Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.     

Seven New Mutations in hMSH2, an HNPCC Gene, Identified by Denaturing Gradient-Gel Electrophoresis

Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of hMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup.     

The response of chicken embryo dermal fibroblasts to cytochalasin B is altered by Rous sarcoma virus-induced cell transformation.

The drug cytochalasin B (CB), which disrupts the cellular microfilament network, allows the identification of as yet unclassified structural differences between normal and Rous sarcoma virus-transformed chicken embryo fibroblasts. When exposed to CB, normal chick fibroblasts attain an arborized or dendritic morphology. This results as the cytoplasm collapses upon the remaining structural and adhesive components of the cell. Rous sarcoma virus-transformed cells did not form or maintain these dendritic-like processes in the presence of CB and, as a result, rounded up but still remained attached to the substrate. With a temperature-sensitive mutant of Rous sarcoma virus, LA24A, it was possible to show that these effects are completely reversible and dependent on the expression of pp60src. The cytoskeleton in these CB-treated cells was examined by both immunofluorescence and electron microscopy. After exposure to CB, the microfilaments were found to be disrupted similarly throughout both the transformed and the nontransformed cells. In the nontransformed cells arborized by exposure to CB, the extended processes were found to contain intermediate filaments in an unusually high concentration and degree of organization. The distribution of these filaments in the central body of the arborized cells was random. This lower concentration and random distribution was similar to that seen throughout the transformed cells rounded up by exposure to CB. The failure of these transformed cells to arborize in CB indicates that the structural component(s) which is necessary for the formation or maintenance or both of the arborized state is altered by the expression of pp60src.     

Cells activated for wound repair have the potential to direct collective invasion of an epithelium

Mechanisms regulating how groups of cells are signaled to move collectively from their original site and invade surrounding matrix are poorly understood. Here we develop a clinically relevant ex vivo injury invasion model to determine whether cells involved in directing wound healing have invasive function and whether they can act as leader cells to direct movement of a wounded epithelium through a three-dimensional (3D) extracellular matrix (ECM) environment. Similar to cancer invasion, we found that the injured cells invade into the ECM as cords, involving heterotypical cell–cell interactions. Mesenchymal cells with properties of activated repair cells that typically locate to a wound edge are present in leader positions at the front of ZO-1–rich invading cords of cells, where they extend vimentin intermediate filament–enriched protrusions into the 3D ECM. Injury-induced invasion depends on both vimentin cytoskeletal function and MMP-2/9 matrix remodeling, because inhibiting either of these suppressed invasion. Potential push and pull forces at the tips of the invading cords were revealed by time-lapse imaging, which showed cells actively extending and retracting protrusions into the ECM. This 3D injury invasion model can be used to investigate mechanisms of leader cell–directed invasion and understand how mechanisms of wound healing are hijacked to cause disease.     

Regional-specific alterations in cell–cell junctions,cytoskeletal networks and myosin-mediated mechanical cues coordinate collectivity of movement of epithelial cells in response to injury

This study investigates how epithelial cells moving together function to coordinate their collective movement to repair a wound. Using a lens ex vivo mock cataract surgery model we show that region-specific reorganization of cell–cell junctions, cytoskeletal networks and myosin function along apical and basal domains of an epithelium mediates the process of collective migration. An apical junctional complex composed of N-cadherin/ZO-1/myosin II linked to a cortical actin cytoskeleton network maintains integrity of the tissue during the healing process. These cells’ basal domains often preceded their apical domains in the direction of movement, where an atypical N-cadherin/ZO-1 junction, linked to an actin stress fiber network rich in phosphomyosin, was prominent in cryptic lamellipodia. These junctions joined the protruding forward-moving lamellipodia to the back end of the cell moving directly in front of it. These were the only junctions detected in cryptic lamellipodia of lens epithelia migrating in response to wounding that could transmit the protrusive forces that drive collective movement. Both integrity of the epithelium and ability to effectively heal the wound was found to depend on myosin mechanical cues.     

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.     

A Highlights from MBoC Selection: A central role for vimentin in regulating repair function during healing of the lens epithelium

Mock cataract surgery provides a unique ex vivo model for studying wound repair in a clinically relevant setting. Here wound healing involves a classical collective migration of the lens epithelium, directed at the leading edge by an innate mesenchymal subpopulation of vimentin-rich repair cells. We report that vimentin is essential to the function of repair cells as the directors of the wound-healing process. Vimentin and not actin filaments are the predominant cytoskeletal elements in the lamellipodial extensions of the repair cells at the wound edge. These vimentin filaments link to paxillin-containing focal adhesions at the lamellipodial tips. Microtubules are involved in the extension of vimentin filaments in repair cells, the elaboration of vimentin-rich protrusions, and wound closure. The requirement for vimentin in repair cell function is revealed by both small interfering RNA vimentin knockdown and exposure to the vimentin-targeted drug withaferin A. Perturbation of vimentin impairs repair cell function and wound closure. Coimmunoprecipitation analysis reveals for the first time that myosin IIB is associated with vimentin, linking vimentin function in cell migration to myosin II motor proteins. These studies reveal a critical role for vimentin in repair cell function in regulating the collective movement of the epithelium in response to wounding.     

The Temperature Sensitivity of the Processes of the Initial Stage of Microbial Decomposition of Woody Litter in Forest Soil

Biophysics - Abstract—Soil organic matter of forest ecosystems is characterized by high sensitivity to increased temperatures, which makes soil organic matter more vulnerable under the...     

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.