Summary The tropomyosins are a family of actin filament binding proteins. In multicellular animals, they exhibit extensive cell type specific isoform diversity. In this essay we discuss the genetic mechanisms by which this diversity is generated and its possible significance to cellular function. introduction The inolecular and biochemical bases for related cellular processes such as cell motility, organelle movement chromosome movement, cytokinesis and the generation of cell shape are important problems in cell biology. These processes are all dependent on a complex macromolecular set of protein fibers found in the cytoplasm, termed the cytoskeleton. The cytoskeleton of eukaryotic cells is composed of three major filamentous systems: actin filaments, intermediate filaments and microtubules. Much progress has been made in recent years in our understanding of the proteins that comprise each of these filamentous systems. What is clear is that each of these filamentous systems are assembled from a number of different proteins, and that different cell types and tissues express specific protein isoforins which comprise these filaments. For example, different isoforms of actin and tropomyosin comprise the actin filaments of skeletal muscle and nonmuscle cells. At present the significance of this isoform diversity is not clear. In some cases, however, distinct isoforms are associated with cell-type specific functions. In this review, we discuss what is currently known about tropomyosin gene structure, expression and function. Although the picture is still incomplete, a great deal of progress has been made in establishing the molecular basis for tropomyosin isoform diversity in a number of different eukaryotic organisms. ~

Tropomyosin Tropomyosins (TM) are a diverse group of proteins found in all eukaryotic cells, with distinct isoforms found in muscle (skeletal, cardiac and smooth). brain and various non-muscle cells. They arc elongated

proteins that possess a simple dimeric &-helical coiledcoil structure along their entire length (reviewcd in ref. 1). The coiled-coil structure is based on a repeated pattern of sevcn amino acids with hydrophobic residues at the first and fourth positions and is highly conserved in all TM isoforms found in eukaryotic organisms from yeast to man. TMs are associated with the thin filaments in the sarcomeres of muscle cells and the microfilaments of non-muscle cells. The TMs bind to themselves in a head-to-tail manner, and lie in the grooves of F-actin, with each molecule interacting with six or seven actin monomers (Fig. 1). The function of TM in skeletal and cardiac muscle i q , in ascociation with the troponin complex (troponins T. C and 1). to regulate the calciumsensitive interaction of actin and myosin (reviewed in ref. 2). Under resting intracellular calcium ion concentrations, the troponin-tropoinyosin complex inhibits actomyosin ATPase activity. When a stimulus induces calcium ion release in the muscle cell, troponin-C binds additional calcium ions and a conformational change is transmitted through the troponin-tropomyosin complex which releases the inhibiticn of actomyosin ATPase activity. resulting in contraction. In contrast with skcletal m u d e and cardiac muscle. the biological functions of smooth muscle and non-muscle TMs are poorly understood. Smooth muscle and non-muscle cells are devoid of a troponin complex and the phosphorylation of thc light chains of myosin appears to be the major calcium-sensitive regulatory mechanism controlling the interaction of actin and myosin (reviewed in ref. 2). These differences in the regulation of the contractile apparatus of various cell types appear

Actin

I

Tropomyosin

Head-to-tail

overlap Pig. 1. A schematic rcprescntation of thc striated muscle thin filament structure. The helical aclin filament is composed of two strands of polymerizcd globular actin monomers. Rod shaped tropomyosin dimers are polymerized in a head-to-tail manncr and lie in both grooves of the actin filament, each spanning seven actin monomers. The troponin complcx consists of threc subunits. Troponin-T is an asymmetric molecule that interacts with tropomyosin over much of its COOH-terminal half including the region of head-to-tail overlap. Troponin-1 contributes to the inhibitory component of the troponintropomyosin complcx. whilc troponin-C is a C'a' ' binding protein that imparts CaZ+sensitivity to the complex. This figure is reprinted, with permission, from Hcclcy, D. H.. Golosinska, K. and Smillie, L. B. (1487) J. Biol. Chem. 262, 9971-9978('").

to require structurally as well as functionally distinct forms of TM. Although TMs from skeletal, cardiac, smooth muscle and various non-muscle cells are highly homologous, structural difference do exist among the various protein isoforms. These divergent regions among different isoforms appear to correspond to functional domains of the proteins. including troponinbinding regions. actin-binding sites, and sequences involved in head-to-tail polymerization.

