Neuron,

Vol.

9, 373-381,

August,

1992,

Copyright

0

1992

by Cell

Press

Plasticin, A Novel Type III Neurofilament Protein from Goldfish Retina: Increased Expression during Optic Nerve Regeneration Eric Glasgow,* Robert K. Druger,* Edward M. Levine,* Chana Fuchs,* and Nisson Schechter*+ *Department of Biochemistry and Cell Biology +Department of Psychiatry and Behavioral Science Health Sciences Center State University of New York at Stony Brook Stony Brook, New York 11794

Summary The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins in this pathway are unexpected and atypical, suggesting these proteins provide a structure that supports growth and plasticity. Using a goldfish retina hgtl0 library, we have isolated a full-length cDNA clone that encodes a novel type III intermediate filament protein. The mRNA for this protein is located in retinal ganglion cells, and its level dramatically increases during optic nerve regeneration. The protein is transported into the optic nerve within the slow phase of axonal transport. W e have named this protein plasticin because it was isolated from a neuronal pathway well known for its plasticity. Introduction The goldfish visual pathway is persistently embryonic in that it displays continuous growth and development. Throughout life a symmetrical increase in the number of retinal ganglion cells connecting to an asymmetrical increase in tectal target cells forces plasticity in order to maintain the retinotopic map (Meyer, 1978; Johns and Easter, 1977; Easter and Stuermer, 1984). Furthermore, a remarkable capacity for functional regeneration occurs after optic nerve injury (Sperry, 1963). These properties are not observed in higher vertebrates where neurogenesis is far more restricted to early development. The goldfish visual pathway is therefore an important system to study molecular components of nerve growth and development (Perry et al., 1987; Benowitz and Lewis, 1983; Heacock and Agranoff, 1976; Quitschke and Schechter, 1983). The cytoskeleton is a particularly prominent structure in neuronal tissue and iscomposed of three morphologically distinct filamentous networks. One major constituent of the cytoskeleton is the intermediate filament (IF) network. The proteins of the IF network are structurally diverse (Osborn and Weber, 1986); furthermore, their expression is cell specific and developmentally regulated (Klymkowsky et al., 1989). The differential expression of IF proteins during development (Shaw and Weber, 1983; Tapscott et al., 1981) and their dynamic organization (Skalli and Goldman, 1991;

Carden et al., 1987; Vikstrom et al., 1989) suggest that these proteins fulfill important physiological requirements of neurogenesis. Although IF proteinsarediverse in structure, amino acid and cDNA sequence analyses show that these proteins belong to a multigene family, all having a common structural organization. IF proteins have a highly conserved a-helical central rod domain that is flanked by an amino-terminal (head) domain and by a carboxy-terminal (tail) domain. These head and tail domains arevariable in size and amino acid sequence and impart the major portion of the structural heterogeneity to IF proteins (Steinert and Roop, 1988; Goldman and Steinert, 1990). The IF proteins expressed in goldfish retinal ganglion cells, which give rise to the optic nerve axons, are different from the conventional IF proteins expressed in the more static retinal ganglion cells of adult higher vertebrates (Quitschke et al., 1985). The structure of the IF proteins expressed in the goldfish visual pathway may reflect its embryonic nature (Giordano et al., 1989; Hall et al., 1990). Here we report the complete sequence of a cDNA for a novel type III IF protein. It is synthesized in retinal ganglion cells and transported into the optic nervewithin the slow phase of axonal transport. Furthermore, the retinal mRNA level for this protein is significantly elevated after optic nervecrush. W e designate this IF protein plasticin, because it was discovered in a neuronal pathway known for its high degree of plasticity. Results Isolation and Sequencing of cDNA Clones Encoding Plasticin A hgtll cDNA library was constructed from goldfish retina poly(A)+ RNA isolated 20 days after optic nerve crush. Approximately IO6 plaques were screened at low stringency with a conserved region of the type I II IF, goldfish vimentin (Figure I), which we have previously isolated from the goldfish retina. Thirty-three positive clones were isolated and subcloned into a Bluescript KS(-) plasmid (Stratagene). Four of these clones represent two sets of overlapping partial cDNAs, pCD2 and pCL3 (Figure I). To obtain a fulllength clone, a goldfish retina, 3 day postcrush, hgtl0 cDNA library (gift of Dr. D. Goldman) was screened at high stringency with clone pCD2. Ninety-eight positive plaques were obtained, 10 of which were further purified. The longest of these, hP5A, contained a 1.8 kb insert and was su bcloned into PBS KS(-) for further characterization. Clone pP5A was sequenced in both directions (see Experimental Procedures). The sequence is 1809 nt long and codes for the entire plasticin protein, 93 nt of 5’ noncoding sequence, and 357 nt of 3’ noncoding sequence. The 3’ noncoding sequence contains three

