LENS CELL ELONGATION IN VITRO AND MICROTUBULES Joram Piatigorsky Laboratory of Molecular Genetics National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20014

The introduction of glutaraldehyde as a fixative for electron microscopy revealed cytoplasmic microtubules in most, if not all, cells.? One year later, Byers and Porter correlated the appearance of longitudinally oriented microtubules, mostly located near plasma membranes, with palisading of cells in the embryonic chick lens rudiment and with further elongation of the posterior cells of the lens vesicle. They suggested a causal relationship between microtubule formation and cell elongation in the developing lens cells, and speculated that microtubules may be of general importance for the development and maintenance of cell shape. Since then, numerous reports have correlated longitudinal arrays of microtubules with elongated cells or cell processes.?-21 Although much has been learned recently about the polymerization of microtubules in cell-free extracts,21-?3it remains unknown at the present time how microtubules assemble in vivo or what role they may play in directing cell elongation. In this report, the relationship between cytoplasmic microtubules and lens epithelial cell elongation will be examined in a tissue culture system first described by Philpott and Coulombre.?'

Description of the Systcin

The test system devised to study elongation of lens cells consists of a square piece of an anterior lens epithelium, attached to its capsule, obtained from a 6-day-old chick embryo. The method used for explanation is diagrammed in FIGURE 1. Approximate values for some characteristic features of the explanted 1. The most important portion of the epithelium are summarized in TABLE feature is, of course, that the cells are initially cuboidal. Some cells beginning to elongate are often present at the edges of the explant; this is due to the difficulty of excluding all the equatorial epithelial cells from the epithelium during the final trimming of the explant. The behavior of the explanted epithelium differs according to the culture 2. Culture of the epithelium in defined medium, as diagrammed in FIGURE medium supplemented with 15% ( v / v ) fetal calf serum promotes elongation of the epithelial cells.?1-tlz The cells approximately double in length within the first 5 hours of culture; subsequent cultivation results in slower, continued cell 27 The peripheral cells elongate faster than those nearer the center. By the third day of culture, the cells are 3 to 4 times their original length, and an outgrowth of flattened cells is present at the periphery of the explant. The cells closest to the explant in the region of outgrowth elongate, and further outgrowth of flattened cells occurs. Histological sections suggest

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FIGURE 1. Diagramatic representation of the explanation of a lens epithelium: axial sections (a-e) and top views (f-h). The ocular lens is removed from the eye of a 6-day-old chick embryo, placed into sterile medium contained in a plastic Petri dish (Falcon Plastics, generally 60 mm x 15 mm), cleaned of vitreous and other adhering debris, and rotated so that the epithelium faces the surface of the dish (a). The capsule (dark line) is torn on the posterior side of the lens with sharpened jeweler's forceps and the fiber mass (F) is removed (b-d), leaving the central (C) and equatorial (E) epithelial cells still attached to the lens capsule. A square piece with sides of approximately 1 mm is cut out from 1he central region of the epithelium by pushing the peripheral regions of the explant into the surface of the plastic culture dish with a scalpel (e, f ) . Next, the equatorial epithelial cells are carefully removed with forceps ( g ) . This yields a piece of isolated central epithelium with the cells adhering to the lens capsule, which is anchored to the dish ( h ) .

TABLE 1 CHARACTERISTICS OF A ~-DAY-OLD EMBRYONIC CHICK LENSEPITHELIAL EXPLANT Size or Characteristic Explant size Cell shape Cell number

1 mm2 cuboidal 23,000

*

Cells in mitosis

2.2%

Cell diameter

10-12 pm

Amount DNA Amount protein

* These data

*

0.056 pg 2.3 P g

Means of Determination

trypsin dissociation; cells counted in hernocytometer paraffin sections; hemotoxylin and eosine stain paraffin sections; microscopically determined with ocular micrometer diphenylamine reaction 'IJ Folin phenol reaction 'I'

were obtained by Dr. Leonard M. Milstone.

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that deposition of a lens capsule beneath the outgrown cells may be necessary for their elongation. Cytochemical studies with explanted fish lens epithelial cells have indicated that the histogenesis of a lens capsule can occur in vitro.2R Elongation of the cultured embryonic chick lens cells is associated with decreased cell division,29-:11as is the formation of fibers in the intact Many features characteristic of terminal lens fiber differentiation are acquired by the lens epithelial cells by 3 to 4 weeks of culture in fetal calf serum. These include extensive cell elongation, pycnosis of nuclei, degradation of DNA, decreased ribosomal RNA synthesis, and increased synthesis of the lens protein, 6-crystallin.?'l

HAM'S F-I0

CELL SERUM/ DIVISION

1

/

'

SERUM

;~?l;;BRYO

PLASMA' CLOT

FIGURE2. Diagramatic representation of the behavior of a 6-day-old embryonic chick lens epithelium cultured in Ham's F-10 medium alone or supplemented with 15% ( v / v ) fetal calf serum. See the text for explanation. Dividing cells possess metaphase chromosomes. Pycnotic nuclei are represented by wavy lines ( 3 weeks in serum); the lens capsule is represented by the thick, black line. ':I

