identified in microvilli of chicken intestine epithelial cells (Matsudaira & Burgess 1979). Biochemical studies have shown that this protein has myosin-like ATPase properties (Collins & Borysenko 1984) and can trans locate along actin filaments in vitro (Mooseker & Coleman 1 989; Collins et al 1 990). The biochemical and cell biological characterization of the Acanthamoeba and intestine brush border myosin proteins have led to the recognition of a new class of myosins that are structurally and functionally distinct from the muscle and cytoplasmic myosin II proteins. The name myosin I has been given to this class of myosins to denote the native state of the proteins as heavy chain monomers, in contrast to the known muscle and cytoplasmic myosins that exist as heavy chain dimers . KINES IN An activity that was thought to form the basis of fast axonal transport was first observed in giant axons of the squid (Allen et a1 1 982; Brady et aI 1982). Further studies indicated that the activity supported movement of organelles and vesicles along microtubules and was ATP-dependent (Allen et al 1 985; Vale et a1 1985c, Schnapp et al 1985). A nonhydrolyzable analogue of ATP, AMP-PNP, was found to inhibit vesicle transport in extruded squid axoplasm by stabilizing vesicle/ATPase/microtubule complexes (Lasek & Brady 1 985). This provided the basis for the biochemical purification of a protein from squid giant axons and optic lobes that could translocate along microtubules (Vale et aI 1985a). The protein, kinesin , was purified using complex formation with microtubules in the presence of AMP-PNP as a primary step, followed by column chromatography, assaying fractions for their ability to support ATP-dependent microtubule gliding on glass surfaces or in solution (Vale et al 1985a). Motility experiments have demonstrated that kinesin translocates in vitro only toward microtubule plus, or fast polymerizing/depolymerizing , ends (Vale et al 1 985b). Its direction of translocation, velocity, sensitivity to ATPase inhibitors , and heavy chain M r of - 1 1OK distinguish kinesin from the dynein microtubule motors . In its native form, kinesin is a tetramer consisting of two heavy and two light chains (Kuznetsov et al 1988) . Molecular cloning and DNA sequence analysis of Drosophila kinesin heavy chain (Yang et a1 1989) have resulted in the identification of a consensus ATP-binding sequence adjacent to a microtubule-binding region in the heavy chain. Expression of this portion of the heavy chain in bacteria as a kinesin/spectrin chimeric protein results in

34

ENDOW & TITUS

an activity that can support gliding of microtubules in vitro (Yang et al 1 990) . This experiment identified the force-generating domain of the kinesin heavy chain. DYNEINS


Axonemal Dynein Dynein was first identified in Tetrahymena as two proteins with sedimentation coefficients of 30S and 14S (Gibbons & Rowe 1 965) . Although the 14S species was initially attributed to 30S degradation, the two proteins are now known to be distinct microtubule-stimulated ATPases (Porter & Johnson 1 983) with probable locations on the outer and inner axoneme arms , respectively (Crossley et aI 1 99 1 ) . The larger dynein species consists of three globular regions (Johnson & Wall 1 983) corresponding to three distinct heavy chains of Mr 400-500K. Other ciliary or flagellar dyneins consist either of two or three heavy chains with associated protein subunits that vary in number and size (reviewed in Witman 1989) . Like the myosins and kinesin , the motor properties of the dyneins reside in the heavy chains (Sale & Fox 1 988) . The large size of the heavy chains has caused the molecular analysis of the dyneins to lag' behind that of the myosins and kinesin. However, the complete sequence of the 13 heavy chain of axonemal dynein from two sea urchin species, Anthocidaris crassispina (Ogawa 199 1 ) and Tripneustes gratilla (Gibbons et al 1 99 1 ) , has recently been reported, with the surprising finding of multiple nucleotide-binding consensus sequences. Only one of the five potential nucleotide-binding sites is likely to correspond to the force-generat ing ATP hydrolysis site. This site has been assigned based on its conformity to amino acid sequences present in known A TPases and its cleavage by UV irradiation in the presence of MgADP and vanadate. The other potential ATP-binding sites may help to regulate the activity of the protein. Outside of the ATP-binding consensus sequences there is no significant similarity between the predicted axonemal dynein and kinesin or myosin heavy chain sequences , which indicates that these motor proteins are probably of indepen dent origin.

Cytoplasmic Dynein The first report of a soluble or cytoplasmic dynein was by Weisenberg & Taylor ( 1 968), who discovered the activity in sea urchin eggs in a search for an ATPase involved in mitotic spindle function. Subsequent work led to the purification of a sea urchin egg ATPase that was similar, but not identical, to axonemal dynein (reviewed in Pratt 1 989) . The possibility that the sea urchin egg dynein might be a precursor of ciliary dynein forestalled its acceptance as a cytoplasmic dynein. Reports of a dynein-like protein associated with


MOLECULAR MOTORS

35

microtubules in vertebrate brain (Bums & Pollard 1 974; Gaskin et al 1 974) , however, again raised the possibility that a cytoplasmic form of dynein might exist. A tubulin-associated , high molecular mass ATPase was purified from bovine brain (Pallini et al 1 982) with sensitivity to the ATPase inhibitors vanadate and EHNA {erythro-9[3-(2-hydroxynonyl)]adenine}, like that re ported for axonemal and sea urchin egg dynein (Pallini et al 1 983) . The microtubule-associated protein, MAP l C , from bovine brain was subsequently demonstrated to be a microtubule-activated ATPase that translocates toward the minus ends of microtubules (Paschal et al 1987; Paschal & Vallee 1987). Based on its electrophoretic mobility relative to MAP 2, the protein purified by Pallini et al ( 1982) probably corresponds to MAP I C . If not, it may represent another cytoplasmic dynein isoform. The cellular role of cytoplasmic dynein has not yet been established, although the protein was initially suspected to have a role in retrograde axonal transport (Vallee et al 1 989) . The localization of cytoplasmic dynein to spindles and kinetochores in dividing avian and mammalian cultured cells (Pfarr et al 1 990; Steuer et al 1 990; Wordeman et al 1 99 1 ) and its trans location toward microtubule minus ends in vitro suggest that the protein functions as a spindle motor and as a chromosome-associated mitotic motor involved in poleward chromosome movement. These results, together with increasing evidence that the forces that drive movement of chromosomes toward spindle poles in meiosis and mitosis are present at the kinetochore (Gorbsky et al 1987; Nicklas 1989; Hyman & Mitchison 1991), have generated intense interest in determining the role of cytoplasmic dynein in chromosome distribution. DISCOVERY OF NEW FAMILY MEMBERS Myosins Molecular analysis of genes with diverse mutant phenotypes has led to the unexpected discovery of proteins related to the previously characterized myosin I and myosin II proteins, but that differ markedly in structure and role in the cell. The new myosin proteins contain ATP-binding consensus sequences and other conserved motor domain sequences, like previously identified myosins, but are otherwise dissimilar in predicted sequence. One of the most unusual new myosins discovered is a protein encoded by the ninaC gene of Drosophila. Mutants of ninaC (!!either inactivation !!or �fterpotential {;;) are defective in photoreception and have abnormal electroretinogram (ERG) recordings, reduced rhabdomere size, and lower rhodopsin content of photoreceptor cells (Matsumoto et al 1987). The rhabdomere is a specialized part of the photoreceptor cell consisting of denselYI DROSOPHILA NINAC


36

ENDOW & TITUS

packed arrays of microvilli that contain cytoskeletal elements , rhodopsin, and other proteins involved in phototransduction . The reduced rhabdomere size appears to result from light- and age-dependent retinal degeneration (Porter et al 1 992)'1 The ninaC gene was identified by P element-mediated rescue of mutants with DNA sequences that mapped to the cytological location of ninaC and were specifically expressed in the eye (Montell & Rubin 1988). Molecular analysis revealed the ninaC gene to encode two proteins of 1 32 and 1 74 kd that differ only in their C terminal 54 and 420 amino acids, respectively. The two proteins arise by alternative usage of AAUAAA sequences required for processing of 3' ends of mRNAs. Sequences encoding the p l 32 C terminus are removed as an intron from the p 174 transcript, which contains additional exons at its 3' end. Comparison of the deduced ninaC protein sequences with sequences of ' known proteins revealed adjacent regions of homology to protein kinases and myosins. An N terminal 266 residue region contains sequences invariant among known serine/threonine, tyrosine, and other protein kinases. The central 725 residue region of the ninaC proteins is homologous to the force-generating domain of myosin (Figure 1) and includes a consensus ATP-binding sequence and predicted actin-binding region. The unique C termini of ninaC p 1 32 and

myosin

II

p190/dilute myosin

myosin

1

ninaC p174

HC

200 kDa

HC

215 kDa

HC

120 kDa

He

174 kDa

1 Structures of representative heavy chains (HC) of different classes of myosin. Structures are based on predictions from primary amino acid sequences (chicken p 190/mouse dilute, Dictyostelium myosin IB, Drosophila ninaC) and rotary-shadowed images (chicken p190) (R. E. Cheney & 1. Heuser, unpublished). Light chains are not shown, Monomer heavy chain relative molecular mass is indicated in kilodaltons (kDa), The conserved force-generating domain of each myosin heavy chain is shaded.

Figure


MOLECULAR MOTORS

37

p 1 74 do not share sequence similarity with each other or any known protein. However, the ninaC p 132 and p 174 C termini contain one or two repeats, respectively, of a -23 amino acid "IQ" motif, characteristic of calmodulin binding proteins (Cheney & Mooseker 1992), and both proteins have been shown capable of binding calmodulin in vitro (1. Porter & c. Montell, unpublished) . Analysis of P element transformants that express only p 132 or p 174 has shown that the phenotype of p 174 null mutants resembles the ninaC null phenotype, while p l32 null mutants appear wildtype (Porter et aI I992). Thus, p174 is the functionally essential form. Expression of the ninaC proteins is specific to the photoreceptor cells of the Drosophila compound eyes and visual organs (Montell & Rubin 1988). The p174 isoform has been localized to rhabdomeres, while p 132 is found in the extra-rhabdomeric cytoplasm of the photoreceptor cell body (Porter et al 1992). ninaC p174 has been proposed to function by phosphorylating, and thus deactivating, rhabdomere proteins involved in phototransduction as it moves along actin filaments present in the rhabdomere (Porter et al 1992). Although the ability of the ninaC proteins to translocate on actin filaments has not yet been demonstrated, the central position of the myosin-like force-generating domain in p 174 (Figure 1) and its location near the C terminus of p 132 distinguish the proteins from previously identified myosins. This difference in structure raises the possibility that motor properties of the ninaC proteins may be unlike those of known myosins. The gilute (d) mutants of the mouse show a lightening of their coat color compared to normal mice. The mutant coat color is associated with melanocytes that have fewer and thinner dendritic processes than normal. The abnormal melanocyte morphology is thought to cause an uneven release of pigment granules into the epidermal cells at the base of the hairshaft, which results in the mutant coat color (Markert & S ilvers 1956). The more severe gilute lethal (d I) mutants show a neurological defect characterized by limb convulsions and seizures, and death at 2-3 weeks of age. Molecular cloning of the d gene was facilitated by the finding that a murine leukemia virus (MuLV) is closely linked to d in the original d mutant, now known as dilute viral (d � (Jenkins et al 198 1) . The finding that reversion of d to d + is associated with loss of MuL V DNA sequences indicates that integration of the viral DNA is the probable basis of the d v mutation and allowed the molecular cloning of the d gene by recovery of viral and flanking DNA sequences (Copeland et al 1983) . DNA sequence analysis of d revealed a predicted protein of Mr 215K consisting of a region homologous to the myosin motor domain attached to a unique C terminus (Mercer et al 1991) (Figure 1). The putative dilute motor MOUSE DILUTE


