The Cytoskelefon of the Cynomolgus Monkey Trobeculor Cell

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1 The Cytoskelefon of the Cynomolgus Monkey Trobeculor Cell //. Influence of Cytoskeleron-Acrive Drugs Robert N. Weinreb,* Mark I. Ryder, f and Jon R. Polansky}: The effects of cytochalasin B (10~ 6 M and 10 s M), taxol (10" 5 M), nocodozole (10~ 5 M), and colchicine (10~ 5 M) on the cytoskeleton of cynomolgus monkey trabecular cells were examined with Nomarski observations, fluorescent labeling as well as extraction, S-l labeling, and critical-point drying. Changes in actin, microtubules, and intermediate filaments, the three major cytoskeletal systems, were correlated with changes in the overall shape and organization of the monkey trabecular cell. Incubation with cytochalasin B caused a marked alteration on actin filament structure, as well as cell shape and cytoskeletal organization. Effects on microtubule structure were noted with taxol, nocodozole, or colchicine; however, no marked changes in overall cell shape or other cytoskeletal structures were observed. These studies demonstrate the importance of actin filaments in regulating the shape and cytoskeletal organization of cynomolgus monkey trabecular cells. Invest Ophthalmol Vis Sci 27: , 1986 Substantial evidence has accumulated during the past several years that the cytoskeleton is an essential component of many cell processes. 1 " 3 In a prior study, 4 transmission electron microscopy was employed to obtain stereo pairs of whole cynomolgus monkey trabecular cells that had been extracted, S-1 labeled, and critical-point dried in order to simultaneously identify actin, microtubules, and intermediate filaments, the three major cytoskeletal systems, and visualize their three-dimensional nature. A double fluorescence technique for actin and microtubules was also used to provide a broad view of cytoskeletal relationships within the cell. In the current investigation, we evaluated the effects of cytoskeleton-active drugs on these systems. Furthermore, we correlated changes in these cytoskeletal systems with changes in the overall shape and organization of the monkey trabecular cell. Materials and Methods Monkey trabecular cells were grown to confluence (7-10 days of culture) on 1 cm 2 glass coverslips (with From the *Departments of Ophthalmology, University of California, San Diego and JSan Francisco, and the fdepartment of Oral Biology, University of California, San Francisco, California. Supported in part by NIH Grants EY05990 (RNW) and DE06681 (MIR), and by a grant from Alcon Laboratories, Inc. Submitted for publication: November 7, Reprint requests: Robert N. Weinreb, MD, University of California, San Diego, Department of Ophthalmology (T-014), La Jolla, CA or without attached grids), using previously described methods. 5 The cells were then incubated for 1 hr at 37 C with either: (1) 0.1 M phosphate buffered saline (PBS) (control); (2) 0.1 M PBS with 0.5% DMSO (control); (3) 10~ 5 M cytochalasin B (Sigma; St. Louis) in 0.1 M PBS with 0.5% DMSO; (4) 10~ 6 M cytochalasin B in 0.1 M PBS with 0.5% DMSO; (5) 10" 5 M taxol in 0.1 M PBS with 0.5% DMSO; (6) 10" 5 M nocodozole (Sigma; St. Louis) in 0.1 M PBS with 0.5% DMSO; or (7) 10" 5 M colchicine (Sigma; St. Louis) in 0.1 M PBS. The cells, attached to the coverslips, were then processed for either (1) Nomarski observations, (2) fluorescent labeling, or (3) extraction, S-l labeling, and critical-point drying using the methods described previously. 46 Results No difference in cell shape (Fig. 1 A) or cytoskeletal organization (Figs. 2A, B) was noted between cells incubated in PBS alone or with PBS with 0.5% DMSO (controls). Light Microscopic Observations Incubation with the antiactin drug, cytochalasin B, caused marked alterations in cell shape and cytoskeletal organization. At 10~ 5 M cytochalasin B the cell body rounded up (Fig. IB) leaving fine branching projections (or "microarborizations"). These morphologic changes were not noted at 10~ 6 M cytochalasin B (Fig. 1C). At both concentrations, a slight increase in blebbing at the free surface was noted. At 10~ 5 M cytochalasin B, 1312

