Delta-crystallin mRNA in chick lens cells: mRNA accumulates during differential stimulation of delta-crystallin synthesis in cultured cells.

Increasing specialization for δ-crystallin synthesis is a prominent feature of the differentiation of chick lens epithelial cells into lens fiber cells and can be studied in cultured embryonic lens epithelia. Quantitation of δ-crystallin mRNA by molecular hybridizaton to a [³H]DNA complementary to δ-crystallin mRNA demonstrates that differentiation, both in ovo and in tissue culture, is associated with the accumulation of δ-crystallin mRNA. In the cultures, there is an overall stimulation of protein synthesis, including δ-crystallin mRNA during the first 5 hr in vitro. Between 5 and 24 hr in vitro there is a differential stimulation of δ-crystallin synthesis and an accumulation of δ-crystallin mRNA that can quantitatively account for this stimulation.     

Sequence Characterization of γ-Crystallins from Lip Shark (Chiloscyllium colax): Existence of Two cDNAs Encoding γ-Crystallins of Mammalian and Teleostean Classes

γ-Crystallin is a common lens protein of most vertebrate eye lenses and the major protein component in lenses of fishes and in many mammalian species during embryonic and neonatal stages. To facilitate the structural characterization of γ-crystallin possessing extensive charge heterogeneity, a cDNA mixture was constructed from the poly(A)⁺ mRNA isolated from shark eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain cDNAs encoding multiple shark γ-crystallins. Sequencing analysis of multiple positive clones containing PCR-amplified inserts revealed the presence of a multiplicity of isoforms in the γ-crystallin class of this cartilaginous fish. It was of interest to find that two shark cDNA sequences coexist, one encoding γ-crystallin (γM₁) of high methionine content (15.5%) and the other encoding one (γM₂) of low methionine content (5.1%), each corresponding to the major teleostean and mammalian γ-crystallins, respectively. Comparison of protein sequences encoded by these two shark cDNAs with published sequences of γ-crystallins from mouse, bovine, human, frog, and carp lenses indicated that there is about 61–80% sequence homology between different species of the piscine class, whereas only 47–66% is found between mammals and shark. A phylogenetic tree constructed on the basis of sequence divergence among various γ-crystallin cDNAs revealed the close relatedness between shark γM₂-crystallin and mammalian γ-crystallins and that between shark γM₁ and teleostean γ-crystallins. The results pointed to the fact that ancestral precursors of γ-crystallins were present in the sharp lens long before the appearance of modern-day mammalian and teleostean γ-crystallins.     

Crystallin synthesis in lens differentiation in cultures of neural retinal cells of chick embryos.

Dissociated cells of neural retinas of 3.5-day-old chick embryos differentiated into “lentoid bodies” within about 10–12 days when cultured in vitro. Protein synthesis of these cultured cells was studied with the use of SDS-polyacrylamide gel electrophoresis, affinity chromatography, and autoradiography combined with immunological techniques. Incorporation of [¹⁴C]leucine into total proteins, α-crystallin, and δ-crystallin was estimated after increasing times of culture up to about 30 days. Isotope incorporation into δ-crystallin was detected at 9 days, and it increased sevenfold after another 17 days. α-Crystallin was also first detected at 9 days, but its relative content reached a maximum at 12 days and then decreased gradually. The ratio of δ-crystallin synthesis to total protein synthesis increased up to 40% at 26 days, while that of α-crystallin synthesis remained 3% throughout the culture period. These results show that lens differentiation from neural retinal cells is associated with the preferential synthesis of lens crystallins, particularly of δ-crystallin.     

Alteration of rat splenic lymphocyte migration in vitro by the state of microtubule integrity

Rat splenic lymphocytes exhibit a positive chemokinetic response to colchicine and vinblastine. Both agents elicit a dose-dependent increase in chemokinesis with their peak effect at 2 to 4 × 10^?7M being 3.5 times baseline random migration. The distance traveled by the leading front and the total movement of rat splenic lymphocytes is maximal in the absence of a gradient at all effective concentrations of colchicine or vinblastine. Checkerboard analysis established this response as entirely chemokinetic without any chemotactic component. That this chemokinetic response was due to a shift in the dynamic state of microtubules toward disassembly was supported by the inactivity of lumicolchicine and the capacity of heavy water to reverse the effect in a dose-response fashion. Cytochalasin B suppressed baseline random migration and reversed the chemokinetic response of the rat splenic lymphocytes to 4 × 10^?7M colchicine. The chemokinetic motility of rat splenic lymphocytes may depend not only on microtubule disassembly but also on the contractile activity of microfilaments.     