list of TM genes that have been identified in various specics is prescnted in Table 1. The a-TM gene is the most complex of the vertebrate TM genes and encodes at least nine i s ~ f o r m s ( ~ -In ~ )the . rat it contains 15 exons, only 5 of which are common to all mRNAs expressed from the gene (Fig. 2). Alternative promoters, associated with exons l a and l b , in this gene result in mRNAs that encode two distinct NH2terminal amino acid sequences. Exon l a is alternatively spliced to exons 2a or 2b, and results in TMs belonging to thc 284 amino acid (high Mr) class, whereas Tropomyosin Gene Structure transcriptional initiation from exon l b results in Many animals including nematodes, flies, frogs, birds mRNAs encoding the 248 amino acid (low Mr) class of TMs. Two internal alternative splice choices are present and mammals possess multiple tropomyosin isoforms. at exons 2a:2b and 6a:6b. Among these splice choices, This isoform diversity is generated by a combination of exons 2b and 6b are the most commonly used. Exon 2a multiple genes, some of which contain alternative is found in the smooth muscle mRNA and 6a is present promoters and some of which exhibit alternative in the fibroblast TM-3 and TM-Sb mRNAs. At the 3’splicing of primary RNA transcripts. The tropomyosin end of the a-TM gene there are four alternatively gene family found in verebrates appears to have arisen spliced exons (9a-9d), each capable of encoding a through duplication of an ancestral gene. There are unique COOH-terminal sequence that is between 25 four different TM genes that have been characterized in and 29 amino acids in length. Exon 9a is used only in the vertebrates. although it is not certain that all species striated muscle (skeletal and cardiac muscle), whereas contain the full complement (Fig. 2). Each of the genes exons 9b and 9c have been detected only in brain have been named after the proteins they encode. The a mRNAs. It is worth noting that in striated muscles exon and /s genes are named after striated muscle 6’ and p9b is spliced to exon 9a and provides only the 3’TMs, respcctively. The TM-4 and TMnm genes are untranslated region, whereas in brain exon 9b is spliced named after the rat fibroblast TM-4 and human to exon 8 and encodes a 29 amino acid COOHfibroblast TM30 nm isoforms, r e ~ p e c t i v c l y ( ~ . ~ ~ A ” - ’ ~ ) directly . Table 1. TM gene distribution”

Organism

Saccliaromyces cerevisiae Caenorhubditis elegans Trichostrotzgylus colubriforints Schistosonia man.soni Drmophila meIunogu.sier Drosophila melanogaster zcbrafish hog quail chicken chicken chicken chicken horse rabbit rabbit mouse mouse rat rat rat human human human human

Gene name

Sequence source

TPMl tmy-1 TMI ( g ~ n e 2 ) ~ TMII (gene l)h a-nrn‘ a a a p (TM-~)~ cardiac‘ TMnm platelet‘ a

genomic genomic cDNA cDNA genomic genomic cDNA cDNA gcnomic cDNA genomic cDNA cDNA protein protein protein cDNA cDNA genomic gcnomic genomic cDNA cDNA cDNA genomic

/I a

B N

P

‘TM-4‘ o(

F

TMpl“ TMnm

Reference 12 13 14 15 16,17 16,17

18 18 19 20,21 7.8 22 20 23 24 24 25 26 3-5 fi

10 27 28 29 9

a Classification of vertebrate genes is based on relative amino acid sequence similarity derived from one or more of genomic, cDNA or protein sequences. In cases where genomic structure is known. we have not cited pcvious cDNA or protein sequences. ’The D. melunoguster genes are named differently by two independent groups. ‘Thc amino acid sequence of the known zebrafish skeletal muscle TM is not significantly more similar to either the a-gene or TMnm gene derived skeletal muscle TMs. “The chicken @-TMgene has also been named TM-1. “The chicken cardiac, horse platelet, rat TM-4, and human TM30pl genes are homologous.

I1

I

’

SMOOTH MUSCLE

P- gene

d M ” f.

@*U,-’’->

lo

2b

ibX

5

4

5 60 60

’ 8 90

9rY

A

SKELETAL MUSCLE

&

’

A

I

‘w

1 M’f

‘ m ’ ~ ’ , \I^r-----~~

SMOOTH MUSCLE RAT FIBROBLAST TM-I

Fig. 2. The introii-exon organization of the vertebrate TM genes and their associated mRNA products. Exons are represented by boxes and introns by horizontal lines. The cxons are numbered from l a through 9d in order to facilitate simple comparison hetween TM genes. Polyadenylation signals are marked with an A . The depicted a-gene structure is that found in The 3‘untraiislated sequence of TMBr-3 includes all of exon Yd. The />gene is a composite from rat and chicken‘6-8). (X) Exons l b is present in the chicken gene hut not in the rat gene. As a consequence the high Mr TM-1 is the only p-gene product in rat fibroblasts while the low Mr TM-Sb is the major p-gene product in chicken fibroblasts. ( Y ) An cxon 9c like sequence is also present in thc chicken but not the rat p-TM

CHICKEN FIBROBLAST TM-3b I0

2b

Ib

3

4

5 6c 6b 7 890 9b

2b‘

Ih

3

4

5

hTMnm gene

GO 7

TM-4 gene FIBROBLAST TM-4 PLATELET

9r!