Lens cell elongation in vitro takes place more quickly if the explants are not flattened upon the culture dish."' Cells in epithelia that are folded so that the lens capsule surrounds the explant can elongate 6 times as much in 3 days of culture as those in explants that are folded so that the capsule faces inward. Implantation of epithelia into lentectomized embryonic chick eyes shows, however, that epithelia folded either way can reconstitute an intact lens, although the mechanism of this morphogenesis differs according to the position of the lens capsule in the folded

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Explanted embryonic chick lens epithelia exhibit neither cell elongation nor cellular outgrowth when cultured at 37" C and 3-5% CO, in a defined medium that lacks serum.z4+29 Furthermore, the lens epithelial cells lose their capacity to elongate when confronted with fetal calf serum if they are precultured for 12 to 24 hours in serum-free medium; instead, the starved cells show enhanced mitosis and outgrowth when supplemented with Fetal calf serum also initiates increased cell division rather than elongation in lens epithelia obtained from 19-day-old chick embryos, even without a period of preculture in serumfree m e d i ~ r n . 3Loss ~ of ability to elongate is not irreversible: a serum-starved 6-day-old embryonic epithelium will undergo considerable lens reconstitution, including marked cell elongation, when implanted into a lentectomized embryonic eye or when cultured in the presence of periocular mesenchyme and neural retina (FIGURE 2). Of particular interest is the observation of an apparent uncoupling of cell enlargement and cell elongation. Serum-starved lens epithelial cells from 6-day-old chick embryos increase in volume, but do not elongate, when surrounded by embryonic neural retina, placed on a Millipore filter that contains a chicken plasma clot underneath, and immersed in 13% fetal calf serum plus 13% chick embryo extract.39 Arrangement of Microtubules During Lens Cell Elongation in Vitro

The ultrastructural changes that occur in the elongating cells of the cultured, embryonic, chick lens epithelium have been described R 7 and shown to resemble the changes that take place during lens fiber differentiation in mammals,40-"' chicks,3. 4 5 , 413 amphibians,"'- 1:) and Microtubules appear randomly arranged in the cuboidal cells at the time of explantation (FIGURE 3a). The elongating cells cultured for 24 hours have numerous longitudinally oriented microtubules, which, in general, are preferentially located along surface membranes (FIGURE 3b). These are evident as soon as 2 hours after explantation, and are still present after 28 days of cultivation." Current studies suggest that more microtubules are present in the apical half of the cultured lens cell than in the basal region. A group of microtubules oriented normal to the axis of elongation is also found at the apical pole of the cell (FIGURE4). It is the longitudinal arrangement of the microtubules present in the columnar lens epithelial cells, then, and the analogous observations reported for many different systems, that suggest the possibility of a causal relationship between microtubules and lens epithelial cell elongation in tissue culture. Further evidence that microtubules are important for lens cell elongation comes from the use of colchicine and vinblastine O 1 both dissociators of cytoplasmic microtubules.21v5 2 The addition of colchicine to the lens epithelia at the time of explantation prevents elongation of the cells (FIGURE 5). Vinblastine sulfate also inhibits cell elongation, although less extensive tests were conducted with this alkaloid because of its apparently greater toxicity. Electron microscopic examination has confirmed the effectiveness of colchicine in dissociating microtubules in the lens epithelia. That colchicine treatment does not inhibit elongation simply by killing the cells is indicated by the steady accumulation of rounded cells in mitosis at the noncapsular surface of the epithelium, and by the quantitatively and qualitatively unaltered pattern of protein synthesis in colchicine-treated explants.?13 Inhibition of elongation by dissociation of microtubules has been observed in many different cells.21 z"i

FIGURE 3. Phase micrographs (insets) and electron micrographs of 6-day-old embryonic chick lens epithelia cultured in Ham’s F-10 supplemented with 15% (v/v) fetal calf serum for I5 min (a, d ) , 24 hours (b, e ) , and 48 hours (c, f ) . Explants were sectioned in the long axis of the cells. Note the longitudinally oriented microtubules (+) in the columnar cells (e, f ) . (Insets, x 512; vertical bar=lO pm. Electron micrographs, x 16,800; horizontal bar= I pm. From Piatigorsky et aI.% Reproduced by permission of Academic Press, Inc.)