38

ENDOW & TITUS

domain is more similar to cytoplasmic myosin II than myosin I proteins . Six tandem imperfect IQ repeats at the junction between the predicted force-gen erating domain and a region of a-helical coiled coil implicate the dilute protein in calmodulin binding (Mercer et al 199 1 ; Cheney & Mooseker 1 992) . The C terminus of the predicted dilute protein d�ffers from that of the myosin I and II proteins. The neurologic phenotype of most d mutants and the presence in mutants of melanocytes with fewer dendritic processes suggest a role for the dilute protein in forming or maintaining neuronal cell processes (Mercer et aI 199 1) . Such a role may involve axonal transport of particles or organelles and is likely to be specific to a subpopulation of neuronal cells, since most neurons of d I mice are dendritic (Mercer et al 199 1) . A protein with striking similarity to the mouse dilute protein has been found in chicken brain. Originally identified as a calmodulin-binding protein associated with brain actomyosin, chicken p 190 has been shown to possess 2+ Ca - and calmodulin-stimulated MgATPase activity (Larson et al 1990) and the ability to translocate along actin filaments in vitro (R. E. Cheney et aI, unpublished). The predicted p 190 sequence is 9 1 % identical along the length of the protein to mouse dilute, and anti-p 190 antibodies stain cultured melanocytes from normal but not d mice (Espreafico et al 1991) . Rotary shadowed images reveal that the native p190 protein is a dimer consisting of two globular regions like muscle myosin, a short stalk, and two terminal regions that do not form an extended rod, and do not associate with other dimers to form filaments (R. E. Cheney & J . Heuser, unpublished) . A temperature-sensitive mutant allele of a yeast gene that encodes a protein similar to mouse dilute and chicken p 190 was recovered in a screen for start-defective cell cycle mutants in Saccharomyces cerevisiae (Prendergast et al 1990) . At nonpermissive temperature , cells of the mutant, renamed myo2-66, accumulate as large unbudded cells with numerous vesicles and delocalized chitin, which is normally deposited at the site of bud emergence (Johnston et al 1991). The accumulation of intracellular vesicles in the myo2-66 mutant implies that My02 is involved in the transport of vesicles in budding cells. Shifting mutant cells to the nonpermissive temperature also causes a change in the distribution of actin, resulting in delocalization of cortical actin patches and reorganization of actin cables into bars. This suggests a role for the My02 protein in stabilization of actin cytoskeletal structures. Molecular cloning of MY02 was achieved by rescue of the myo2-66 temperature-sensitive cell cycle defect (Johnston et a1 199 1 ) . DNA sequence analysis revealed a protein that resembles mouse dilute and chicken p 190 in two distinctive features: the deduced My02 protein contains a myosin-like mechanochemical domain adjacent to a region with six IQ repeats (Cheney & Mooseker 1 992) . My02 shows 52% identity to mouse dilute and chicken p l 90 in its N terminal myosin-like motor domain, and 22-36%

MOLECULAR MOTORS

39

identity along the remaining length of the protein (Espreafico et al 1991; R . E. Cheney, personal communication). Co-localization of myosin I and actin to the leading edge of migrating cells (Fukui et al 1989) provided evidence that myosin I functions in cell locomotion in Dictyostelium and stimulated attempts to clone the gene. Molecular cloning experiments resulted in the surprising finding that Dictyostelium contains genes that encode more than one type of myosin I protein (lung et al 1989a; Titus et al 1989). 2 There are at least three genes that encode proteins (myoB , myoC , myoD) that are structurally similar to the Acanthamoeba myosin I proteins along their length (Hammer 199 1; M . A. Titus, unpublished) . Two further genes encode smaller proteins (myoA, myoE) (Titus et al 1989; Urrutia et al 1990) that contain myosin-like mechanochemical domains and typical myosin I mem brane-binding sites, but lack the GPA- (gly-pro-ala) rich region in the C terminus that corresponds to a second, ATP-insensitive actin-binding domain typical of amoeba myosin I proteins (for reviews, see Hammer 1991; Pollard et al 1991) . Dictyostelium is not unusual in having multiple myosin I genes since three myosin I genes have been found both in Acanthamoeba (lung et al 1989b) and rat (Sherr & Greene 199 1). Present evidence suggests that there may be as many as six myosin 1 genes in Acanthamoeba (Jung et al 1989b) . The genes that encode myosin I differ from genes for non-muscle myosin II proteins in that single cells have been found to express multiple myosin I proteins while different myosin II proteins are usually expressed in different cell types.


DICTYOSTEUUM MYOSIN I AND SMALL MYOSIN I PROTEINS

Kinesins For several years after its discovery kinesin was thought to be unique among the motor proteins in being the only member of its class , but the entry into the DNA database of the first kinesin heavy chain gene sequence (Yang et al 1989) provided the basis for the discovery of a family of related proteins. Proteins similar to kinesin heavy chain have been identified by molecular genetic analysis of mutants in several organisms. BIMC Among the first of the new kinesin proteins to be identified was the product of the bimC (Qlocked in mitosis g gene in Aspergillus nidulans. bimC4 was isolated as a temperature-sensitive mitotic mutant that is unable to complete nuclear division. The mutant shows normal duplication of spindle pole bodies at nonpermissive temperature, but the spindle pole bodies fail to 2The Dictyostelium myosin I protein names have been changed from abmA. abmB, abmC (Titus et al 1989) to myoA, etc.


40

ENDOW & TITUS

separate and form spindles (Enos & Morris 1990). Wildtype gene sequences were recovered by transformation of mutant cells with a plasmid library and rescue of the bimC4 temperature-sensitive phenotype . DNA sequence analysis revealed the predicted bimC protein to be highly similar to Drosophila kinesin heavy chain in its N terminus (Figure 2). An amino terminal region of 4 19 amino acid residues is 42% identical to kinesin heavy chain and corresponds to the predicted globular domain in kinesin that contains an ATP-binding consensus sequence and a region that can bind microtubules (Enos & Morris 1990) . The C terminal half of the bimC protein, however, shows no significant sequence similarity to kinesin heavy chain. Proteins encoded by two further genes, Schizosaccharomyces pombe cutl (Hagan & Yanagida 1990) and Xenopus laevis Eg5 (Le Guellec et al 1991), have also been found to be similar to kinesin heavy chain, but are more similar to bimC than to each other or other members of the kinesin family. The bimC, cut7 and Eg5 proteins form a subfamily of kinesin proteins that may be related in cellular function. Identification of cut7 was made by analysis of the cutl-446 mutant, a temperature-sensitive mutant that shows abnormal spindle formation at restrictive temperature. The observed abnormalities in spindle morphology are consistent with the interpretation that spindle pole body

kinesin HC

110 kDa

bimC

132 kDa

Kar3

84kDa

ned

78kDa

Figure 2 Structures of kinesin heavy chain (HC) and three kinesin proteins. Amino termini are to the left and carboxy termini to the right. Kinesin light chains are not shown. Structures of Aspergillus bime, S. cerevisiae Kar3, and Drosophila ncd are based on predictions of primary amino acid sequences. Predicted monomer relative molecular mass is indicated in kilodaltons (kDa). The conserved force-generating domains of each protein are shaded (reprinted from Endow 1991, with permission).


MOLECULAR MOTORS

41

duplication can occur, but normal sliding apart of microtubules for spindle formation is defective (Hagan & Yanagida 1 990), similar to the defect in bimC4. The N terminal 415 amino acid residues of the predicted cut7 protein are 39% identical to Drosophila kinesin heavy chain, but show 57% identity to the bimC N terminus. Similarly, the N terminal 358 residues of the predicted Xenopus Eg5 protein exhibit greater identity to bimC than to Drosophila kinesin heavy chain (Le Guellec et al 1991). Eg5, bimC, and cut7 contain further regions of similarity to one another along the length of the proteins (Le Guellec et al 1991 ), which suggests a common function for the proteins. Eg5 is the first of the vertebrate kinesin-related proteins to be reported. It was identified as an RNA that undergoes changes in adenylation during early Xenopus development, becoming adenylated in the mature egg, and dea denylated after fertilization. Eg5 RNA is not detectable in adult tissues, which suggests that it is only required during egg maturation or very early in embryonic development. The gene was isolated from a cDNA library prepared from unfertilized eggs in a differential screen for sequences that undergo changes in adenylation after fertilization. Although the deduced bimC and cut7 proteins were originally reported to lack a central region capable of coiled coil formation (Enos & Morris 1990; Hagan & Yanagida 1990), alignment with the Eg5 sequence reveals conserved residues, most of which are leucines , separated by six residues in the central regions of all three proteins (Le Guellec et al 1991). This seven-residue or heptad repeat pattern is reminiscent of the leucine repeat motif (Landschulz et al 1 988), which has been demonstrated to be a protein dimerization domain (Turner & Tjian 1989). The EgS protein is presumed to function in early mitosis, based on its similarity to bimC and cuO, and the presence of the Eg5 RNA in mature eggs but not adult tissues (Le Guellec et al 1 991). The second kinesin identified by sequence similarity to the Drosophila protein is the product of the Saccharomyces cerevisiae KAR3 gene. Mutants of Kar3 exhibit reduced efficiency of nuclear fusion during conjugation and are consequently kIDogamy-defective. The mutant kar3-1 has a semi-domi nant defect in karyogamy and also shows slow mitotic growth (Meluh & Rose 1990) . KAR3 was cloned by transformation of kar3-1 with a yeast plasmid library and rescue of the karyogamy defect (Meluh & Rose 1990) . Gene disruption to produce a null mutant showed kar3 null cells to be severely defective in karyogamy. Although KAR3 is non-essential for mitotic growth, cells of the null mutant grow slowly like kar3-1 cells, which indicates that KAR3 is required for normal growth. Cultures of the null mutant accumulate large, unbudded cells with a single nucleus and short spindle, consistent with a defect in spindle elongation (Meluh & Rose 1 990). DNA sequence analysis revealed a 328 residue region at the C terminus of

KAR3


42

ENDOW & TITUS

Kar3 with 39% identity to D rosophila kinesin heavy chain. The region of identity corresponds to the motor domain of kinesin and includes an ATP binding consensus sequence and a region that can bind microtubu1es. The remainder of the predicted Kar3 protein shows no significant similarity to kinesin heavy chain. The presence of the kinesin-like force-generating domain of Kar3 at the C terminus of the protein (Figure 2) distinguishes Kar3 from kinesin heavy chain, as well as from subsequently identified kinesin family members . The reversed structure suggests that motor properties of Kar3 may differ from those of kinesin heavy chain . Movement of haploid nuclei during karyogamy is mediated by microtubules that extend from the two spindle pole bodies and probably involves sliding of antiparallel microtubules. The role of the Kar3 protein may be to crosslink antiparallel microtubules and move toward the minus ends of microtubules, causing nuclei to move together (Meluh & Rose 1990). Motility experiments will be required to determine Kar3 motor properties and direction of translocation on microtubules. Study of a mutant that was known to affect both eyecolor and meiotic chromosome distribution in Drosophila led to the identification of a third kinesin family member. The mutant, ed'd C£lflret !1on fiisjunetional) (Lewis & Gencarella 1952) , has abnormal dark red eyecolor and also shows severe egg inviability, and produces offspring that are frequently nondisjunc tional, aneuploid, or somatic mosaics for the X and/or 4th chromosome. The d egg viability and defect in chromosome distribution in ea o are attributed to frequent nondisjunction and loss of chromosomes during meiosis , and chro mosome loss in the mitotic divisions of the early zygote, based on studies of the corresponding mutant in D. simulans (Sturtevant 1929) . Molecular cloning of the ea locus revealed the existence of two genes that were identified as the ned (flon !;.laret gisjunetional) chromosome segregation gene and the ea eyecolor gene based on P element-mediated rescue of the defect in chromo some distribution and the presence or absence of RNAs in mutants that affect d eyecolor, chromosome distribution, or both (Yamamoto et al 1989). ea n was found to be a null mutant both for ca and ned (Yamamoto et al 1989). DNA sequence analysis resulted in the finding that the ncd segregation protein is related to kinesin heavy chain (Endow et al 1990). The predicted ncd protein contains a 323 residue region in its C terminus that is 41 % identical to the motor domain of Drosophila kinesin heavy chain. The structure of the ned protein, with the mechanochemical region of the protein at its C terminus, is similar to Kar3 (Figure 2), but differs from kinesin heavy chain, bimC, and all other members of the kinesin family reported to date. Motility experiments using bacterially-expressed ncd protein showed an unexpected difference in motor properties compared with kinesin. In contrast to kinesin, the ncd motor protein translocates toward the minus ends of microtubules in in vitro assays

DROSOPHILA NCD


MOLECULAR MOTORS

43

(Walker et al 1990; McDonald et a11990) and rotates around the microtubule as it moves along it (Walker et al 1990) . Movement toward the minus ends of microtubules suggests a potential function for the protein in poleward chromosome movement during cell division. The discovery that ncd is homologous to kinesin heavy chain (Endow et al 1 990) prompted a comparison of the sequence of nod (no �istributive disjunction) to kinesin heavy chain (Zhang et al 1990), which resulted in the finding that nod too is a kinesin protein. Like mutants of ncd, nod causes nondisjunction and loss of chromosomes in meiosis. However, nod affects predominantly nonexchange chromosomes (Carpenter 1973), in contrast to ncd mutants, which cause nondisjunction and loss both of exchange and nonexchange chromosomes (Davis 1969). The observation that recombination can compensate for loss of nod function suggests that nod may act, like chiasmata, to hold chromosomes on the metaphase plate (Hatsumi & Endow 1992) . If nod is chromosome-associated, this predicts that nod moves toward the plus ends of microtubules, in the opposite direction as ncd. Motility experiments will be required to establish nod as a microtubule motor protein and to determine the direction of its movement on microtubules . The high' degree o f motor domain sequence conservation among newly identified kinesin proteins suggests the use of the polymerase chain reaction (PCR)to identify further kinesin family members. The first report of the successful,application of this approach (McDonald & Goldstein 1990) was in Drosophila with the recovery of DNA sequences that were identified as ned by sequence comparison, after the homology between ncd and kinesin had been discovered (Endow et al 1 990). The discovery of further kinesin family members' has permitted the redesign of PCR primers, leading to the recovery of sequences from Drosophila that may encode as many as six kinesin proteins (Stewart et al 199 1) . A somewhat different approach has been to hybridize gel-purified, PCR amplified DNA to polytene chromosomes of Drosophila and to map sites of hybridization (Endow & Hatsumi 1991). The number of hybridization sites provides an indication of the number of kinesin genes in the genome, and the cytological locations can provide clues to function when they correspond to map positions of known mutants. Amplification of sequences from cDNA libraries constructed from RNA of staged embryos or specific tissues provides information regarding the developmental time and tissue specificity of expression. The use of these methods resulted in the detection of more than 30 sites of hybridization in the Drosophila genome (Endow & Hatsumi 1991). The map positions of the amplified DNA sequences corresponded to sites encoding the three previously identified kinesin proteins-- kinesin heavy chain, ned, and nod. A fourth site of, hybridization in the zeste- white region resulted PCR SCREENS


44

ENDOW & TITUS

ncd, and nod. A fourth site of hybridization in the zeste- white region resulted in the discovery of a new kinesin protein that was initially thought to be the product of the lethal complementation group, 1(1 )zw4 [lethal(1) zeste- white 4], but now appears to be produced from an adjacent gene (B-: Williams & M. Goldberg, unpublished) . Sequences corresponding to a fifth site were partially analyzed and found to' contain amino acid sequence motifs charac teristic of the kinesin motor domain. Finally, four further sites of hybridization match those of DNAs that have been partially analyzed and shown potentially to encode kinesin proteins (Stewart et al 1991). Results of the PCR and in situ hybridization screens thus provide evidence that the Drosophila kinesin family of microtubule motor proteins is likely to be very large.

