inhibition of cytoskeletal reorganization stimulates actin and tubulin syntheses during...

Inhibition of Cytoskeletal Reorganization StimulatesActin and Tubulin Syntheses During Injury-InducedCell Migration in the Corneal EndotheliumSheldon R. Gordon* and Renee M. Buxar

Department of Biological Sciences, Oakland University, Rochester, Michigan 48309

Abstract A single layer of squamous epithelial cells termed the ‘‘endothelium’’ resides upon its natural basementmembrane (Descemet’s membrane) along the posterior surface of the vertebrate cornea. A well-defined circular freezeinjury to the center of the tissue exposes the underlying basement membrane and results in the directed migration ofsurrounding cells into the wound center. This cellular translocation is characterized by the reorganization of the actinand tubulin cytoskeletons. During migration, circumferential microfilament bundles are replaced by prominent stressfibers while microtubules, observed as delicate lattices in non-injured cells, become organized into distinct web-likepatterns. To determine whether this cytoskeletal reorganization requires actin or tubulin synthesis, injured rabbitendothelia were organ cultured for various times and metabolically labeled with 35S-methionine/cystine (250 µCi/ml) forthe final 6 h of each experiment. Analysis of actin and tubulin immunoprecipitates indicated no significant increases in35S incorporation occurred during the course of wound repair when compared to isotope incorporation in noninjuredtissues. However, when cytoskeletal reorganization was hampered, either by pre-treating tissues with 7 µM phalloidin tostabilize their circumferential microfilament bundles, or culturing in the presence of 1028M colchicine to dissociatemicrotubules, 35S incorporation increased significantly into both actin and tubulin immunoprecipitates at 48 hpost-injury. Furthermore, in both cases, exposure to actinomycin D substantially suppressed isotope incorporation.These results indicate that cytoskeletal rearrangement of microfilaments and microtubules during wound repair, incorneal endothelial cells migrating along their natural basement membrane, utilizes existing actin and tubulin subunitsfor filament reorganization. Disrupting this disassembly/reassembly process prevents cytoskeletal restructuring andleads to the subsequent initiation of actin and tubulin syntheses, as a result of increased transcriptional activity. J. Cell.Biochem. 67:409–421, 1997. r 1997 Wiley-Liss, Inc.

Key words: corneal endothelium; actin; tubulin; upregulation; autoregulation; migration; wound repair

The posterior surface of the vertebrate cor-nea is lined by a single layer of very squamousepithelial cells that are termed the corneal ‘‘en-dothelium.’’ They reside on their natural base-ment membrane, termed Descemet’s mem-brane. These cells serve as a transport epitheliawhose function is to maintain corneal deturges-cence and hence transparency.

Cells of the adult corneal endothelium aremitotically inactive, and the entire cell popula-tion resides in the Go phase of the cell cycle[Gordon and Rothstein, 1978]. Trauma to thetissue, such as a circular freeze injury, provides

the stimulus for cells around the wound regionto initiate macromolecular synthesis, traversethe cell cycle, divide, and migrate into thewound, along Descemet’s membrane [Gordon,1994]. Previous studies have shown that cellmigration is the major mechanism involved incorneal endothelial wound repair [Doughmanet al., 1976; Gordon and Rothstein, 1982]. How-ever, the underlying mechanisms governing cellmovement along natural basement membranesare less well characterized. For example, ratcorneal endothelial cells synthesize and secreteextracellular matrix proteins during wound re-pair [Gordon, 1988; Munjal et al., 1990] despiteresiding on and moving along Descemet’s mem-brane after an injury.

The initiation of endothelial migration is alsoaccompanied by cytoskeletal alterations. Theactin cytoskeleton of these cells is character-ized by the presence of a distinct circumferen-

Contract grant sponsor: Oakland University Research Ex-cellence Program in Biochemistry and Biotechnology.*Correspondence to: Sheldon R. Gordon, Ph.D., Depart-ment of Biological Sciences, Oakland University, Rochester,MI 48309-4476. E-mail: [email protected] 23 July 1997; Accepted 13 August 1997

Journal of Cellular Biochemistry 67:409–421 (1997)

r 1997 Wiley-Liss, Inc.