2 No. 9 TRABECULAR CELL CYTOSKELETON: II. CYTO5KELETON-ACTIVE DRUGS / Weinreb er ol Fig. 1. Nomarski micrographs of cynomolgus monkey trabecular cells after 1 hr of incubation with selected cytoskeletal drugs. A, After incubation with phosphate buffered saline (control), the cells are flat and polygonal shaped. B, After treatment with 10" 5 M cytochalasin B, the cell body has rounded up, leaving fine branching peripheral cell processes ("microarborizations"). Some blebbing (arrows) is noted on the cell surface. C, Although the cell shape is similar to the control after treatment with 1O~ 6 M cytochalasin B, there is some cell surface blebbing (arrow). D, Although cell shape has not significantly changed after treatment with 1O" S M taxol, blebbing is present (arrows). E, After treatment with 10~ s M nocodozole, cell shape has not markedly changed and there is pronounced blebbing (arrows). F, After treatment with 10" 5 M colchicine, there is marked blebbing without change in cell shape (as with nocodozole) (all micrographs X2,600). double labeled fluorescence views of the organization of actin and microtubules revealed marked changes in the cytoskeleton. There was a near total alteration of the actin filament structure into globular condensations with a few residual stress fibers (Fig. 3A). At 10~ 6 M cytochalasin B, these globular condensations were also noted within intact stress fibers (Fig. 4A). A peripheral reduction and condensation of microtubules was also noted at 10~ 6 M (Fig. 4B). No marked changes in cell shape were noted with taxol, nocodozole, or colchicine, drugs which are known to effect microtubules (Figs. 1D-F). However, a marked increase in free surface blebbing was noted, especially with nocodozole and colchicine (Figs. IE, F). The overall actin stress fiber organization remained intact with these three drugs (Figs. 5A, 6A, 7A). However, each drug had a marked effect on microtubule organization. With 10~ 5 M taxol there was a reduction in microtubule staining at the periphery, with a concentration at the cell nucleus (Fig. 5B). With 10~ 5 M colchicine or nocodozole, there was a marked reduction in all microtubule staining (Figs. 6B, 7B). Rather, microtubule staining was seen in globular and granular condensations as well as within short lengths of microtubules radiating outward from residual centrioles. Electron Microscopic Observations Low power transmission electron microscopic (TEM) observations of extracted, critical-point dried

3 1314 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / September 1986 Vol. 27 Fig. 2. Double fluorescence for actin (A) and microtubules (B) after incubation with PBS for 1 hr (control). A, The prominent stress fiber organization of actin (arrows) is apparent with a concentration of actin around the periphery. B, Microtubules are seen to concentrate around the nuclear region (N) and to radiate outwards in overlapping arcs <X2,100). Fig. 3. Double fluorescence for actin (A) and microtubules (B) after incubation with 1O~ S M cytochalasin B for 1 hr. A, The stress fiber organization has been almost completely obliterated into globular condensations (arrows). B, With change in cell shape, the microtubule staining pattern is condensed (X2,100). Fig. 4. Double fluorescence for actin (A) and microtubules (B) after incubation with 10"* M cytochalasin B for 1 hr. A, Both stress fibers and globular condensations of actin (arrows) can be seen, B, The microtubule staining pattern appears more condensed at the periphery when compared to the control (2B) (X2,100). Fig. 5. Double fluorescence for actin (A) and microtubules (B) after incubation with 10~ s M taxol for 1 hr. A, The stress fiber organization appears intact. B, The microtubule staining pattern appears condensed towards the nucleus (X2,l00). cells at 10 5 M also revealed the near complete alteration of stress fiber organization (Fig. 8). In addition, the distribution of granules and vesicles was homogenous throughout the cytoplasm, as opposed to concentrated around the nucleus as seen in controls. TEM observations of cells treated with 10~ 5 M taxol revealed no marked changes in the structure of microtubules, intermediate filaments, and actin filaments. With 1CT 5 M nocodozole or colchicine (Fig. 9), the overall stress fiber structure remained intact. High power TEM observations of cells incubated with cytochalasin B demonstrated that actin condensed primarily into globular condensations (Fig. 10). However, both microtubules and intermediate filaments remained intact. With 10~ 5 M colchicine or nocodozole (Fig. 11), there was an absence of microtubules among the intact stress fibers, actin filaments, and intermediate filaments. Discussion Cytochalasins B and D, actin disrupting drugs, have been reported previously by Kaufman to increase outflow facility after intracameral infusion in cynomolgus monkeys. 7 " 9 Morphologic evaluation of the trabecular meshwork of cynomolgus and rhesus monkeys under