Dyneins The dyneins exist both as cytoplasmic and axonemal forms within a given organism. In the cilia and flagella that have been studied, multiple axonemal forms have been found. Sperm flagella of the sea urchins Strongylocentrotus purpuratus and Tripneustes gratilla contain an outer arm dynein consisting of two different heavy chains associated with intermediate and light chains and two or more inner arm dyneins that are dimers or trimers of four or five different heavy chains, respectively, with intermediate and light chains (reviewed in Sale et al 1989). The number of sea urchin axonemal dyneins and their heavy chain composition is comparable to that of Chlamydomonas axonemes. Chlamydomonas flagella have one outer arm dynein consisting of three distinct heavy chains and at least five inner arm dyneins formed from six different inner arm dynein heavy chains (reviewed in Witman 1992) . Although the relationship between the forms of dynein within an organism and the number of genes that encode them is not known, multiple dynein genes are likely to be required, based on the number of dyneins needed for cytoplasmic and axonemal function, and their heteromultimeric subunit composition. Evidence for multiple dynein heavy chain genes in the sea urchin genome has recently been obtained using PCR. Degenerate primers corresponding to sequences that flank the proposed force-generating ATP hydrolysis site in sea urchin axonemal dynein \3 heavy chain were used in reverse transcription experiments followed by PCR to amplify sequences from unfertilized egg and blastula RNA (Asai et al 1991) . Cloning and sequence analysis of PCR-am plified DNA resulted in the identification of four distinct dynein heavy chain genes, one of which corresponds to the previously sequenced gene for axonemal dynein 13 heavy chain. Parallel experiments in Drosophila have led to the identification of multiple gene sequences that potentially encode dynein heavy chains and map to different sites in the genome (T. Hays et aI, personal communication) . Further analysis of the sea urchin and Drosophila sequences

MOLECULAR MOTORS

45

should provide much needed information concerning the number of dynein heavy chain genes in eukaryotes, the relationship of the genes to one another, and the cellular functions of these proteins. Although several of the DNAs are expected to encode the multiple dynein heavy chains of the sea urchin and Drosophila axonemes, genes for cytoplasmic dynein heavy chain are also expected to be present. The number of cytoplasmic dyneins is not known, nor


are the cellular roles of these proteins, although evidence for a potential role as a kinetochore and/or spindle motor has been obtained, as noted previously. These studies are anticipated to provide important information regarding both the number and cellular functions of the dynein microtubule motor proteins.

MUTANT PHENOTYPES AND MOTOR PROTEIN INTERACTIONS The mutant phenotypes and genetic interactions of known or presumed motor proteins may help to establish the cellular functions of newly identified family members. Because of their diversity, discussion of mutant phenotypes is limited to a few examples for each motor protein family. These are summarized in Table 1, together with phenotypes of some of the mutants previously discussed.

Myosins MUSCLE MYOSIN MUTANTS

Mutants of muscle myosin have been studied in

C. elegans and D. melanogaster. There are two predominant muscle types in C. elegans. Body wall muscle is used for several species, including

locomotion, and pharyngeal muscle is used for ingestion of food. The major body wall myosin heavy chain is defective in

unc-54 mutants; however.

mutants of unc-54 are viable because of the existence of two separate heavy chain genes for pharyngeal myosin (Miller et al 1986). Pharyngeal myosin is presumed to be essential for feeding behavior and survival of individuals

198 1) . In contrast to the four myosin heavy chain genes of C. elegans (Miller et al 1986), the muscle myosins of D. melanogaster are

(MacLeod et al

encoded by a single heavy chain gene, and isoforms are generated by alternative RNA splicing (Bernstein et al

1986; Rozek & Davidson 1986) . (Muscle myosin

Differential use of the 29 exons present in the Drosophila Mhc

lleavy f.hain) gene results in muscle-specific heavy chain isoforms. Dominant, flightless mutants with defects in the Mhc gene have been identified and shown to be homozygous lethal. A second class of dominant, flightless, homozygous viable mutations has been found to affect exons used specifically in adult muscles (reviewed in Bernstein et al 1992) . The phenotypes both of C. elegans and Drosophila mutants can therefore reflect the loss o f function o f specific myosin heavy chain isoforms.

46 Table 1

ENDOW & TITUS Motor protein mutant phenotypes and proposed cellular roles

Motor protein

Mutant phenotype

Muscle myosi n HC

Uncoordinated; flightless

Muscle contraction

Cytoplasmic myosin

Multinucleate or polyploid

Cytokinesis

Proposed cellular role

HC, LC


M yosin I

Slower movement

Cell locomotion

ninaC

Abnormal ERG recordings

Phototransduction

dilute

Lighter coat color

Vesicle transport needed

for dendrite outgrowth

Kinesin HC

Embryonic or larval lethality, im paired movement; 1st mitotic

unc l 04

Vesicle accumulation, impaired motor function Spindle pole bodies fail to sepa rate, no bipolar spindles

Vesicle transport in neuronal cells

ned, nod

Meiotic chromosome nondisjunc

Chromosome distribution; spin

Kar3

tion and loss Karyogamy-defective

Movement of nuclei together

Axonal transport; mitosis (unc 1 1 6)

division defective

bimC (cut? , Cin8, urchin)

Spindle formation, spindle pole

function dle function (ncd) for fusion

Axonemal dynein

Paralyzed?

Flagellar motility

Cytoplasmic dynein

Lethal?

Mitotic chromosome distribution

Cellular functions of many of the newly discovered motor proteins are inferred from their mutant phenotypes. Examples are shown for each motor protein family. HC

=

heavy chain, ERG

=

electroretinogram.

CYTOPLASMIC MYOSIN MUTANTS The role of cytoplasmic myosin in forming the cleavage furrow during cytokinesis has been confirmed by examining Dictyostelium cells that are null mutants for the heavy chain (De Lozanne & Spudich 1 987; Manstein et al 1 989b). Heavy chain null cells fail to undergo cytokinesis and, because DNA synthesis is not impaired, the cells become large and multinucleate. Disruption of the yeast heavy chain gene similarly results in cells that are large and multinucleate (Watts et al 1 987). Reduced function of cytoplasmic myosin regulatory or essential light chains also causes a cytokinesis-defective phenotype. In Drosophila, the late larval lethal mutant sqh (spaghetti-squash) causes mitotically dividing cells to become extremely polyploid. The defect in sqh is attributed to reduced levels of cytoplasmic myosin regulatory light chain (Karess et al 1 99 1 ) . Dictyostel ium with reduced expression of cytoplasmic myosin essential light chain become large and multinucleate (Pollenz et al 1 992), indicating a defect in cytokinesis. The mutant phenotype of cells deficient for cytoplasmic myosin regulatory or essential light chain underscores the critical role of the light chains in protein function. This is also likely to be true for the intermediate and/or light chains associated with other motor proteins.


MOLECULAR MOTORS

47

Although biochemical and cell biological experiments suggested that cytoplasmic myosin most likely functions in cell locomotion, the genetic experiments that generated Dictyostelium cells deficient for cytoplasmic myosin heavy chain do not support this role. Myosin heavy chain null cells retain the ability to undergo chemotaxis in response to cAMP, although at reduced efficiency, and to extend and retract filopodia and pseudopodia (Spudich 1 989) . The present observations, therefore, indicate that cytoplasmic myosin is not essential for cell locomotion. Disruption of the myoB gene (Jung & Hammer 1 990) has provided information concerning the role of the protein in Dictyostelium motility. The myoB null cells ingest bacteria at slower rates, consistent with the localization of the myoB protein to the phagocytic cup (Fukui et al 1 989) , and move across surfaces with decreased velocity and increased frequencies of turning and formation of lateral pseudopodia (Wessels et al 1 99 1 ) . However, the myoB null cells exhibit a normal response to cAMP, which includes changes in the distribution of actin (Wessels et al 1 99 1 ) . The absence of a nonmotile null mutant phenotype, together with the existence of multiple myosin I genes , suggest overlapping gene function. Consistent with this possibility, the null phenotype of myoA appears to resemble that of myoB (M. A . Titus et aI, unpublished). Analysis of strains multiply deficient for the myosin I proteins, or introduction into cells of gene constructs that antagonize myosin I activity, is likely to be necessary to determine the roles of the proteins in the cell. MYOSIN I NULL MUTANTS

Melanocytes in the dilute mutants of the mouse have fewer and thinner dendrites than normal , which suggests a role for the dilute protein in transport of vesicles or intracellular particles that are needed to form or maintain neuronal cell processes. Other murine coat color mutants such as £eade!1 (In) and ashen (ash) similarly reduce the number and size of melanocyte processes, causing irregular distribution of pigment granules along the hairshaft (Markert & Silvers 1 956; Lane & Womack 1 979) . Migration of melanoblasts from the neural crest and the formation of melanin granules are apparently normal both in d and In (Markert & Silvers 1 956) . The mutant dsu (dilute suppressor) restores normal coat color to d, In and ash mice (Moore et al 1 988b). Partial or near complete suppression of the mutant effect on coat color is accompanied by the appearance of melanocytes that are partially or completely dendritic. The dsu mutant is thought to act by producing an altered product that can compensate for the defect in d, In or ash mice by interacting directly or indirectly with the proteins with which the mutant proteins normally interact (Moore et al 1 988b) . This mechanism of suppression differs from the extracopy suppression described below for

DILUTE MUTANTS

48

ENDOW & TITUS

and CIN9 (Figure 3). In the case of dsu, a mutated gene can suppress the effects of other mutant genes . Previously described suppressor mutations in Drosophila act at the level of transcription (e. g . see Parkhurst & Corces 1 985; Zachar et al 1 985) . This does not appear to be true for dsu, since dsu can suppress a null allele of dilute that is deleted for d DNA sequences, and d RNA is absent both in suppressed and nonsuppressed mice (Moore et al 1 988a). The dsu mutation acts specifically to compensate for the loss of function of d, In and ash and fails to suppress the abnormal coat color of other mutants that affect pigment deposition, but not melanocyte morphology. The observations that d, In and ash cause similar effects on melanocyte morphology and that all three can be suppressed by dsu suggest that the genes share a common function. Analysis of the In, ash and dsu genes may reveal a family of proteins that are functionally similar to d. Differentiation of melanocytes and pigmentation of the hair involve a series of developmental steps that includes migration of melanoblasts from the neural crest, differentiation of melanoblasts into melanocytes , and pigment granule formation and release into epithelial cells at the base of the hairshaft. These processes offer the possibility of involvement of further myosins , which might be identified by careful selection of mutants for study among the large number of coat color mutants available in the mouse.