tial microfilament bundle (CMB) [Gordon et al.,1982; Gordon, 1990; Gordon and Staley, 1990]that is associated with apical junctional com-plexes [Barry et al., 1995]. Injury to the tissueresults in the reorganization of CMBs as cellsinitiate movement into the wound region toreestablish the monolayer [Gordon and Staley,1990; Petroll et al., 1995; 1997]. The extent ofactin reorganization and migration in this sys-tem correlates with the size of the wound [Ichi-jima et al., 1993]. In small endothelial injuries,cells maintain contact with adjacent neighborsduring the repair process and those borderingthe wound edge display decreased F-actin whileretaining their CMBs [Petroll et al., 1995], remi-niscent to that reported for small wounds incultured vascular endothelium [Wong and Got-lieb, 1988]. In some regions along the woundedge, microfilament arcs around the injury havealso been reported [Petroll et al., 1995], sugges-tive of ‘‘purse string’’ wound closure [Martinand Lewis, 1992; Bement et al., 1993]. In con-trast, larger transcorneal freeze injuries resultin the loss of cell-to-cell contacts [Munjal et al.,1990], that are associated with a dynamic reor-ganization of the actin cytoskeleton to formstress fibers in migrating cells [Gordon et al.,1982; Gordon and Staley, 1990; Ichijima et al.,1993]. Similar microfilament changes have beenconfirmed in vascular endothelium [Gabbianiet al., 1983; Gotlieb et al., 1984; Rogers et al.,1989; Ettenson and Gotlieb, 1993b], intestinaland respiratory epithelium [Zahm et al., 1991;Nusrat et al., 1992], and in the organ culturedretinal pigment epithelium [Hergot et al., 1989].Recently, Lee et al. [1996] described three se-quential stages of microfilament reorganiza-tion in response to the wounding of vascularendothelial monolayer cultures. Initially, actinwas arranged as CMBs, but following an injurythese disappear and central microfilaments ap-pear that parallel the wound. This stage istransient and soon microfilaments assume aperpendicular orientation to the wound as move-ment is initiated. Collectively, these data indi-cate that in many systems, the cellular re-sponse to wounding is accompanied by adistinctive rearrangement of the actin cytoskel-eton into stress fibers that are associated withcell migration.

Alterations in the microtubule pattern of cor-neal endothelial cells during the transition froma resting to a migratory state are not as dra-matic as microfilament changes. Initially, micro-tubules appear in a delicate filamentous lattice

that, following injury, forms a web-like pattern[Gordon and Staley, 1990]. Accompanying thischange is a translocation of the microtubuleorganizinging center (MTOC) to the anteriorportion of the cell [Gordon, 1994]. Similar re-sults are observed in vascular endothelium,both in vitro [Gotlieb et al., 1984] and in theorgan cultured system [Rogers et al., 1989].Mechanisms underlying MTOC translocationare not fully understood, although it has beenshown to depend on transcriptional activityoccurring shortly after wounding [Ettenson andGotlieb, 1993a]. The relocation of the MTOCappears to correlate with directed cell migra-tion and interfering with this process retardswound repair [Ettenson and Gotlieb, 1993a].Similarly, in the corneal endothelium, MTOCtranslocation may be coupled not only withdirected migration but also to the polarizedsecretion of extracellular matrix protein. Dis-rupting microtubule integrity with colchicineprevents not only the deposition of fibronectin,but results in the restriction of cell movement[Sabet and Gordon, 1989; Gordon and Staley,1990]. In this present study, we have investi-gated cytoskeletal changes in the organ cul-tured corneal endothelium during wound re-pair. Specifically, we examined whethermicrofilament and microtubule alterations dur-ing cell migration resulted from the reorganiza-tion of the pre-existing filaments or the synthe-sis of new actin and tubulin.

MATERIALS AND METHODSInjury and Organ Culture

Eyes were isolated from New Zealand whiterabbits (2.2 kg) and corneas given a circulartranscorneal freeze injury (approximately 5 mmdiameter) using a tapered hollow brass conecontaining an ethanol/dry ice mixture (270°C).Corneas were carefully dissected away from theglobe, maintaining a 1-mm wide scleral rim tofacilitate handling. In phalloidin pre-treatmentexperiments, endothelia were given a directfreeze injury using a stainless steel circularprobe pre-cooled to 270°C in ethanol and dryice. Corneas were cultured endothelial side upin basal medium Eagle (BME) (Sigma CemicalCo., St. Louis, MO) supplemented with 10%rabbit serum (Sigma) and 40 µg/ml gentamicin(Sigma) and incubated at 37°C in a humidifiedatmosphere of 5% CO2 and 95% air. For someexperiments involving tubulin cytoskeletal reor-ganization, the medium was also supplementedwith 1028M colchicine (Sigma). For other experi-

410 Gordon and Buxar

ments, tissues were incubated in mediumsupplemented with 0.05 µg/ml actinomycin D(Sigma) from 18–48 h post-injury. For meta-bolic labeling, tissues were transferred intominimal essential medium Eagle without cys-tine or methionine (ICN Pharmaceuticals, CostaMesa, CA) containing 250 µCi/ml 35S Transt(ICN) for the final 6 h of incubation.