NeuVXl

374

PSA

5’

CL3

5’

A”

Figure 1. Schematic ticin Clone5

Representation

of Plas-

A”

Schematic depiction of the cDNA clones compared with the olasticin orotein. The E x RR6 1 scale denotes kilobase pairs and indicates 5' A" the size of the cDNA clones. The head and tail domains and coils la, lb, and 2 in the Ill’ i’ llIl/ll II a-helical rod region are shown in the sche1.0 1.5 2.0 0 0 5 matic diagram for the protein. The depitted E&RI (E) to Xbal (X) fragment of the TAIL HEAD Ib H COIL2 la -c N-m vimentin-like clone pRR6.1 was used to screen a xgtll cDNA library at low stringency. This screening resulted in the partial plasticin cDNA clones CD2 and CL3. The CD2 insert was used to screen a @IO cDNA library at high stringency. This screening resulted in the isolation of a full-length plasticin cDNA clone, P5A. The entire CD2 insert was used to generate the antisense riboprobes used in the Northern and RNAase protection analyses. The pCD2 clone, linearized with Mval (M), was used to generate the antisense riboprobe used in the in situ hybridizations. 5’

M

CD2

3’

potential polyadenylation addition signals, 16, 104, and 127 nt upstream from the putative polyadenylation site (Figure 2). The predicted amino acid sequence of the cDNA is 453 aa long, with a calculated molecular mass of 52,632 daltons. Classification of Plasticin as a Novel Type III IF Protein The amino acid sequence of plasticin displays a structure characteristic of all IF proteins (Figure 3). It has variable head and tail domains, which flank a conserved a-helical central rod domain. A large portion of the amino acid sequence of the rod domain is arranged in heptad units (a-b-c-d-e-f-g),, where a hydrophobicaminoacid is usually in theaand d position and the amino acids in the other positions are either polar or charged. Two small linker regions (Ll and Ll-2) of variable amino acid sequence interrupt the rod domain to form threedistinct a-helical tracts (coils la, lb, and 2). Additionally, there is another small linker sequence in the amino-terminal region of coil 2 and a single amino acid insertion causing a stagger in the heptad repeats near the middle of this coil (Steinert and Roop, 1988). IF proteins have been categorized into six classes (type I-VI), based upon the sequence homology of the rod domains. In addition, within the classes there are often regions of homology in the variable head and tail domains. Comparison of the plasticin amino acid sequence with the other type III IF proteins, rat peripherin, hamster vimentin, and hamster desmin shows a high degree of similarity throughout the a-helical rod domain (Figure 3). Plasticin also contains the type Ill-specific consensus sequence IKTVETRDG in the tail domain (Leonard et al., 1988). Although plasticin shares the highest overall sequence similarity with peripherin, it is no higher than the sequence similarity that peripherin shares with desmin or vimentin. In addition, plasticin lacks the sequence SSYRR(T/M)FG, which is found in the head domains of peripherin, vimentin, and desmin. Northern Northern

Blot Analysis blot analysis indicates

the size of plasticin

mRNA and its response to optic nerve crush. One microgram of poly(A)+ RNA from normal retina and retina IO days after optic nerve crush was probed with a [32P]UTP-labeled antisense RNA transcript synthesized from the clone pCD2. Under high stringency conditions a single band is detected at approximately 1.9 kb. This message shows a large increase in response to optic nerve crush (Figure 4). At lower stringency a second band is detected at approximately 2.4 kb that does not respond to optic nerve crush. Plasticin mRNA Is Expressed in Goldfish CNS Tissues RNAase protection analysis was used to assay for the presence of plasticin mRNA in tissues of the goldfish. A p2P]UTP-labeled 373 nt antisense RNA probe was synthesized from the pCD2 clone. Annealing of this probe to 20 ug of total RNA from several goldfish tissues, followed by RNAase A digestion, results in the protection of a 277 nt fragment (Figure 5). Plasticin mRNA was detected only in CNS tissues. There is a low level of plasticin mRNA in normal retina, with a large increase of mRNA 20 days after optic nerve crush. Also, there is a small amount of mRNA in brain and a moderate amount in spinal cord. No mRNA was detected in eye lens, skin, liver, oocytes, or optic nerve. Plasticin mRNA Expression Increases during Optic Nerve Regeneration The expression of plasticin mRNA in retina was assayed by RNAase protection (as above) at several time points after optic nerve crush (Figure 6A). Twenty micrograms of total retina RNA was assayed from the left nonoperated eye and the right operated eye at 0, 5, 10, 20, 27, 37, and 120 days after optic nerve crush. The day 0 time point and the left nonoperated retina time points remain essentially unchanged and serve as a baseline control. The operated right retinas show a dramatic increase in plasticin mRNA levels. Plasticin mRNA levels increase 3-fold by day 5, peak approximately 15fold by day 20, and then decrease to precrush levels by 120 days after optic nerve crush (Figure 6B).