SMYI

Kinesins KINESIN HEAVY CHAIN MUTANTS Mutants that affect kinesin heavy chain have recently been recovered in D rosophila and C. elegans. Drosophila 8 carrying khc , an EMS-induced presumed null allele of kinesin heavy chain, together with a deficiency that covers the khc region, die as second or third instar larvae (Saxton et al 1 99 1 ) . Survival of the khc mutants to the larval stage is attributed to the nonessential function of kinesin in embryonic cells. The null mutant larvae are reduced in size and show slow growth, sluggish movement, and loss of posterior touch sensitivity. The effect of the null mutant on larval growth and sensitivity to touch is interpreted to mean that kinesin has an essential role in axonal transport in neuronal cells . Antibody staining experiments show that kinesin heavy chain is present in the cortical region of preblastoderm embryos and the apical cytoplasm of postblastoderm cells (Saxton et al 1991). The protein is uniformly distributed in the cellular cytoplasm of later stage embryos, but is not present in mitotic spindles or chromosomes . The presence of the protein in the cortical region of pre blastoderm embryos suggests a role for kinesin in localization of substances needed during embryogenesis, a possibility that has not been addressed by studies to date . The absence of antibody staining of chromosomes and spindles, and apparently normal early embryonic development of mutants, support its

MOLECULAR MOTORS

wildtype


dsu

mutant

49

suppressed mutant

8 o

� �

SMYl

e

Figure 3

Mechanisms of suppression. Genetic suppressors suppress the effects of mutations in unlinked genes via several different mechanisms, three of which are shown. Mouse dsu (dilute suppressor) encodes an altered protein that can replace the function of an unlinked mutant motor protein. Chlamydomonas sup prl (suppressor of paralyzedjlagella 1) encodes an altered (motor) protein that compensates for a structural change in an unlinked mutant nonmotor protein. Extra copies of S. cerevisiae SMY1 (suppressor of myosin 1) permit the wildtype motor protein to carry out the function of an unlinked motor protein. S. cerevisiae CIN9 (KIP I) acts as an extracopy suppressor in a manner similar to that of SMY1 . Not shown are suppressor mutations that act at the level of transcription. Wildtype proteins are denoted by + and nonfunctional proteins are crossed out.

nonessential role, if any , in mitotic chromosome movement or spindle function (Saxton et al 1 99 1 ) . This is consistent with the demonstration that kinesin localized to spindles of early sea urchin embryos is membrane- or vesicle-as sociated (Wright et al 1991) and may not function in mitosis. In C. eiegans, unc-1 16 encodes kinesin heavy chain (Patel & Mancillas, cited in Hall et al 1991). Mutants of unc- 1 1 6 show mild uncoordination to paralysis , and severe alleles result in early embryonic lethality. In contrast to 8 khc , which has no apparent effect on mitosis , lethality of unc-1 l 6 mutants is attributed to defects in the first cleavage division (Hall et al 1 99 1 ) . Defective divisions include failure of pronuclei to meet prior to the first cleavage division, abnormal positioning of the spindle, and the generation of cytoplasts or multinucleate cells. The unc- 1 1 6 protein may may be involved in proper positioning of the pronuclei, the spindle, or daughter nuclei at various stages of the first mitotic division, and differs from Drosophila khc in having


50

ENDOW & TITUS

an apparent role in mitosis . The unc- 1 1 6 protein is also required for normal axon outgrowth and secretory cell morphology. Unexpectedly, C. elegans contains a second kinesin protein with neuronal cell function. Mutants of unc-1 04, a kinesin family member (Otsuka et al 1 99 1 ) , exhibit a phenotype that differs from that of unc- 1 1 6 mutants. While synaptic vesicle populations are normal in unc- 1 16 mutants , unc- 104 mutants show the accumulation of numerous vesicles in the neuronal cell body (Hall & Hedgecock 1 99 1 ) . unc-1 04 mutants affect the function of many classes of neurons in C. elegans, which results in impairment of complex motor behaviors such as feeding and locomotion. Neuromuscular junctions are rare or absent in mutants, and few axonal vesicles are present. B ased on these observations and the assumption that axonal microtubules are oriented with plus ends away from the cell body , the unc- l04 protein is predicted to function as a neuron-specific , synaptic vesicle translocator that moves toward micro tubule plus ends (Hall & Hedgecock 1 99 1 ) . The finding i n C . elegans o f two kinesin proteins with distinct roles in neuronal transport raises the question of the number of such proteins required solely for this function. Cell-type specific proteins that carry out specialized transport functions could account for the multiplicity of possible kinesin genes detected in PCR-based screens (Endow & Hatsumi 1 99 1 ; Stewart et aI 1991). KINESIN MUTANTS THAT AFFECT CHROMOSOME DISTRIBUTION AND NUCLEAR

A surprising feature of the newly identified kinesin proteins is their involvement, based on their mutant phenotypes, in chromosome distri bution and nuclear function. Predicted roles include chromosome segregation (ncd, nod) , spindle pole function (bimC , cut7 , ncd), and nuclear fusion during karyogamy (Kar3) . These functions are related to the proposed roles of kinesin heavy chain in axonal transport and neuronal cell differentiation only in being microtubule-based. Mutants of ncd and nod show abnormal meiotic chromosome segregation in Drosophila oocytes and a maternal effect on chromosome distribution in the early embryo. B ecause meiosis is nonessential for viability, female Drosophila carrying these mutations are phenotypically normal . Null mutants of ned lay many eggs that fail to hatch and produce frequent aneuploids among their few offspring . The absence of mutant effect in male Drosophila carrying ned and nod mutations suggests that functionally similar proteins are present in males. The mutant pal (paternal !oss) shows loss specifically of paternally derived chromosomes in the early embryo (B aker 1 975) that parallels the loss of maternal chromosomes observed in early embryos of ncd mutants . This characteristic of the pal mutant suggests that the gene may encode a protein similar to ncd. Proteins with roles in mitotic chromosome distribution similar to those of ncd and nod may also exist, but mutations in these proteins are expected to be lethal. FUNCTION


MOLECULAR MOTORS

51

Evidence for the involvement of the ned microtubule motor protein in spindle function is based on the presence in mutant oocytes of broad, diffuse, or multipolar meiosis I spindles (Wald 1 936; Kimble & Church 1 983 ; Hatsumi & Endow 1 992) . The abnormal spindles in mutant oocytes and the localization of ned to the meiotic spindle (M. Hatsumi & S . A. Endow , unpublished) suggest a role for the protein in establishing or maintaining spindle poles during meiosis (Hatsumi & Endow 1 992) . A role in spindle pole function has also been proposed for the Aspergillus bimC and S. cerevisiae cut7 kinesin proteins. Spindle pole bodies in bimC and cut7 mutant cells duplicate but fail to separate, which results in failure of spindles to form and nuclei to divide. This causes bimC mutants to produce growth tubes containing a single large nucleus instead of the usual multiple nuclei (Enos '& Morris 1 990) . The spindle pole bodies in cut? mutant cells remain together, but nucleate microtubules that extend into the cytoplasm as two separate clusters. This forms a characteristic V-shaped spindle with the spindle pole bodies at the base of the V (Hagan & Yanagida 1990) . Although nuclear division is arrested, further incubation at the nonpermissive temperature results in the appearance of large cells with two sets of chromosomes. The phenotype of the urchin mutant in DrosphUa most closely resembles the phenotype of the mutant in the bimC gene of Aspergillus (P. G. Wilson & M. T. Fuller, personal communication) urchin causes high degrees of polyploidy in mitotically dividing tissue. Unlike the polyploidy observed in the previously discussed Drosophila sqh mutant, which has reduced levels of cytoplasmic myosin regulatory light chain (Karess et al 1 99 1 ) , the polyploidy in urchin arises in the absence of bipolar anaphase spindles (Wilson & Fuller 1 99 1 ) . urchin causes larval lethality when homozygous, thus indicating that normal function of the bimC-like urchin protein is essential for viability. The proposed role of Kar3 in nuclear fusion during karyogamy is based on the inefficient fusion of nuclei in mutants (Meluh & Rose 1 990) . A model in which sliding of antiparallel microtubules moves nuclei together for fusion predicts the involvement of a motor protein that moves toward microtubule minus ends; however, a model involving a plus end-directed motor has also been proposed (Meluh & Rose 1 990) . The determination of Kar3 motor properties will be of great interest because it will allow the formulation of models for nuclear movement during karyogamy that are based on the characteristics of a protein that is required for the process.

Dyneins Mutants with defects in dyneins have been described to date only in Chlamydomonas. In Chlamydomonas reinhardtii, analysis of the pf mutants was used to assign the multiple axonemal dyneins to specific structures of the axoneme. The pf mutants of Chlamydomonas were found among strains with short, J!aralyzed flagella recovered after treatment of wildtype cells with


52

ENDOW & TITUS

ultraviolet light (Lewin 1 954) , nitrosoguanidine , or methylmethanosulfate (Huang et al 1979) . Electron microscopic analysis showed that some of the pf mutants lacked the outer or inner arms of the peripheral microtubule doublets , known to be the site of the axonemal dyneins . The axonemal dyneins cause adjacent microtubule doublets to slide along one another, which results in the bending or beating of the flagella. The absence of arm structures in pf mutants was correlated with the absence of specific proteins of Mr > 300K that could be separated by gel electrophoresis. These high molecular mass proteins were also present in the subunits released upon salt extraction of axonemes. The subunits were identified as dyneins based on their ATPase activity (Piperno & Luck 1979). The presence or absence of the high Mr proteins in inner or outer arm pf mutants, together with careful biochemical analysis of the subunits released by salt extraction (Piperno & Luck 1 979; Pfister & Witman 1 984) , led to the identification of the three outer arm dynein ATPases. A similar combined genetic, cell biological , and biochemical approach has been used to identify as many as five inner arm dyneins present in Chlamydomonas flagella (reviewed in Witman 1 992). Further outer arm flagellar mutants were found subsequently , with a motile, but slow swimming, phenotype (Kamiya & Okamoto 1985; Mitchell & Rosenbaum 1 985) . A screen for such mutants (Kamiya 1 988) led to the recovery of 35 independent strains that represented ten complementation groups . Together with pf13 and pj22 , the mutants identify 1 2 genes involved in outer arm structure or assembly . The assumption that the pf and oda (Quter �ynein 9,rm) mutants affect single proteins, but cause the loss of entire outer or inner arms , gave rise to the idea that arm assembly requires the presence of specific component polypeptides that may or may not be dyneins. Among the oda mutants , oda4 is thought to affect the outer arm dynein f3 heavy chain, based on the failure of recombination experiments (cited in Luck & Piperno 1989) to separate oda4 and the suppressor mutation , SUPpfl . SUppfl (�pressor ofraralyzed.f}agella !) was recovered as a UV-induced revertant of the paralysis of pj24 (Huang et al 1982) . Analysis of the revertant strain revealed the presence of pj24 together with an unlinked suppressor mutation, designated suppfl . suppfl restores flagellar motility to pj24 and other pf mutants that are defective in axoneme radial spokes or central microtubule doublets, but not to mutants defective in outer or inner axoneme arms. Suppression of the paralyzed flagella phenotype occurs not by correcting the pf defect in axoneme structure, but by altering another component of the axoneme. The basis of suppfl suppression differs from that of the previously described dilute suppressor, which restores normal melanocyte morphology to the mutant (Figure 3 ) . The suppressor effect of suppd is correlated with a decrease in Mr of the outer arm dynein 13 heavy chain , consistent with suppression resulting from an internally deleted protein (Huang et al 1982) . Properties of the altered dynein heavy chain have not yet been described .


MOLECULAR MOTORS

53

DNA sequence analysis of the outer arm dynein heavy chain genes is underway in several laboratories (Wilkerson et al 199 1 ; Mitchell & Brown 199 1 ) . Sequence analysis of the !3 heavy chain gene and analysis of the oda4 and sUp pr l mutant alleles will be necessary to establish the nature of the mutations. If the mutants affect the protein, they should be useful in the analysis of 13 heavy chain function. The £Y, 13 , "I, and inner arm dynein heavy chain sequences are anticipated to yield information regarding structural features of the axonemal dyneins that may help to explain their relative roles in flagellar motility. Studies regarding function will be facilitated by the use of transfor mation in Chlamydomonas (e. g. see Mitchell & Kang 1 99 1 ) and aided by molecular analysis of the dynein intermediate and light chain genes. This should provide a start in unraveling the complexities of axonemal dynein function .

Genetic Evidence for Motor Protein Interactions Genetic evidence for motor protein interactions is limited at present, but even now suggests unexpected interactions between motor proteins . These interac tions provide an opportunity for designing genetic screens or tests to identify further proteins involved in a specific cellular process . The interactions that have been observed include extracopy suppression, synthetic lethality, extrage nie noncomplementation, and functional redundancy. These interactions are diagrammed in Figure 4 and discussed below. EXTRACOPY SUPPRESSION Unexpected evidence for the interaction between proteins of two different molecular motor families was provided by analysis of a multicopy suppressor of myo2-66 in S. cerevisiae. SMY1 (Iuppressor of lliYosin) was recovered as a high copy number suppressor of the large, unbudded, temperature-lethal phenotype of myo2--66 (Lillie & Brown 1992). DNA sequence analysis led to the surprising finding that SMYl encodes a protein of M r -74K with significant similarity in its N terminus to the force-generating domain of kinesin heavy chain. Although Smy l is the most divergent of the kinesin proteins described to date, alignment of the predicted Smyl amino acid sequence with similar regions in the previously discovered bime, cut7, Kar3 , ncd, and nod proteins reveals conservation of motifs characteristic of kinesins. Wildtype SMYl in multiple extrachromosomal copies suppresses the temperature lethality of myo2--66, which suggests that the Smy l protein can interact with , or substitute for, Myo2 (Figures 3, 4) . The possibility that Smyl and My02 interact or carry out a similar function is supported by the observation that spores that are doubly mutant for myo2--66 and a smyl null allele are nonviable at room temperature, even though the myo2-66 and smyl null single mutants can sporulate and grow normally at room temperature. This represents a case of synthetic lethality, in which the single mutants appear wildtype, but the double mutant is lethal (Figure 4) .

