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K K Svoboda

Publications and source records attributed to K K Svoboda.

At least 19 recordsLinked to original sources
PubMed2000

Hyperbaric oxygen downregulates ICAM-1 expression induced by hypoxia and hypoglycemia: the role of NOS.

Hyperbaric oxygen (HBO) is being studied as a therapeutic intervention for ischemia/reperfusion (I/R) injury. We have developed an in vitro endothelial cell model of I/R injury to study the impact of HBO on the expression of intercellular adhesion molecule-1 (ICAM-1) and polymorphonuclear leukocyte (PMN) adhesion. Human umbilical vein endothelial cell (HUVEC) and bovine aortic endothelial cell (BAEC) induction of ICAM-1 required simultaneous exposure to both hypoxia and hypoglycemia as determined by confocal laser scanning microscopy, ELISA, and Western blot. HBO treatment reduced the expression of ICAM-1 to control levels. Adhesion of PMNs to BAECs was increased following hypoxia/hypoglycemia exposure (3. 4-fold, P < 0.01) and was reduced to control levels with exposure to HBO (P = 0.67). Exposure of HUVECs and BAECs to HBO induced the synthesis of endothelial cell nitric oxide synthase (eNOS). The NOS inhibitor nitro-L-arginine methyl ester attenuated HBO-mediated inhibition of ICAM-1 expression. Our findings suggest that the beneficial effects of HBO in treating I/R injury may be mediated in part by inhibition of ICAM-1 expression through the induction of eNOS.

Animals↗
PubMed2000

Erk and PI-3 kinase are necessary for collagen binding and actin reorganization in corneal epithelia.

PURPOSE: It was recently shown that phosphatidylinositol-(PI)3 kinase is upregulated in wounded rabbit corneal epithelia. Extracellular signal-regulated kinase (erk)-1 and -2 proteins and PI-3 kinase were activated in embryonic corneal epithelia after 1-hour stimulation by type I collagen. In the current investigation specific inhibitors of PI-3 kinase and mitogen-activated kinase-kinase (MEK-1 kinase) were used to determine the role of these signaling molecules in actin reorganization and collagen binding to isolated sheets of corneal epithelial tissue. METHODS: Effects of specific PI-3 kinase and MEK-1 inhibitors (LY294002, PD98059, respectively) were investigated in embryonic corneal epithelial tissues. Avian embryonic corneal epithelia were isolated as tissue sheets, organ cultured in the presence of these specific inhibitors, and stimulated with type I collagen. The tissues were evaluated for collagen-stimulated actin reorganization, erk-1 and -2 and PI-3 kinase activity, total filamentous actin accumulation, and collagen binding. RESULTS: The MEK-1 inhibitor PD98059 decreased erk-1 and -2 phosphorylation and blocked actin reorganization in a dose-dependent manner. The PI-3 kinase 85-kDa subunit was decreased 25% in LY294002-treated tissue, and collagen binding also decreased significantly in tissues treated with MEK-1 and PI-3 kinase inhibitors compared with control tissues. In addition, both inhibitors blocked actin cortical mat reorganization. CONCLUSIONS; PI-3 kinase and erk-1 and -2 signaling pathways are activated and necessary for collagen binding and integrin-mediated actin reorganization in embryonic avian corneal epithelium.

Actins↗
PubMed1999

Zyxin and vinculin distribution at the cell-extracellular matrix attachment complex (CMAX) in corneal epithelial tissue are actin dependent.