Phalloidin Pre-Treatment

In some experiments, endothelial microfila-ments were exposed to phallotoxins prior towounding. For this, corneal endothelia wereorgan cultured in BME containing 7.5 µM phal-loidin, either non-labeled or labeled with rhoda-mine (TRITC) for 20 h prior to receiving aninjury. At the conclusion of the experiments,tissues exposed to non-labeled phalloidin werefixed and stained with TRITC-phalloidin. Inother cases, tissues pre-treated with labeledphalloidin were fixed and processed for observa-tion by fluorescence microscopy.

Histological Observations

For the histological observation of the endo-thelium, corneas were fixed in Carnoy’s solu-tion overnight and rehydrated through a de-scending alcohol series. Corneas were dissectedfree of their surrounding scleral rim, and theendothelial cell layer, along with Descemet’smembrane, were gently separated from the restof the tissue. Isolated tissues were flat mountedon albumin-coated glass slides and received4–5 radial cuts to allow them to lie flat. Tissueswere then stained with 1% cresyl violet acetate(Eastman Kodak Co., Rochester, NY), andmounted in Permount (Fisher Scientific, FairLawn, NJ).

Cytoskeletal Visualization

Microfilaments were visualized with eitherTRITC or FITC phalloidin (Molecular Probes,Junction City, OR) as described [Gordon andStaley, 1990]. Briefly, cultured tissues were gen-tly rinsed in 0.1M PBS and fixed in 3.7% formal-dehyde (Fisher Scientific) for 10 min at roomtemperature. Following a 30-min PBS wash,flat mounts were prepared and allowed to airdry. Tissues were then extracted in acetone(210°C) and subsequently rinsed in 0.1M PBSfor 10 min. Endothelia were exposed to 1.67 31027M phalloidin in 0.1M PBS for 30 min, rinsedand mounted in a p-phenylenediamine/glycerolanti-fading solution [Johnson andAraujo, 1982].

For microtubule localization, fixation and ex-traction of the tissue were identical to thatdescribed for microfilaments. Microtubules werestained with Tu-27B, a monoclonal antibody tob-tubulin (a generous gift of Dr. L.I. Binder,Northwestern University), followed by stainingwith a 1:16 dilution of rabbit anti-mouse IgG-rhodamine (ICN) using standard immunocyto-chemical protocols. Endothelial flat mountswere sealed using the anti-fading medium de-scribed above.

Biochemical Analysis

For analysis of the endothelial cytosolic pro-tein profile, tissues were isolated from corneasand homogenized on ice for 15 min in 0.1M Trisbuffer containing 0.1% SDS and the followingprotease inhibitors: aprotinin (1 mg/ml), leupep-tin (1 µg/ml), pepstatin (0.7 µg/ml), and phenyl-methylsulfonylfluoride (PMSF; 2 mM). Follow-ing protein determination using the DC proteinassay (Bio-Rad Laboratories, Hercules, CA),b-mercaptoethanol was added to the sample togive a final concentration of 2%. Samples wereboiled for 5 min, cooled, and centrifuged toremove any particulate material. Between 70–100 µg of cytosolic protein was loaded onto andseparated on 10% polyacrylamide mini gels(0.375M Tris HCl, pH 8.8) with an overlying 4%stacking gel and subsequently stained with Bril-liant Blue G concentrate (Sigma). To identifythe actin and tubulin band within the cytosolicprofile, proteins were electrophoretically trans-ferred onto Immobilon Pt membranes (Milli-pore Corporation, Bedford, MA) and exposed toeither anti-actin (ICN) or Tu-27B, respectively.This was followed by incubation in goat anti-rabbit IgG-HRP or Protein G-HRP (both fromBio-Rad) and visualized with diaminobenzidineas the substrate.

Immunoprecipitation

Endothelia were isolated and homogenizedfor 30 min on ice in 10 mM Tris HCl containing0.05% Nonidet P-40 (pH 7.2) with 0.15M NaCl,1 mM CaCl2, 1 mM EDTA, 4 mM PMSF, and0.02% sodium azide. Following centrifugationto remove Descemet’s membrane, actin or tubu-lin was immunoprecipitated using either anti-actin (IgG fraction; Biomedical Technologies,Stoughton, MA) or Tu-27B, respectively.Samples were incubated overnight at 4°C withagitation to optimize antibody/antigen interac-tion. Immune complexes were then incubatedin Protein A-sepharose CL-4B beads (Pharma-

Cytoskeletal Proteins and Endothelial Repair 411

cia LKB, Uppsala, Sweden) and isolated bycentrifugation. The ensuing pellet was washedand dissolved in Tris sample buffer containing1% SDS without b-mercaptoethanol. Followingthe determination of protein concentration, ap-proximately 7 µg of protein was loaded ontogels as described above and subsequently elec-trophoresed by SDS-PAGE.