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FIGURE4. Electron micrograph of a 6-day-old embryonic chick lens epithelium cultured in Ham’s F-10supplemented with 15% (v/v) fetal calf serum for 10 hours. The explant was sectioned normal to the long axis of the cells in the apical region. In this region, microtubules (+) are oriented perpendicular to the cell axis. The microtubule designated by the arrow may be associated with the centriole, cut tangentially, and located to the right of the microtubule. Note the band of microfilaments (mf) beneath the region of cell junctions, which surround the apical pole. The jurictions appear to be of the zonula adherens type. ( x 28,000. Micrograph by courtesy of Dr. Gerald W.Robison.) The addition of colchicine after 10 or 24 hours of culture inhibits further lens cell elongation. The elongated epithelial cells treated with colchicine retain their columnar shape, although the cells appear increasingly disorganized. Similar retention of an elongated shape without microtubules was observed in primary mesenchyme of sea urchin embryos,Xand in lens placode cells of chick embryos; l1 this suggests that microtubules are more directly involved in the process of elongation than in the maintenance of a columnar shape. This appears to be true for closely apposed cells, which can probably support each other for some time. By contrast, individual cells that are elongated or possess elongated processes in vitro, such as neurons,l2#13, B 3 fibroblasts,54-5Gor unicellular organism^,^^ 5 7 retract when their microtubules are dissociated. Search f o r Specific Factors That Control Lens Cell Elongation in Vitro

The chemical complexity of serum increases the difficulty of determining which specific substances stimulate or control elongation of the cultured lens

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cells. Several different compounds were therefore tested for their ability to promote lens cell elongation, in the hope of simplifying the chemical environ2 ) . Only insulin preparations have proven successful in proment (TABLE and alignment of microtubules.5s This may not be moting cell elongation surprising, inasmuch as insulin can replace serum as a growth requirement in Insulin can also stimulate microtubule a variety of culture assembly in explanted rat adipocytesGS In contrast to native insulin, reduced and carboxymethylated A and B chains of insulin d o not cause lens cell elongation, either individually at 1 pglml, or together, each at 1 pg/ml. Zinc and glucagon (both contaminants of insulin preparations) are also ineffective.5s The relatively high concentration of insulin required to initiate lens cell elongation cannot be reduced by the addition of bovine serum albumin to the medium in order to prevent adsorbtion of the insulin to the culture dishes. The necessity for such a high insulin concentration suggests that it is functioning with the insulinlike activity found in serum, which is not suppressible with antiinsulin antibody, rather than with the insulin activity observed at lower, more physiological concentrations, which is suppressible with anti-insulin antibody.69 Since insulin and nerve growth factor are structurally related and may share a common evolutionary precursor,7o the latter was tested for its ability to promote lens cell elongation in vitro. Nerve growth factor, however, in contrast to its promotion of axonal elongation in cultured dorsal root ganglia of embry-

FIGURE5 . Effect of colchicine on cell elongation in 6-day-old embryonic chick lens epithelia cultured in Ham’s F-10supplemented with 15% (v/v) fetal calf serum. The arrows denote the time of colchicine addition; the numbers in parentheses indicate the number of explants examined at each point. Each explant was fixed, embedded in paraffin, and serially sectioned. Mean cell lengths were determined by averaging the cell length, measured with an ocular micrometer, in every fifth serial section in the central third of the explant. Unpublished data were added to those of Pietigorsky et al.”

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TABLE2 ABILITY OF DIFFERENT COMPOUNDS TO PROMOTE LENSCELL IN 10 HOURSOF CULTIVATION ELONGATION Treatment Fetal calf serum Porcine insulin Bovine serum albumin Hydrocortisone Nerve growth factor t cGMP dBu-CGMP cAMP mBu-CAMP dBu-CAMP Theophylline CAMP+ dBu-CAMP CAMP+ theophylline dBu-CAMP+ theophylline dBu-cAMP+ papaverine

Concentration 15% (v/v) 1 pg/ml 5-20 mg/ml 10." M 0.01-1 pg/ml

1 0 - ~ - 1 0M -~~ 10-~-10-~~ M 3 mM 1 mM 1 mM 0.1-1 mM 3 mM 1 mM 1 mM 0.1 mM 1 mM 0.1-1 mM

1 mM 0.1 mM

Cell Elongation

+ +-

-

-

-

~

_ __. _ _.

* Six-day-old embryonic chick lens epithelia were explanted (FIGURE1 ) and cultured at 37" C and 5% CO, in Ham's F-10 medium, with the specified supplements. t This was generously provided by Dr. Eric Shooter; the explants were cultured for 24 hours. onic chicks," did not stimulate lens cell elongation under the present conditions. Next, guanosine-3':5'-cyclic monophosphate (cGMP) and its dibutyryl derivative were examined with respect to their ability to promote lens cell elongation, since an increase in cGMP has been observed in isolated fat cells and liver slices of rats when treated with insulin in culture.'? These compounds, like nerve growth factor, did not cause the lens cells to elongate. Finally, adenosine-3': 5'-cyclic monophosphate (CAMP) and its butyrated derivatives were tested for their ability to stimulate lens cell elongation, since experiments with cultured Chinese hamster ovarian cells,i3. i1 sarcoma cells,i5 7 7 and embryonic chick sensory ganglia isindicated neuroblastoma that a high intracellular cAMP concentration decreases cell division and might be a direct regulator of microtubule assembly and cell elongation. But neither cAMP nor its butyrated derivatives stimulated elongation of the cultured lens epithelial cells. Supplementing cAMP or dibutyryl cAMP (dBu-CAMP) with theophylline or papaverine, both phosphodiesterase inhibitors that increase the intracellular concentration of CAMP, produced the same negative results. Theophylline and papaverine have been shown to inhibit phosphodiesterase activity in the cultured lens cells; nonetheless, final conclusions must be reserved until the cAMP concentrations in these cells have been That treatment with cAMP or dBu-CAMP does not initiate lens cell elongation, as it does in a number of other cultured cells, is consistent with the observations that insulin and serum lower the cAMP concentrations in a variety of