Avian embryonic corneal epithelia are two cell layers thick. If isolated without (-) basal lamina, the basal cells have unorganized actin and project cytoplasmic protrusions termed blebs. The actin-based cytoskeleton at the cell-extracellular matrix junction (termed the actin cortical mat) is disrupted. These epithelia respond to soluble extracellular matrix molecules by reorganizing the actin cortical mat. Sheets of epithelia were isolated + or -basal lamina. Epithelia isolated -basal lamina were cultured +/- laminin-1 and/or +/- cytochalasin D (CD). The intracellular localization of zyxin, vinculin, paxillin, focal adhesion kinase, and tensin was determined using indirect immunohistochemistry. Protein levels were determined by Western blot analysis. Zyxin and vinculin were concentrated in two areas of the tissue. The interface between the upper cell layer (periderm) and the basal cells. The second area of concentration was at the inferior 1-4 microns of the basal cells in an area with multiple actin bundles termed the actin cortical mat. The actin bundles align toward zyxin and vinculin that were located near basal lateral membranes. Zyxin was displaced from the basal compartment of blebbing basal cells. In contrast tensin, vinculin and focal adhesion kinase were found diffusely throughout the blebs. Zyxin and vinculin redistributed to the basal-lateral membranes as actin bundles reorganized in laminin-stimulated epithelia. In contrast to the altered protein distribution, extractable protein levels were similar in blebbing and laminin-stimulated epithelia. Zyxin, vinculin, and other associated proteins were disrupted in the CD-treated tissues and do not colocalize with each other or CD-induced actin aggregates. The intracellular localization of zyxin and vinculin were concentrated in distinct areas along the inferior basolateral membranes of basal cells termed the cell-extracellular matrix attachment complex (CMAX). The distribution of CMAX proteins was dependent upon actin bundle organization.

Actins↗
PubMed1999

ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent.

Previous studies demonstrated that corneal epithelial cells isolated without basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorganizes. We hypothesize that the actin reorganization is initiated by intracellular signaling mechanisms that includes tyrosine phoshporylation and activation of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. Our goals were to develop a morphological assay to test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have the same configuration in response to different ECM molecules? 2) What is the minimum time ECM molecules need to be in contact with the tissue for the actin to reorganize? 3) Will blocking tyrosine phosphorylation inhibit reorganization of the actin? 4) Are known signal transduction proteins phosphorylated in response to soluble matrix molecules? The actin cortical mat demonstrated distinct bundle configurations in the presence of different ECM molecules. Soluble fibronectin accumulated at the basal cell surfaces 75-fold over 30 min in a clustered pattern. The cells need contact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In contrast, two substances that bind to heptahelical receptors to stimulate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the actin bundles in 15-30 min. Focal adhesion kinase, p190 Rho-GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk-1, erk-2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorganization induced by ECM molecules. In conclusion, we have developed two morphological assays to examine the response of corneal epithelial cells to ECM molecules. In addition, actin bundle reorganization involved tyrosine phosphorylation, MAP kinase, and PI3 kinase activation.

Actins↗
PubMed1998

Chondrocyte-matrix attachment complexes mediate survival and differentiation.

Integrin mediated cell-extracellular matrix interactions are required for survival and differentiation of many cell types. In this review, the cell-matrix attachment complex (CMAX) is described for chondrocytes. The evidence that integrin-mediated signal transduction is necessary for normal chondrocyte differentiation and survival in various culture conditions and in vivo are reviewed. The possible signal transduction pathways stimulated by the extracellular matrix components are discussed with a review of current data from chondrocyte experiments. In addition, the influence of parathyroid hormone and transforming growth factor beta on chondrocyte survival has been included as they may function in concert with integrin mediated signal transduction. Finally, specific changes in gene expression preceding apoptosis are discussed. The current understanding of how integrin-mediated signals prevent apoptosis and implications of anchorage-dependent survival for development and differentiation of the chondrocyte phenotype are discussed.

Animals↗
PubMed1998

Characterization of Bsk mice: I. The Bsk mutation does not involve a recombination of cornea-specific keratin 12 and skin-specific hair keratin genes.