4 No. 9 TRADECULAR CELL CYTOSKELETON: II. CYTOSKELETON-ACTIVE DRUGS / Weinreb er ol Fig. 6. Double fluorescence for actin (A) and microtubules (B) after incubation with 10" s M nocodozole for 1 hr. A, There are no marked changes in stress fiber organization. B, Microtubule staining has been almost completely reduced to granular or globular condensations. Short lengths of microtubules are seen to radiate from residual centriole regions (arrows) (X2,100). Fig. 7. Double fluorescence for actin (A) and microtubules (B) after incubation with 10" 5 M colchicine for 1 hr. A, As with nocodozole, there is no marked change in stress fibers. B, Granular condensations and short lengths of microtubules (arrows) from the centriole region are observed (X2.100). Fig. 8. Low power TEM of an extracted, critical-point dried cell incubated with 10" s M cytochalasin B, Stress fiber organization is not visible. Granules and/or vesicles (N = nucleus) are homogenously distributed (X4.900). Fig. 9. Low power TEM of an extracted, critical-point dried cell incubated with 10~ 3 M colchicine. Stress fibers (arrows) are prominent in this cell. Granules and/or vesicles are concentrated around the nucleus (N) (X4.900). these conditions revealed separation of trabecular cells, washout of extracellular material, and disruptions of the inner wall of Schlemm's canal These effects were fully reversible and there were no detectable alterations in the trabecular meshwork 6 days after treatment. 10 Based on this physiologic and morphologic data, Kaufman has likened the effects of cytochalasin on the trabecular meshwork to a "pharmacologic trabeculocanalotomy" 9 and suggested that they may be useful for normalizing outflow facility and intraocular pressure in glaucoma. Analogous morphologic effects occurred in vitro. Using scanning electron microscopy, we have previously reported that 10~ 5 M cytochalasin B or D distorted the morphology of cultured human trabecular cells within 10 min and that a reversal of this effect was observed beginning 1 hr after the drug was removed. 12 Similar morphologic and cytoskeletal effects were observed in the current study when cultured cynomolgus monkey trabecular cells were exposed to 10~ 5 M cytochalasin B. Through the use of double fluorescence and critical-point drying techniques, we were also

5 1316 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Seprember 1986 Vol. 27 Fig. 10. High power TEM of an extracted, S-1 labeled, and critical-point dried cell incubated with 10" 5 M cytochalasin B. Although some remnants of actin filaments (AF) with their attached S-l arrowheads (arrow) are visible, most of the actin has condensed into globular condensations (G). However, both microtubules (MT) and intermediate filaments (IF) are still prominent. Stereo pair±3 tilt(x32,ooo). Fig. 11. High power TEM of an extracted, S-l labeled, and critical-point dried cell incubated with 10~ 5 M nocodozole. Both single actin filaments (AF), bundles of actin filaments in stress fibers (SF), and intermediate filaments (IF) are prominent. However, microtubules cannot be discerned (X65.000). able to observe the effects of cytochalasin B on the intracellular organization of microtubules and intermediate filaments as well as intracellular granules and vesicles. At a lower dose of cytochalasin B (10~ 6 M) there was no marked change in cell shape. However, a partial dissolution of the stress fiber organization into globular condensations was observed. These observations suggest that changes in cytoskeleton may precede actual changes in overall cell shape. Observations of critical point-dried cells treated with 10~ 5 M or 10~ 6 M cytochalasin B indicated intact microtubules and intermediate filaments. However, fluorescence studies demonstrated a condensation of the microtubule staining pattern at the periphery, while TEM studies revealed a redistribution of granules from the perinuclear region in controls to a diffuse distribution throughout the cytoplasm. Thus, we have demonstrated the critical role of actin in regulating trabecular cell shape, as well as the organization of other cytoskeletal elements and intracellular organelles. We also investigated the effects on cell shape and cytoskeletal organization of colchinine and nocodozole, microtubule depolymerizing drugs, and taxol, a microtubule stabilizing drug. Colchicine has been reported to reduce intraocular pressure, 13 although the basis for this effect is not known. The cytoskeletal effects of these three drugs were similar to their effects in vitro in other cells. Taxol induced a perinuclear condensation of mi-