Scintillation Analysis

Actin and tubulin immunoprecipitates wereanalyzed by liquid scintillation counting usinga 1214 Rackbeta liquid scintillation counter(LKB/Wallac). Seven micrograms of each pro-tein sample were dissolved in 10 ml of ACSscintillation fluid (Dupont/NEN, Boston, MA).Counts were adjusted for background and givenas counts/µg protein.

Autoradiography

Gels containing 35S-labeled proteins wereplaced in cellophane (BioDesign, Inc., Carmel,NY), dried overnight and placed in film cassetteholders with RX medical X-Ray film (Fuji,).Film was exposed up to 1 week at 240°C anddeveloped with Kodak D-19 developer.

RESULTSEndothelial Wound Repair

The entire adult noninjured corneal endothe-lium is composed of a monolayer of non-cycling,tightly packed polyhedral cells containing ovalto kidney shaped nuclei (Fig. 1A). A freeze in-jury to the tissue (Fig. 1B,C) produces a distinctwound border between the cells and the de-nuded injury zone, where the underlying Desce-met’s membrane is exposed. At 24 h after injury(Fig. 1D), cells adjacent to the wound havebegun to separate from the rest of the mono-layer and initiate movement into the injuryzone. By 48 h after wounding, extensive inwardmovement has occurred within the injury area(Fig. 1E) and Descemet’s membrane has nearlybeen re-epithelialized by the cells.

Biochemical examination of the Coomassieblue stained cytosolic protein profile (Fig. 2,lanes 2–4) indicated variations in the pattern ofendothelial proteins between noninjured tis-sues and tissues undergoing wound repair. Au-toradiographic analysis of metabolic labeled tis-sues (Fig. 2, lanes 5–7) shows that moreextensive changes were apparent relative tothe synthesis of various proteins within thebands.

Cytoskeletal Changes AccompanyingWound Repair

The injury-induced migratory response of cor-neal endothelial cells is accompanied by cyto-skeletal reorganization. Morphologically, micro-filament pattern changes are much moreextensive than are those of microtubules. In thenoninjured endothelium (Fig. 3A) phalloidinstaining reveals a pattern of circumferentialmicrofilament bundles within the cells that isobserved throughout the tissue. Twenty-fourhours after a circular freeze wound, cells adja-cent to the wound border begin to migrate intothe injury. By this time microfilaments havereorganized into stress fibers (Fig. 3B) that areorientated in the direction of the cells broad-based lamellipodia. By 48 h post-injury (Fig.3C), these cells have migrated far into thewound region, nearly covering the defect, andstill display prominent stress fibers.

Microtubule rearrangements in migrating en-dothelial cells appear much more subtle. Immu-nofluorescent microscopy of the noninjured tis-sue demonstrates that cells are tightly packedand exhibit an extensive microtubular network(Fig. 3D). By 24 h after an injury, cells surround-ing the wound display distinct MTOCs that areoriented towards the wound region (Fig. 3E).When observed at 48 h after injury, the microtu-bule patterns are somewhat masked due to theextensive disorganization within the wound re-gion (Fig. 3F), but spindles, where present, arereadily visible (Fig. 3F, inset).

Phalloidin Effect on Cytoskeletal ReorganizationDuring Wound Repair

Incubation of organ cultured endothelium in7.5 µM TRITC-phalloidin for 20 h (‘‘pre-treated’’), results in toxin uptake and the stain-ing of the cells circumferential microfilamentnetworks (Fig. 4A) in a pattern reminiscent tothat seen after the staining of fixed prepara-tions. Pre-treated tissues that were subse-quently injured and cultured for up to 48 h andexamined after fixation showed dramaticchanges relative to microfilament reorganiza-tion. When observed at 24 and 48 h after injury,endothelial cells exhibited few, if any, stressfibers within their cytoplasm (Figs. 4B,C). Inorder to investigate whether the lack of visiblestress fibers could be explained simply as a lossof label, experiments were repeated using non-labeled phalloidin followed by post-fixationstaining with fluorochrome conjugated phalloi-din. When these tissues were examined, only


diffuse fluorescence could be observed at 24 hpost-injury (Fig. 4D), but, after 48 h, stainingrevealed the presence of distinct stress fiberswithin the cells (Fig. 4E). Similar results wereobtained with preparations that were pre-

treated with TRITC-phalloidin before injuryand post-stained with FITC-phalloidin after theconclusion of the experiment. Here, cells showeda distinct lack of TRITC-phalloidin stainedstress fibers at 48 h post-injury (Fig. 4F),

Fig. 1. Wound response in the organ-cultured rabbit cornealendothelium. A: Flat mount of the noninjured endothelium.Note the monolayer, composed of closely packed polyhedralcells, does not contain any cells undergoing mitosis. B: Theinjury zone (IZ) is clearly delineated following a circular freezewound. C: Higher magnification of rectangular region seen in B.