PURPOSE: Bsk (bare skin) is an autosomal dominant mutation linked to the Krt 1 (type 1 keratin) locus of mouse chromosome 11. The adult Bsk mouse manifests hair loss and corneal opacity. To identify and characterize the keratin genes involved in this mutation, we examined the hypothesis proposing that the Bsk mutation might involve a recombination event between cornea-specific (K12) and hair-specific (mHa 1, 2, 3 and 4) type I keratin genes. METHODS: The Bsk phenotype was examined by histochemical analysis, using light and electron microscopy. RFLP was used for their genotyping, and possible keratin gene expression was examined by immunohistochemical staining, Western analysis, RT-PCR and Northern hybridization. RESULTS: Northern hybridization, RT-PCR and Western blot analysis revealed that mHa 1, 2, 3 and 4 keratins are expressed in the skin, but not in cornea, whereas the expression of K12 is limited to the corneas of the Bsk mice. These data ruled out the hypothesis that Bsk phenotype results from a recombination event between K12 and mHa 1, 2, 3 and 4. Ultrastructural and biochemical analyses also indicated that Bsk does not involve negative dominant mutations of keratin 12, mHa 1, 2, 3 and 4, epidermal-specific keratin 10, or basal cell-specific keratin 14. Expression of an acidic 50 kD keratin, recognized by monoclonal antibody AK 2, was up-regulated in the injured corneas of normal mice as well as Bsk corneas. CONCLUSION: The gene linked to the Bsk mutation remains unknown. The pathological changes in the skin and corneas may be secondary to the loss of protecting hairs and lashes by an unknown mechanism.

Animals↗
PubMed1997

Chondrocyte survival and differentiation in situ are integrin mediated.

Chondrocytes in specific areas of the chick sternum have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix prior to bone deposition. Middle and caudal chondrocytes remain cartilaginous throughout development and continue to secrete collagen types II, IX, and XI. The interaction of integrin receptors with extracellular matrix molecules has been shown to affect cytoskeleton organization, proliferation, differentiation, and gene expression in other cell types. We hypothesized that chondrocyte survival and differentiation including the deposition into interstitial matrix of type X collagen may be integrin receptor mediated. To test this hypothesis, a serum-free organ culture sternal model that recapitulates normal development and maintains the three-dimensional relationships of the tissue was developed. We examined chondrocyte differentiation by five parameters: type X collagen deposition into interstitial matrix, sternal growth, actin distribution, cell shape, and cell diameter changes. Additional sterna were analyzed for apoptosis using a fragmented DNA assay. Sterna were organ cultured with blocking antibodies specific for integrin subunits (alpha2, alpha3, or beta1). In the presence of anti-beta1 integrin (25 microg/ml, clone W1B10), type X collagen deposition into interstitial matrix and sternal growth were significantly inhibited. In addition, all chondrocytes were significantly smaller, the actin was disrupted, and there was a significant increase in apoptosis throughout the specimens. Addition of anti-alpha2 (10 microg/ml, clone P1E6) or anti-alpha3 (10 microg/ml, clone P1B5) integrin partially inhibited type X collagen deposition into interstitial matrix; however, sternal growth and cell size were significantly decreased. These data are the first obtained from intact tissue and demonstrate that the interaction of chondrocytes with extracellular matrix is required for chondrocyte survival and differentiation.

Animals↗
PubMed1996

Intracellular distribution of type II collagen mRNA and prolyl 4-hydroxylase in embryonic avian corneal epithelia.

BACKGROUND: The distribution of endoplasmic reticulum (ER) proteins and type II collagen mRNA in whole mount preparations of embryonic corneal epithelia was investigated. We asked, do transmission electron microscopy (TEM) and confocal microscopy of chick prolyl 4-hydroxylase (CPH) show similar ER distributions as CPH is necessary for collagen biosynthesis? METHODS: The overall distribution of ER was analyzed by TEM. Collagen secreting ER was localized with antibodies to chick prolyl 4-hydroxylase (CPH, CPH-alpha, or CPH-beta subunits). Type II collagen mRNA distribution was determined by in situ hybridization. RESULTS: The ER localized with TEM, and immunohistochemistry using anti-CPH antibodies, showed similar distribution patterns. Immunostaining for CPH and CPH-beta was prominent in both periderm and basal cells and also appeared to stain the periderm apical surface. CPH-alpha was less intense, only localizing to ER regions. The basal cell CPH distribution appeared perinuclear in optical sections that contained nuclei, but occupied nearly all the cytoplasm in a reticular pattern above and below nuclei, similar to that seen with TEM cross-sections. Epithelia double labeled with propidium iodide and CPH showed overlapping cytoplasmic staining. The distribution of type II collagen mRNA was similar to the ER staining pattern, appearing to represent a subset of total ER. CONCLUSIONS: This study demonstrates that ER markers have a similar distribution as type II collagen mRNA in embryonic avian corneal epithelia. In addition the CPH subunits had distinct, but overlapping distributions, suggesting that they may act independently.