+I

L?

m’ U L ’t’-

_-

IIomTever, mRNAs containing this sequence have not vet been characterized. The depicted hTMnm gene structurc is that found in human(’). The skeletal muscle isoforrn expressed from this gene is thought to be present. mainly in slow twitch fibers and is morc similar in sequence to a-skeletal than B-skeletal TMs and is thcreforc referred to as slow twitch wTM. The TM-4 gene structure has been determined only in rat(”). (Z) Non-functional sequences similar to exons 2h and 9a havc been lound in the rat gene. Evidence from human and chicken indicate that these exons are functional in the homologous genes from other vertebrates(22.2g). genes“,8).

m

\-

1

I

1

~-

terminal sequence. Exon 9d is used in smooth muscle and a variety of non-muscle cells including fibroblasts, and at least certain cells of liver, kidney, and intestinal epithelium. The p-TM gene has been extensively characterized in both rat and (Fig. 2). Relative to the a-TM gene, the chicken @-TMgenc docs not contain exons 2a and 9b(7.8),while the rat p-TM gene does not contain exons 2a, 9b, and 9c and the internal promoter associated with the use of exon l b (ref. 6 and unpublished). Both chicken and rat @-TMgenes encode skeletal muscle and smooth muscle isoforms. In rat, the smooth muscle /?-TM is alyo identical to a major

fibroblast isoform termed TM-l“.”). In chicken fibroblasts, the NH2-terminus of the ma‘or 6-TM gcne isoform (TM3b) is encoded by exon Ibh’. At present, the TMiim gene has been characterized only in humand’). It is similar in structure to the a-TM gene but the presence or absence of altei-nate exons 2a and Yc has not yet been determined (Fig. 2). The products of the TMnm gene include a low Mr fibroblast TM, tcrmcd TM30nm, and a high Mr isoform expressed in slow twitch skeletal muscle. The rat TM-4 gene is the simplest of the vertebrate TM genes that has been characterized(’”). It contains 8 functional exons and encodes a single non-muscle isoform, fibroblast TM-4.

However, remnants of what appear to h a w been alternatively spliced exons 2b and 9a are found within the TM-4 gene. Thus it appears that the ancestral precursor of t h e TM-4 gene contained alternative promoters and exons. Evidence that this is indeed the case comes from recent findings that the chicken cardiac muscle TM was shown to be expressed from a gene which also encodes the avian homolog of rat TM-4("). In addition, the homologous TM30pl gene of humans is thought to possess alternatively spliced ex on^(*^). Thus it appears that each of the four vertebrate TM genes are alternatively spliced in at least some species. The strong similarities between the vertebrate genes indicate that they likely evolved through gene duplication from a common ancestral gene that possessed an extensive array of alternatively spliced exons. In addition, the intron/exon organization of TM genes exhibit extensive conservation between invertebrates and vertebrates. For example. in the Drosophila melanogaster TMII gene (also known as gene 1) the position of the intron/exon junctions relative to the final transcript are identical to vertebrate TM enes, and the gene contains alternative However, unlike the vertebrate genes, the Drosophila TMII gene does not contain duplications of exons 2 and 6, but contains alternative exons 4a:4b, 5a:5b and 7a:7b. In addition, although the Drosophila genc contains four alternatively spliced exons at the 3'-end of the gene, exon 9a encodes the COOH-terminus of a non-muscle TM, while exons 9b, 9c and 9d encode the COOH-termini o f striated muscle tropomyosins. This situation is directly opposite to that found in the vertebrates. A recent brief report on the structure of thc Cuenorhuhditis elegans TM gene and cDNA sequences from the skeletal muscle of another nematode Trichostrongylus colubriformis and a trematode Schistosoma munsorii have revealed some additional aspects of TM gene evolutio~i('~-'~). The C. ekgans gene appears to encode both muscle and non-muscle transcripts through the use of altcrnative promoters and splicing. Internal alternative splices are found at exons 3a:3b which is unique to C. elegans and at exons 4a:4b