54

ENDOW & TITUS

Genetic interactions similar to those described for SMY1 and myo2-66 have been observed for the CIN9 and cin8 (chromosome instability) genes of S . cerevisiae (Saunders et a1 1 99 1 ; Hoyt etaI 1 992). Multiple temperature-sen sitive alleles of cin8 were recovered in a screen for mutants that showed

Phenotype


Genotype Extracopy

sis

Suppression

s s s sis s s s

Synthetic

+

mlm

+

m/m

Lethality

m l /m l

Extragenic

Noncomplementation

m 1 /+

Functional

+

Redundancy

m l /m l

Dominant

+

Negative

(Antimorph)

+

mutant

wildtype

m Um 1

wildtype

m21m2

wildtype

m2/m2

lethal

m U+

wildtype

m21+

wildtype

m2/+

mutant

m Um 1

wildtype

m21m2

wildtype

m2/m2

mutant

nul/I+

wildtype

mI+

mutant

Figure 4 Genetic interactions between motor proteins. The genetic interactions are depicted for diploid cells, however, the same principles apply to haploid cells. Extracopy suppression and synthetic lethality can be regarded as special cases of functional rcdundancy. A higher Icvel of functional redundancy would result in wildtype, rather than mutant, phenotype for the mllml +

m21m2 double mutant. m +

=

wildtype gene.

=

mutant gene,

S =

wildtype suppressor gene, null

=

null allele of gene,


MOLECULAR MOTORS

55

elevated frequencies of mitotic chromosome loss at 37°C (Hoyt et al 1990) . CIN9 was identified as a low copy extrachromosomal suppressor of cin8 (Hoyt et al 1 992) and was recovered independently as KIPl in a PCR screen for kinesin genes in yeast (Roof et aI 1 992). CIN8 and CIN9 both encode kinesin proteins that are more similar to the bimC subfamily than to other kinesin proteins (Hoyt et al 1992) . At nonpermissive temperature, spindle pole body duplication occurs in cin8 cells, but spindles fail to form and cells arrest at mitosis. The absence of mitotic spindles in cin8 mutants is attributed to the failure of spindle pole bodies to separate and form spindles, which parallels the defects in bimC4 and cut7-446. Consistent with this interpretation, the Cin8 protein has been localized to spindle microtubules, but is not present on cytoplasmic microtubules (Hoyt et al 1 992). The Cin8 and Cin9 proteins are postulated to act as plus-end microtubule motors that mediate separation of spindle pole bodies by causing antiparallel microtubules to slide along one another (Hoyt et al 1 992). The two proteins also act to maintain spindle pole separation. Extra copies of ClN9 can suppress the temperature-sensitive spindle defect of cinS mutants, which indicates that the Cin8 and Cin9 proteins overlap in function. A common function for the two proteins is also indicated by the observation that haploid cells that are null both for CIN8 and CIN9 are inviable at room temperature, although singly mutant cin8 or cin9 null cells are viable at room temperature. Thus, deletion of the single genes gives viable cells, but deletion of both genes results in a synthetic lethality like that observed for myo2-66 (ts) smyl null haploid cells. The genetic interactions between SMY1 and myo2 , and ClN9 and cinS, suggest the use of extracopy suppressor or synthetic lethal genetic screens to identify further motor proteins involved in a given process . Tests for these genetic interactions can also be used to assign roles to proteins with sequence similarity to known motor proteins, but of unknown function.

Most combinations of recessive mu tations give a nonmutant phenotype in heterozygotes, i . e. ml!+ , m2!+ cells are normal in phenotype where ml and m2 are mutations and + is the wildtype allele. However, certain combinations of recessive mutations cause a mutant phenotype in heterozygotes. The observation that recessive mutations in unlinked genes can give a mutant phenotype in heterozygotes is referred to as extragenic noncomp1ementation (Figure 4) (Regan & Fuller 1 988; Steams & B otstein 1 988) . Genetic screens for unlinked noncomplementing mutants of l3-tubulin mutants have resulted in the recovery of a-tubulin mutants (Steams & Botstein 1 988; Hays et al 1 989). Analysis of the newly recovered a-tubulin mutants has shown that unlinked noncomplementation is gene and allele specific , indicating that protein-protein interactions are likely to be EXTRAGENIC NONCOMPLEMENTATION

,


56

ENDOW & TITUS

affected. These observations suggest that unlinked noncomplementation may be a generally useful method for identifying interacting proteins. Tests for unlinked noncomplementation of mutants that affect complex processes may provide insight into the process. An example of such a process is chromosome distribution, which is likely to involve several microtubule motor proteins with functions in the spindle or kinetochore , or both (for review, see McIntosh & Pfarr 1 99 1 ) . Two Drosophila kinesin proteins, ncd and nod, have mutant phenotypes that implicate the wildtype proteins in meiotic chromosome distribution. The roles of the proteins in chromosome distribution have not yet been established. nod acts early in meiosis and affects primarily nonexchange chromosomes, while ncd may have a role in meiotic spindle pole function, as discussed previously. d Extragenic noncomplementation tests have been carried out for can , an ned null mutant (Yamamoto et al 1 989), and six nod mutants that are either known or presumed null (Knowles & Hawley 1 99 1 ) . Results of these tests show that the nod mutants fail to complement ed'd in double heterozygotes; d producing higher frequencies of nondisj unctional offspring than can /+ or nodl+ females (Knowles & Hawley 1 99 1 ) . However, noncomplementation of null mutations indicates a mutant effect resulting from insufficient product levels rather than an effect of allele-specific interactions (Steams & Botstein 1 988) . Evidence for protein-protein interactions is based on noncomplementa tion of missense mutations (Regan & Fuller 1 988; Steams & Botstein 1 988; Hays et al 1 989). The existence of specific interactions between ncd and nod has therefore not been addressed by the reported experiments and will require tests for noncomplementation of ned and nod missense mutations. Although the experiments with eand and nod mutants are not informative with regard to possible interactions of the ned and nod proteins, the use of unlinked noncomplementation as a genetic screen or test is a potentially powerful method of identifying interacting proteins. A further, as yet untested, possibility is that noncomplementation of unlinked null mutations could be used to screen for mutants that affect a specific process. Evidence now shows that the molecular motor proteins are encoded in eukaryotic genomes as large multigene families. The multiplicity of myosins, kinesins , or dyneins detected in a given organism suggests that some of these proteins have similar or overlapping functions . Mutations i n functionally redundant genes may not cause an observable phenotype because they are complemented by genes that encode functionally similar proteins (Figure 4). This has been found to be true of the myosin I genes of Dictyostelium and some of the kinesin genes of yeast, such as the previously discussed SMYl and C1N9 genes. In Dictyostelium , cytological evidence implicating myosin I in cell locoREDUNDANCY OF FUNCTION

MOLECULAR MOTORS

motion has been obtained. However, null mutants of

57

myoA or myoB , genes

that encode myosin I proteins, are unexpectedly motile, although the reduced

velocity of the null mutants supports a role for the proteins in cell movement (Wessels et al

that the

1 99 1 ; M. A. Titus et aI, unpublished) . Conclusive evidence Dictyostelium myosin I proteins function in cell locomotion may

require the use of antimorphic or "dominant negative" mutations that


antagonize the effect of the normal gene allele(s) and are therefore dominant in their action (Figure

4) (Muller 1 932; Herskowitz 1 987) . Examples of antimorphic or dominant negative mutations are the C. elegans unc-54(d) mutants that disrupt assembly of wildtype unc-54 protein in heterozygotes ,

& Anderson 1 988). DNA sequence analysis of 3 1 independent unc-54(d) mutations resulted in the striking finding that all 3 1 mutations map to the myosin motor

thus causing a dominant, muscle-defective phenotype (Bejsovec

domain and are missense mutations, most of which affect conserved residues

& 1 990). The mutations described in these studies can be used to direct the construction of dominant negative mutations in Dictyostelium myosin I genes for use in gene replacement experiments. Analysis of the

in the ATP-binding consensus sequence or actin-binding region (Bejsovec Anderson

mutants bearing these constructs may provide genetic evidence for the role of the myosin I proteins in cell locomotion .

FUTURE PROSPECTS Roles in the Cell The use of molecular genetics has led to the identification of a large number

of potential motor proteins, based on their sequence similarity to known motor proteins. Many of these potential motor proteins are of unknown function .

The expanding number o f these proteins suggests that some may b e involved in previously unrecognized roles in the cell . An area in which

increasing evidence suggests the involvement of

molecular motor proteins is the localization of morphogens in oocytes or eggs. The requirement for myosins or microtubule motors in localizing morphogenic determinants in early development has not yet been demonstrated. However, substantial evidence now exists that cytoskeletal elements play a role .

Microtubules appear to be required for proper localization of RNA during oogenesis in

bed (bieoid ) Drosophila (Pokrywka & Stephenson 199 1 5: �hTch

raises the possibility that microtubule motors are involved. The bed protein

is a determinant of anterior structures of the embryo. It is translated after

fertilization and forms an anterior-to-posterior gradient in early embryos as a

consequence of its earlier localization as RNA (Driever & Niisslein-Volhard 1988). Dependence on microtubules includes transport of bed RNA from its


58

ENDOW & TITUS

site of synthesis in nurse cells into the developing oocyte, localization at the anterior cortex of the oocyte, and maintenance of localization. Similar observations have been made for Xenopus Vgl RNA, a vegetally localized maternal RNA (Melton 1 987). The Vg l protein is a member of the TGF� family and may function in mesoderm induction (Yisraeli et al 1 990). Localization of Vgl RNA in Xenopus oocytes requires intact microtubules, while anchoring of the RNA at the vegetal cortex is dependent on micro filaments (Yisraeli et al 1990). In C. elegans, segregation of P granules to the posterior cortex of the one-cell zygote requires intact microfilaments (Hill & Strome 1988); however, in contrast to VgJ RNA, anchoring of P granules in place after segregation has occurred is not dependent on microfilaments . Morphogenic events that occur later in development or are inducible have also been shown to be dependent on intact cytoskeletal components and may require the activity of molecular motors. A well studied example is the change in retinal photoreceptor cell shape in teleost fish in response to changes in light. Although neither microtubules nor actin filaments are required for maintenance of the elongated or contracted state, elongation of cone cells in the dark and their contraction in response to light require intact microtubules and microfilaments , respectively (reviewed in Burnside 1 989). Cone cell cytoplasmic microtubules are oriented along the length of the cell and increase in total length during cone cell elongation. The sensitivity of elongation to ATPase inhibitors such as vanadate and EHNA in lysed cell model systems and the dependence of elongation on intact microtubules suggest the involve ment of a microtubule motor protein. In addition to possible roles in localizing early cytoplasmic determinants and mediating changes in cell shape, newly detected proteins related to the known molecular motors may function in cytoplasmic transport. Evidence that movement of organelles in squid axoplasm is cytochalasin-sensitive (Kuznetsov et al 1 992) suggests the involvement of myosin-like motors in axoplasmic organelle/vesicle transport. The velocity of the observed move ment corresponds to fast axonal transport, which raises the question of the relative roles of the myosins and kinesin in fast axonal transport in neuronal cells. The association of a Drosophila myosin I with particles present in the cortical cytoplasm in syncytial blastoderm embryos (Miller et al 1 99 1 ) suggests a possible role in particle transport or localization that may be essential for further development.

Mechanisms of Motor Protein Action One of the exciting prospects that has come with the expansion of the motor protein families is the possibility of understanding their mechanism of action. Substantial progress has been made in the development of in vitro motility assays (Kron & Spudich 1 986; Harada et al 1 987; Howard et al 1 989) , and


MOLECULAR MOTORS

59

application of laser technology has allowed the measurement of motor force generation in vivo (Ashkin et al 1990) and improved the precision of in vitro assays (Block et al 1990). But despite these advances, critical information is lacking. The missing information includes structural data and information concerning the changes in motor properties that occur with alterations in structure. The expression of the mechanochemical domain of kinesin in bacteria (Yang et al 1990) and myosin in Dictyostelium (Manstein et a1 1989a) now permits the purification of sufficient protein for structural studies using NMR (nuclear magnetic resonance) or X-ray diffraction. Structures determined in the presence of ATP or ADP may reveal changes in conformation that occur upon hydrolysis of ATP. Point mutations can be introduced into the ATP hydrolysis site or polymer-binding region, and proteins can be tested in vitro for changes in velocity and rate of ATP hydrolysis, as well as analyzed for structure. Changes in the motor domain may cause changes in the postulated weak and strong binding states of the motor (reviewed in Cooke 1986) , thereby altering the duration of the strongly bound state per ATP hydrolysis cycle (Uyeda et al 199 1) . Such alterations in the ATP hydrolysis or force cycle may provide important clues about the energy transduction mechanism, including the determination of motor directionality . Structural analysis of the force-generating domain of rabbit skeletal muscle myosin is underway (Rayment & Winkelmann 1984); however, the use of protein synthesized using expression systems offers the advantage of allowing mutations to be introduced into the protein, as noted above. Structural analysis of mutated proteins, together with in vitro motility assays and tests of function in vivo, should result in working models for motor protein function. The use of molecular genetics in combination with structural studies therefore offers the possibility of gaining much anticipated information regarding the mech anisms of motor function.