Animals↗
PubMed1995

Distribution of F-actin, vinculin and integrin subunits (alpha 6 and beta 4) in response to corneal substrata.

Our goal was to determine the early response of corneal epithelial cells to living modified stromal substrates. We examined the distribution of integrin subunits (alpha 6 and beta 4), vinculin and the organization of F-actin in epithelial cells after cell-matrix and cell-cell hypothesized that the distribution of proteins in the cell matrix attachment complex would be altered if the substrate was modified. Integrin subunits, alpha 6 and beta 4, were chosen as they play a role in cell matrix adhesion and adhesion site formation. Corneal epithelial cells were cultured from explants and seeded on three corneal substrates (a stroma containing an intact basal lamina, a cornea lacking a basal lamina and a cornea treated with alkali). After 3 h of incubation, the tissue was fixed and stained with monoclonal antibodies specific for vinculin and for the integrin subunits alpha 6 and beta 4. The organization of F-actin was assessed using rhodamine phalloidin. The localization of the proteins was recorded with confocal laser scanning microscopy. Filamentous actin delineated the lateral cell membranes of corneal epithelial cells. The organization of actin and distribution of vinculin and integrin subunits of epithelial cells cultured on stromal substrates containing a basal lamina mimicked a simple epithelial organization. In contrast, when cells are cultured on the substrate lacking a basal lamina and alkaline treated corneal substrates the distribution of the specific proteins examined was altered. Vinculin and alpha 6 were present along membranes of cells cultured on substrates lacking a basal lamina and were diffuse in cells cultured on the alkaline substrates. These studies demonstrate that changes in the distribution of adhesion and cytoskeletal proteins in response to different surfaces may contribute to the healing dynamics in different wounds.

Actins↗
PubMed1994

Cellular invasion and collagen type IX in the primary corneal stroma in vitro.

During different stages in the development of the avian cornea, various collagen types have been shown to participate in matrix formation and have been implicated in morphogenesis. One of these is the fibril-associated collagen type IX. This molecule is present when the primary corneal stroma is in a compact state, but rapidly disappears just prior to stromal swelling and its invasion by mesenchymal cells. The temporospatial pattern of the disappearance of type IX collagen in the developing cornea suggests that this molecule may be involved in stabilizing the primary corneal stromal matrix by interacting either with other type IX collagen molecules or with other matrix components. To explore further whether the removal of type IX collagen is involved in stromal swelling, we have employed an in vitro culture system in which swelling of the primary stroma and mesenchymal cell invasion can be experimentally manipulated by culturing chick corneal explants on a Nuclepore filter support in the presence or absence of an associated lens. We have also examined the effect of exogenously added human recombinant tissue inhibitor of metalloproteinases (TIMP-1) on the presence of type IX collagen and cellular invasion. When stage 25-26+ corneal explants were cultured with an associated lens, the primary stroma did not swell; immunohistochemically detectable type IX collagen was still present, and mesenchymal cell invasion failed to occur. Conversely, when the same stages of corneal explants were cultured without an associated lens, the primary stroma swelled; type IX collagen disappeared, and mesenchymal cell migration occurred. Under both conditions, however, the type II collagen of the stroma, which is known to be a component of the striated fibrils, remained clearly detectable and with time even seemed to increase in amount. This result is consistent with the proposition that type IX collagen is one factor involved in maintaining the primary stroma as a compact matrix, possibly by functioning as a bridging/stabilizing factor. When TIMP was added to cultures of corneal explants, type IX collagen remained detectable in focal regions, suggesting that one or more metalloproteinases are involved in the removal of the type IX collagen. In addition, some of these type IX-containing regions contained mesenchymal cells, suggesting that in addition to type IX collagen other factors are likely to be involved in regulating mesenchymal cell migration.