Rates of protein synthesis in explanted embryonic chick lens epithelia: differential stimulation of delta-crystallin synthesis.

Cells in the central region of 6-day-old embryonic chick lens epithelia display morphological and biochemical changes, when cultured in medium supplemented with fetal calf serum, comparable to those of lens fiber cells differentiating in vivo. In the present study the rates of synthesis of total protein and of δ-crystallin were quantitated during the first day of culture by measuring (1) ³H-valine incorporation into bulk proteins and into δ-crystallin (isolated by quantitative immunoprecipitation), (2) the specific radioactivity of picomolar amounts of intracellular valine (determined by analysis of the ¹⁴C-dansyl-derivative of ³H-valine), (3) the amount of protein degradation occurring during the labeling period (estimated by “pulse-chase” experiments with cycloheximide), and (4) the number of cells in the explants (counted following dispersal with trypsin-EDTA). The results showed that total protein synthesis increased 1.7-fold per cell during the first 24 hrs in vitro. In contrast, δ-crystallin synthesis increased 2.8-fold per cell during this time. These experiments establish that δ-crystallin synthesis is differentially stimulated in epithelia cultured in serum-supplemented medium, and provide the basis for quantitative analysis of the mechanism controlling differential protein synthesis during lens fiber differentiation in vitro.     

Effect of cytochalasins on surfactant release from alveolar type II cells

Cytochalasins enhanced surfactant secretion from primary cultures of [³H]choline-labeled type II epithelial cells from the rat. Cytochalasins A, B, C, D and dihydrocytochalasin B enhanced secretion of phosphatidyl-[³H]choline ([³H]PC) in a dose-dependent manner with EC₅₀ values of 1, 2, 0.5, 0.1 and 1 μM for cytochalasins A, B, C, D and dihydrocytochalasin B, respectively. Only cytochalasin A caused significant cytotoxicity as determined by release of the intracellular enzyme lactate dehydrogenase (EC 1.1.1.17). Dose responses of surfactant release induced by cytochalasins B, C and D were biphasic; maximal release was observed between 0.1–1.0 μM for cytochalasins C and D between 1 and 10 μM for cytochalasin B. Secretion decreased toward control levels at concentrations of cytochalasin above these maximal concentrations. Increased rates of [³H]PC release were noted between 1 and 3 h after exposure to cytochalasin D. Increased rates of surfactant release induced by cytochalasin D were additive to release induced by the β-adrenergic agonist, terbutaline, or forskolin, although cytochalasin D had no direct effect on cytosolic cyclic AMP levels. Changes in cell shape and microfilament organization were observed by phase-contrast microscopy and fluorescence microscopy using rhodamine-conjugated phalloidin after exposure of the isolated type II cells to cytochalasin D. Disruption of microfilaments associated with lamellar bodies of the purified type II cells occurred after treatment with cytochalasin D. Cytochalasin D augmented surfactant release from purified type II cells and disrupted the microfilament structure of those cells, supporting the hypothesis that alterations in microfilaments are associated with surfactant release.     