D: Twenty-four hours after injury, cells surrounding the woundhave begun to migrate inward toward the center of the wound.E: By 48 h post-injury, the wound is nearly filled in andextensive migration is observed. Original magnifications:A, 3130; B, 310; C, 332; D, E, 380.


whereas, FITC-phalloidin staining revealed nu-merous stress fibers within the cytoplasm (Fig.4F).

Actin and Tubulin Synthesis During EndothelialCell Migration

Rabbit corneal endothelial cell actin and tubu-lin were characterized using SDS-PAGE, anti-actin and anti-tubulin immunoblotting andimmunoprecipitation. When immunoblottedcytosolic proteins were compared to molecularweight standards, two bands were detected atapproximately 45 kD and near 50 kD, corre-sponding to the molecular weights of actin andtubulin, respectfully (Fig. 5). Electrophoresis ofimmunoprecipitated actin and tubulin from en-dothelial cells resulted in bands whose migra-tion paralleled results obtained by Western blots(Fig. 5). Densitometric scans (data not shown)of the Coomassie blue stained cytoplasmic pro-file determined that actin comprised approxi-mately 10% of the total soluble protein, whereas,tubulin constituted about 4.8%.

Metabolically labeled actin and tubulin wereimmunoprecipitated from pooled tissues of ei-ther noninjured, 24- or 48-h post-injured endo-thelial preparations. Immunoprecipitates of ac-tin and tubulin from noninjured tissuesexhibited very little incorporation of 35S (TablesI and II) and neither protein was detected onautoradiograms of the gels (Fig. 6). When analy-sis was performed on pooled preparations from24 or 48 h post-injured tissues, once again,neither actin nor tubulin immunoprecipitateswere detected by autoradiographic imaging (Fig.

6). Low isotope incorporation was verified byliquid scintillation counting (Tables I and II)that indicated 35S uptake into actin and tubulindid not significantly differ during wound repairfrom those levels observed in noninjured tis-sues.

When actin and tubulin reorganization wasimpaired using either phalloidin pre-treatmentor colchicine, respectively, high levels of 35Sincorporation were detected at 48 h post-injuryfor each protein (Tables I and II) and theirrespective immunoprecipitates were clearly vis-ible on autoradiograms (Fig. 6). Furthermore,incubating injured tissues in media containing0.01 µg/ml AMD for the final 30 h of culture (fora total culture time of 48 h), greatly surpressed35S incorporation into both actin and tubulin(Tables I and II). Fluorescent observations ofactin in cells treated by this protocol show thatcells contained some microfilaments (Fig. 7A),but far less than their control counterparts(Fig. 7B).

DISCUSSION

Results presented here indicate that cornealendothelial cytoskeletal reorganization duringcell migration, in response to a circular freezeinjury, is not dependent on de novo synthesis ofactin or b-tubulin, since labeled material wasnot detected following immunoprecipitation ofeither protein. However, the synthesis of eachprotein was heightened by interfering with thereorganization of its respective filament sys-tem. Pre-incubating tissues in phalloidin priorto injury prevented subsequent microfilamentreorganization and resulted in the synthesis ofactin. Likewise, incubation of endothelia in me-dium containing colchicine also resulted in asignificant incorporation of label into immuno-precipitated tubulin. In both cases, treatmentwith actinomycin D suppressed isotope incorpo-ration, suggesting that the synthesis of eachprotein resulted, at least in part, from new geneexpression rather than post-transcriptionalchanges of pre-existing m-RNA.