PlasticIn, Neurofilamrnt

Protein in Retina

375

PLASTICIN AAG CTT CM ACA ACA

78

79

MSHSTFSHLFSPHFGAPVYSP ACT AGA AGT ATA ACC ATG ACT CAC TCT ACA TTT TCG CAC CTC TTT TCA CCG CAC TTC GGT GCA CCT GTC TAT AGC CCT

21 156

22 157

VSSRIGGRYVSSSVPTRSVDFRSRSS GTG TCC ACT CGC ATA GGG CCC CCC TAC GTC TCC TCC TCT GTT CCC ACC CGC TCT GTG GAC TTC AGG AGC CGC TCC AGT

47 234

40 235

APAPRLSYDKVDFSSAEAINCIEFFAT GCT CCC GCC CCC CGT CTC TCT TAT GAT AAA GTG GAC TTC TCG TCG GCA GAG GCC ATC MC

CAG GAG TTC TTT GCC ACA

73 312

74 313

RSNEKRELPELNDRFASFIEKVRHLE CGC AGC MT GAG AAA AGA GM CTA CAG GM CTC MT

GAC CCC TTT GCC AGC TTC ATA GAG AAG GTG CGG CAT TTA GAG

99 390

100 391

PPNSKLILELGPYKDPHPGSTGRINE CAG CAG MC TCG AAG CTG ATT CTG GAG CTG GGT CAG TAC AAG GAC CAG CAT CM GGG TCG ACA GGC CCC ATC AAT GAG

125 468

126 469

L C P Cl E M R E L R R P L E L H A K D R D Cl M Cl V E CTC TGC CAG CAG GAG ATG AGA GM CTG CCC AGG CAG CTG GAG CTG ATG CCC AAA GAT CCC GAC CAG ATG CAG GTG GAG

151 546

152 547

RDNLAEDVALLNQRLNEEMGKRPEAE AGA GAC MC CTG GCC GM GAT GTG GCC CTG CTC AAT CAG AGG TTA AAT GAG GAG ATG GGG AAA AGA CAG GAA GCT GAG

177 624

178 625

NNLTLFRKDVDDATLARLELERKIES AAT AAT TTG ACT CTC TTT CGC AAG GAT GTG GAT CAT GCC ACA CTT GCT CGT CTG GM CTG GAG AGA AAG ATC GAG TCT

203 702

204 703

LMDEIEFLKKMHDEEIPD~PVSVPSP CTG ATG GAT GAG ATT GAG TTC CTC MG AAG ATG CAT GAT GAG GM ATT CAG GAT GTA CAG GTG ACT GTT CAG AGT CAG

229 780

230 781

PMKHEVMETSSRPDLTGALRDIRAPY CAG ATG MC ATG GAG GTG ATG GAG ACG TCC AGC CGG CCT GAT CTG ACC CGA GCT CTT CGA GAC ATT AGA GCT CAG TAT

255 a58

256 a59

ESIATKNMPESEEUYKSKFADLTDSA GAG AGC ATC GCC ACC MA AAC ATG CAG GAA TCT GAA GAG TGG TAC AAG TCC MC

TTT GCT GAC CTG ACT GAT TCG GCC

281 936

282 937

KRNAEAMRPGKPENNDLRRPIPAPNC AAG CGC AAT GCT GAG GCC ATG AGA CAG GGT AAA CAA GAG MC AAT GAC CTG AGG AGA CAA ATT CAA GCC: CAG AAC TGT

307 1014

308 1015

DIDSLKRTNEALLRPMREMEEPFAAE GAT ATC GAT TCT CTC AAG AGG ACT AAT GAG GCT CTG CTG AGG CAG ATG AGA GAA ATG GM GAG CAG TTT GCT GCA GAG

333 1092

334 1093

ARNYPDTVSRLEDEIRNLKEEMSRHL GCC AGA AAC TAT CAA GAT ACT GTG TCC CGT CTG GM GAC GAG ATA CGA AAC CTG AAG GAG GAG ATG TCk CCC CAC CTG

359 1170

360 1171

REYPDLLNVKMALDIEIATYRKLLEG CCC GAA TAC CAG CAT CTG CTC MC

GTT MG ATG GCC CTA GAT ATT GAG ATT CCC ACC TAC AGG AAA CTC CTG GAG GGA

385 1248

386 1249

EENRlVVPIMKMPSMSGYSGDYGQFS GAG GM MC CGA ATT GTG GTT CCT ATC ATG AAA ATG CCT TCA ATG AGT GGA TAC AGT GGT GAT TAT GGT CAG TTT TCT