α-Crystallin: molecular chaperone and protein surfactant

Bovine lens α-crystallin has recently been shown to function as a molecular chaperone by stabilizing proteins against heat denaturation (Horwitz, J. (1992) Proc. Natl. Acad. Sci. USA, 89, 10449–10453). An investigation, using a variety of physico-chemical methods, is presented into the mechanism of stabilization. α-Crystallin exhibits properties of a surfactant. Firstly, a plot of conductivity of α-crystallin versus concentration shows a distinct inflection in its profile, i.e., a critical micelle concentration (cmc), over a concentration range from 0.15 to 0.17 mM. Gel chromatographic and ¹H-NMR spectroscopic studies spanning the cmc indicate no change in the aggregated state of α-crystallin implying that a change in conformation of the aggregate occurs at the cmc. Secondly, spectrophotometric studies of the rate of heat-induced aggregation and precipitation of alcohol dehydrogenase (ADH), β_L- and γ-crystallin in the presence of α-crystallin and a variety of synthetic surfactants show that stabilization against precipitation results from hydrophobic interactions with α-crystallin and monomeric anionic surfactants. Per mole of subunit or monomer, α-crystallin is the most efficient at stabilization. α-Crystallin, however, does not preserve the activity of ADH after heating. After heat inactivation, gel permeation HPLC indicates that ADH and α-crystallin form a high molecular weight aggregate. Similar results are obtained following incubation of β_L- and γ-crystallin with α-crystallin. ¹H-NMR spectroscopy of mixtures of α- and β_L-crystallin, in their native states, reveals that the C-terminus of βB2-crystallin is involved in interaction with α-crystallin. In the case of γ- and α-crystallin mixtures, a specific interaction occurs between α-crystallin and the C-terminal region of γ_B-crystallin, an area which is known from the crystal structure to be relatively hydrophobic and to be involved in intermolecular interactions. The short, flexible C-terminal extensions of α-crystallin are not involved in specific interactions with these proteins. It is concluded that α-crystallin interacts with native proteins in a weak manner. Once a protein has become denatured, however, the soluble complex with α-crystallin cannot be readily dissociated. In the aging lens this finding may have relevance to the formation of high molecular weight crystallin aggregates.     

The role of calcium in depigmentation of iris epithelial cells during cell-type conversion

During the conversion of newt iris epithelial cells into lens cells, melanosomes disappear from the cytoplasm. In this “depigmentation,” exocytosis of melanosomes is involved. The role of Ca²⁺ in this process has been the subject of this work. The intracellular Ca²⁺ concentration of cultured iris epithelial cells was increased by three methods: microinjection of 10^?3, M CaCl₂ into the cytoplasm, fusion of phospholipid vesicles containing 10^?3, M CaCl₂ with the cell membrane, and exposure to the calcium ionophore A23187. Each of these treatments caused an increase in the release of melanosomes. Further experiments suggest that cAMP stimulates exocytosis probably by liberating Ca²⁺ from intracellular stores.     

Differences in the G/total actin ratio and microfilament stability between normal and malignant human keratinocytes

The state of polymerization of actin and the organization of actin filaments is widely believed to be related to cellular transformation. Since the intracellular monomer (G) and filamentous (F) actin content reflects the state of microfilament polymerization, we measured the G/total actin ratio in primary cultures of normal and malignant human keratinocytes. In normal keratinocytes the mean value of this ratio was 0·30 ± 0·03 (mean ± SE, n = 15), while in basal cell carcinoma (BCC) keratinocytes it was 0·49 ± 0·03 (n = 8) and in squamous cell carcinoma keratinocytes (SCC) 0·5 ± 0·07 (n = 4), indicating a 1·7-fold increase of the G/total actin ratio in malignant cells. These results imply that the proportion of polymerized actin is decreased markedly in malignant keratinocytes, suggesting alterations of microfilament structures which probably occur during the transformation process. This was supported by the morphological changes of microfilament structures as assessed by fluorescence microscopy. A different distribution of actin filaments in normal and malignant cells became evident; stress-fibres were converging in patches at several points in SCC cells, when compared to normal keratinocytes. Furthermore, incubation of normal and malignant keratinocytes with cytochalasin B indicated differences in the resistance of their microfilament networks. After 1 h exposure to 10^?6 and 10^?5 M cytochalasin B, microfilaments in normal cells appeared to be less affected than their counterparts in neoplastic cells. Even in a high excess of cytochalasin B (10^?4 M ), normal keratinocytes preserved their shape, while both basal cell and SCC were totally disrupted. We concluded that the G/total actin ratio was significantly increased in malignant keratinocytes. This seems to be correlated with altered microfilament morphology and resistance to cytochalasin B treatment. Our results suggest that the process of malignant transformation may be characterized by changes in the state of the polymerization of actin and in the stability of the microfilament network indicating that both features could potentially serve as markers determine the transformed state of keratinocytes.     