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cell types,Ss85 possibly by the inhibition of adenyl cyclase RZ, 8 7 and stimulation of phosphodiesterase 88-9n activities. Therefore, cAMP and its derivatives were tested for an antagonistic effect upon lens cell elongation, stimulated by serum or insulin. The results will be reported in detail elsewhere.llG It may be stated, however, that cell elongation still occurs and longitudinally arranged microtubules are observed in epithelia cultured in the presence of cAMP or dBu-CAMP, alone or in combination with theophylline or papaverine (FIGURE 6). Thus, no role has been demonstrated for cGMP or cAMP in lens epithelial cell elongation in vitro and microtubule alignment at the present time. Eflect of Inhibition of Transcription and Translation on Lens Cell Elongation in Vitro

Inhibition of RNA synthesis with actinomycin DS1 or of protein synthesis with cycloheximideZGat the time of explantation does not prevent the initial, rapid phase of lens cell elongation stimulated by fetal calf serum (FIGURE7). In fact, after 5 hours of culture the mean length of cells in actinomycin D and in cycloheximide was 1.5 times and 1.2 times greater respectively than that of cells in serum alone. By 10 hours of cultivation, the mean cell length in epithelia in the presence of actinomycin D was 1.6 times that of untreated cells, while cells in the presence of cycloheximide were similar in length to the controls. Further culture in the presence of either chemical resulted in cell shrinkage, detachment from the capsule, and presumably death.31 Addition of cycloheximide to columnar lens cells after 10 or 20 hours of culture inhibited further elongation and caused the cells to diminish in length.

FIGURE 6. Phase micrograph (inset) and electron micrograph of a 7-day-old cmbryonic chick lens epithelium cultured for 10 hours in Ham’s F-10 supplemented with 15% ( v / v ) fetal calf serum, 3 mM dBu-CAMP and 0.1 mM papaverine. The explant was sectioned in the long axis of the cells. The cells were the same length as those cultured in serum without dB-CAMP and papaverine. Note the longitudinally oriented microtubules (+). Microtubules are also present along the nuclear membrane. (Inset, x 512; Electron micrograph, x 16,800. Micrograph by courtesy of Dr. Gerald W. Robison.)

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It is not known why the greater cell length follows treatment with actinomycin D or cycloheximide than treatment with serum alone. Possibly a different mechanism is operating, although the elongated cells in cycloheximide are associated with longitudinally oriented microtubules.26 Furthermore, the cultured lens cells resemble cultured neuroblastoma cells with respect to their ability to elongate in the presence of inhibitors of protein synthesis. The initial formation of axons and longitudinal alignment of cytoplasmic microtubules can take place in the absence of protein synthesis,'?. l6 while continued axonal outgrowth requires protein synthe~is.~? I

I

I

I

I

I

-

38 -

/ (13)

-

-

-

Actinomycin D ( l . 2 5 p q / m l ) b - 4 -or C y c l o h e x i m i d e ( 2 0 ~ / m l ) ~ - 4

-

2I

I

I

1

I

I

I

I

I

I

I

I

Synthesis of Microtubule Protein During Lens Cell Elongation in Vitro

It is not known if elongation of cultured lens cells is associated with significant changes in the number of microtubules, or only with their orientation. This is under investigation at the present time.116 The general impression obtained by inspection of the electron micrographs is that the more elongated cultured lens cells do not contain appreciably more microtubules than the