411 1326

412 1327

DTRAGPKVVIKTVETRDGEVVKESTK CAT ACT AGA GCT GGA CAG AAA GTC GTA ATC AAG ACC GTT GAG ACT CGT CAT CGA GAG GTG GTG AAA GAA TCG ACA AAA

437 1404

438 1405 1491 1594 1697 la00

E K G R D E K K D S Ii G P G K D l GM MC GGC AGA CAT GAG AAA AAA GAT TCA CAT GGT CAA GGC AM CAT TM ATCTGACMAGATTAGAGCGTGCAGATMATGAAT GCTTGATGAGTTATTMTATGTGTTGCAGTTTTMGGACCGTMCAGTGTTTACGATGATGCTGTAGAGTGTGAATAATCAGCTMAATGTGACTCAGTATTG TGTATGATATGGTGCTATTACTATATATGAGCATMCATGACACTGCCTTMCTMTMATCACTGTTTGCAGACCTMT~GTCACTAACMCATGCCCAA CAAATMTCACTTACAGACMCAAAAATAATATTTCAGAAGTTCACGTCAATATMTCTTCAMTMAAGTGAATGGAAACTGAAAAAAAAAAAAAAAAAAAA MAAAAAMA

454 1490 1593 1696 1799 la09

GAG CTA CTA GAG ACA AAA CAG CCA GAC GM GAG GM GAT ACA MC AGG CAT ATC ATT AM

Figur .e 2. The Sequence pP5A encodes the entire shown in boldface.

of pP5A cDNA plasticin

protein

and the Predicted of 453 aa with

Amino

Acid

a molecular

Plasticin Is Slowly Transported during Optic Nerve Regeneration Plasticin protein was identified by in vitro transcription of sense RNA, synthesized from the T, promoter of the pP5A clone, followed by in vitro translation of the RNA in a rabbit reticulocyte system containing [35S]methionine. The labeled translation product was

Sequence

TM

of Plasticin

mass of 52.6 kd. The three

polyadenvlation

addition

signals

are

then mixed with cytoskeletal proteins from the optic nerveandanalyzed bytwo-dimensionalelectrophoresis and autoradiography. In vitro transcribed plasticin RNA directed the synthesis of a single protein that corn&rated with a protein of approximately 53 kd in agreement with the predicted molecular mass of plasticin (Figures 7A and 78). Transport of protein into

NeUWl

376

AMINO ACID SEQUENCE

PLASTICIN PERIPHERIN VIMENTIN DESMIN PLASTICIN PERIPHERIN VIMENTIN DESMIN PLASTICIN PERIPHERIN VIMENTIN DESMIN PLASTICIN PERIPHERIN VIMENTIN DESMIN PLASTICIN PERIPHERIN VIMENTIN DESMIN

OF PlASTlClN

AND OTHER TYPE Ill IF PROTEINS

HEAD --------------------------------MBHSTFSHLFSpHFGApV MSHHSSGLRSSISkTSYRRTFGbP------------PSLSPGAFSYSSSS ------MSTRSVS SSYRRMF PGTSNRQSSNRSYVTTSTRTYSLGS-L ---MSQAYSSSQ SSYRRTF --APSFSLG-SPLSSPVFPRAGFGTKG = ROD YBPVBSRIGGRYVSSSVPTRSVDFRSRSSAPAP RLSY----DKVDFSSAEA RFSSSRLLGSGSP--SSSARLGSFRAPRAG-AL RLPS----ERL.DFSMAEA RPSTSRSLYSSSPGGAYVTRSSAVRLRSSMPGV RLLQ----DSVDFSLADA SSSSVTSRVYQVSRTSGGAGGLGSLRASRLGST BAPSYGAGELLDFSLADA COIL la Ll INQBF FATRSNEKRBLQBLNDBFASFIBKVBHLEQQNSKLILELGQY KDQ LNQEF LATBBNBKQBLQBLNDRFANFIBKVRFLBQQNAALRGBLSQA RGINTEF KNTRTNEKVBLQBLNDRFANYIDKVRFLBQQNKILLAELEQL KGVNQEF LATRTNEKVELQELNDRFANYIEKVRFLBQQNAALAAEVNRL KGCOIL lb HQGSTGRINEL CQQEMBELFlRQLELMAKDRDQMQVERDNLAEDVALLNQRL --QEPARADQL CQQBLRBLRRELELLGRSAYRVQVERDGLAEDLGALKQRL --QGKSRLGDL YEEEMRELBBQVDQLTNDKARVEVERDNLAEDIMRLREKL --REPTRVAEL YEEENRELRRQVEVLTNQRARVDVERDNLIDDLQRLKAKL