Injured corneal endothelial cells adjacent tothe wound edge undergo striking modificationsin their actin organization. Initially, these cellsexhibit apical CMBs, but as migrating cells,actin is reorganized into distinct stress fibers[Gordon and Staley, 1990; Ichijima et al., 1993],implying that actin reorganization may involvethe depolymerization of the apical microfila-ment bundle. Our results suggest that CMBdepolymerization is central to actin reorganiza-tion and is probably sufficient enough to in-

Fig. 2. Detection of rabbit corneal endothelial tissue proteinsby 10% SDS-PAGE and 35S metabolic labeling. Lanes 1–4 arestained with Coomassie blue. Lane 1, protein standards; lanes2–4, 0, 24, and 48 h post-injuries, respectively. Lanes 5–7, thecorresponding autoradiographic patterns from 0, 24, and 48 hpost-injuries.


crease the monomeric G-actin pool size, thuscurtailing additional actin synthesis, and pro-mote stress fiber assembly. Indeed, reversibledynamics between G-actin, F-actin, and theirassociated proteins serve as the basis for actin

function [Korn, 1982; Cooper, 1991]. Endothe-lia pre-treated with TRITC-phalloidin displayedno labelled stress fibers at either 24 or 48 hpost-injury, suggesting that the toxin stabilizedthe CMB, thereby preventing its depolymeriza-

Fig. 3. Microfilament and microtubule changes accompany-ing rabbit corneal wound repair. In noninjured tissue (A), thecircumferential microfilament bundles of the cells can be ob-served following rhodamine phalloidin staining. B: Twenty-fourhours after a circular freeze wound, cells migrating into theinjury area exhibit stress fibers but not circumferential microfila-ment bundles. C: Forty-eight hours after injury, cells havemigrated into the wound region as the monolayer becomes

reestablished. D: Immunofluorescence of microtubule distribu-tion in noninjured tissue. The closely packed cells display acrowded distribution of microtubules. E: Twenty-four hours afterinjury, cells preparing to migrate into the wound orientateMTOCs in the direction of their movement. F: By 48 h post-injury, microtubule patterns are difficult to see because of thedisorganization within the injury zone. Inset: mitotic spindle ofa dividing cell. Original magnifications: A, C–F, 3128; B, 3200.


Fig. 4. Effect of phalloidin pre-treatment on corneal endothe-lial actin reorganization following a circular freeze wound. A:Incubating tissues with rhodamine labeled phalloidin for 20 hstains the circumferential microfilament bundles similar to thatseen using post-fixation staining. B,C: Fluorescent micrographsof endothelial cells pre-incubated for 20 h in rhodamine phalloi-din, injured, and fixed at 24 and 48 h after injury, respectively.In both cases, cells exhibit very few rhodamine phalloidinpositive stress fibers in their cytoplasm. D,E: Fluorescent micro-graphs of tissues pre-treated in non-labeled phalloidin for 20 h,

injured, and cultured for either 24 or 48 h, then fixed andpost-stained with labeled phalloidin. At 24 h after wounding (D)only diffuse fluorescence is observed and stress fibers are notevident; however, cells in 48-h post-injured preparations (E),display prominent stress fibers. F,G: Preparations that wereinitially pre-treated in rhodamine phalloidin for 20 h, injured,and fixed after 48 h show no evidence of stress fibers (F).However, when the same tissue is subsequently stained withfluorescein phalloidin (G), distinct stress fibers are now de-tected. Original magnifications: A–G, 3200.

tion and subsequent actin reorganization. Simi-lar work by Serpinskaya et al. [1990], demon-strated that phalloidin loading into culturedembryonic mouse fibroblasts increased polymer-ized actin within cells while decreaseing mono-meric levels. This change was accompanied bya 2–3-fold increase in actin synthesis. Reuneret al. [1995a,b] demonstrated that phalloidintreatment promotes F-actin assembly by driv-ing the polymerization of the pre-existingG-actin, and provided evidence that the G-actinlevel may serve to regulate actin synthesis in anegative feedback manner [Reuner et al., 1991,1996]. Hepatocytes treated with phalloidin de-creased their G-actin/F-actin ratio but increasedtheir actin mRNA levels, whereas treatmentwith Clostridium botulinum C2 toxin increasedthe G-actin/F-actin ratio but decreased actinmRNA [Reuner et al., 1991]. Furthermore, anyincreases in actin mRNA levels were blocked byactinomycin D exposure [Reuner et al., 1995a],suggesting that increased actin synthesis wasthe result of new transcription. The results ofthese studies taken together indicate that anyagent capable of shifting the G-actin/F-actinratio should significantly affect the levels ofactin synthesis. Recently, Cappelletti et al.[1996] showed this to be the case with A549

human lung carcinoma cells treated with theoxidant agent paraquat. Paraquat was shownto promote the assembly of numerous singlemicrofilaments throughout the cytoplasm witha parallel decrease in the monomeric actin level,creating a shift in the G-actin/F-actin ratio.This ratio change was then followed by strongde novo actin synthesis [Cappelletti et al., 1996].