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shorter cells. Even after 28 days of culture, epithelial cells that have elongated some 30- to 50-fold do not appear to have accumulated numerous microtubules,?7 as is observed, for example, in elongating neuronal cells.9*l o ,9 3 Current biochemical experiments on cultured lens epithelia, briefly summarized below, are consistcnt with the electron micrographs. Pulse-labeled microtubule protein ( tubulin) was quantitatively precipitated with vinblastine sulfate D? from postribosomal fractions of homogenates of cultured, embryonic lens epithelia in the presence of excess carrier tubulin from embryonic chick brain. The precipitate was dissolved in 1 % sodium dodecylsulfate, heated to 100" C for 1 min in the presence of 2-mercaptoethanol, and chromatographed from a sodium dodecylsulfate-hydroxyapatite column with a gradient of sodium phosphate.9' Tubulin eluted at a sodium phosphate concentration of 0.27 M, and was completely separated from 6crystallin, the predominant protein of the 6-day-old embryonic chick lens epithelium,zs, 957 9 6 which eluted at approximately 0.32 M sodium phosphate. The amount of tubulin in the fraction isolated from the column was quantitated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Lens epithelial cell tubulin labeled with [3H]valine was identified on the gel by its molecular weight of 53,000-55,00097 and by coelectrophoresis with l4CC-labeledtubulin from embryonic chick brain. The amount of 3H-radioactivity found in the purified tubulin could be related to molecules of tubulin synthesized per sec per cell, assuming that tubulin has a molecular weight of 120,000 and contains 66 residues of ~ a I i n e . ~This ~ - ~calculation ~ is possible because both the cell number and intracellular specific activity of valine have been determined for the cultured lens cells,115and because the experiments showed negligible degradation of tubulin during the labeling time of 75 min. In 2 experiments an average of 28 molecules of tubulin were synthesized per second per cell in epithelia cultured in 15% fetal calf serum for 2.5 hours; 23 molecules were synthesized per second per cell after 24 hours of culture. By contrast, the rate of bulk protein synthesis increased approximately 1.3-fold per cell. This is due mostly to a specific increase in 6-crystallin Thus, lens cell elongation in vitro is not associated with an increasing rate of tubulin synthesis. These biochemical findings are consistent with a quantitative electron microscopic study, which showed that elongation of epithelial cells during neurulation in amphibians is associated with a decrease in the number of longitudinally oriented microtubules, although the total cumulative length of the microtubules per cell appears to be conserved.", ?" D o Microtubules Directly Prorriote Lens Cell Elongation in Vitro? Since the number of microtubules observed in the elongating lens cells is not impressive, the most convincing evidence that microtubules are required for cell elongation in the cultured embryonic chick lens epithelium is derived from the colchicine inhibition experiments. The mechanism by which the microtubules influence cell elongation is not known. Possibilities for direct microtubular control of cell elongation, including such alternatives as microtubule elongation by subunit addition, active sliding of adjacent microtubules, and microtubule-directed cytoplasmic flow, have been evaluated recently for epithelial cells during neurulation in amphibians.'" The favored hypothesis was directed cytoplasmic flow.

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Another possibility consistent with the present observations is that increased population pressure results in elongation of the cells. This has been advanced as a possible mechanism that controls cell elongation and invagination of the embryonic chick lens rudiment.lnO In the case of the lens rudiment, the pressure is believed to result from continuing cell division in a limited area. An approximate 2.5-fold increase in cell density was correlated with an approximate 2.5-fold increase in cell height. Although such cell population pressure may be an important factor contributing to the observed cell elongation in the cultured lens epithelium, several arguments speak against this idea as the major directive force. First, and perhaps most important, cell number increases only by about 1.1 times after 5 hours, and by 1.4 times after 24 hours in culture,115while cell length increases approximately 2 times after 5 hours and 2.5 times after 24 hours in vitraZG Furthermore, the area of the explants is not limited and does spread slowly.2i Another fact inconsistent with the notion that increases in cell population density cause cell elongation is that serum-starved lens epithelia from 6-day-old embryos 29 or explanted epithelia from 19-day-old embryos 3n undergo cell division rather than cell elongation when cultured with 15% fetal calf serum. In these experiments, however, the cell population density at the time of serum treatment is not known, and may be less than that of the 6-day-old embryonic epithelium at the time of explantation. A fourth argument against the idea that increase in cell number causes lens cell elongation in vitro is this: culture conditions that favor a high intracellular cAMP concentration inhibit division in most cells,1n1-104including lens epithelial cells,ln5Vlo6 but do not suppress lens cell elongation in the cultured epithelium. It will be important to confirm that cAMP inhibits cell division in the cultured embryonic chick lens epithelium under the present conditions. Increased population pressure caused by cell division, then, does not appear to be a major factor in cell elongation in the cultured lens epithelium. POSsibly increase in cell volume, perhaps by uptake of fluid from the medium, raises the pressure between the cells and forces an elongated cell shape. This is not supported by visual inspection of trypsin-dissociated cells after 5 hours of culture, although the volume of these cells has not been determined; by contrast, after 24 hours of culture the dissociated cells do give the impression of having a significantly larger volume.11' It should be noted that trypsindissociated lens cells do assume a spherical shape. An additional argument against the hypothesis that increased cell volume produces pressures that result in cell elongation is that colchicine inhibits elongation in all the cultured lens cells, regardless of their position in the cell cycle, without apparent metabolic effects on the treated cells. Naturally, colchicine may be fortuitously inhibiting cell volume increase as well as microtubule assembly. Finally, whatever the population pressure in the cultured epithelium, it is insufficient to prevent the rounding of mitosing cells,1nO+1 0 7 even when the surrounding epithelial cells are very long. Taken together, the evidence suggests (but does not prove) that specific cellular factors control cell elongation in the cultured lens epithelium. Experiments reviewed here suggest the importance of microtubules, although their role remains elusive. Until their mechanism of action becomes understood, it would be wise to continue serious consideration that other factors are also involved, such as possibly contractile elements s*, 5R, I o h and cell junctions,3i*l n I f - l l l and also to probe more deeply into the mechanisms of microtubule action.

