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Keith E Mostov

Publications and source records attributed to Keith E Mostov.

At least 19 recordsLinked to original sources
PubMed2006

Phosphoinositide 3-kinase regulates the role of retromer in transcytosis of the polymeric immunoglobulin receptor.

Retromer is a multimeric protein complex that mediates intracellular receptor sorting. One of the roles of retromer is to promote transcytosis of the polymeric immunoglobulin receptor (pIgR) and its ligand polymeric immunoglobulin A (pIgA) in polarized epithelial cells. In Madin-Darby Canine Kidney (MDCK) cells, overexpression of Vps35, the retromer subunit key for cargo recognition, restores transcytosis to a pIgR mutant that is normally degraded. Here we show that pIgA transcytosis was not restored in these cells when treated with the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Likewise, the decrease in pIgA transcytosis by wild-type pIgR seen upon PI3K inhibition was not reverted by Vps35 overexpression. PI3K inhibition reduced membrane association of sorting-nexins (SNX) 1 and 2, which constitute the retromer subcomplex involved in membrane deformation, while association of the Vps35-Vps26-Vps29 subcomplex, involved in cargo recognition, remained virtually unaffected. Colocalization between the two retromer subcomplexes was reduced upon the treatment. Whereas the interaction among the subunits of the Vps35-Vps26-Vps29 subcomplex remained unchanged, less Vps35 was found associated with pIgR upon PI3K inhibition. In addition, colocalization of internalized pIgA with subunits of both retromer subcomplexes throughout the transcytotic pathway was substantially reduced by LY294002 treatment. These data implicate PI3K in controlling retromer's role in pIgR-pIgA transcytosis.

Animals↗
PubMed2006

Simulating properties of in vitro epithelial cell morphogenesis.

How do individual epithelial cells (ECs) organize into multicellular structures? ECs are studied in vitro to help answer that question. Characteristic growth features include stable cyst formation in embedded culture, inverted cyst formation in suspension culture, and lumen formation in overlay culture. Formation of these characteristic structures is believed to be a consequence of an intrinsic program of differentiation and de-differentiation. To help discover how such a program may function, we developed an in silico analogue in which space, events, and time are discretized. Software agents and objects represent cells and components of the environment. "Cells" act independently. The "program" governing their behavior is embedded within each in the form of axioms and an inflexible decisional process. Relationships between the axioms and recognized cell functions are specified. Interactions between "cells" and environment components during simulation give rise to a complex in silico phenotype characterized by context-dependent structures that mimic counterparts observed in four different in vitro culture conditions: a targeted set of in vitro phenotypic attributes was matched by in silico attributes. However, for a particular growth condition, the analogue failed to exhibit behaviors characteristic of functionally polarized ECs. We solved this problem by following an iterative refinement method that improved the first analogue and led to a second: it exhibited characteristic differentiation and growth properties in all simulated growth conditions. It is the first model to simultaneously provide a representation of nonpolarized and structurally polarized cell types, and a mechanism for their interconversion. The second analogue also uses an inflexible axiomatic program. When specific axioms are relaxed, growths strikingly characteristic of cancerous and precancerous lesions are observed. In one case, the simulated cause is aberrant matrix production. Analogue design facilitates gaining deeper insight into such phenomena by making it easy to replace low-resolution components with increasingly detailed and realistic components.

Cell Shape↗
PubMed2006

A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma.

The PI3 kinase family of lipid kinases promotes cell growth and survival by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate. To define targets critical for cancers driven by activation of PI3 kinase, we screened a panel of potent and structurally diverse drug-like molecules that target this enzyme family. Surprisingly, a single agent (PI-103) effected proliferative arrest in glioma cells, despite the ability of many compounds to block PI3 kinase signaling through its downstream effector, Akt. The unique cellular activity of PI-103 was traced directly to its ability to inhibit both PI3 kinase alpha and mTOR. PI-103 showed significant activity in xenografted tumors with no observable toxicity. These data demonstrate an emergent efficacy due to combinatorial inhibition of mTOR and PI3 kinase alpha in malignant glioma.

Animals↗
PubMed2006

Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures.