which is also found in D. melanogaster. Alternative splicing at the 3'-end is similar to that in D. melanogaster, with exon 9a expressed in non-muscle cells and exons 9b and 9c in skeletal muscle. The amino acid sequences of T. colubriformis and S . munsoni TMs indicate that overall thcy are about e ually similar to fly and vertebrate skelctal muscle TMJ4.lS).However, at their COOH-terminal ends they are distinctly more similar to the D. melanogaster TMT gene isoforms. These findings indicate that the COOH-terminus is the region of TM which has undergone the most significant alterations during the evolution of vertebrates from their distant ancestors. TM lsoform Diversity and Cellular Function As described abovc, multicellular organisms exhibit a multiplicity of TM isoforms. For example, at least 12 isoforms have been identified in rat on the basis of primary sequence differences (Table 2). While these may encompass the entire range of rat TM diversity, some equivocal evidence from two-dimensional gel electrophoresis and northern blot analysis suggests that other isoforms may e ~ i s t ( ~ ~ 3In~ addition, '). humans and chickens possess TM isoforms that have not been found in rats. All vertebrate tropomyosins possess a highly conserved core sequence encoded by exoiis 3.4,5,7 and 8. The major differences between TM isoforms results from the use of alternative exons. Thus, exons 9a and 9d of the rat a-TM gene encode only four identical amino acids out of twenty-seven (Fig. 3). However, sequences contained within some alternatively spliced exons have been highly conserved among different species. For example, the COOH-terminal sequence encoded by exon 9a of the rat a-TM genc is identical to that encoded by the equivalent gene in fish (3,18). The extent of our knowledge concerning the structure/function relationship of vertebrate TM isoform diversity is presented below. Striated Muscle One-dimensional electrophoresis of striated muscle

Table 2. Rat TM isoform diversity

lsoform

Apparent Mr"

No. of amino acids

Fibroblast TM-1 smooth muscle /3 Fibroblast TM-6 smooth muscle N Skeletal muscle P Skeletal muscle N Fihrohlast TM-2 Braiii TMBr-1 Fibroblast TM-3 Fibroblast TM-4 Fibroblast TM-Sa Brain TMBr-2 Fibroblast TM-5b Brain TMBr-3

40 000 39 OW 39 000 3h 500 36 500 3s 800 35 000 32 500 32 000 32 000 31 500 31 000

284 284 284 284 284 281 284 248 248 25 1 248 245

Gcne

Rcfercncc

P

6.30 3,32 and unpublished 6 3 32 S 32 10,33 32

CC

P N

n a

n TM-4 N N

5

N

32

a

5

a The apparent molecular wcights of the TMs are based on SDS-~polyacrylainidegel clectrophoresis. The molecular weights of the fibroblast TMs were obtained from reference 34.

Fig. 3. comparison of thc amino acid sequenccs cncodcd by alternative exons l a and Ib, 2e and 2h, ha and 6h. and 9a.9b,9c and 9d from vertebrate TMs. The 7 residue repeated pattern is indicated by a,b,c,d.e,f,g. Amino acid sequence numbers for 284 amino acid TMs are indicated at the top of each alignment. The sequences are ordered mjith thosc expressed in striated musclc first. Identities to thc first sequence in each series is indicated (-). Several known vertehrate scqucnces have not bceii included due to their high degree of sequence identity with one or more or the presented sequence.;. Rcfercnccs for cach of thcse sequences arc given in Table 1. Abbreviations include, (HUM) human, (CHK) chicken, (QUA) quail, (ca) cardiac, (4) TM-4, (nin) TMnm.

RAT-CI 2 b QUA-U 2 b HUMnm 2b RAT-/?

2D

C H K - f ? 2b

CHKca 2b RAT-II

2a

Q3A-01 2a

RAT-CI

6b

3 9

HLiMnm 6h

RAT-^

fib

CHK-p

6b

RAT4

6b

C S K c a 6b RAT-II

6a

HUMnm 6 a

R A T - P 6a CHK-P 6a

258 270 284 fqabcdefqabcdefgabcdefgabcd

DELYAQKLKYKAISEELDHALNDMTSI

3

HUMnm 9 a

...........................