Fig. 5. Characterization of rabbit corneal endothelial tubulinand actin by SDS-PAGE and immunoblotting. Lane 1, Coo-massie brilliant blue G stained protein standards; lane 2, endo-thelial cell protein profile; lane 3, immunoblotting of tubulin;lane 4, immunoblotting of actin; lane 5, immunoprecipitation ofendothelial tubulin; lane 6, immunoprecipitation of endothelialactin.

TABLE I. Actin Synthesis*

Hoursafterinjury Treatment

CPM/µgprotein

n-value

0 None 36.16 6 11.47 424 None 33.37 6 11.99 348 None 31.25 6 4.59 248 7.5 µM

phalloidin16,418.65 6 9,884.62 2

48 7.5 µM phal-loidin 1 AMD

232.69 6 44.46 2

*Corneas were cultured in 10% rabbit serum for all experi-ments and treated as indicated. Tissues were labeled with35S for the final 6 h of culture and subsequently immunopre-cipitated. Where noted, 0.01 µg/ml actinomycin (AMD) wasadded to cultures at 18 h post-injury for the duration of theexperiment. During wound repair (0, 24, 48 h post-injury)only minimal 35S incorporation into actin was observed.When corneas were pre-loaded with 7.5 µM phalloidin for20 h, injured and cultured under normal conditions, asignificant increase in incorporation occurs. Corneas pre-loaded with 7.5 µM phalloidin for 20 h, injured and culturedin the presence of AMD, show a significant decrease in 35Sincorporation. Each n-value represents 10 pooled endothe-lium.

TABLE II. Tubulin Synthesis*

Hoursafterinjury Treatment

CPM/µgprotein

n-value

0 None 26.76 6 21.23 424 None 25.99 6 24.47 348 None 32.71 6 15.96 248 1028M colchicine 3,195.94 6 1,928.93 248 1028M colchi-

cine 1 AMD 85.97 6 1.39 2

*Corneas were cultured in 10% rabbit serum for all experi-ments and treated as indicated. Tissues were labeled with35S for the final 6 h of culture and subsequently immunopre-cipitated. Where noted, 0.01 µg/ml actinomycin (AMD) wasadded to cultures at 18 h post-injury for the duration of theexperiment. During wound repair (0, 24, 48 h post-injury)only minimal 35S incorporation into tubulin was observed.When corneas were injured and cultured in the presence of1028M colchicine, a significant increase in incorporationoccurs. Corneas injured and cultured in the presence of1028M colchicine and AMD show a significant decrease in35S incorporation. Each n-value represents 10 pooled endo-thelium.


Our actin results parallel the above findings.Although we did not measure G-actin pools inour system, phalloidin treatment probablycauses a similar decline in the G-actin/F-actinratio by inducing actin polymerization, leadingto a dramatic upregulation in actin synthesisand probably actin mRNA transcription. Onthe other hand, the increased availability ofG-actin provided by the disassembly of the CMB

probably suppresses actin mRNA levels andexplains why very low levels of radiolabelledactin immunoprecipitate were detected duringthe course of wound repair in non-phalloidin-treated tissues. In contrast, disrupting CMBdepolymerization with phalloidin probably leadsto decreased G-actin levels and concomitantincreases in actin mRNA levels. This wouldaccount for the large incorporation of radiolabel

Fig. 6. Autoradiographs of 35S-Transt incorporation into immu-noprecipitated actin and tubulin during organ cultured cornealendothelial wound repair. Lane 1, Coomassie brilliant blue Gstained standards; lane 2, immunoprecipitation of actin, lanes3–5 autoradiographs of actin immunoprecipitates at 0, 24, and48 h after injury; lane 6, autoradiograph of actin immunoprecipi-

tate 48 h post-injury following pre-treatment with 7.5 µMphalloidin prior to wounding; lane 7, tubulin immunoprecipita-tion; lanes 8–10, autoradiographs of tubulin immunoprecipi-tates at 0, 24, and 48 h after injury; lane 11, autoradiograph oftubulin immunoprecipitated 48 h post-injury. Tissues were or-gan cultured in the presence of 1028M colchicine.

Fig. 7. Actinomycin D exposure for the final 30 h of organ culture, in tissues pre-treated with phalloidin prior toinjury. Note the greatly reduced numbers of stress fibers in the actinomycin D exposed tissue (A) when compared tothe control tissue (B) that did not receive the drug.


into actin immunoprecipitates as well as theappearance of new stress fibers (as detected bypost-fixation staining) in phalloidin-treated cellsat 48 h post-injury. Furthermore, because acti-momycin D treatment suppresses 35S incorpora-tion into actin immunoprecipitates, it appearslikely that increased synthesis is due primarilyto new transcription and not necessarily theresult of any longed-lived mRNA.