Rho GTPases are critical regulators of epithelial morphogenesis. A powerful means to investigate their function is three-dimensional (3D) cell culture, which mimics the architecture of epithelia in vivo. However, the nature of 3D culture requires specialized techniques for morphological and biochemical analyses. Here, we describe protocols for 3D culture studies with Madin-Darby Canine Kidney (MDCK) epithelial cells: establishing cultures, immunostaining, and expressing, detecting, and assaying Rho proteins. These protocols enable the regulation of epithelial morphogenesis to be explored at a detailed molecular level.

Animals↗
PubMed2005

The role of syntaxins in the specificity of vesicle targeting in polarized epithelial cells.

In polarized epithelial cells syntaxin 3 is at the apical plasma membrane and is involved in delivery of proteins from the trans-Golgi network to the apical surface. The highly related syntaxin 4 is at the basolateral surface. The complementary distribution of these syntaxins suggests that they play a role in the specificity of membrane traffic to the two surfaces. We constructed a chimeric syntaxin where we removed the N-terminal 29 residues of syntaxin 3 and replaced it with the corresponding portion of syntaxin 4. When expressed in polarized epithelial cells, this chimera was exclusively localized to the basolateral surface. This indicates that the N-terminal domain of syntaxin 3 contains information for its polarized localization. In contrast to the apical localization of syntaxin 3, the basolateral localization of syntaxin 4 was not dependent on its N-terminal domain. Syntaxin 3 normally binds to Munc18b, but not to the related Munc18c. Overexpression of the chimera together with overexpression of Munc18b caused membrane and secretory proteins that are normally sent primarily to the apical surface to exhibit increased delivery to the basolateral surface. We suggest that syntaxins may play a role in determining the specificity of membrane targeting by permitting fusion with only certain target membranes.

Animals↗
PubMed2004

Beta1-integrin orients epithelial polarity via Rac1 and laminin.

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.

Animals↗
PubMed2004

The mammalian retromer regulates transcytosis of the polymeric immunoglobulin receptor.

Epithelial cells have separate apical and basolateral plasma membrane domains with distinct compositions. After delivery to one surface, proteins can be endocytosed and then recycled, degraded or transcytosed to the opposite surface. Proper sorting into the transcytotic pathway is essential for maintaining polarity, as most proteins are endocytosed many times during their lifespan. The polymeric immunoglobulin receptor (pIgR) transcytoses polymeric IgA (pIgA) from the basolateral to the apical surface of epithelial cells and hepatocytes. However, the molecular machinery that controls polarized sorting of pIgR-pIgA and other receptors is only partially understood. The retromer is a multimeric protein complex, originally described in yeast, which mediates intracellular sorting of Vps10p, a receptor that transports vacuolar enzymes. The yeast retromer contains two sub-complexes. One includes the Vps5p and Vps17p subunits, which provide mechanical force for vesicle budding. The other is the Vps35p-Vps29p-Vps26p subcomplex, which provides cargo specificity. The mammalian retromer binds to the mannose 6-phosphate receptor, which sorts lysosomal enzymes from the trans-Golgi network to the lysosomal pathway. Here, we show a function for the mammalian Vps35-Vps29-Vps26 retromer subcomplex in promoting pIgR-pIgA transcytosis.

Adenoviridae↗
PubMed2004

ERK and MMPs sequentially regulate distinct stages of epithelial tubule development.

Epithelial cells undergo tubulogenesis in response to morphogens such as hepatocyte growth factor (HGF). To organize into tubules, cells must execute a complex series of morphogenetic events; however, the mechanisms that underlie the timing and sequence of these events are poorly understood. Here, we show that downstream effectors of HGF coordinately regulate successive stages of tubulogenesis. Activation of extracellular-regulated kinase (ERK) is necessary and sufficient for the initial stage, during which cells depolarize and migrate. ERK becomes dispensable for the latter stage, during which cells repolarize and differentiate. Conversely, the activity of matrix metalloproteases (MMPs) is essential for the late stage but not the initial stage. Thus, ERK and MMPs define two regulatory subprograms that act in sequence. By inducing these reciprocal signals, HGF directs the morphogenetic progression of tubule development.

Animals↗
PubMed2003

Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity.