6 No. 9 TRABECULAR CELL CYTOSKELETON: II. CYTOSKELETON-ACTIVE DRUGS / Weinreb er ol crotubules, 14 while colchicine and nocodozole obliterated the peripheral microtubule structure, leaving a residual centriole region Besides changing the overall cell shape minimally, these drugs had little effect on the organization of actin filaments in stress fibers, intermediate filament structure, and intracellular granule and vesicle organization. A marked "blebbing" of the upper surface of cells exposed to colchicine and nocodozole was observed, however. Such blebbing was also observed to a lesser extent with taxol and cytochalasin B. This may be the result of subtle changes in the underlying cytoskeletal organization, or may represent a release of granules and vesicles due to the elimination of microtubule or actin structure. This latter hypothesis is supported by pharmacological studies which demonstrate the intracellular redistribution or release of pigment granules, lysosomal granules, and fibroblast secretory vesicles after treatment with antimicrotubule or antiactin drugs Thus, although microtubules may not affect the shape of the trabecular cell, they may directly influence its phagocytic and secretory functions. Such a hypothesis may be confirmed by correlating the comprehensive morphologic techniques of this study with functional and biochemical studies. Key words: trabecular cells, cytochalasin B, taxol, nocodozole, colchicine Acknowledgments The authors gratefully acknowledge Dr. Mark Kirschner for providing the antitubulin antibodies and taxol, and Dr. Richard Niederman for providing the S-1 label. We also appreciate the assistance of Donna Chesnut and Carol Fiuren in preparing the manuscript. References 1. Pollard TD: Cytoskeletal functions of cytoplasmic contractile proteins. J Supramol Struct 5:317, Goldman RD, Milsted A, Schloss JA, Starger J, and Yerna MJ: Cytoplasmic fibers in mammalian cells: cytoskeletal and contractile elements. Ann Rev Physiol 41:703, Geiger B: Membrane-cytoskeleton interaction. Biochim Biophys Acta 737:305, Ryder MI and Weinreb RN: The cytoskeleton of the cynomolgus monkey trabecular cell: I. General considerations. Invest Ophthalmol Vis Sci 27: , Polansky JR, Weinreb RN, Baxter JD, and Alvarado J: Human trabecular cells. I. Establishment in tissue culture and growth characteristics. Invest Ophthalmol Vis Sci 18:1043, Ryder MI, Weinreb RN, and Niederman R: The organization of actin filaments in human polymorphonuclear leukocytes. Anat Rec 209:7, Kaufman PL and Barany EH: Cytochalasin B reversibly increases outflow facility in the eye of the cynomolgus monkey. Invest Ophthalmol Vis Sci 16:47, Kaufman PL and Erickson KA: Cytochalasin B and D postoutflow facility response relationships in the cynomolgus monkey. Invest Ophthalmol Vis Sci 23:646, Kaufman PL: The effects of drugs on the outflow of aqueous humor. In Glaucoma: Applied Pharmacology in Medical Treatment, Drance SM and Neufeld AH, editors. Orlando, Grune and Stratton, Inc., 1984, pp Svedbergh B, Lutjen-Drecoll E, Ober M, and Kaufman PL: Cytochalasin B-induced structural changes in the anterior ocular segment of the cynomolgus monkey. Invest Ophthalmol Vis Sci 17:718, Johnstone M, Tanner D, Char B, and Kopecky K: Concentrationdependent morphologic effects of cytochalasin B in the aqueous outflow system. Invest Ophthalmol Vis Sci 19:835, Polansky JR, Bloom E, Konami D, Weinreb RN, and Alvarado JA: Cultured human trabecular cells: evaluation of hormonal and pharmacological responses in vitro. In Recent Advances in Glaucoma, Ticho V and David R, editors. Amsterdam, Excerpta Medica, 1984, pp Williams RN and Bhattacherjee P: Reduction in intraocular pressure induced by colchicine and related compounds. European J Pharmacol 77:17, Green KT and Goldman RD: The effects of taxol on cytoskeletal components in cultured fibroblasts and epithelial cells. Cell Motility 3:283, Anderson DC, Weibel LJ, Hughes BJ, Smith CW, and Brinkley BR: Cytoplasmic microtubules in polymorphonuclear leukocytes: Effects of chemotactic stimulation and colchicine. Cell 31:719, Snyder JA, Vogt SI, and McLelland SL: Nocodozole pretreatment in anaphase selectively reduces anaphase B in ptkl cells. Cell Motility 3:79, Hoffstein S and Weissman G: Microfilaments and microtubules in calcium ionophore induced secretion of lysosomal enzymes from human polymorphonuclear leukocytes. J Cell Biol 78:769, Byers HR and Porter KR: Transformations in the structure of the cytoplasmic ground substance in erythrophores during pigment aggregation and dispersion I. A study using whole cell preparations in stereo high voltage electron microscopy. J Cell Biol 75:541, 1977.