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A Sue Menko

Publications and source records attributed to A Sue Menko.

11 recordsLinked to original sources
PubMed2006

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.

Actin Cytoskeleton↗
PubMed2006

Phosphatidylinositol 3-kinase is necessary for lens fiber cell differentiation and survival.

PURPOSE: To determine the mechanisms of action of phosphatidylinositol 3-kinase (PI3K) in lens cell differentiation and survival. METHODS: Primary quail lens cell cultures were treated at different stages of differentiation with the PI3K inhibitor LY294002, and expression of survival proteins and differentiation markers were determined by immunoblot analysis. The connection between PI3K regulation of lens differentiation and actin cytoskeleton reorganization was examined by fluorescent-phalloidin staining and Rac activity assay. Survival in the absence of PI3K signaling was examined by TUNEL and DAPI staining. Phosphorylation of the PI3K effector glycogen synthase kinase-3 (GSK3) in the absence of PI3K signaling was induced with lithium chloride. RESULTS: Exposure to LY294002 blocked lens epithelial cell differentiation initiation. This result was linked to attenuation of Rac activity and inhibition of actin filament reorganization from stress fibers to cortical fibers, which has been shown to signal lens differentiation initiation. The survival of lens epithelial cells in the absence of PI3K signaling correlated with induction of numerous survival factors, including Bcl-2. In contrast, inhibition of PI3K signaling in differentiating lens fiber cells induced apoptosis by blocking inactivation of GSK3, showing that PI3K/GSK3 signaling has a protective role in the late stages of differentiation as nuclei and organelles are lost. CONCLUSIONS: PI3K signaling regulates lens cell differentiation initiation through its ability to signal reorganization of the actin cytoskeleton from stress fibers to cortical fibers. In differentiating lens fiber cells, PI3K has a protective function, signaling survival through inactivation of its downstream effector GSK3.

Actins↗
PubMed2005

The canonical intrinsic mitochondrial death pathway has a non-apoptotic role in signaling lens cell differentiation.

The mitochondrial cell death pathway is known for its role in signaling apoptosis. Here, we describe a novel function for the mitochondrial cell death pathway in signaling initiation of differentiation in the developing lens. Most remarkably, we induced lens cell differentiation by short-term exposure of lens epithelial cells to the apoptogen staurosporine. Activation of apoptosis-related pathways induced lens epithelial cells to express differentiation-specific markers and to undergo morphogenetic changes that led to formation of the lens-like structures known as lentoids. The fact that multiple stages of differentiation are expressed at a single stage of development in the embryonic lens made it possible to precisely determine the timing of expression of proteins associated with the apoptotic pathway. We discovered that there was high expression in the lens equatorial epithelium (the region of the lens in which differentiation is initiated) of pro-apoptotic molecules such as Bax and Bcl-x(S) and release of cytochrome c from mitochondria. Furthermore, we found significant caspase-3-like activity in the equatorial epithelium, yet this activity was far lower than that associated with lens cell apoptosis. These apoptotic pathways are likely regulated by the concurrent expression of prosurvival molecules, including Bcl-2 and Bcl-x(L); phosphorylation of Bad; and high expression of inhibitor of apoptosis proteins chicken IAP1, IAP3, and survivin. This finding suggests that prosurvival pathways allow pro-apoptotic molecules to function as molecular switches in the differentiation process without tipping the balance toward apoptosis. We call this process apoptosis-related Bcl-2- and caspase-dependent (ABC) differentiation.

Actins↗
PubMed2004

Coordinate signaling by Src and p38 kinases in the induction of cortical cataracts.

PURPOSE: The goals of this study were to determine whether MAP kinase signaling pathways play a role in the formation of lens cataracts and to examine the potential signaling relationship between Src and MAP kinases in signaling the induction of lens opacities. METHODS: Embryonic day (E)10 chick lenses were cultured in Medium 199 containing 10% fetal bovine serum. The activation state of Src kinases and the MAP kinases extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in the lens epithelium was determined over a time course from 10 minutes to 10 days in culture by immunoblot analysis. Src kinase activation was suppressed by exposure to the Src family kinase-specific inhibitor PP1. To examine the role of specific MAP kinases in the development of lens opacities, lenses were grown for 10 days in the presence or absence of inhibitors of ERK (U0126), JNK (SP600125), and p38 (SB203580). Lenses were observed and photographed daily, and the degree of opacification was quantified by using image-analysis software. RESULTS: Within a short time after placing embryonic lenses in culture conditions that induce the formation of cataracts, there occurred a great increase in the activation state of the MAP kinase ERK. Activation of ERK was both rapid and transient. No activation of the MAP kinase JNK was observed in the cataract cultures beyond that which occurred in normal lens epithelium, even though JNK activation is often linked to the cellular response to stress. In contrast, although p38 activation was barely detected in the normal embryonic lens, this stress-activated protein kinase exhibited a robust activation in cataract cultures that was sustained throughout the culture period. Studies conducted to map the cataract signaling pathways indicate that the p38 MAP kinase functions upstream of the Src kinase. To analyze the potential role of ERK, JNK, and p38 in cataract induction, lenses were cultured in the presence of specific MAP kinase inhibitors. Although the inhibitors of ERK and JNK did not interfere with the formation of cataract, p38 inhibitors blocked the development of lens opacities with an efficacy similar to that of the Src kinase inhibitor PP1. CONCLUSIONS: Activation of both Src and p38 kinases lead to the induction of cataract.