9

R A T - 8 9a

--V----M----------N----I--L

6

C H K c a 9a

- - - _ - - _ - _ _ _ _ _ _ _ _ _ _L_ _ _ _ _ _ _ _

22

RAT-Q

9a

5

HUMnm 9d RAT-B9d

-Q--H-LEQNRRLTN--KL---ED ERSRQPAE-NRVLTN--RVI-TELNN EKVAHA-EENLSMHQM--QT-LELNNM EKVAYA-EENLNMHQM--QT-LELNNM -K-KCT-EEHLCTQRM--QT-L-LNEM ET-ASA-EENW-HQ?--QT-LEL"L

6

C H K - ~ 9d

ES-ASA-EENVG-HQV--QT-LEL"L

HUMpl 9 d

EK-AQA-EENVSLbQT--QT--ELNC-

7,8 29

RAT4

EK-AQA-EENVGLHQT--QT--EINC-

10

-K-AQA-EENLGLHQT--QT--ELNC-KFLCFSPPKTPS-SRMS-LSELCICLLS S

22

RAT-(Y 9 C

CHK-8 9~ RAT-Cl

9d

Q U A 4 9d

9d

CHKca 9 d RA?-(r

9b

5,8 3 19

3

3.5

TMs identified two isoforms which were named a (lower apparent Mr) and /$"). The a isoform is more prominent in the cardiac muscle of mammals and in fast twitch skeletal muscles, while is abundant in slow twitch muscles. Two additional isoforms termed y and 6 have been identified in slow twitch muscles of rabbit

and rat by two-dimensional electrophoresis(3s). At least one of these isoforms is also Found in chicken, cat and cow (reviewed in ref. 33). It is likely that one of the slow twitch muscle isoforms identified by gel electrophoresis is encoded by the TMnm gene which has been characterized in humans('). Since the skeletal muscle product encoded by the TMnm gene is more similar to a than to B-TM, it is referred to as slow twitch a-TM. The partial se uence of a similar isoform has been found in chicken(20q. As mentioned above, a fourth gene encodes the chicken cardiac TM(22).To date, all striated muscle TM sequences that have been obtained, including those from frog and fish(l8), appear to be encoded by the exon sequence la, 2b, 3, 4, 5 , bb, 7, 8, 9a. Of these the COOH-terminal coding exon 9a is the only one used exclusively in striated muscle TMs. In rat and quail, RNA hybridization analysis has not shown any deviation from this patter^^(^,^,'^.^') . Further isoform diversity in striated muscle is created by phosphorylation of serine 283 by a tropomyosin kinase. This modification is known to affect head-to-tail polymerization of TMS(? The tissue-specific exons used in strialcd muscle appear to encode distinct functional domains of the TM molecule. In skeletal and cardiac muscle, troponin-T interacts with two regions on the tropomyosin molecule, one located in the proximity of Cys-190 and the other at the COOH-terminal end of the protcin (Fig. l)(ia,40! These two troponin T-binding domains correspond to two cell-type specific splice choices involving exons 6b and exons 9a. In addition, sequences at the amino- and COOH-termini, which are encoded by alternative exons l a and 9a, respectively, are important for head-to-tail polymerization between TM molecules(1). The entire sequence of striated muscle TMs including regions encoding both the constitutively exprcsscd exons 3,4,5,7 and 8, and alternative exons 1a, 2b, 6b and 9a has been highly conserved (>85 % amino acid sequence identity) within all known vertebrate genes (Fig. 3). As to the differences that are present, nothing is known as to their effect on the functional properflies of the four striated muscle isoforms. The amino acid sequence differences that occur between striated muscle TMs and those found in other tissues are more profound and result from use of alternative promoters and alternative RNA splicing as discussed below.