Animals↗
PubMed1994

Precursors of macrophages in embryonic rat lungs fail to exhibit granulocyte-forming potential.

BACKGROUND: Mesenchyme-like macrophage (M) precursors called angular cells are present in rat lungs on the thirteenth day of gestation and by then can differentiate into outright macrophages. Based on studies of bone marrow-derived cells, it is widely believed that the macrophage line necessarily proceeds from a colony-forming unit with dual granulocyte-macrophage potential (CFU-GM). In embryos this seems doubtful since macrophages are already scattered throughout the body before the first granulocytes appear. We examined the question in organ cultured 14 day prenatal rat lungs after having shown earlier that the macrophage population developed in explants is increased by exposure to M- and GM-colony-stimulating factors (CSFs) but is unaffected by multi (IL-3)- or granulocyte (G)-CSF. Reportedly retinoic acid (RA) shifts CFU-GM strongly towards granulocytic differentiation and inhibits mitosis of unipotential macrophage precursors but not differentiated cells. Transforming growth factor beta 1 (TGF) inhibits multipotential blood progenitors but allows proliferation of committed precursors, and TGF together with GM-CSF induces granulocytopoiesis from CFU-GM. METHODS: Lung pairs were grown on a serum-containing medium or one supplemented either by RA, TGF, or TGF/GM-CSF to form a control and three experimental groups. A fourth experiment compared responses to M-CSF exposure and M-CSF/TGF. Macrophage population growth was estimated by measuring the areas of coronas formed by macrophages emerged from the explants. F-actin was stained with fluorescein-labeled phalloidin. RESULTS: In all experiments macrophages were produced unmixed with granulocytes. By +8 days they had largely emerged to form coronas about the lungs. In cultures exposed to RA, macrophages were less intensely stained for actin and slower to emerge than controls. At +8 days, however, coronal areas were not significantly different from controls, as was also true for the TGF group. In contrast, coronal areas of cultures grown with TGF/GM-CSF were much larger. At +17 days, mean coronal area of TGF cultures was about half that of controls (P < 0.05), whereas mean coronal area of the TGF/GM-CSF group was 5.4 times greater (P < 0.001). Macrophages from control and TGF-exposed cultures responded to M-CSF by an increase in coronal area which was greater among cultures given M-CSF alone than those given TGF + M-CSF (both P < 0.005). CONCLUSIONS: Macrophage precursors in embryonic lungs are distinct from CFU-GM.

Animals↗
PubMed1994

Intracellular distribution of beta-actin mRNA is polarized in embryonic corneal epithelia.

The intracellular distribution of filamentous actin (F-actin), all actin isoforms and beta-actin mRNA were analyzed in whole-mount preparations of freshly isolated corneal epithelia. Filamentous actin distribution was analyzed with fluorescently tagged phalloidin. An antibody that recognizes an epitope on both globular (G-actin) and F-actin was used in an immunohistochemical analysis of actin protein distribution. Whole-mount epithelial tissues were examined with a confocal laser scanning microscope (CLSM). Biotinylated oligonucleotide probes specific for the beta-actin mRNA were used, and visualized with avidin-FITC. The intracellular localization of the beta-actin mRNA was similar to the F-actin protein distribution. In the most apical optical sections of embryonic cornea, actin staining delineated the cell borders and microvilli of the periderm cells. The actin is also detected as an organized network at the interface between the basal and periderm cells. At the level of the basal cell nucleus, F-actin is sparse, associating only with the lateral cell membranes. However, at the optical plane below the nuclei, the actin forms an elaborate actin cortical mat. Actin mRNA staining was visualized as discrete punctate areas. The beta-actin mRNA was positive at the optical plane just below the periderm cell apical membrane surface, similar to actin in microvilli. These cells also contained punctate staining near the cell membranes and in the periderm-basal cell junction area. At the level of the basal cell nucleus the actin mRNA was present in a punctate pattern along the cell membranes. Below the basal cell nuclei the actin mRNA staining increased at the level of the actin cortical mat. These experiments are the first demonstration that actin mRNA is polarized in embryonic corneal epithelia and co-localized with actin protein in an intact tissue.