Animals↗
PubMed2002

A signaling role for the uncleaved form of alpha 6 integrin in differentiating lens fiber cells.

Many alpha integrin subunits are cleaved during their processing to yield heavy and light chains, which remain associated by disulfide bonds. While uncleaved alpha integrin subunits can form functional receptors that sometimes have distinct signaling roles from their better-characterized endoproteolytically cleaved counterparts, their expression at the cell surface and their association with signaling complexes have yet to be determined in vivo. In this study, we demonstrate that, in differentiating lens fiber cells, the uncleaved form of alpha 6 integrin was expressed at the cell surface. This form of alpha 6 integrin coimmunoprecipitated with both the signaling adaptor molecule Shc and its downstream effector Grb2, suggesting that, in lens fiber cells, uncleaved alpha 6 integrin was associated with a Shc-mediated signaling complex. We show that expression of the cleaved form of alpha 6 integrin progressively decreased relative to uncleaved alpha 6 integrin as the state of lens cell differentiation increased, resulting in the predominance of uncleaved alpha 6 integrin in the lens fiber cell zones. Interestingly, we previously have shown that alpha 6 integrin is localized principally along the extensive cell-cell interfaces of these lens fiber cells, in the absence of its extracellular matrix ligand laminin. While we found that the cleaved form of alpha 6 integrin contained both high mannose and complex sugars, the uncleaved form of alpha 6 integrin contained only high mannose sugars. These properties suggest that the uncleaved form of alpha 6 integrin may have a unique role in the embryonic lens. Its high association with Shc and Grb2 in the differentiating cortical fiber cell zone indicates that alpha 6 integrin may provide a cell survival signal in the presence of the apoptotic-like processes that are initiated in this region of the embryonic lens to clear the lens cells of their organelles.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Role for alpha 6 integrin during lens development: Evidence for signaling through IGF-1R and ERK.

We show that alpha 6 integrin function was required for normal lens cell differentiation by using an antisense construct to suppress alpha 6 integrin expression. To elucidate the mechanism by which this integrin functions in the regulation of the lens cell differentiation process, we determined the molecular composition of alpha 6 integrin signaling complexes at distinct stages of differentiation in vivo. Because both alpha 6 integrin and insulin-like growth factor-1 (IGF-1) have been implicated in signaling lens cell differentiation, we examined the possibility that they formed a signaling complex in the embryonic lens. Coprecipitation analysis revealed that alpha 6 integrin/IGF-1 receptor complexes were present and that their association was greatest in the equatorial zone, the region of the embryonic lens in which lens cells proliferate and then initiate their differentiation. These results provide in vivo support for the formation of integrin/growth factor receptor signaling complexes. We also found that extracellular signal-regulated kinase (ERK), a downstream effector of both integrin and growth factor receptor signaling pathways, was associated with the alpha 6 integrin signaling complexes in the embryonic lens. This result was supported by our findings that activated ERK, in addition to its nuclear location, localized to lens cell membranes in specific regions of cell-matrix and cell-cell contact. A connection between integrin ligand engagement and ERK activation was shown in vitro after lens cell attachment to laminin. These results demonstrate that alpha 6 integrin function is required for the early stages of lens cell differentiation most likely through its association with the IGF-1 receptor and the activation of ERK.

Animals↗
PubMed2002

Regulation of cadherin junctions during mouse submandibular gland development.