Results obtained with b-tubulin paralleledthose of actin in that the initiation of newsynthesis appears dependent on interfering withmicrotubule reorganization during wound re-pair. In the normal repair process, immunopre-cipitation of labelled b-tubulin gave virtuallylittle or no S35 incorporation. In contrast, expo-sure of endothelia during wound repair to 1028Mcolchicine, which is sufficient to cause the totalloss of immunofluorescent microtubular stain-ing [Gordon and Staley, 1990], prompted a largeincrease in isotope incorporation into immuno-precipitates by 48 h post-injury. It thereforeappears that colchicine induced depolymeriza-tion of microtubules, in a temporal dependentmanner, can subsequently upregulate b-tubu-lin synthesis.

Microtubules are characterized by ‘‘dynamicinstability,’’ the assembly and disassembly ofmicrotubules that involve rapid exchanges ofsubunits between the polymerized and non-polymerized (soluble) tubulin pool [Cassimeris,1993; Gelfand and Bershadsky, 1991; Cassime-ris et al., 1987; Mitchison and Kirschner, 1984].Recent work [Panda et al., 1994] suggests thatthe dynamic properties and functions of micro-tubules may be, in part, dictated by the relativeamounts of different tubulin isotypes. The syn-thesis of b-tubulin is under autoregulatory con-trol, whereby its synthesis is dependent on theratio between free tubulin subunits and thepolymerized state [Caron et al., 1985b; Cleve-land et al., 1983; Ben’Zev et al., 1979], with thefree tubulin, in turn, capable of regulating thestability and levels of its own mRNA [Bachur-ski et al., 1994; Cleveland et al., 1981; Pachteret al., 1987]. In contrast, a-tubulin appears tolimit its own synthesis by repressing its transla-tion [Gonzalez-Garay and Cabral, 1996] in aprocess that may act as part of a mechanism tomaintain the coordinated syntheses of a- andb-tubulins, thus ensuring that appreciable lev-els of free a- or b-tubulins do not occur [Gonza-lez-Garay and Cabral, 1995, 1996].

Previous short-term (#6 h) studies with col-chicine [Ben Ze’ev et al., 1979; Caron et al.,1985b] demonstrated that depolymerizing mi-crotubules increased the level of soluble tubu-lin and depressed its synthesis. This was subse-quently confirmed by microinjecting purifiedtubulin subunits into cultured cells [Clevelandet al., 1983]. Longer treatment with low dosesof colcemid resulted in reduced microtubularlengths, thus lowering polymer mass and de-creasing tubulin synthesis by a small percent-age that remained proportionate to soluble tu-bulin levels [Caron et al., 1985a]. In contrast,vinblastine exposure disrupts microtubules butforms aberrant aggregations of tubulin as‘‘paracrystalline’’ structures that decrease thesubunit pool and stimulate synthesis [Ben’Zevet al., 1979].

Our data initially appear to contradict theabove findings but, in fact, do support them.There is no doubt that short-term incubationwith colchicine initially increases the free tubu-lin pool and reduces tubulin synthesis. Find-ings presented here demonstrate the reverseeffect, that is, alteration of free tubulin sub-units with colchicine, so that they become inef-fective in microtubular dynamics, initiates theupregulation of tubulin synthesis. Since micro-tubules play pivitol roles for cell movement inthe corneal endothelium during wound repair[Sabet and Gordon, 1989], and in other systemsas well [Liao et al., 1995], there is a criticalneed to maintain a viable subunits pool. Colchi-cine-tubulin complexes are known to bind micro-tubules at either end in vitro, reducing theirgrowth rate [Vandecandelaere et al., 1994].Long-term colchicine incubation produces atransmuted pool of tubulin subunits that ef-fects the free/polymerized subunit ratio, andsubsequently results in the upregulation of tu-bulin transcription, analogous to vinblastinetreatment [Ben’Zev et al., 1979]. Thus, in re-sponse to colchicine, cells initially react to in-creased free tubulin levels and decrease tubu-lin synthesis. However, since colchicine-tubulincomplexes neither polymerize nor support mi-crotubular dynamic instability [Vandecan-delaere et al., 1994], and effectively decreasethe viable free tubulin pool, the cell has nochoice but to upregulate tubulin synthesis.

ACKNOWLEDGMENTS

The authors thank Dr. Lester I. Binder, De-partment of Cell and Molecular Biology, North-


western University, for his generous gift of Tu-27B antibody.

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