Regulation of Motor Protein Activity The observations that a presumed motor protein can complement the loss of function of a motor protein of a different family , as in the case of Smyl and Myo2, and that motor proteins of different families may be present on individual vesicles or organelles (Kuznestov et al 1992) suggest that regulation of motor protein activity will be important in understanding cellular transport. The presence of actin and microtubule networks throughout the cell and the bidirectional organelle movement seen on single microtubules (Schnapp et a1 1985) indicate that trafficking of motor proteins will be involved in the regulation of motor protein activity . Understanding this process is likely to require the imaginative use of available techniques of cell and molecular biology, and may benefit from the use of genetic methods to identify interacting components of the transport networks .

60

ENDOW & TITUS

CONCLUDING REMARKS


The use of classical , molecular, and cytogenetics has greatly expanded our knowledge of the molecular motor protein families. Its continued application promises to extend the limits of cell biology, biochemistry, and biophysics by providing information that will lead to the understanding of the cellular roles of molecular motor proteins and their mechanisms of action. AFfERWORD The writing of this review was guided by the recognition that, for the field of molecular motors, "genetics may have come to the rescue" (Sawin & Mitchison 1 990). ACKNOWLEDGMENTS

We thank R . Cheney, M . Goldberg, G . Langford, S . Lillie and S . Brown, C . Mantell, W . Saunders and A . Hoyt, T. Hays, P. Wilson and M . Fuller for communicating results prior to publication. K . Collins, S . Garrett, R . Joshi, G. Witman, and members of the Witman laboratory provided valuable comments on the manuscript. Special thanks to M . Langan for Figure 1 and the TIBS editorial office for a drawing of Figure 2. Supported by United S tates Public Health Service grants (GM3 1 279 & GM46225) to S . A.E. , and grants from the United States Public Health Service (GM46486) and American Cancer Society (CD495 & JFRA378) to M . A.T. Literature Cited A1banesi, J. P . , Fujisaki, H . , Hammer, J. A. III, Korn, E. D. , Jones, R. , et al. 1 985. Monomeric Acantharnoeba myosins I sup port movement in vitro. J. Bioi. Chern. 260:8649-52 Allen, R . D . , Metuzals, 1., Tasaki, l . , Brady , S. T . , Gilbert, S. P. 1 982. Fast axonal transport in squid giant axon. Science 2 1 8;

1 1 27-29 Allen, R. D . . Weiss. D. G . . Hayden, 1. H Brown, D . T . , Fujiwake, H . , et al. 1985.

. •

Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic trans port. J. Cell BioI. 100; 1 736-52 Asai, D. J . , Tang, W.-J. Y . , Ching, N. S . , Gibbons, 1. R . 1 99 1 . Cloning and sequencing of the ATP-binding domains of novel isoforms of sea urchin dynein. J. Cell BioI. 1 1 5(3, pI. 2);369a (Abstr.) Ashkin, A . , Schutze, K . , Dziedzic, J. M . , Euteneuer. U . , Schliwa, M . 1 990. Force

generation of organelle transport measured in vivo by an infrared laser trap. Nature 348;346-48 Baker, B . S. 1975. paternal loss (pal); a meiotic mutant in Drosophila melanogaster causing loss of paternal chromosomes. Ge netics 80:267-96 Bejsovec, A . , Anderson, P. 1 988. Myosin heavy-chain mutations that disrupt Caenorhabditis elegans thick filament as sembly . Genes Dev. 2 : \ 307-1 7 Bejsovec, A . , Anderson, P . 1990. Functions of the myosin ATP and actin binding sites are required for C. elegans thick filament assembly. Cell 60: 1 33-40 Bernstein, S . I . , Hansen, C. 1. , Becker, K . D . , Wassenberg, D . R . II, Roche, E . S . , et al. 1 986. Alternative RNA splicing gener ates transcripts encoding a thorax- specific isoform of Drosophila melanogaster myosin heavy chain. Mol. Cell. Bioi. 6;25 1 1-19 Bernstein, S . I., O'Donnell, P . T., Cripps , R . M . 1 992. Molecular genetic analysis of


MOLECULAR MOTORS muscle development, structure and function in Drosophila . /nt. Rev. Cytol. In press Block, S. M . , Goldstein, L. S . B . , Schnapp , B . 1. 1990. Bead movement by single kinesin molecules studied with optical tweezers. Nature 348:348-52 Brady, S. T. , Lasek, R. J . , Allen, R. D. 1982. Fast axonal transport in extruded axoplasm from squid giant axon. Science 2 1 8: 1 12931 Burns, R. G. , Pollard, T. D . 1974. A dynein-like protein from brain. FEBS Lett. 40:274-80 Burnside, B. 1989. Microtubule sliding and the generation of force for cell shape change. In Cell Movement, ed. F. D. Warner, J. R. McIntosh, 2: 1 69-89. New York:Liss. 478 pp. Carpenter, A . T. C. 1 97 3 . A meiotic mutant defective in distributive disjunction in Dro sophila melanogaster. Genetics 73:393428 Chen, M. S . , Obar, R. A . , Schroeder, C. C . , Austin, T . W. , Poodry, C. A . , e t al. 1 99 1 . Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature 35 1 :583-86 Cheney , R. E . , Mooseker, M. S. 1992. Un conventional myosins. Curro Opin. Cell Bioi. 4:27-35 Collins, 1. H . , Borysenko , C. W. 1984. The I IO,OOO-dalton actin- and calmodulin-bind ing protein from intestinal brush border is a myosin-like ATPase . 1 . Bioi. Chem. 259: 1 4 1 28-35 Collins, K . , Sellers, J . R., Matsudaira, P. 1990. Calmodulin dissociation regulates brush bor der myosin I ( I lO-kD-calmodulin) mech anochemical activity in vitro. 1. Cell Bioi. 1 10 : 1 1 3 7-47 Cooke, R. 1 986. The mechanism of muscle contraction. CRC Crit. Rev. Biochem. 2 1 : 53- l l 8 Copeland, N . G . , Hutchinson, K . W . , Jenkins, N. A. 1983. Excision of the DBA ecotropic provirus in dilute coat-color revertants of mice occurs by homologous recombination involving the viral LTRs. Cell 33:379-87 Crossley, E. M . . Hyman, S. C . , Wells. C . 1 99 1 . Axonemal dynein from Tetrahymena. J. Cell Sci. (Suppl.) 1 4: 1 17-20 Davis, D. G. 1969. Chromosome behavior under the influence of claret-nondisjunc tional in Drosophila melanogaster. Genetics 6 1 :577-94 De Lozanne, A. , Spudich, J. A. 1 987. Dis ruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science 236: 1 086-9 1 Driever, W . , Niisslein-Volhard, C. 1988 . The bicoid protein determines position in the Drosophila embryo in a concentration-de pendent manner. Cell 54:95-104 Endow, S. A. 199 1 . The emerging kinesin

61

family o f microtubule motor proteins. Trends Biochem . Sci. 16:221-25 Endow, S. A . , Hatsumi, M. 199 1 . A multi member kinesin gene family in Drosophila . Proc. Nat!. Acad. Sci. USA 88:4424-27 Endow, S . A . , Henikoff, S . , Soler-Niedziela, L. 1 990. Mediation of meiotic and early mitotic chromosome segregation in Dro sophila by a protein related to kinesin. Nature 345:8 1-83 Enos, A . P . , Morris, N. R . 1990. Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans. Cell 60: 1 0 1 9-27 Espreafico , E. M . , Cheney, R. E . , Matteoli, M., Nascimento, A . A. c., De Camilli, P. V . , et al. 1 99 1 . Chicken brain p 1 90: a member of a new class of unconventional myosins that includes its mouse homolog, dilute. and the MY02 gene product of yeast. 1. Cell Bioi. 1 1 5(3 , pt. 2):332a (Abstr.) Fujiwara, K . , Pollard, T. D. 1978. Simultan eous localization of myosin and tubulin in human tissue culture cells by double anti body staining . 1. Cell Bioi. 77: 1 82-95 Fukui , Y . , Lynch , T. 1 . , Brzeska, H . , Korn, E. D. 1989. Myosin I is located at the leading edges of locomoting Dictyostelium amoebae. Nature 34 1 : 328-3 1 Gaskin, F. , Kramer, S. B . , Cantor, C. R . , Adelstein, R . S . , Shelanski, M . L . 1974. A dynein-like protein associated with neuro tubules. FEBS Lett. 40:28 1-86 Gibbons, I. R . , Gibbons, B. H . , Mocz, G . , Asai, D . I . 1 99 1 . Multiple nucleotide binding sites in the sequence of dynein J3 heavy chain. Nature 352:640-43 Gibbons , I. R . , Rowe , A. 1965. Dynein: a protein with adenosine triphosphatase ac tivity from cilia. Science 149:424-26 Gorbsky , G. 1 . , Sammak, P. J . , Borisy , G. G . 1 987. Chromosomes move poleward i n anaphase along stationary microtubules that coordinately disassemble from their kin etochore ends. J. Cell Bioi. 104:9- 1 8 Hagan, I . , Yanagida, M . 1 990. Novel potential mitotic motor protein encoded by the fission + yeast cut7 gene. Nature 347:563-66 Hall, D. H . , Hedgecock, E. M . 1 99 1 . Kinesin related gene unc-/04 is required for axonal transport of synaptic vesicles in C. elegans. Cell 65:837-47 Hall, D. H . , Plenefisch, J . , Hedgecock, E. M . 1 99 1 . Ultrastructural abnormalities of kin esin mutant unc- 1 1 6 . J. Cell BioI. 1 1 5(3 , pI. 2):389a (Abstr . ) Hammer, 1. A. III 1 99 1 . Novel myosins. Trends Cell BioI. I :S�56 Harada. Y . , Noguchi , A . , Kishino, A . , Yan agida, T. 1987. S liding movement of single actin filaments on one-headed myosin fila ments . Nature 326:805-8

62

ENDOW & TITUS

Hatsumi , M . , Endow, S. A. 1992. Mutants of the microtubule motor protein, nonclaret disjunction�l, affect spindle structure and chromosome movement in meiosis and mi tosis. J. ell Sci. 1 0 1 :547-59 Hays, T. S . , Deuring, R. , Robertson, B . , Prout, / M . , Fuller, M. T. 1989. Interacting proteins identifi�d by genetic interactions: a missense mutation in (l-tubulin fails to complement alleles of the testis-specific j3-tubulin gene of rosophila melanogaster. Mol. Cell. Biol! 9:875-84 6 Hersk witz, I. 1987. Functional inactivation of / enes by dominant negative mutations. Nature 329:2 1 9-22 Hil\! D. P . , Strome, S . 1 988. An analysis of the role of microfilaments in the estab lishment and maintenance of asymmetry in Caenorhabditis elegans zygotes. Dey. BioI. 125:75-84 Howard, J . , Hudspeth, A. J . , Vale, R. D. 1 989 . Movement of microtubules by single kinesin molecules. Nature 342: 1 54-5 8 Hoyt, M: A . , He, L . , Loo, K. K . , Saunders, W. S. 1 992 . Two S. cereyisiae kinesin-re lated gene products required for mitotic spindle assembly. 1. Cell Bioi. 1 1 8 : 1 09-20 Hoyt, M . A . , Stearns, T . , Botstein, D. 1990. Chromosome instability mutants of Sac charomyces cerevisiae that are defective in microtubule-mediated processes. Mol.

F


I1

g

Cell. Bioi. 10:223-34

Huang, B . , Piperno, G . , Luck, D. J. L. 1979. Paralyzed flagella mutants of Chlamydom onas reinhardtii defective for axonemal doublet microtubule arms. 1. BioI. Chem. 254:3091-99 Huang, B . , Ramanis, Z . , Luck, D. 1. L. 1982. Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for flagellar function. Cell 28: 1 1 5-24 Hyman, A. A . , Mitchison, T. J. 199 1 . Two different microtubule-based motor activities with opposite polarities in kinetochores. Nature 351 :206-1 1 Jenkins, N . A . , Copeland, N . G . , Taylor, B . A . , Lee, B . K . 1 98 1 . Dilute (d) coat colour mutation of DBA/2J mice is asso ciated with the site of integration of an ecotropic MuLV genome . Nature 293:37074 Johnson, K. A . , Wall, J. S. 1983. Structure and molecular weight of the dynein ATPase. 1. Cell Bioi. 96:669-78 Johnston, G. C . , Prendergast, J. A . , Singer, R. A. 1 99 1 . The Saccharomyces cerevisiae MY02 gene encodes an essential myosin for vectorial transport of vesicles. 1. Cell Bioi. 1 1 3 :539-51 Jung, G . , Hammer, J. A. III 1990. Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform.