Smooth Muscle In vertebrates. the smooth muscle cy and p TMs are encoded by the same genes that encode the striated muscle a and /j isoforms (Fig. 2). Expression of the ac gene in smooth muscle is associated with the use of exons 2a and 9d, whereas exons 2b and 9a are used in striated muscle. Likewise, for the b-TM gene, exons 6a and 9d are used in smooth muscle, whilc exons 6b and 9a are used in striated muscle. This results in proteins having different internal and COOH-terminal amino acids sequences (Fig. 3). The differences in exon usage between striated and smooth muscle TMs may be related to the mechanism of contraction in the respective cell types. The smooth muscle actomyosin is not subject to regulation by the interaction of TM with the troponin complcx. Instead, it is activated through the phosphorylation of a myosin light chain by the Ca*+-calmodulin sensitive myosin light chain kinase (reviewed in ref. 2). Thus the sequences encoded by the alternatively spliced exons may be required to carry out specific functions in ditferent cell types e.g., striated verwy smooth muscles. Although smooth muscle contains two TlM isoforms, it remains to be determined whether each isoform has a unique role. Comparing their amino acid sequences reveals that there are distinct differences between the smooth muscle a- and P-TMs. Although they contain highly conserved COOH-terminal coding sequcnccs (exon 9d), they exhibit substantial differences between amino acids 39-80 (exons 2a and 2b) and amino acids 189-213 (cxons 6a and 6b) (Fig. 3). The level of encoded amino acid sequence identity is only 33 % for exons 2a vs. 2b and 35 % for exons 6a vs. 6b (Fig. 3). It is worth noting that sequences encoded by exon 2a have not been conserved throughout vertebrate evolution. A comparison of the amino acid3 sequences encoded by exon 2a from rat and quail reveals that there are 15 substitutions, only six of which are conservative (Fig. 3). By contrast, there are only three substitutions, all of which are conservative, between sequences encoded by exons 2b from rat and from quail. At present, actin is the only protein that is known to bind to this region of tropomyosin. The reason for the large divergence in amino acids 39 to 80 of smooth muscle a-TMs therefore remains a mystery, Fibroblast Tropomyosins On the basis of migration on one- and two-dimensional polyacrylamide gels, as many as six distinct isoforms of TM have been detected in cultures of non-muscle cells from human, mouse, rat and chicken (reviewed in refs 32,41). For example. based on their relative abundance, rat fibroblasts were reported to contain three major tropomyosins termed TM-1, TM-2, and TM-4 (apparent M,=40 000. 36 500, and 32 400, respectively) and two minor TM isoforms termed TM-3 and TM-5 (apparent M,=35 000 and 32 000, re~pectively)(~"). We now know that these isoforms are encoded by three

distinct genes (see also Fig. 2). The rat fibroblast TM-3 isoform, which is identical to smooth muscle p-TM, is encoded by the p-TM gene(6).The TM-4 isoform is the sole isoform encoded by the TM-4 gene('"). TM-2, TM3, TM-Sa, TM-Sb are all encoded by the a-TM gene via the use of alternate promoters and alternative RNA splicing('*). The TM-Sa and TM-Sb isoforms are difficult to separate by one- and two-dimensional gel electrophoresis and have been referred to as TM-5 in the literature. The existcncc of TM-Sa and TM-Sb has not yet been demonstrated in humans, but at least a portion of the TM-5 protein detected by twodimensional gel electrophoresis (referred to as TM30nm) is encoded by the TMnm gene('). It is worth noting that rat fibroblasts have been reported to express an additional isoform termed TM-6 (apparent M,= 39000), which is identical to smooth muscle a-TM (refs 3, 31 and our unpublished observations). The function of each of the individual TM isoforms expressed in fibroblasts remains to be established. Why, for example, do rat fibroblasts contain at least six forms of TM, when skeletal muscle contains two isoforms, smooth muscle contains two isoforms, and rat heart contains a single isoform? As indicated in Fig. 2, the major distinguishing feature of fibroblast and smooth muscle isoforms relative to striated muscle and brain specific isoforins is the COOH-terminus, which is encoded by exon 9d. The functional significance of the COOH- terminal isoform-specific regions (exons 9a-9d) in different cell types is not presently understood. In addition, why differences exist between isoforms with respect to the internal sequences encoded by exons 6a and 6b are not known. Immunofluorescence studies of fibroblasts have revealed that TM is associated with the actin-containing microfilaments. Microfilaments of fibroblasts represent dynamic structures that can exist in different supramolecular forms such as microfilament bundles (stress fibers), microfilament meshworks, polygonal networks, and contractile rings. The particular TM isoforins involved in these higher ordered actin structures are poorly defined. The multiplicity of TM isoforms in fibroblarts raises the possibility that specific associations of given isoforins with themselves and other cellular proteins lead to distinct actin structures. Differences in TM isoform localization have been demonstrated with antibodies recognizing only the high Mr TMs versus those recognizing only the low Mr TMs("). The low Mr forms were found in both ruffles and stress fibers while the high Mr forms were found only in stress fibers. The amino acid sequences at the NH*-termini of high Mr and low Mr TMs. which are encoded by cxons l a and l b , respectively, are highly divergent (Fig. 3). At the biochemical level, high Mi- fibroblast TMs were found to have a greater affinity for filamentous actin and a greater protective effect against actin filament severing ~). by gelsolin than do the low Mr i s ~ f o r r n s ( ~These findings are in agreement with many studies demonstrating that low Mr isoforms generally have weaker