Actins↗
PubMed1993

Intracellular relationship between actin and alpha-actinin in a whole corneal epithelial tissue.

Alpha-actinin is an actin crosslinking protein that may be one of the proteins involved in the attachment of the actin cytoskeletal framework to the plasma membrane. We investigated the distribution of alpha-actinin in whole-mount embryonic chick corneal epithelia using confocal laser scanning analysis. The intracellular alpha-actinin distribution was compared with F-actin using phalloidin, or total actin using an anti-actin antibody. Corneal epithelial tissues were isolated with or without the basal lamina (+ or -BL), and fixed immediately. In addition, epithelia isolated -BL were cultured for 2 hours with either control medium, laminin-supplemented medium or laminin and cytochalasin D (CD)-containing medium. The single- and double-labeled epithelia showed that alpha-actinin delineated the cell borders and microvilli of the periderm cells in the most apical optical sections of control and laminin-treated epithelia. At the optical plane through the basal cell nuclei, the alpha-actinin was distributed diffusely throughout the cytoplasm, whereas the actin was sparse, only associated with the lateral cell membranes. Epithelia (-BL) cultured in control medium had cytoplasmic protrusions or blebs on the basal cell surface. The blebs contained both actin and alpha-actinin. In epithelial cultured with laminin, the basal cell surface was flat. The actin cortical mat became reorganized within two hours. Actin and alpha-actinin were colocalized in the re-formed basal cytoskeletal network. In cells cultured with cytochalasin D (CD) and laminin the actin cortical mat was not reorganized. Actin networks from both cell layers were eliminated and replaced by aggregates scattered throughout the cytoplasm. The alpha-actinin remained diffusely distributed in the cytoplasm and failed to colocalize with the actin aggregates. The alpha-actinin appeared closer to the basal cell membrane than the actin in cross-sectional views of the tissue. Results from these double-labeling experiments confirmed the intimate association of alpha-actinin and actin in the laminin-stimulated actin cortical mat reorganization. This study is the first to demonstrate that CD-aggregated F-actin does not capture the alpha-actinin. The alpha-actinin appeared to remain diffuse throughout the cytoplasm and separate from F-actinin; however, there was some overlap with G-actin.

Actinin↗
PubMed1992

Sheets of embryonic epidermis can be isolated and cultured with or without the basal lamina.

In the current study, we determine the concentration of Dispase II, a neutral protease, and the incubation time necessary for isolation of embryonic avian epidermis. The epidermis can be isolated from the dermis consistently with or without the basal lamina. Because the action of Dispase II is selective and easily controlled, this study suggests that Dispase can serve as a powerful tool for studying epidermal-extracellular matrix interactions. In further experiments we study the response of sheets of epidermis isolated with and without its basal lamina to extracellular matrix. When the epidermis is isolated without the basal lamina, the basal surface blebs and the actin in the basal cytoplasm are disrupted. Epidermis isolated without the basal lamina and cultured on extracellular matrix reorganizes the basal actin mat and retracts the blebs. However, epidermis isolated without the basal lamina and cultured in the absence of extracellular matrix continues to have blebs and a disorganized actin cortical mat. In contrast, the control epidermis isolated with the basal lamina retains a flat basal surface and organized actin cortical mat after culturing either with or without extracellular matrix. The extracellular matrix has been shown by many investigators to play an important role in cytoskeletal organization, metabolism, and differentiation. How the extracellular matrix interacts and influences these functions has not been completely elucidated. The ability to isolate tissue consistently with and without its basal lamina using Dispase II will facilitate addressing these and other epithelial cell biology questions.