I

PLASTICIN PERIPHERIN VIMENTIN DESMIN

NEEMGKRQEAENNLTLFRKDVDDATLARLELERKIESLMDEIEFLKKMRDEE EEETRKREDAEHNLVLFRKDVDDATLSRLBLERKIESLMDEIBFLKKLHEEE QEEHLQREEAESTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHDEE QEEIQLKEEAENNLAAFBADVDAATLARIDLEBRIESLNEEIAFLKKVHEEE COIL 2 Ll-2 IQDV QVSVQSQQMKMEVMETSSRPDLT GALRDIRAQYESIATKNMQESEE LRDL QVSVESQQVQQVEVEATVKPELT AALBDIRAQYBNIAAKNLQEAEE IQEL QAQIQEQHVQIDVDV--SKPDLT AALRDVRQQYESVAAKNLQEAEE IREL QAQLQEQQVQVEMDM--SKPDLT AALRDIRAQYETIAAKNISEAEE

PLASTICIN PERIPHERIN VIMENTIN DESMIN

WYKSKFADLTDSAKRNAEAMRQGKQENNDLBFtQIQAQNCDIDSLKRTNEALL WYKSKYADLSDAANRNHBALKQAXQEMNESRRQIQSLTCEVDGLRGTNEALL WYKSKF?sDLSEAANRNNDALRQAKQESNEYBRQVQSLTCEVDALKGTNESLE WYKSKVSDLTQAANIZNNDALKQAKQEMMEYRHQIQSYTCEIDALKGTNDSLM

PLASTICIN PERIPHERIN VIMENTIN DESMIN

RQMREMBEQFAAEAFtNYQDTVSRLEDEIRNLKBENSRHLREYQDLLRVKMAL RQLRELEBQFALEAGGYQAGAARLEEELRQLKREMARRLREYQELLNVKMAL RQMRBMEENFALEAANYQDTIGRLQDEIQNMKEEMARRLREYQDLLNWU4AL RQMREL.EDRFASEASGYQDNIARLEEEIFUiLKDEMARHLKBYQDLLNVKMAL TAIL DIEIATYRKLLEGEENRIV VPIMKMPSMSGYSGDYGQFSDTRAGQ-K--W DIEIATYRKLLEGEESRIS VPVHSFASLSLKTTVPEVEPPQD-SHSRKMVL DIEIATYRKLLEGEESRIS LPLPNFSSLNLRETNLESLPLVD-THSKRTLL DVEIATYRKLLEGEESRIN LPIQTFSALNFRETSPEQR-GSE-VHTKKT'JM

PLASTICIN PERIPHERIN VIMENTIN DESMIN

-WKESTKEKGRDEKKDSHGQGKD* KWTESQKEQHSELDKSSIHSY* -VINET-SQHHDDLE* -WSEA-TQQQHEVL*

PLASTICIN PERIPHERIN VIMENTIN DESMIN Figure 3. Comparison

of Plasticin

with

Other

Type

III IF Proteins

The predicted amino acid sequence of plasticin manually aligned with those of rat peripherin (Thompson and Ziff, 1989), hamster vimentin (Quaxet al., 1983), and hamster desmin (Quaxet al., 1985). Vertical lines demarcate structural regions common to all IF proteins. Boldfaced amino acids represent sequences that are identical to the plasticin sequence. The sequence shared by peripherin, vimentin, and desmin in the head domain is boxed. The sequence shared by all type III IF proteins in the tail domain is also boxed. Dashes are inserted to optimize alignment.

the optic nerve during regeneration was examined by injecting the retina with P5S]methionine 30 days after optic nerve crush. After 15 days the optic nerves were processed, and the labeled proteins were analyzed by two-dimensional electrophoresis and autoradiography. The plasticin protein is labeled and transported into the optic nerve during the slow phase of axonal transport (Figure 7C). Although plasticin is a relatively minor protein in the nonregenerating optic nerve, a

considerable amount of this protein is transported during optic nerve regeneration (Figures 7A and 7C). Plasticin mRNA Is localized Retinal Ganglion Cell layer

to the

The site of plasticin mRNA synthesis in the retina was examined by in situ hybridization using an [35S]UTPlabeled antisense RNA probe to the variable tail region of plasticin (Figure 1). Significant hybridization is seen

Plasticin, Neurofilament 377

Protein

in Retina

A

1 2

1 2

3

4

5

6

0

Figure 4. Northern

Blot Analysis

of Plasticin

Figure 6. Analysis of Plasticin tic Nerve Regeneration

mRNA

A [a*P]UTP-labeled antisense RNA plasticin probe was synthesized from the pCDP clone and hybridized to a Northern blot at high stringency. Lane 1, goldfish retina 20 days after optic nerve crush, 1 fig of poly(A)+ RNA. Lane2, nonoperated goldfish retina, 1 pg of poly(A)’ RNA. Arrowheads denote the positions of the 28s and 18s rRNAs.