1. Cell Bioi. 1 10: 1 955-64

Jung, G . , Saxe, C. L. III, Kimmel, A. R . , Hammer, J . A . III 1989a. Dictyostelium discoideum contains a gene encoding a myosin I heavy chain. Proc. Natl. Acad. Sci. USA 86:61 86-90 Jung, G . , Schmidt, C. J . , Hammer, J . A. III 1 989b . Myosin I heavy-chain genes of Acanthamoeba castellanii: cloning of a second gene and evidence for the existence of a third isoform. Gene 82:269-80 Kamiya, R. 1988 . Mutations at twelve inde pendent loci result in absence of outer dynein arms in Chlamydomonas reinhardtii. 1. Cell Bioi. 107:2253-58 Kamiya, R . , Okamoto, M. 1985. A mutant of Chlamydomonas reinhardtii that lacks the flagellar outer dynein arm but can swim. J. Cell Sci. 74: 1 8 1-91 Karess, R. E . , Chang, X . , Edwards, K. A . , Kulkarni, S . , Aguilera, I . , et al. 1 99 1 . The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila. Cell 65: 1 177-89 Karn, J . , Brenner, S . , Barnett, L. 1 98 3 . Protein structural domains in the Caenorhabditis elegans unc-54 myosin heavy chain gene are not separated by introns. Proc. Natl. Acad. Sci. USA 80:4253-57 Kiehart, D. P . , Mabuchi, I . , Inoue, S. 1982 . Evidence that myosin does not contribute to force production in chromosome move ment. 1. Cell Bioi. 94: 1 65-78 Kimble, M . , Church, K. 1983. Meiosis and early cleavage in Drosophila melanogaster eggs: effects of the claret-non-disjunctional mutation. J. Cell Sci. 62:30 1-18 Knowles, B. A . , Hawley, R . S . 1 99 1 . Genetic analysis of microtubule motor proteins in Drosophila: a mutation at the ned . locus is a dominant enhancer of nod. Proc. Natl. Acad. Sci. USA 88:7 1 65-69 Korn, E. D. 1 99 1 . Acanthamoeba myosin I: past, present, and future. Curro Topics Membr. 38: 1 3-30 Korn, E . D . , Hammer, J . A . III 1988. Myosins of nonmuscle cells. Annu. Rev. Biophys . Biophys. Chem. 17:23-45 Kron, S. J . , Spudich, J. A. 1 986. Fluorescent actin filaments move on myosin fixed to a glass surface . Proc. Natl. Acad. Sci.

USA 83 :6272-76

Kuznetsov, S. A . , Langford, G. M . , Weiss , D. G. 1992. Actin-dependent organelle movement in squid axoplasm. Nature. 356:722-25 Kuznetsov, S. A . , Vaisberg, E. A . , Shanina, N. A . , Magretova, N. N . , Chernyak, V . Y . , e t a l . 1 9 8 8 . The quaternary structure of bovine brain kinesin. EMBO 1. 7:35356 Landschulz, W. H . , Johnson, P. F . , McKnight, S. L. 1 988. The leucine zipper: a hypo-


MOLECULAR MOTORS thetical structure common to a new class of DNA binding proteins. Science 240: 1 759--64 Lane, P. W . , Womack, J. E. 1979. Ashen, a new color mutation on chromosome 9 of the mouse. 1. Hered. 70: 1 33-35 Larson, R. E . , Espindola, F. S . , Espreafico, E. M. 1990. Calmodulin-binding proteins and calcium/calmodulin-regulated enzyme activities associated with brain actomyosin. 1. Neurochem . 54: 1 288-94 Lasek, R. J . , Brady, S . T. 1985. Attachment of transported vesicles to microtubules in axoplasm is facilitated by AMP-PNP. Nature 3 1 6:645--47 Le GueUec, R . , Pari s , J . , Couturier, A . , Roghi, C . , Philippe, M. 1 99 1 . Cloning by dif ferential screening of a Xenopus eDNA that encodes a kinesin-related protein. Mol. Cell. Bioi. 1 1 :3395-98 Lewin, R. A. 1954. Mutants of Chlamydo monas moewusii with i mpaired motility. J. Gen. Microbial. 1 1 :358-63 Lewis, E. B . , Genearella, W. 1 9 52 . Claret and non-disjunction in Drosophila mel anogaster. Genetics 37:600-1 (Abstr.) Lillie, S . H., Brown, S . S . 1 99 2 . Suppression of a myosin defect by a kinesin-related gene. Nature. 356:358-61 Luck, D. J . L . , Piperno , G. 1989. Dynein ann mutants of Chlamydomonas. In Cell Movement, ed. F. D. Warner, P. Satir, 1. R. Gibbons, 1 :49-60. New York:Liss. 337 pp. Mabuchi, I . , Okuno, M. 1977. The effect of myosin antibody on the division of star fish blastomeres. J. Cell Bioi. 74 : 2 5 1 63 MacLeod, A. R . , Karn, J . , Brenner, S. 1 98 1 . Molecular analysis of the Imc-54 myosin heavy-chain gene of Caenorhabditis ele gans. Nature 291 :386--90 MacLeod, A. R . , Waterston, R. H . , Brenner, S. 1977a. An internal deletion mutant of a myosin heavy chain in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 74: 5336--40 MacLeod, A. R . , Waterston, R. H . , Fishpool, R. M . , Brenner, S . 1977b. Identification of the structural gene for a myosin heavy chain in Caenorhabditis elegans. J. Mol. BioI. 1 1 4: 1 33--40 Manstein, D. J . , Ruppel, K. M . , Spudich, J. A . 1 989a. Expression and characterization of a function al myosin head fragment in Dictyostelium discoideum. Science 246: 656--5 8 Manstein, D. J . , Titus, M. A . , De Lozanne , A . , Spudich, J. A. 1 989b. Gene replace ment in Dictyostelium: generation of myosin null mutants. EMBO J. 8:923-32 Markert, C. L. , Silvers, W. K. 1 956. The effects of genotype and cell environment

63

on melanoblast differentiation in the house mouse. Genetics 4 1 :429--50 Matsudaira, P. T . , Burgess, D. R. 1 979. Iden tification and organization of the compon ents in the isolated microvillus cytoskeleton. 1. Cell Bioi. 83:667-73 Matsumoto, H . , Isono, K . , Pye, Q., Pak, W. L. 1987. Gene encoding cytoskeletal pro teins in Drosophila rhabdomeres. Proc. Natl. Acad. Sci. USA 84:985-89 McDonald, H. B . , Goldstein, L. S. B . 1990. Identification and characterization of a gene encoding a kinesin-like protein in Dro sophila. Cell 61 :99 1 - 1 000 McDonald, H. B . , Stewart, R. J . , Goldstein, L. S. B. 1990. The kinesin-Iike ncd protein of Drosophila is a minus end-directed micro tubule motor. Cell 63: 1 1 59-65 McIntosh, J. R . , Pfarr, C. M. 1 99 1 . Mitotic motors. J. Cell Bioi. 1 1 5:577-85 Melton, D. A. 1987. Translocation of a localized maternal mRNA to the vegetal pole of Xenopus oocytes. Nature 328:80-82 Me1uh, P. B . , Rose, M. D. 1990. KAR3, a kincsin-related gene required for yeast nu clear fusion. Cell 60; 1 029--41 Mercer, J. A . , Seperaek, P. K . , Strobel, M . c . , Copeland, N. G . , Jenkins, N . A. 1 99 1 . Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 349:709--1 3 Miller, D. M . , Stockdale, F . E . , Karn, J. 1 986. Immunological identification of the genes encoding the four myosin heavy chain isoforms of Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 83:2305-9 Miller, K. G . , McNally , J. G . , Mermall,V. 1 99 1 . In vitro activities and in vivo function of a myosin I from Drosophila. 1. Cell Biol. 1 15(3 , pt. 2):370a (Abstr.) Mitchell , D . R . , Brown, K . 1 99 1 . Sequence analysis of dynein heavy chain genes. J. Cell Bioi. 1 1 5(3, pt. 2): 167a (Abstr.) Mitchell, D. R . , Kang, Y. 1 99 1 . Identification of oda6 as a Chlamydomonas dynein mutant by rescue with the wild-type gene. 1. Cell Bioi. 1 1 3:835--42 Mitchell, D. R . , Rosenbaum, J. L. 1 985. A motile Chlamydomonas flagellar mutant that lacks outer dynein arms. J. Cell Bioi. 1 00: 1 228-34 Montell, C . , Rubin, G. M. 1988 . The Droso phila ninaC locus encodes two photo receptor cell specific proteins with domains homologous to protein kinases and the myosin heavy chain head. Cell 52:757-72 Moore, K. J . , Seperack, P. K . , Strobel, M . C . , Swing, D . A . , Copeland, N . G . , e t al. 1988a. Dilute suppressor dsu acts semi dominantly to suppress the coatdi&lor phe notype of a deletion mutant, d , of the murine dilute locus. Proc. Natl. Acad. Sci. USA 85:8 1 3 1-35


64

ENDOW & TITUS

Moore, K. J . , Swing, D. A . , Rinchik, E. M . , Mucenski, M. L. , Buchberg, A. M . , e t al. 1988b. The murine dilute suppressor gene dsu suppresses the coat-color phenotype of three pigment mutations that alter mel anocyte morphology, d, ash and In. Genetics I I 9:933-41 Mooseker, M. S . , Coleman, T. R. 1 989. The I IO-kD protein-calmodulin complex of the intestinal microvillus (brush border myosin I) is a mechanoenzyme . J. Cell BioI. 108:2395-400 Muller, H. J. 1 932 . Further studies on the nature and causes of gene mutations. Proc. 6th Int. Congr. Genet. 1 : 2 1 3-55 Nicklas, R. B . 1 989. The motor for poleward chromosome movement in anaphase is in or near the kinetochore . J. Cell Bioi. 1 09:2245-55 Obar, R . A . , Collins, C. A . , Hammarback, J . A . , S hpetner, H. S . , Vallee, R . B . 1 990. Molecular cloning of the microtubule-as sociated mechanochemical enzyme dyna min reveals homology with a new family of GTP-binding proteins. Nature 347:25661 Ogawa, K. 1 99 1 . Four ATP-binding sites in the midregion of the �heavy chain of dynein. Nature 352:643-45 Otsuka, A. 1 . , Jeyaprakash, A . , Garcia Aiioveros, J . , Tang, L. Z. , Fisk, G . , et al. 1 99 1 . The C . elegans unc-J04 gene encodes a putative kinesin heavy chain-like protein. Neuron 6: 1 1 3-22 Pallini, V. , Bugnoli , M . , Mencarelli, C . , Scap igliati, G. 1982. Biochemical properties of ciliary, flagellar and cytoplasmic dyneins. Symp . Soc. Exp . Bioi. 35:339-52 Pallini, V. , Mencarelli, C . , Bracci, L. , Contomi, M . , Ruggiero, P., et a!. 1983 . Cytoplasmic nucleoside-triphosphatases similar to axon emal dynein occur widely in different cell types. J. Submicrosc. Cytol. 1 5:229-35 Parkhurst, S. M . , Corces, V. O. 1985 . forked, gypsys, and suppressors in Drosophila. Cell 4 1 :429-37 Paschal, B . M . , Shpetner, H. S . , Vallee, R . B . 1 987 . MAP l C is a microtubule-act ivated ATPase which translocates micro tubules in vitro and has dynein-Iike properties. J. Cell BioI. 105: 1 273-82 Paschal, B. M . , Vallee, R. B. 1987. Retrograde transport by the microtubule-associated pro tein MAP Ie. Nature 330: 1 8 1-83 Pfarr, C. M . , Coue, M . , Grissom, P. M . , Hays, T. S . , Porter, M. E . , et al. 1 990. Cyto plasmic dynein is localized to kinetochores during mitosis . Nature 345:263-65 Pfister, K. K . , W itman , G. B . 1984. Sub fractionation of Chlamydomonas 18 S dy nein into two unique subunits containing ATPase activity. J. Bioi. Chem. 259:

1 2072-80

Piperno, G . , Luck, D. J. L. 1 979. Axonemal adenosine triphosphatases from flagella of Chlamydomonas reinhardtii. Purification of two dyneins. J. Bioi. Chern. 254:3084-90 Pokrywka. N . J . , Stephenson , E . C. 1 99 1 . Microtubules mediate the localization of bicoid RNA during Drosophila oogenesis. Development 1 1 3:55-66 Pollard, T. D . , Doberstein, S . K . , Zot, H . G . 1 99 1 . Myosin-I. Annu. Rev. Physiol. 53: 653-81 Pollard, T. D . , Korn, E. D. 1 973. Acanthamoeba myosin. I . Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. J. BioI. Chem. 248:4682-90 Pollenz, R. S . , Chen, T. L. L . , Trivinos-Lagos, L . , Chisholm, R. L. 1 992. The essential light chain is required for Dictyostelium myosin function in vitro and in vivo. Cell 69:951-62 Porter, J . , Hicks, 1. L . , Williams, D. S . , Montell, C . 1992. Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells. J. Cell Bioi. 1 16:683-93 Porter, M. E . , Johnson, K. A. 1983 . Char acterization of the ATP-sensitive binding of Tetrahymena 30 S dynein to bovine brai n microtubules. J. Bioi. Chem. 258: 6575-8 1 Pratt, M. M. 1989. Dyneins in sea urchin eggs and nerve tissue. See Burnside 1989, pp. 1 25-40 Prendergast, J. A . , Murray, L. E . , Rowley , A . , Carruthers, D. R . , Singer, R. A . , et al . 1990. Size selection identifies new genes that regulate Saccharomyces cerevisiae cell proliferation. Genetics 1 24:81-90 Rayment, I . , Winkelmann, D. A. 1 984. Crys tallization of myosin subfragment 1 . Proc.