actin binding capabilities (reviewed in ref. 44). One hypothesis that has been proposed is that changes in the relative levels of different forms of TM in the cell may alter the organization of microfilaments. Accordingly, the alterations in microfilament structure in transformed cells have been suggested to be the direct result of the altered patterns of TM expression observed in these cells (reviewed in refs 32, 41). However, at this time there is no direct evidence establishing a causal relationship between changes in TM expression and alterations in cytoarchitecture. Several of the fibroblast-type TMs are expressed in a variety of non-muscle cell types. In rat, the mRNA for TM-4 has been found in liver, kidney, brain, stomach. and uterus, and its equine homolog is the major isoform in platelets(23733).Messenger RNA for TM-Sb is prominent in kidney, liver and intestinal epithelium of rats, while mRNA for TM-Sa is present in brain and kidney(32).TM-Sb differs from TM-Sa and other low Mr brain tropomyosins in that amino acids 154-177 are encoded by exon 6a rather than 6b. The functional significance of the use of exons 6a or 6b are unclear at this time. However, one possibility is that these sequences may play a role in the interaction of TMs with actin. For example, it is known that in chicken, the low Mr TMs from intestinal epithelium have a greater affinity for actin filaments than those from brain(45). This finding correlates well with the differential use of exons 6a and 6b in these tissues. In the future, it will be of interest to determine if TM-Sb doe5 in fact have a higher affinity for actin than TM-Sa. Brain Until recently there was little indication in the literature that brain TMs would be significantly different from the low Mr isoforms found in platelets or fibroblasts. Unexpectedly, the a-TM gene was found to express a unique set of isoforms in the rat brain, which were named TMBr-1, TMBr-2 and TMBr-3(5). These TMs are distinguished from all other isoforms by their COOH-terminal sequences which are encoded by exons 9b or 9c (Fig. 2). Exon 9b encodes a COOH-terminal sequence that docs not contain the typical 7 residue repeated pattern, and is not likely to be a-helical due to multiple prolines (Fig. 3). Exon 9c encodes a sequence that is distinctive from that of exons 9a and 9d but which maintains a typical 7 residue repeated pattern (5). In addition to these brain-type isoforms, both high Mr (TM-1 and TM-6) and low Mr TMs (TM-Sa and TM-4) have been detected in brain. TMBr-3 and TM-4 are the predominant isoforms detected in brain tissue. Since brain is a heterogeneous tissue comprised of multiple cell types, it remains to be determined if these isoforms are cell type or brain region specific. In addition, the functional significance of these isoforms remains to be determined. Actin-based filaments are associated with a variety of neural structures, such as growth cones, dendritic spines, axoplasm, prcsynaptic termini, post

synaptic densities, and the actin filament network found beneath the plasma membrane. These structures are thought to play a role in motile processes such as axoplasmic transport. growth cone movement, synaptic rearrangement, and vesicle transport. Antibodies with general specificity detect a broad distribution of TM in In the future the neurons including the growth development of antibodies directed to the brain-specific isoforms (TMBr-1, TMBr- 2 and TMBr-3) should assist in determining their function in brain. Tropomyosin Regulation Clearly, much work remains to be done on the regulation of TM expression, as well as on the function of TM isoform diversity. Our understanding of the regulation of TM gene expression is now in it5 infancy. It is well known that the expression of striated muscle TM isoforms is turned on during myogenesis, and it is expected that transcriptional regulators such as myo D and myogcnin will affect TM expression. In fibroblasts, the (u-TM gene has been shown to be induced by the addition of serum to quiescent cultures, indicating that it may be regulated by second messenge;4$athways similar to that of genes such asfos and jun . At the level of RNA splicing, several cis-acting elements have been found which regulate the alternate use of exons(48-5"). However. the cellular factors that control tissue-specific splicing have yet to be identified. Concluding Remarks Our knowledge of TM isoform diversity has progressed rapidly in the last five years, primarily through the use of molecular biology to idcntify and characterize a large number of cell type-specific isoforms. In the future a greater understanding of the cell biology and genetics of TMs will be required in order to understand the significance of TM isoform diversity. The expression of a diverse group of TM isoforms in a highly tissuespecific manner strongly suggests that each isoform is required to carry out specific functions in the actinbased filaments of various muscle and non-muscle cells. It will be a challenge to understand how the various isoforms function together with other cytoskeletal elements. The development of isoform-specific antibodies and the use of molecular biology will provide the tools with which thcse problems can be approached. In D. melnnognster, a tropomyosin mutant which destro s flight muscle function is being studied in detail(51-5) , The mutation, which blocks expression of only one of two alternatively spliced TM mRNAs, results in disruption of the myofilainent lattice. Knockout of a yeast TM gene leads to a loss of actin cables(l'l. These studies demonstrate the importance of tropomyosin for normal actin filament function. Non-vertebrate genetics should provide key information as to whether the high Mr and low Mr isoforins can replacc each other in a living system. A large number of vertebrate TMs do not