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A ckno wledgrnents

In thank Dr. G. W. Robison for contributing FIGURES 4 and 6, Ms. S. S. Rothschild for histological help, Ms. N. Goor for drawing FIGURE1, Drs. L. M. Milstone and W. Glinsmann for many productive and critical discussions, Dr. A. J. Coulombre for critically reading the manuscript, and Ms. C. Kunkle for typing the manuscript. Referetices I . SAEATINI, D. D., K. BENSCH& R. J . BARRNEIT.1963. J. Cell Biol. 17: 19-58. 2. PORTER, K. R. 1966. Cytoplasmic microtubules and their functions. 111 Principles of Biornolecular Organization. Ciba Foundation Symposium. G. E. W. Wolstenholme & M. O’Connor, Eds. : 308-345. Little, Brown and Co. Boston, Mass. 3. BYERS,B. & K. R. PORTER.1964. Proc. Nat. Acad. Sci. U S . 52: 1091-1099. 4. OVERTON, J. 1966. J. Cell Biol. 29: 293-305. 5. PERRY,M. M. & C. H. WADDINGTON. 1966. J. Enibryol. Exp. Morphol. 15: 3 17-330. 6. FISCHMAN, D. A. 1967. J. Cell Biol. 32: 557-575. 7. TILNEY,L. G . 1968. Develop. Biol. 2(Suppl.): 63-102. 8. TILNEY,L. G . & J. R. GIBBINS.1969. J. Cell Biol. 41: 227-250. C . BARON]Rr M. COHN.1969. Proc. Nat. Acad. 9. &HUBERT, D., s. HUMPHREYS, Sci. U.S. 64: 316-323. 10. OLMSTED, J. B., K. CARLSON, R. KLEBE,F. RUDDLE& J. ROSENBAUM. 1970. Proc. Nat. Acad. Sci. US.65: 129-1 36. 1 1 . SCHROEDER, T. E. 1970. J. Ernbryol. Exp. Morphol. 23: 427-462. T. AMANO& M. W. NIRENBERG. 1970. Proc. 12. SEEDS,N. W., A. G. GILMAN, Nat. Acad. Sci. U S . 6 6 160-167. 1 3 . YAMADA, K. M., B. S. SPOONER & N . K. WESSELLS. 1970. Proc. Nat. Acad. Sci. U.S. 66: 1206-1212. 14. PEARCE, T. L. & J. ZWAAN.1970. J. Embryol. Exp. Morphol. 23: 491-507. IS. YAMADA, K. M., B. S. SPOONER & N. K. WESSELLS.1971. J. Cell Biol. 49: 6 14-635. 16. YAMADA, K. M. & N. K. WESSELLS.1971. Exp. Cell Res. 66: 346-352. 17. BURNSIDE, B. 1971. Develop. Biol. 26: 416-441. 18. HANDEL, M. A. & L . E. ROTH. 1971. Develop. Biol. 25: 78-95. 19. KARFUNKEL, P. 1971. Develop. Biol. 25: 30-56. B. 1973. Amer. Zool. 13: 989-1006. 20. BURNSIDE, J . B. & G. G. BORISY. 1973. Ann. Rev. Biochem. 42: 507-540. 21. OLMSTED, R. C. 1972. Science 177: 1104-1 105. 22. WEISENBERG, 23. BORISY,G . G., I. B. OLMSTED, J. M. MARCUM & C. ALLEN. 1974. Federation Proc. 33: 167-174. 24. PHILPOTT,G . W. & A. J. COULOMBRE. 1965. Exp. Cell Res. 38: 635-644. J., H. DEF. WEBSTER & S. P. CRAIG.1972. Develop. Biol. 27: 25. PIATIGORSKY, 176-189. 26. PIATIGORSKY, J . , H. DEF. WEBSTER& M. WOLLBERC. 1972. J. Cell Biol. 55: 82-92. 27. PIATIGORSKY, J., S. S. ROTHSCHILD & L. M. MILSTONE.1973. Develop. Biol. 34: 334-345. 28. VON SALLMAN, L., P. GRIMES& D. ALBFRT. 1969. Amer. J. Ophthalmol. 68: 435-438. 29. PHILPOTT, G . W. 1970. Exp. Cell Res. 59: 57-68. J . & S. S. ROTHSCHILD. 1972. Develop. Biol. 28: 382-389. 30. PIATIGORSKY, 31. WATANABE, H. & I. KAWAKAMI. 1973. Develop. Growth Diff. 15: 101-112. R. & E. BELL. 1965. Science 150: 71-72. 32. REEDER.

346 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.

56. 57. 58. 59.

60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.