Physicochemical characterization of γ-crystallins from bovine lens—Hydrodynamic and biochemical properties

A detailed hydrodynamic study has been made on the γ-crystallin of the bovine lens. Sedimentation study indicates that γ-crystallin shows a nearly gaussian peak throughout the course of sedimentation at high speed, using a synthetic boundary cell. The diffusion and sedimentation coefficients are 10.3×10^?7 cm²/sec and 2.51 S, respectively. The weight-average molecular weight of the unfractionated γ-crystallin calculated from sedimentation equilibrium is 21,800. The four major subfractions of γ-crystallin show similar hydrodynamic properties with an intrinsic viscosity of 2.50 ml/g and a Stokes radius of 21 Å. The distinct electrophoretic mobilities exhibited by the four subfractions show gel-concentration dependence and similar slopes in the Ferguson plot, indicative of being charge isomers of the same molecular species. Amino acid analysis of these four subfractions corroborated the conclusions that these γ-crystallin polypeptides are closely related and comprise a multigene family of crystallins. Based on the sedimentation and intrinsic viscosity data, γ-crystallin can be modeled as a prolate ellipsoid with an axial ratio of approximately 3.0 and a hydration factor of 0.27 g water per gram protein. The circular dichroism data for γ-crystallins showed a minimum at about 217 nm, characteristic of a β-sheet conformation. These structural characteristics are in good accord with those derived from X-ray diffraction data for γ-crystallin II.     

Modulation of interleukin 1 production by activated macrophages: in vitro action of hydrocortisone, colchicine, and cytochalasin B

In vitro cultured irradiated rat peritoneal macrophages (XPCs) secreted markedly more Interleukin 1 (IL-1) when pretreated with carrageenan. Exposure of cultures of carrageenanpretreated as well as untreated XPCs to hydrocortisone (OHC) produced a dose-dependent inhibition of IL-1 secretion. OHC at concentrations from 10^?4 to 10^?6M completely blocked the enhanced IL-1 secretion induced by carrageenan pretreatment. These observations suggest that the complex immunosuppressive effects of glucocorticoids could partly result from their capability to control IL-1 production. Colchicine, a microtubule depolymerizing agent, markedly enhanced IL-1 secretion in cultures of both carrageenan-pretreated and untreated XPCs. Stimulatory effects were dose dependent and maximum stimulation occurred at 10^?4M, i.e., at a concentration known to profoundly affect the microtubular function. Lumicolchicine, however, was inactive. Cytochalasin B, a microfilament disrupting agent and inhibitor of phagocytosis, elicited a dose-dependent increase of IL-1 secretion in cultures of carrageenanactivated XPCs but was inhibitory for unstimulated XPCs. Although mechanisms by which Colchicine and Cytochalasin B act in stimulating IL-1 production remain unclear, our study demonstrates that production and secretion of IL-1 do not require participation of intact components of the cytoskeleton.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: II. A tumor-promoter (TPA) delays differentiation and promotes cell proliferation

The tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) delayed pigmentation in developmentally homogeneous quail neural crest cell populations in vitro. Doses below 5 × 10^?8M resulted only in a delay in pigmentation for up to 2 days without changing the rate of pigmentation, whereas intermediate doses (5 × 10^?8–10^?7M) delayed the initiation and decreased the rate of pigmentation but had no effect on the ultimate fate. Doses of TPA above 2 × 10^?7M irreversibly prevented pigmentation in some of the cells. TPA did not influence growth of the undifferentiated cell population. Since TPA delayed the decline in the cell proliferation rate that normally occurred with pigmentation, however, the final number of pigmented cells could be increased up to 10-fold in TPA-treated cultures without changing the homogeneity of the cultures. A part from the effects on cell differentiation, TPA caused an immediate change in cellular association within the clusters.     