Hepatocyte growth factor (HGF) induces mitogenesis, motogenesis, and tubulogenesis of cultured Madin-Darby canine kidney (MDCK) epithelial cells. We report that in addition to these effects HGF stimulates morphogenesis of tight, polarized MDCK cell monolayers into pseudostratified layers without loss of tight junction (TJ) functional integrity. We tested TJ functional integrity during formation of pseudostratified layers. In response to HGF, the TJ marker ZO-1 remained in morphologically complete rings and functional barriers to paracellular diffusion of ruthenium red were maintained in pseudostratified layers. Transepithelial resistance (TER) increased transiently two- to threefold during the morphogenetic transition from monolayers to pseudostratified layers and then declined to baseline levels once pseudostratified layers were formed. In MDCK cells expressing the trk/met chimera, both HGF and NGF at concentrations of 2.5 ng/ml induced scattering. However, 2.5 ng/ml HGF did not affect TER. The peak effect of HGF on TER was at a concentration of 100 ng/ml. In contrast, NGF at concentrations as high as 25 microg/ml had no effect on TER or pseudostratified layer morphogenesis of trk/met-expressing cultures. These results suggest that altered presentation of the stimulus, such as through HGF interaction with low-affinity sites, may change the downstream signaling response. In addition, our results demonstrate that HGF stimulates pseudostratified layer morphogenesis while inducing an increase in TER and maintaining the overall tightness of the epithelial layer. Stimulation of epithelial cell movements by HGF without loss of functional TJs may be important for maintaining epithelial integrity during morphogenetic events such as formation of pseudostratified epithelia, organ regeneration, and tissue repair.

Animals↗
PubMed2003

Pak1 and PIX regulate contact inhibition during epithelial wound healing.

Wound healing in epithelia requires coordinated cell migration and proliferation regulated by signaling mechanisms that are poorly understood. Here we show that epithelial cells expressing constitutively active or kinase-dead mutants of the Rac/Cdc42 effector Pak1 fail to undergo growth arrest upon wound closure. Strikingly, this phenotype is only observed when the Pak1 kinase mutants are expressed in cells possessing a free lateral surface, i.e. one that is not engaged in contact with neighboring cells. The Pak1 kinase mutants perturb contact inhibition by a mechanism that depends on the Pak-interacting Rac-GEF PIX. In control cells, endogenous activated Pak and PIX translocate from focal complexes to cell-cell contacts during wound closure. This process is abrogated in cells expressing Pak1 kinase mutants. In contrast, Pak1 mutants rendered defective in PIX binding do not impede translocation of activated Pak and PIX, and exhibit normal wound healing. Thus, recruitment of activated Pak and PIX to cell-cell contacts is pivotal to transduction of growth-inhibitory signals from neighboring cells in epithelial wound healing.

Animals↗
PubMed2003

An ecdysone and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling.

Regulated expression systems are invaluable for studying gene function, offer advantages of dosage-dependent and temporally defined gene expression, and limit possible clonal variation when toxic or pleiotropic genes are overexpressed. Previously, establishment of inducible expression systems, such as tetracycline- and ecdysone-inducible systems, required assessment of the inducible characteristics of individual clones by tedious luciferase assays. Taking advantage of a green fluorescent protein (GFP) reporter controlled by tetracycline- or ecdysone-responsive element and fluorescence-activated cell sorting, we propose a simple and efficient strategy to select highly inducible cell lines according to their fluorescence profiles after transiently transfecting the candidate cell pools with a surrogate GFP reporter. We have demonstrated that tetracycline- and ecdysone-inducible systems could be set up in Madin-Darby canine kidney and HEK-293 cells by employing this selection scheme. Importantly, this dual regulatory expression system is applied in studying the complex interplay between two Ras-related small GTPases, Cdc42 and Rac1, on detachment-induced apoptosis. Furthermore, establishment of two tightly regulated expression systems in one target cell line could be of great advantage for dissecting small GTPase Rac1-transduced signaling pathways by using global gene expression approaches such as proteomic assays.

Animals↗
PubMed2003

The exocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum.