Natl. Acad. Sci. USA 81 :4378-80 Regan, C. L . , Fuller, M. T. 1 988. Interacting genes that affect microtubule function: the nc2 allele of the haywire locus fails to complement mutations in the testis-specific l3-tubulin gene of Drosophila . Genes Dev. 2:82-92 Rozek, C. E . , Davidson, N. 1986. Differential processing of RNA transcribed from the single-copy Drosophila myosin heavy chain gene produces four mRNAs that encode two polypeptides. Proc. Natl. Acad. Sci. USA 83:21 28-32 Sale, W. S . . Fox, L. A. 1 988. Isolated l3-heavy chain subunit of dynein translocates micro tubules in vitro . J. Cell Bioi. 1 07 : 1 793-97 Sale, W. S . , Fox, L. A . , Milgram, S . L. 1 989. Composition and organization of the inner row dynein arms. See Luck, 1989, pp. 89-

102

Saunders , W. S . , He, L . , Hoyt, M. A. 1 99 1 . Role of two kinesin-like proteins i n the


MOLECULAR MOTORS separation of the mitotic spindle pole bodies of S. cerevisiae . J. Cell B ioi . 1 15(3, pt. 2):348a (Abstr.) Sawin, K . , Mitchison, T. 1990. Motoring in the spindle. Nature 345:22-23 Saxton, W. M . , Hicks , J . , Goldstein, L. S . B . , Raff, E. C . 1 99 1 . Kinesin heavy chain is essential for viability and neuromuscular functions in D rosophila , but mutants show no defects in mitosis. Cell 64: 1 093-102 Schnapp, B. J . , Vale, R. D . , Sheetz, M. P . , Reese, T. S . 1 985. Single m icrotubules from squid axoplasm support bidirectional movement of organelles. Cell 40:455-62 Sherr, E. H . , Greene, L. A. 1 99 1 . Detection and partial sequencing of novel myosin-I genes in mammalian nervous tissue. J. Cell Bioi. 1 1 5(3, pt. 2) :33 I a (Abstr . ) Shpetner, H. S . , Vallee, R. B. 1989. Iden tification of dynamin , a novel mechano chemical enzyme that mediates interactions between microtubules. Cell 59:421-32 Shpetner, H. S . , Vallee, R. B. 1992. Dynamin is a GTPase stimulated to high levels of activity by microtubules. Nature 355:73335 Spud ich , J. A. 1989. In pursuit of myosin function. Cell Regul. 1 : 1- 1 1 Stearns, T. , Botstein, D. 1988. Unlinked non complementation: isolation of new condi tional-lethal mutations in each of the tubulin genes of Saccharomyces cerevisiae. Gen etics 1 1 9:249-60 Steuer, E. R . , Wordeman, L . , Schroer , T. A . , Sheetz, M . P . 1 990. Localization of cyto plasmic dynein to mitotic sp indles and kinetochores. Nature 345:266-68 Stewart, R. J . , Pesavento, P. A . , Woerpel , D . N . , Goldstein, L. S . B . 1 99 1 . Identification and partial characterization of six new members of the kinesin superfamily in Drosophila . Proc. Natl. Acad. Sci. USA 88: 8470-74 Sturtevant, A. H. 1929. The claret mutant type of Drosophila simulans: a study of chromosome elimination and of ce ll-lineage . Z. Wiss. Zool. 1 35:323-56 Sutoh, K. 1983. Mapping of actin-binding sites on the heavy chain of myosin sub fragment 1 . Biochemistry 22: 1 579-85 Titus, M. A . , Warrick, H. M . , Spudich , J. A . 1989 . Multiple actin-based motor genes in Dictyostelium. Cell Regul. 1 :55-63 Toyoshima, Y. Y . , Miki-Noumura, T . , Shimizu , T . , Kokubu, T. , Hisanaga, S . , e t al. 1 99 1 . Dynamin is a microtubule pl us end-directed motor. J. Cell Bioi. 1 1 5(3, pt. 2) :35a (Abstr.) Turner, R., Tj ian, R . 1 989. Leucine repe ats and an adjacent DNA binding domain med iate the formation of functional cFos-c1un heterodimers. Science 243:1689-94 Urrutia, R. A . , Jung, G . , Hammer, J. A . III

65

1990. Cloning and characterization of the Dictyostelium myosin IE gene. J. Cell B ioi . 1 1 1 (5 , pt. 2): 1 68a (Abstr.) Uyeda, T. Q. P . , Warrick, H. M . , Kron, S .

J . , Spudich, J . A . 1 99 1 . Quantized velocities at low myosin densities in an in vitro motility assay. Nature 352:307-1 1 Vale, R. D . , Reese, T. S . , Sheetz, M. P. 1985a. Identification of a novel force-gen erating protein, kinesin, involved in mi crotubule-based moti l i t y . Cell 4 2 : 3950 Vale, R . D . , S chnapp , B. J. , Mitchison, T. , S teuer , E. , Reese, T. S . , et al. 1 985b. Different axoplasmic proteins generate movement in opposite directions along mi crotubules in vitro. Cell 43:623-32 Vale, R. D . , Schnapp , B . J . , Reese, T. S . , Sheetz, M. P . 1985c. Movement of organ elles along filaments dissociated from the axoplasm of the squid giant axon. Cell 40:449-54 Vallee, R. B . , Shpetner, H. S . , Paschal , B . M . 1989. The role of dynein in retrograde axonal transport. Trends Neuro. Sci. 1 2:6670 van der Bliek, A. M . , Meyerowitz, E. M . 199 1 . Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic . Nature 35 1 :4 1 1 - 1 4 Wald, H . 1936. Cytologic studies o n the abnormal development of the eggs of the claret mutant type of Drosophila simulans. Genetics 2 1 :264-8 1 Walker, J. E . , Saraste, M . , Runswick, M. J . , Gay, N . J . 1982. Distantly related se quences in the (X- and J3-subuni ts of ATP synthase , myosin, kinases and other ATP requiring enzymes and a common nucleotide binding fold. EMBO J. 1 :945-5 1 Walker, R. A . , Salmon, E. D . , Endow, S. A . 1990. The Drosophila claret segregation protein is a minus-end directed motor mol ecule. Nature 347:780-82 Warrick, H. M . , Spudich, J. A. 1 987. Myosin structure and function in cell motility . Annu . Rev. Cell Bioi. 3:379-42 1 Watts, F. Z. , Shiels , G . , Orr, E. 1 987 . The yeast MYO] gene encoding a myosin-like protein required for cell division . EMBO J. 6:3499-505 Weisenberg , R . , Taylor, E. W. 1968. Studies on ATPase activity of sea urchin eggs and the isolated mitotic apparatus. Exp. Cell

Res. 53:372-84 Wessels, D . , Murray , J . , Jung, G. , Hammer, J. A. III , Soli, D. R. 1 99 1 . Myosin IB null mutants of Dictyostelium exhibit abnor malities in motility. Cell Moti!. Cyto skeleton 20 :30 1 - 1 5 Wi lkerson , C. G . , King, S. M . , Witman, G . B . 1 99 1 . Sequence analysis of the "y heavy chain from the Chlamydomonas outer arm


66

ENDOW & TITUS

dynein. J. Cell Bioi. 1 15(3, pt.2) : 1 67a (Abstr.) Wilson, P. G . , Fuller, M. T. 1 99 1 . Genetic analysis of spindle function in Drosophila melanogaster. J. Cell Bioi. 1 1 5(3, pt. 2):347a CAbstr. ) Witman, G . B . 1 989. Composition and molec ular organization of the dyneins. See Luck, 1 989 pp. 25-35 Witman , G. B . 1992. Axonemal dyneins. Curro Opin . Cell Bioi. 4:74--70 Wordeman, L. , Steuer, E. R . , Sheetz, M . P . , Mitchison, T . 1 99 1 . Chemical subdomains within the kinetochore domain of isolated

CHO mitotic chromosomes. 1. Cell BioI.

1 14: 285-294 Wright, B . D . , Henson, J. H . , Wedaman, K . P. , Willy, P. J . , Morand, J . N ., et al. 1 99 1 . Subcellular localization and sequence of sea urchin kinesin heavy chain: evidence for its association with membranes in the mitotic apparatus and interphase cytoplasm. J. Cell Bioi. 1 1 3 : 8 1 7-33 Yamamoto, A. H . , Komma, D. J . , Shaffer, C. D . , Pirrotta, V . , Endow, S. A. 1989. The claret locus in Drosophila encodes products required for eyecolor and for meiotic chromosome segregation. EMBO J. 8: 3543-52 Yang, J. T . , Laymon, R. A . , Goldstein, L. S . B . 1989. A three-domain structure of k inesin heavy chain revealed by DNA sequence and microtubule binding analyses. Cell 56: 879-89

Yang, J. T . , Saxton, W. M . , Stewart, R. J . , Raff, E . c . , Goldstein, L . S . B . 1 990. Evidence that the head of kinesin is suf ficient for force generation and motility in vitro. Science 249:42-47 Yeh, E . , Driscoll, R. , Coltrera, M . , Olins, A . , Bloom, K . 1 99 1 . A dynamin-like protein encoded by the yeast sporulation gene SP015. Nature 349: 7 1 3- 1 5 Yisraeli, J . K . , Sokol, S . , Melton, D . A . 1990. A two-step model for the localization of maternal mRNA in Xenopus oocytes: In volvement of microtubules and micro filaments in the translocation and anchoring of Vg l mRNA. Development 1 08 : 2 8 998 Zachar, Z . , Davison, D. , Garza, D . , B ingham , P. M. 1 985. A detailed developmental and structural study of the transcription al effects of insertion of the copia transposon into the white locus of Drosophila me/ano gaster. Genetics 1 1 1 :495-5 1 5 Zhang, P . , Knowles, B . A . , Goldstein, L . S . B . , Hawley , R . S . 1 990. A kinesin-Iike protein required for distributive chromo some segregation in Drosophila. Cell 62: 1053-62 NOTE ADDED IN PROOF

Roof, D. M . , Meluh, P. B . , Rose, M. D. 1 992.

Kinesin-related proteins required for assem

bly of the mitotic spindle. J. Cell Bioi. 1 1 8:95- 108

Roop Mallik: From machines to molecular motors.

Molecular biological approaches to genetic disorders in prenatal diagnosis.

Molecular genetic approaches to understanding the comorbidity of psychiatric disorders.

Molecular genetic approaches to the study of cellular aging.

Molecular genetic approaches to the study of cellular senescence.

Molecular genetic approaches to the analysis of colorectal cancer.

Molecular motors: Hook-ing up early endosomes.

Molecular motors in the nervous system.

Molecular motors: on track with nanotubes.

Molecular motors: DNA takes control.

Introduction to the ECR Special Issue on Molecular Motors.

Genetic variants in Alzheimer disease - molecular and brain network approaches.

Molecular approaches to nerve regeneration.

Molecular motors: Shifting gears with light.

Rotation of artificial rotor axles in rotary molecular motors.

Tuning the rotation rate of light-driven molecular motors.

Genetic variation and nutrition in obesity: approaches to the molecular genetics of obesity.

Genetic approaches to understanding muscle development.

Genetic Approaches to Facilitate Antibacterial Drug Development.

New approaches to establish genetic causality.

Neuroimaging genetic approaches to Posttraumatic Stress Disorder.

Longitudinal analytical approaches to genetic data.

Traffic jams and shocks of molecular motors inside cellular protrusions.

"Cargo-mooring" as an operating principle for molecular motors.