Animals↗
PubMed1992

Embryonic corneal epithelial actin alters distribution in response to laminin.

In the current study, laser scanning confocal image analysis was used to investigate the actin distribution in whole-mount preparations of freshly isolated and cultured corneal epithelia. Actin staining defined the cell borders and microvilli of the periderm cells. The actin was prominent as an organized network at the interface between the basal and periderm cells and in the basal compartment of the basal cells (actin cortical mat) when isolated with the basal lamina (BL). In epithelia isolated without BL, the actin in the periderm cells and the network at the periderm-basal cell interface was the same as in epithelia isolated with BL. However, the actin in the basal compartment of the basal cells was localized in the cellular blebs that projected from the basal cell surface. Epithelia isolated without BL and cultured in the presence of laminin reorganized the actin cortical mat within 2 hours. However, epithelia isolated without BL and cultured without BL proteins continued to have basal cell projections, sometimes into the pores of the filter. Treatment of epithelia with 2 microM cytochalasin D prevented the reorganization of the actin by laminin. In conclusion, the use of confocal analysis increased our understanding of actin distribution in the epithelial sheets. These results confirmed and extended previous studies using electron microscopy to determine that corneal epithelial cells respond to extracellular matrix molecules by an actin-dependent mechanism.

Actins↗
PubMed1991

Intracellular localization of types I and II collagen mRNA and endoplasmic reticulum in embryonic corneal epithelia.

The intracellular distribution of endoplasmic reticulum (ER) and types I and II collagen mRNA was analyzed in whole-mount preparations of freshly isolated corneal epithelia using in situ hybridization combined with confocal laser scanning analysis. The ER stained with DiOC6 (3) was prominent in both the periderm and basal cells. The basal cell ER distribution was perinuclear in the center of the cells, but below the nucleus the ER occupied nearly all of the cytoplasm in a reticular pattern similar to that seen with TEM cross-sections. Initial single label in situ hybridization studies showed that both the periderm and basal cells were positive for both types I and II collagen mRNA. The collagen cDNA probes appeared perinuclear in the center of the basal cells, similar to the DiOC6(3) staining pattern. In double-labeling experiments, the two mRNAs that translate chains of type I collagen, alpha 1 and alpha 2, colocalized within the same cell. However, the hybridization of probes specific for type I and II collagen mRNAs had separate, but overlapping, distributions within the same cell.

Animals↗
PubMed1988

Embryonic chicken cornea and cartilage synthesize type IX collagen molecules with different amino-terminal domains.

We have analyzed embryonic chicken cornea for the presence of type IX collagen mRNA and protein. Using RNA transfer blot analysis, we demonstrate that alpha 1(IX) and alpha 2(IX) mRNAs are expressed by corneal epithelial cells at the time that the primary stromal components are synthesized. The levels of the mRNAs decrease with increasing developmental age and are barely detectable at day 11 of development. In contrast, type IX collagen protein is detectable by immunofluorescence at days 5 and 6 and undetectable by day 8. Using probes specific for alpha 1(IX) and alpha 2(IX) mRNAs, we demonstrate that the size of alpha 2(IX) mRNA is the same in cornea as in chondrocytes, the major source of type IX collagen. However, the alpha 1(IX) mRNA is about 700 nucleotides shorter in the cornea than in cartilage because the corneal form of the mRNA does not contain the 5' region that encodes the non-triple-helical amino-terminal globular domain of cartilage type IX collagen. Therefore, corneal type IX collagen must lack this domain. This structural modulation of an extracellular matrix protein is likely to contribute to the functional differences between cartilage matrix and the early corneal stroma, both of which are rich in type II collagen.

Amino Acid Sequence↗