in the retinal ganglion cell layer of retinas 16 days after optic nerve crush (Figure 8). The hybridization is observed as discontinuous clusters of activity in the retinal ganglion cell layer. In noncrushed control retinas the low level of plasticin mRNA, which is detect-

P LR RR SC B 1 S LI 0

Figure

5. Analysis

of Plasticin

Y ON

RNA Levels in Goldfish

Tissues

RNAase protection analysis was used to investigate the levels of plasticin RNA in various goldfish tissues. A 373 nt, [3zP]UTPlabeled antisense RNA probe was synthesized from the T3 promoter of the pCD2 clone in PBS (see Figure 1). This probe was incubated with 20 pg of total RNA from various goldfish tissues, digested with RNAase A, and run on a 6% polyacrylamide-urea gel. The protected fragment is 277 nt. P, 150 cpm undigested probe; LR, left retina, nonoperated control; RR, right retina, 20 days after optic nerve crush; SC, spinal cord; B, brain; L, lens; S, skin; LI, liver; 0, oocytes; Y, yeast tRNA (negative control); ON, optic nerve.

7

8

9

20 40 DAYS

IO 11 12 13 14 15

120

mRNA Levels in Retina during

Op-

A 373 nt, P*P]UTP-labeled antisense RNA probe was synthesized from the T3 promoter of the pCD2 clone in PBS (see Figure 1). This probe was incubated with 20 Bg of total RNA from retina, which innervates either the crushed or the opposite noncrushed (control) optic nerve. (A) The retina RNA was digested with RNAase A and separated on a 6% polyacrylamide-urea gel. The protected fragment is 277 nt. Lane 1,150cpm undigested probe; lane2, day0 nonoperated retina; lanes 3, 5, 7, 9,11, 13, nonoperated (control) retina 5, IO, 20,27, 37,120 days after optic nerve crush, respectively; lanes 4, 6,8,10,12,14, operated retina 5,10,20,27,37,120 days after optic nerve crush, respectively; lane 15, yeast tRNA (negative control). (B) Graphic representation of the RNAase protection data. The relative optic density (Relative OD) was determined for each protected band by densitometry. Relative optic density is plotted against days after optic nerve crush. Operated eye (closed circles). Nonoperated, control eye (open circles). The smooth curve was drawn by hand.

able by Northern ure 4; Figure 5; by this method. not be detected crush when the RNAase A prior was hybridized

and RNAase protection analysis (Figand Figure 6), could not be detected Additionally, plasticin mRNA could in retinas 16 days after optic nerve sectionswere incubated in pancreatic to hybridization or when the tissue with the sense RNA probe.

Discussion

We present the nucleotide and predicted amino acid sequence of a cDNA coding for a novel type III IF protein, plasticin, which we have isolated from goldfish retina. The amino acid sequence of plasticin is consistent with the overall structure of IF proteins. Comparison of plasticin with other IF proteins clearly

NWKNl 378

A

Figure7. Plasticin Nerve Crush

Protein

Is Slowly

Transported

after

Optic

(A) Coomassie blue-stained cytoskeletal preparation of optic nerve proteins. In vitro transcribed/translated plasticin was mixed with optic nerve cytoskeletal proteins and separated by two-dimensional electrophoresis. Brackets indicate 58 kd ONs. Arrowhead indicates plasticin. (B) Full-length sense plasticin RNAwas transcribed in vitro from the T, promoter of clone pP5A in Bluescript. This RNA was used to direct the synthesis of plasticin protein in the rabbit reticulocyte in vitro translation system in the presence of [?S]methionine. The products were mixed with optic nerve cytoskeletal proteins and analyzed by two-dimensional electrophoresis and autoradiography. Arrowhead indicates in vitro synthesized plasticin. (C) Autoradiogram of slowly transported proteins in the optic nerve after nerve crush. [%]methionine (50 PCi) was injected into the right eye 30 days after optic nerve crush. After 15 days, nerves were isolated and analyzed by two-dimensional electrophoresis andautoradiography.Arrowhead indicatestransported plasticin.