Annals New York Academy of Sciences HANNA, C. & H. C. KEATTS.1966. Exp. Eye Res. 5: 111-115. MODAK, S. P., G. MORRIS& T. YAMADA. 1968. Develop. Biol. 17: 544-561. PERSONS, B. J. & S. P. MODAK.1970. Exp. Eye Res. 9: 144-151. ZWAAN, J. & T. L. PEARCE. 1971. Develop. Biol. 25: 96-118. 1974. Can. J. Zool. 52: 345-352. MCLEAN,B. G. & C. V. FINNEGAN. J. L. & A. J. COULOMBRE. 1971. J. Exp. Zool. 176: 15-24. COULOMBRE, PHILPOTT,G. W. & A. J. COULOMBRE. 1968. Exp. Cell Rcs. 52: 140-146. T. & M. A. GAVIN. 1959. J. Biophys. Biochem. Cytol. 6: 97-102. WANKO, COHEN,A. I. 1958. Amer. J. Anat. 103: 219-245. COHEN, A. I. 1961. Develop. Biol. 3: 297-316. RABAEY,M. & A. LAGASSE.1965. Electron microscopic observations on the developing lens of the golden hamster. I n Eye Structure I1 (Symposium). J. W. Rohen, Ed.: 371-382. Schattauer. Stuttgart, West Germany. KUWABARA, T. 1968. Arch. Ophthalmol. 79: 189-195. & A. BRINI. 1968. Arch. Ophthalmol. 28: 681-706. PORTE,A., M. E. STOECKEL T. S., G. EGUCHI& M. TAKEICHI. 1973. Develop. Biol. 34: 321-333. OKADA, EGUCHI,G. 1963. Bull. Marine Biol. Sta. Asamushi Tohoku Univ. 11: 223228. G. 1964. Embryologia 8: 247-287. EGUCHI, J. 1965. J. Cell Biol. 24: 21 1-222. OVERTON, ARNOLD, J. M. 1966. J. Ultrastruct. Res. 14: 534-539. J., H. DEF.WEBSTER& S. P. CRAIG. 1971. J. Cell Biol. 47: 158a. PIATGORSKY, S. B. MIZEL,L. M. GRISHAM & K. M. CRESWELL. WILSON,L., J. R. BAMBURG, 1974. Federation Proc. 33: 158-166. DANIELS, M. P. 1972. J. Cell Biol. 53: 164-176. 1969. Exp. Cell Res. 57: 263-276. GOLDMAN, R. D. & E. A. C. FOLLETT. JOHNSON, G. S., R. M. FRIEDMAN & 1. PASTAN.1971. Proc. Nat. Acad. Sci. U S . 68: 425-429. & A. W. HSIE. 1972. Proc. Nat. Acad. Sci. U.S. PUCK,T. T., C. A. WALDREN 69: 1943-1947. TILNEY, L. G. 1968. J. Cell Sci 3: 549-562. PIATIGORSKY, J. 1973. Develop. Biol. 30: 214-216. J., S. S. ROTHSCHILD & M. WOLLBERG.1973. Proc. Nat. Acad. PIATIGORSKY, Sci. U.S. 70: 1195-1 198. I. & P. OVE. 1959. J. Biol. Chem. 234: 2754-2758. LIEBERMAN, WAYMOUTH, C. & D. E. REED. 1965. Texas Rep. Biol. Med. 23(Suppl. I ) : 41 3-419. TEMIN,H. M. 1967. J. Cell Physiol. 69: 377-384. SCHWARTZ,A. G. & H. AMOS. 1968. Nature 219: 1366-1367. BLAKER, G. J., J. R. BIRCH& S. J. PIRTS. 1971. J. Cell Sci. 9: 529-537. HARDING, C. V., J. R. REDDAN, N. J. UNAKER & M. BAGCHI.1971. Intern. Rev. Cvtol. 31: 215-300. & G. M. TOMKINS. 1971. Nature 232: HER~HKO, A., P. MAMONT, R. SHIELDS 206-211. M. ANDERSON,J. MOLSON& M. B. DAVIDSON. GERSCHENSON, L. E., T. OKIGAKI, 1972. Exp. Cell Res. 71: 49-58. SOWER,D., T. BRAUN & 0. HECHTER.1971. Science 172: 269-271. MEGYESI,K., C. R. KAHN,J. ROTH,E. R. FROESCH,R. E. HUMBEL,J . ZAPF & D. M. NEVILLE, JR. 1974. Biochem. Biophys. Res. Commun. 57: 307-3 15. FRAZIER,W. A., R. HOGUEANGELETTI & R. A. BRADSHAW. 1972. Science 1 7 6 482-488. LEW-MONTALCINI, R. & P. U. ANGELETTI.1968. Physiol. Rev. 68: 534-569. G., G. P. E. TELL,M. I. SIEGEL & P. CUATRECASAS. 1973. Proc. Nat. ILLIANO, Acad. Sci. US. 7 0 2443-2447. HSIE, A. &T.T. PUCK. 1971. Proc. Nat. Acad. Sci. U S . 68: 358-361. HSIE,A., C. JONES& T. T. PUCK.1971. Proc. Nat. Acad. Sci. U.S. 6 8 16481652.