Y

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have equivalent isoforms in non-vertebrate organisms, indicating the need for a good vcrtebratc genctic system, such as that of the mouse, to study these forms. Elucidating the role of each isoform will help us to understand the role of actin-based filamentous systems in eukaryotic cells.

of a-tropomnyosin exom enforced hy an unurunl lariat \>ranch point location: implications for conntitutivr spl~cing.C'dl 56, 739-758. 50 GOLX-PFLLI!'IAN. M.. LIURI,D.. D'ALIUCNTON-CAR.4I.A. Y..FIS2M.W. M.. UKOUU,E. AYD MARIO. J . (19YO). I n vitro splicing of mutually cxclusive exoiis from the chicken [i-troponivosin gcnc: rolc of the branch point location and vcr-y long pyrimidine stretch. EMRO J . 9, 241-249. 51 KARLIK.C. C. . 4 h ~FYRBFRG. E. A. (1Y8S). An in\ertion within a variably spliced Urosophila lropomyosin gene blocks accumulation of only one encoded isoform. Cell 41, 57-66. 52 T4NSFY, T.. MlliuS, M. D., DUMOULIN, M. AND STORTI. R . V. (1587). Transformation and rcscuc of a flightless Drosopphtlu tropomyosin mutant. EMBO .I. 6 , 1375-1385.

modulation of actin-scvcring activity of gelsoliii by inultiplc isoforins of cultured . 7490-7497. rat ccll tropomyosin. .I. R i d . C h ~ m264. 44 COTE,C;. P. (1983). Structural and functional properties of the non-muscle tropomyosins. Mol. Cell. B d .57. 127-116. 45 BROSCIIAT, K. 0. A N D BURGESS. W . R. (1586). Low Mr tropomyosin isoformi from chicken brain and intestinal epithelium have distinct actinbinding properties. J. B i d Chem. 261. 13 350-13355. 46 I . E T ~ ~ R N E AP.L IC. . AND SHAITUCK, T. A . (1989). Distribution and possible interactions of actin-associated protein?; and cell adhesion molecules of nerve growth cones. Deidopntenr 105, 505-519. 47 RusErrc, R.-P.. hfACDONAI.D-BKAVOIH., Z E R 1 4 1 . bf. A N D BRAVO, R. (1989). Coordinate induction of fibronectin. fihronzcti~ireceptor. tropomyosin, and 48 HELFMAN, D. M., ROSCXGNO. R. F., MULLIGAN, G. J . , FINN.L. A . A ~ WEBER.K. S . (1990). Idciitification of two distinct inrroii clcnientq involved in alternative splicine of 0-tropomvosin nrc-niRNA. Gcnrs Dev. 4, 98-1 10. . . 49 SMITH, C. W. J. AYD NAUAL-CPIARD. B. (1989). Mutually exclusive splicing

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James P. Lees-Miller and David M. Helfman are at the Cold Spring Harbor Laboratory, P.O. Box IOU, Cold Sorine Harbor. New York. NY 11724. USA. I

L,

THE INTERNATIONAL FEDERATION OF SOCIETIES FOR HISTOCHEMISTRY AND CYTOCHEMISTRY ANNOUNCES THE

9th International Congress of Histochemistry and Cytochemistry MAASTRICHT, THE NETHERLANDS August 30-September 5, 1992 Plenary Lcrtuw topics: In situ hybridization ; Intravital microscopy; Cytochemistry at the EM level; Enzyme histochemistry: lmmunocytochemistry: Intracellular Transport: Nerve-immune system interactions; Peroxisomes. Symposia will highlight the impact of lzisto- and cytochernistry In the u r e u 08 Developmental biology and ageing; Cell growth and differentiation; Neurosciences; Plant Cell Biology; Diagnostic Pathology; Toxicology; Receptors and Iigands; Intracellular calcium. Workshop will focus on new lrerzds in: Tissue processing techniques; Flow and image cytomctry; Morphometry ; Autoradiography ; Confocal microscopy. Organizing Secretariat: Prof. Dr. F. C. S. Ramaekers, Department of Molecular Cell Biology; University of Limburg; P.O. Box 616; 6200 MD MAASTRICHT, The Netherlands; Tel. 31-43-888642; Fax. 31-43-437640.

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