places it within the type III class of IF proteins. The type III classof IF proteins presentlyconsistsof vimentin, glial fibrillary acidic protein, desmin, and peripherin (Goldman and Steinert, 1990). The rod domain of plasticin showsextensive homologyto these proteins. Plasticin also shares with the type III proteins the highly conserved hallmark sequence IKTVETRDG, which is found only in type III proteins. The amino terminal head domain is the most divergent in amino acid sequence when compared with other type III IF proteins. Notably, the highly conserved sequence SSYRR(T/M)FG, which is shared by peripherin, vimentin,desmin,and NF-L(neurofilament light), is not present in plasticin. However, plasticin is evolutionarily related to these proteins in that there is considerable homology in the a-helical rod domain. In contrast, the significant divergence in the amino-terminal head domain and the extended tail domain indicates that plasticin is a new member of the type III class of IF proteins. In addition to their structural diversity, the expression of IF proteins is developmentally regulated and cell specific (Mall et al., 1982; Klymkowskyet al., 1989). This is clearly observed during neurogenesis, when almost all of the IF protein types are expressed from the earliest stages of stem cell differentiation to the elaboration of a mature neuron (Steinert and Liem, 1990). During the earliest stages of the process, the type I and type II keratins, K8 and K18, are expressed in neuroepithelial cells (Jackson et al., 1980). As neurogenesis proceeds there is an orchestrated expression of IF proteins. Vimentin and nestin are expressed in neuronal stem cells (Bignami et al., 1982; Cochard and Paulin, 1984; Lendahl et al., 1990). As the neuronal precursor cells become postmitotic, nestin expression is suppressed. Coincident with the end of migration and the beginning of neuronal differentiation, a-internexin expression begins, soon to be followed by NF-L and NF-M (neurofilament medium) expression in most CNS neuronal cell populations (Kaplan et al., 1990; Chiu et al., 1989). Alternatively peripherin, NF-L and NF-M expression begins in peripheral and some CNS neuronal cell populations (Troyet al., 1990; Gorham et al., 1990). The expression of NF-H (neurofilament heavy) lags somewhat behind the expression of NF-L and NF-M as the neurons mature (Shaw and Weber, 1982). Thus, a large range of IF protein types is utilized during neurogenesis and then restricted to a stereotyped set of major proteins in the fully differentiated mature mammalian neuron. The IF protein composition in the goldfish visual pathway is different and displays greater complexity than that of the more static adult mammalian visual system. The major proteins of the optic nerve consist of the 58 kd optic nerve IF proteins (ONs), ONI through ON4, and a 48 kd protein. The goldfish optic nerve also contains the NF-L and NF-M proteins with apparent molecular masses of 145 kd and 80 kd, respectively(Quitschke eta/., 1985). Results from molec-

Plastrcin, Neurofilament 379

Protein in Retina

Figure 8. Plasticin mRNA Localization Goldfish Retina by In Situ Hybridization

in

(A) Goldfish retina 16 days after optic nerve crush. (6) Uncrushed control retina. CCL, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PE, pigment epithelium. Bar, 100 pm.

cloning studies indicate that there are several minor IF proteins expressed in this system that are homologous to developmentally expressed mammalian IF proteins (R. K. Druger, E. M. Levine, E. Glasgow, P. S. Jones, and N. Schechter, submitted). Plasticin is a minor component of the goldfish optic nerve, significantly induced after optic nerve crush, and not homologous to any known mammalian protein. The recent elucidation of an orchestrated expression of IF proteins during neuronal development in higher vertebrates results from the molecular cloning of novel IF proteins from tissues and cultured cells at specific stages of neuronal differentiation. All of the more recently identified neuronal IF proteins, i.e., peripherin (Leonard et al., 1988; Parysek et al., 1988), a-internexin (Fliegner and Liem, 1991), and nestin (Lendahl et al., 1990), are major constituents of the neuronal cytoskeleton, and their initial expression reflects specific stages of differentiation. Our results indicate that there may be additional IF proteins that are expressed or coexpressed during neuronal differentiation or in restricted cell populations. It is likely that the type, stoichiometry, and degree of phosphorylation of IF proteins expressed define and regulate the physiological properties of the IF network (Goldman and Steinert, 1990). Additional IF proteins may further expand this repertoire, fulfilling structural requirements of the cytoskeleton during neuronal development. The question remains if this newly identified type III IF protein, plasticin, will correspond to an unidentified mammalian homolog and at what stage during neurogenesis this putative protein is expressed. Alternatively, plasticin may represent an evolutionarily divergent type III protein, which has structural attributes that support thecapacity for continuous development and plasticity in the goldfish. ular

Experimental

Procedures

Animals Common goldfish (Carassius auratus, 8-12 cm) were obtained from Mt. Parnell Fisheries, Mercersburg, PA and maintained in 40 gallon tanks at approximately 18OC. lntraorbital nerve crush was performed on the right optic nerve after anesthetization by immersion in 0.05% tricaine methanesulfonate. The left optic nerve was left intact to serve as a control.