We previously showed that the exocyst complex specifically affected the synthesis and delivery of secretory and basolateral plasma membrane proteins. Significantly, the entire spectrum of secreted proteins was increased when the hSec10 (human Sec10) component of the exocyst complex was overexpressed, suggestive of post-transcriptional regulation (Lipschutz, J. H., Guo, W., O'Brien, L. E., Nguyen, Y. H., Novick, P., and Mostov, K. E. (2000) Mol. Biol. Cell 11, 4259-4275). Here, using an exogenously transfected basolateral protein, the polymeric immunoglobulin receptor (pIgR), and a secretory protein, gp80, we show that pIgR and gp80 protein synthesis and delivery are increased in cells overexpressing Sec10 despite the fact that mRNA levels are unchanged, which is highly indicative of post-transcriptional regulation. To test specificity, we also examined the synthesis and delivery of an exogenous apical protein, CNT1 (concentrative nucleoside transporter 1), and found no increase in CNT1 protein synthesis, delivery, or mRNA levels in cells overexpressing Sec10. Sec10-GFP-overexpressing cell lines were created, and staining was seen in the endoplasmic reticulum. It was demonstrated previously in yeast that high levels of expression of SEB1, the Sec61beta homologue, suppressed sec15-1, an exocyst mutant (Toikkanen, J., Gatti, E., Takei, K., Saloheimo, M., Olkkonen, V. M., Soderlund, H., De Camilli, P., and Keranen, S. (1996) Yeast 12, 425-438). Sec61beta is a member of the Sec61 heterotrimer, which is the main component of the endoplasmic reticulum translocon. By co-immunoprecipitation we show that Sec10, which forms an exocyst subcomplex with Sec15, specifically associates with the Sec61beta component of the translocon and that Sec10 overexpression increases the association of other exocyst complex members with Sec61beta. Proteosome inhibition does not appear to be the mechanism by which increased protein synthesis occurs in the face of equivalent amounts of mRNA. Although the exact mechanism remains to be elucidated, the exocyst/Sec61beta interaction represents an important link between the cellular membrane trafficking and protein synthetic machinery.

Animals↗
PubMed2003

Role of rab proteins in epithelial membrane traffic.

Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such may define functional organelle identity. Most rabs are ubiquitously expressed. However, some rabs are preferentially expressed in epithelial cells where they appear intimately associated with the epithelial-specific transcytotic pathway and/or tight junctions. This review discusses the role of rabs in epithelial membrane transport.

Animals↗
PubMed2003

Epithelial polarity and tubulogenesis in vitro.

The most fundamental type of organization of cells in metazoa is that of epithelia, which comprise sheets of adherent cells that divide the organism into topologically and physiologically distinct spaces. Some epithelial cells cover the outside of the organism; these often form multiple layers, such as in skin. Other epithelial cells form monolayers that line internal organs, and yet others form tubes that infiltrate the whole organism, carrying liquids and gases containing nutrients, waste and other materials. These tubes can form elaborate networks in the lung, kidney, reproductive passages and vasculature tree, as well as the many glands branching from the digestive system such as the liver, pancreas and salivary glands. In vitro systems can be used to study tube formation and might help to define common principles underlying the formation of diverse types of tubular organ.

Animals↗
PubMed2003

Hepatocyte growth factor switches orientation of polarity and mode of movement during morphogenesis of multicellular epithelial structures.

Epithelial cells form monolayers of polarized cells with apical and basolateral surfaces. Madin-Darby canine kidney epithelial cells transiently lose their apico-basolateral polarity and become motile by treatment with hepatocyte growth factor (HGF), which causes the monolayer to remodel into tubules. HGF induces cells to produce basolateral extensions. Cells then migrate out of the monolayer to produce chains of cells, which go on to form tubules. Herein, we have analyzed the molecular mechanisms underlying the production of extensions and chains. We find that cells switch from an apico-basolateral polarization in the extension stage to a migratory cell polarization when in chains. Extension formation requires phosphatidyl-inositol 3-kinase activity, whereas Rho kinase controls their number and length. Microtubule dynamics and cell division are required for the formation of chains, but not for extension formation. Cells in the monolayer divide with their spindle axis parallel to the monolayer. HGF causes the spindle axis to undergo a variable "seesaw" motion, so that a daughter cells can apparently leave the monolayer to initiate a chain. Our results demonstrate the power of direct observation in investigating how individual cell behaviors, such as polarization, movement, and division are coordinated in the very complex process of producing multicellular structures.

Actins↗
PubMed2002

Exocytosis: the many masters of the exocyst.

The exocyst is a conserved eight-subunit complex involved in the docking of exocytic vesicles. The exocyst has now been identified as an effector for five small GTPases, including Sec4, Rho1, Rho3, Cdc42 and, most recently, RalA.

Animals↗