Submandibular gland (SMG) development involves branching morphogenesis of the salivary epithelium into the surrounding mesenchyme, accompanied by proliferation and differentiation of immature salivary cells along acinar and ductal cell lineages. During development, salivary cell sorting and cell-cell adhesion are likely to be directed by cadherin adhesion receptors. We show that two classic cadherins, N- and E-cadherin, participate in SMG development. Early in embryonic morphogenesis, both cadherins displayed diffuse staining with regionalized localization to cell-cell borders. At this stage, significant pools of N- and E-cadherins were Triton-soluble, suggesting that fractions of these molecules were not localized to stable junctional complexes associated with the actin cytoskeleton. With cytodifferentiation, cadherins became progressively Triton-insoluble, and this correlated with their organization at cell-cell interfaces. In the cytodifferentiated SMG, N-cadherin was absent, whereas E-cadherin remained at cell-cell interfaces. Early in morphogenesis, beta-catenin was also primarily Triton-soluble, and its association with the actin cytoskeleton and localization to the adherens junctions increased with cytodifferentiation. Greater recruitment of cadherins and beta-catenin to cell-cell borders was paralleled by changes in membrane association of two Rho GTPases, Cdc42 and RhoA. N-cadherin was detected only at early stages of postnatal development, whereas E-cadherin and beta-catenin became progressively Triton-insoluble during differentiation. Our results indicate that N-cadherin functions transiently in SMG development. On the other hand, E-cadherin and beta-catenin appear to play different roles during tissue organization and cytodifferentiation. In early morphogenesis, E-cadherin and beta-catenin are likely to participate in SMG remodeling, whereas during cytodifferentiation, they form stable cell-cell contacts, and may collaborate with Rho GTPases in the establishment and maintenance of salivary cell polarity.

Animals↗
PubMed2002

Transition between proliferation and differentiation for lens epithelial cells is regulated by Src family kinases.

As in many cell types, lens cells must withdraw from the cell cycle before they initiate their differentiation. The involvement of Src family kinases (SFKs) in this key initiating event in cell differentiation was examined in lens epithelial cell cultures. SFK activity was suppressed with the specific inhibitor PP1. This induced expression of the cyclin-dependent kinase (CDK) inhibitors p27 and p57 and suppressed lens epithelial cell proliferation. Therefore, inhibition of SFK activity created conditions permissive for undifferentiated lens epithelial cells to withdraw from the cell cycle. Growth of the lens epithelial cell cultures in the presence of PP1 induced expression of filensin and CP49, lens differentiation-specific intermediate filament proteins, providing evidence that suppression of SFK activity also promoted the initiation of lens cell differentiation. The mechanism by which PP1 signaled cell cycle withdrawal and commitment to differentiation was shown to involve induction of N-cadherin cell-cell junction assembly and reorganization of the actin cytoskeleton from stress fibers to cortical filaments. This result was supported by the compaction of the epithelial monolayer in response to PP1, a morphogenetic change that we have previously shown to be dependent on N-cadherin function and a hallmark of the commencement of the lens differentiation program in culture. The results presented in this study suggest that the decision of lens epithelial cells to withdraw from the cell cycle and initiate differentiation requires inhibition of SFKs and the formation of N-cadherin cell-cell junctions.

Actins↗
PubMed2002

The role of Src family kinases in cortical cataract formation.

PURPOSE: The goal of this study was to determine the role of Src family kinases (SFKs) in the development of lens cataract. This question was particularly significant, because these tyrosine kinases mediate the stress pathways known to lead to cataract formation. The experiments were focused on whether the inhibition of SFK activity suppresses the formation of lens opacities. METHODS: A whole-lens culture system was developed, in which cortical opacities formed within 5 days, in embryonic day (E)10 lenses grown in medium containing 10% fetal bovine serum. SFK activity was blocked in the cultured lenses by growth in the presence of the SFK-specific inhibitor PP1. Control cultures were grown in medium without inhibitor or in the presence of PP3, the inactive analogue of PP1. Lenses were cultured for 10 days, observed, and photographed daily. Opacification was quantified with image-analysis software. Tissue architecture was determined after hematoxylin and eosin staining and cellular organization by fluorescent localization of filamentous actin with fluorescein-conjugated phalloidin. RESULTS: Almost all lenses in the control cultures developed cortical opacities covering approximately 50% of the lens area by day 10. Similar to control cultures, PP1-treated lenses showed mild posterior opacities during the first 5 days in culture, but then became strikingly transparent. Only 7% of the PP1-treated lenses showed development of cortical cataract, and the average area of opacity was just 0.5% by culture day 10. In all cultured lenses, even in the presence of the PP1 inhibitor, the bow region of the lens extended to the posterior pole, and distribution of nuclei from the posterior pole toward the anterior aspects of the lens suggested that newly added fiber cells were misdirected. However, neither this feature, nor the presence of vacuoles appeared to correlate with the development of opacity in the cultured lenses. Instead, the lens opacities appeared to result from gross abnormalities in the shape and organization of cells in the equatorial and cortical fiber zones, as observed by F-actin staining. Culturing the lenses in the presence of the SFK inhibitor prevented these lens cell aberrations as well as the development of lens opacity. CONCLUSIONS: The formation of cataract can involve activation of SFK-mediated pathway(s) leading to disorganization of developing lens fiber cells, and inhibiting these tyrosine kinases blocks cataract progression.

Animals↗