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G. S., R. M. FRIEDMAN & I. PASTAN.1971. Proc. Nat. Acad. Sci. U.S. 75. JOHNSON, 68: 425-429. 76 PRASAD, K. N. & A . W. HSIE. 1971. Nature 233: 141-142. & M. LUBIN. 1971. Nature 233: 413-415. 77. FURMANSKI, P., D. J. SILVERMAN 78. ROISEN,F. J., R. A. MURPHY, M. E. PICHICHFRO & W. G. BRADEN. 1972. Science 175: 73-74. & W. G. BRADEN.1972. Science 177: 809-811. 79. ROISEN,F. J., R. A. MURPHY 80. KRAM,R., P. MAMONT & G. M. TOMKINS.1973. Proc. Nat. Acad. Sci. U.S. 70: 1432-1436. 81. OTTEN,J., G. S. JOHNSON & I. PASTAN.1972. J. Biol. Chem. 247: 7082-7087. 1973. Proc. Nat. 82. DEASUA, L. J., E. S. SURIAN, M. M. FLAWIA & H. N. TORRES. Acad. Sci. U.S. 70: 1388-1392. 83. SHEPPARD, J. R. 1972. Nature 236: 14-16. 84. ROZENGURT, E. & L. J. DEASUA.1973. Proc. Nat. Acad. Sci. U S . 70: 36093612. 85. KONO,T. & F. W. BARHAM.1973. J. Biol. Chem. 248: 7417-7426. 86. HEPP, K. D. 1972. European J. Biochem. 31: 266-276. 87. ILLIANO, G. & P. CUATRECASAS. 1972. Science 175: 906-908. 1970. Biochem. J. 1 2 0 187-193. 88. LOTEN,E. G. & J. G. T. SNEYD. 89. HOUSE,P. D. R., P. POULIS& M. J. WELDEMANN. 1972. European J. Biochem. 24: 429437. 90. MANGANIELLO, V. & M. VAUGHAN.1973. J. Biol. Chem. 248: 7164-7170. 1973. Biochim. Biophys. Acta 299: 642-653. 91. CRAIG,S. P. & J. PIATIGORSKY. 92. SCHUBERT, D., S. HUMPHREYS, F. DE VITRY& F. JACOB.1971. Develop. Biol. 25: 514-546. 93. HIER,D. B., B. G . W. ARNASON & M. YOUNG. 1972. Proc. Nat. Acad. Sci. U.S. 69: 2268-2272. 94. MOSS,B. & E. N. ROSENBLUM. 1972. J. Biol. Chem. 247: 5194-5198. 95. ZWAAN,J. & A . IDEKA. 1968. Exp. Eye Res. 7: 301-311. 96. YOSHIDA,K. & A. KATOH.1971. Exp. Eye Res. 11: 122-131. 97. BRYAN, J. 1974. Federation Proc. 33: 152-157. 98. LUDUENA, R. F. & D. 0. WOODWARD. 1973. Proc. Nat. Acad. Sci. U S . 70: 3594-3598. 99. BRYAN,J. & L. WILSON. 1971. Proc. Nat. Acad. Sci. U.S. 68: 1762-1766. 100. ZWAAN, J. & R. W. HENDRIX.1973. Amer. Zool. 13: 1039-1049. 101. WOLFE,J. 1973. J. Cell Physiol. 82: 39-48. 102. WILLINGHAM, M. C., R. A. CARCHMAN & I. H. PASTAN.1973. Proc. Nat. Acad. Sci. US.70: 2906-2910. 103. OEY, J., A. VOGEL& R. POLLACK.1974. Proc. Nat. Acad. Sci. U.S. 71: 694698. 104. FROEHLICH, J. E. & M. RACHMELER.1974. J. Cell Biol. 60: 249-257. 1972. Invest. Ophthalmol. 11: 231-235. 105. GRIMES,P. & L. VON SALLMAN. L. & P. GRIMES. 1974. Invest. Ophthalmol. 13: 21C218. 106. VON SALLMAN, 107. HENDRIX, R. W. & J. ZWANN.1974. Nature 247: 145-147. 108. WESSELLS,N. K., B. S. SPOONER,J . F. ASH, M. 0. BRADLEY, M. A. LUDUENA, E. L. TAYLOR, J. T. WRENN& K. M. YAMADA.1971. Science 171: 135-143. 109. LEESON,T. S. 1971. Exp. Eye Res. 11: 78-82. D. H. & G. W. CROCK. 1972. Invest. Ophthalmol. 11: 809-815. 110. DICKSON, 1 1 1. FARNSWORTH, P. N., S. C. J. Fu. P. A. BURKE& I. BAHIA.1974. Invest. Ophthalmol. 13: 274-279. 112. HAM,R. G. 1963. Exp. Cell Res. 29: 515-526. 113. GILES,K. W. & A. MYERS. 1965. Nature 206: 93. 114. LOWRY,0. H., N. J. ROSEBROUGH, A. L. FARR & R. J . RANDALL.1951. J. Biol. Chem. 193: 265-275. 115. MILSTONE, L. M. & J. PIATIGORSKY. 1975. Develop. Biol. In press. 116. PIATIGORSKY, J., G. W. ROBISON& G. J. CHADER.In progress. 117. MILSTONE, L. M. Unpublished data.

Lens cell elongation in vitro and microtubules.

LENS CELL ELONGATION IN VITRO AND MICROTUBULES Joram Piatigorsky Laboratory of Molecular Genetics National Institute of Child Health and Human Develop...
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