RNA Isolation Tissue was immediately frozen in liquid nitrogen, and total cellular RNA was isolated with RNAZasol 8 according to the manufacturer’s instructions (Tel-Test, Inc.). Poly(A)+ RNA was selected from total RNA by passage over a column of oligo(dT)-cellulose (Aviv and Leder, 1972).

cDNA Library Construction A retina kgtll cDNA library was constructed by standard techniques (Sambrook et al., 1989) using poly(dTJ-primed reverse transcription of 5 pg of poly(A)+ RNA isolated from retinas 20 days after optic nerve crush. The primary plating gave an initial titer of 5 x IO5 pfu with an average insert size of approximately 1 kb. Subsequently, thelibrarywasamplified toatiterof approximately 1 x 108 pfu/kI.

Screening of cDNA Libraries Initially,a338 bpEcoRI-Xbalfragmentofagoldfishvimentin-like partial cDNA clone (see Figure 1) was labeled with PZP]dCTP (NEN) by random primer synthesis (Amersham) and used to screen the kgtll cDNA library at low stringency (Sambrook et al., 1989). The final wash was in 0.2x SSC, 0.1% SDS at 42°C for 20 min. A lgtl0 cDNA library was screened with the [‘ZP]dCTP-labeled plasticin partial cDNA clone pCD2 (see Figure 1) at high stringency. The final wash was in 0.2x SSC, 0.1% SDS at 68°C for 30 min. After plaque purification, the 1 clone inserts were subcloned into PBS KS(-) (Stratagene). Internal restriction sites and synthe sized primerswere utilized to sequence the inserts in both directions using the Sequenase I I kit (USE). Sequences were analyzed by computer using DNASIS and the CCC programs (Devereux et al., 1984).

NWKNl 380

Northern Blot Analysis One microgram of retina poly(A)+ RNA from regenerating and nonregenerating eyes was separated on a 1.3% agarose-formaldehyde gel, transferred to Nytran (Schleicher and Schuell), and probed as previously described (R. K. Druger, E. M. Levine, E. Glasgow, P. S. Jones, and N. Schechter, submitted). The final wash was in 0.1 x SSC, 0.1% SDS at 68OC for 1 hr. RNAase Protection Assays RNAase protection assays were performed essentially as described by Melton et al. (1984). Labeled riboprobe was purified on a 5% polyacrylamide gel. Total RNA (20 pg) was hybridized with 5 x IOZ cpm probe at 4S°C overnight. Single-stranded RNA was digested with RNAase A (4 pglml) for 1 hr at 37OC. The protected RNA fragments were analyzed on a 6% polyacrylamideurea gel. Autoradiography was performed with an intensifying screen at -80°C overnight. In Vitro Transcription and Translation RNA transcripts of plasticin were synthesized in vitro using the T, and T, promoters after linearization, with BamHl or Hindlll, of the pBS plasmid containing the 1.8 kb plasticin insert pP5A. Transcribed RNA was translated in a rabbit reticulocyte lysate containing [?S]methionine according to the manufacturer’s instructions (Promega). The translation reactions were mixed with 30 pg of goldfish optic nerve cytoskeletal preparation in 15 ~rl of CHES buffer (SigmaJand analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Cytoskeletal Preparation and Two-Dimensional Electrophoresis Cytoskeletal proteins were isolated from goldfish optic nerve as described previously (Jones et al., 1986b). Two-dimensional electrophoresis was performed essentially as described by Q’Farrell (1975) with slight modifications (Quitschke and Schechter, 1980). Autoradiography was performed as described previously (Ciordano et al., 1990). In Situ Hybridizations Goldfish were dark adapted and anesthetized in ice prior to tissue removal. Retinas were rinsed in phosphate-buffered saline and fixed with 4% paraformaldehyde in phosphate-buffered saline for 2 hr. Regenerating and control retinas were cryoprotected overnight in 30% sucrose and em bedded in a I:1 mixture of OCT (Miles Labs) and Aquamount (Lerner Labs) as previously described (Jones et al., 1986a). Cryostat sections (10 Frn) were collected on Superfrost plus slides (Fisher Scientific). Slides were processed as previously published (Sternini et al., 1989) with the following modifications. Retinas were deproteinated with proteinase K for 7.5 min at room temperature. A 263 nt plasticin riboprobe was labeled with P5S]LJTP by in vitro transcription of pCD2 using the Ts (antisense RNA) or T, (sense RNA) promoters. Slides were incubated overnight at60°C in 40 PI of hybridization buffer containing 4 ng of probe. Additional controls were incubated in pancreatic RNAase A (50 ug/ ml) for 30 min at 37OC prior to prehybridization. Acknowledgments We thank Dr. Dan Goldman for the hgtl0 goldfish retina library. We thank Dr. N. Brecha and Dr. S. Yazulla for their assistance with the in situ hybridization experiments. We thank Dr. Andrew Francis for helpful comments on the manuscript. This work was supported by a grant from the National Institutes of Health (EY05212) to N. S. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

31, 1992; revised

June 10, 1992.

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The accession is M90532.

number

for the sequence

reported

in this paper