Endocytosis in Chang liver cells. Quantitation by sucrose- 3 H uptake and inhibition by cytochalasin B

The addition of 0.08 M sucrose to a culture medium containing Chang-strain human liver cells causes intense cytoplasmic vacuolation. Electron microscopy of these cells grown inferritin, time-lapse cinematography, and radioautography reveal that the vacuoles arise by endocytosis and that the sucrose is taken into the cell and localized in the vacuoles. Tracer studies demonstrate that sucrose-³H provides a marker for quantitation of endocytosis and that it neither induces nor stimulates endocytosis. Electron micrographs of vacuolated liver cells show microfilaments in close proximity to the inside of the plasma membrane, in the pseudopodia, and to the cytoplasmic side of the membrane surrounding endocytosis vacuoles. Cytochalasin B (CB), a mold metabolite that inhibits various types of cell motility, has a dose-dependent inhibitory effect on the uptake of sucrose-³H by these cells. This inhibition is accompanied by a cessation of the movement of ruffles and pseudopodia on the surface of the cells and the formation of blebs which arise from the cell''s surface. These morphological changes are quickly reversible upon removal of CB. Alterations in the appearance and location of microfilaments are also observed in CB-treated cells.     

Inhibitory effects of dibutyryl cyclic AMP and theophylline on the melanosome transformation in the embryonic chick pigmented retina cultured in vitro.

When the retinal pigment epithelial cells of chick embryo are cultured in monolayer conditions, the pigment granules are lost from the cytoplasm. The first structural change in depigmentation is the transformation of pigment granules into the degradative organelles designated as the dense body and melanosome complex. The cells are grown in medium containing DBcAMP of various doses from 10^?5 to 10^?2M. Cell proliferation is retarded by treatment with DBcAMP (10^?3M). The transformation of pigment granules is almost completely prevented in all 1-day cultured cells. In 5-day cultured cells continuously treated with more than 10^?4M, the transformation is not only prevented, but the synthesis of pigment granules is stimulated. A similar result is obtained by the administration of 10^?3M theophylline. 5′-AMP does not prevent the transformation of pigment granules but seems to stimulate the synthesis of pigment granules. On the other hand, cGMP is ineffective both on prevention of transformation and on synthesis of pigment granules. The mechanisms of the transformation of pigment granules are discussed.     

Chicken embryo lens cultures mimic differentiation in the lens

Embryonic chicken lenses, which had been disrupted by trypsin, were grown in culture. These cultures mimic lens development as it occurred in vivo, forming lens-like structures known as lentoids. Using a variety of techniques including electron microscopic analysis, autoradiography, immunofluorescence, and polyacrylamide gel electrophoresis, it was shown that the lentoid cells had many characteristics in common with the differentiated cells of the intact lens, the elongated fiber cells. These characteristics included a shut off of DNA synthesis, a loss of cell organelles, an increase in cell volume, an increase in δ-crystallin protein, and the development of extensive intercellular junctions. The cultures began as a simple epithelial monolayer but then underwent extensive morphogenesis as they differentiated. This morphogenesis involved three distinctive morphological types which appeared in sequence as an epithelial monolayer of polygonal shaped cells with pavement packing, elongated cells oriented end to end, and the multilayered, multicellular lentoids. These distinct morphological stages of differentiation in culture mimic morphogenesis as it occurs in the lens.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Actin filament organization regulates the induction of lens cell differentiation and survival

The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

Organization of intestinal epithelial cells into multicellular structures requires laminin and functional actin microfilaments

Epithelial cell organization into multicellular structures is a critical biological process required for both organogenesis and repair following injury. The basement membrane and the cytoskeleton have important roles in this process; however, the functions of individual components of basement membrane and cytoskeleton are poorly understood. We used IEC-6 cells, a rat intestinal crypt cell line, grown on a three-dimensional gel of reconstituted basement membrane as a model system to determine which extracellular matrix and cytoskeletal components mediate intestinal epithelial cell organization. The cells entered the gel and formed hollow, tubular structures that resembled intestinal crypts. These structures were characterized by a single layer of polarized cells with apical tight junctions and microvilli on the luminal surface. Antiserum to laminin and the pentapeptide Tyr-Ile-Gly-Ser-Arg (which prevents cell attachment to laminin) inhibited this organization, but a control pentapeptide (Tyr-Tyr-Gly-Asp-Ala) and antiserum to collagen IV did not. Cytochalasin B, which interferes with actin microfilament polymerization, also inhibited organization of cells into multicellular structures, but vinblastine and Colcemid, which disrupt microtubules, and cycloheximide, which inhibits protein synthesis, did not. We conclude that organization of intestinal epithelial cells on a basement membrane into multicellular structures results from specific interactions between cells and laminin and requires intact actin microfilaments.