Our research objectives are to understand cellular mechanisms involved
in development and maintenance of cell polarity. Recent studies indicate
that development of epithelial cell polarity is a multistage process
requiring instructive extracellular cues (eg. cell-cell and cell-substratum
contact) and reorganization of proteins in the cytoplasm and on the
plasma membrane. Once established, polarity is maintained by targeting
and retention of proteins to functionally distinct apical and basal-lateral
plasma membrane domains.
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Last Modified: Monday, 31-Jan-2011 14:04:10 PST
|Determining the role of Nek2 in regulating beta-catenin function
b -catenin is a multifunctional protein with critical roles in cadherin mediated cell-cell adhesion, Wnt-induced transcriptional activation of genes essential for differentiation and bipolar spindle formation during cell division. b -catenins role(s) in cell-cell adhesion and Wnt-signaling has been extensively studied. In contrast, the mechanism(s) for how b -catenin regulates bipolar spindle formation is poorly understood. The centrosomal NIMA-related protein kinase 2 (Nek2) binds and phosphorylates b -catenin and stimulates centrosome disjunction. Nek2, like b -catenin, is required for bipolar spindle formation. Abnormalities in bipolar spindle formation, can contribute to disease states: extra centrosomes result in multipolar spindles while the failure of centrosomes to separate induces monopolar spindles. Biochemical and cell biological approaches will be used to further investigate the mechanism of Nek2 regulation on b -catenin stability and function in cell division, signaling and adhesion.
APC regulation of microtubules
Microtubule interactions with the cell cortex are important for many fundamental cellular processes, including epithelial cell polarization, membrane extension and retraction during cell motility, and vesicle trafficking to the plasma membrane. One protein that appears to mediate microtubule-membrane interactions is adenomatous polyposis coli (APC). APC is a large scaffolding protein with many binding partners that together stimulate microtubule assembly and stabilization, however, the molecular mechanism(s) by which they do so remain unclear. To better understand how the APC multi-protein complex regulates microtubules, we are using a rigorous biochemical approach to dissect protein-protein interactions in the APC multi-protein complex and determine effects of APC and APC binding proteins on microtubule dynamics and organization. In addition, we are examining how disruption of the APC multi-protein complex in cells effects microtubule dependent processes such as cell migration.
|-Elizabeth S. Harris
|Septin function in cell development and signaling in mamallian cells
Septins are a novel family of filamentous GTPases associated with cell membranes and the cytoskeleton, and play essential roles in cell division and cellular morphogenesis. In the budding yeast Saccharomyces cerevisiae, septins are localized to the neck between daughter and mother cells and function as scaffolds and diffusion barriers to localize many proteins required for bud site selection, cell-cycle progress and cell division. However, the function of septins in mammalian cells is poorly understood. I am interested in the septins' function in cell development and signaling in interphase cells and cell cycle progression and division in mitotic cells. Our results show that septins are essential for post-Golgi vesicle trafficking during epithelial biogenesis and we identify a drug, Forchlorfenuron which affects septin organization and dynamics in mammalian cells. Currently, I am working on the mechanisms by which septins regulate certain cell development and signaling events and cell division.
|Deciphering the molecular and cellular activation of Rac1 at initial cell-cell contacts
Epithelial cell adhesion is a dynamic process that is fundamental to development and the establishment of cell polarity. Cell-cell contacts are established by the homophilic interaction of E-cadherin cytoplasmic domains and become stabilized by interaction of E-cadherin with the cytoskeleton. Although many molecules have been identified during the process of cell adhesion, the mechanisms involved in initiating, strengthening and stabilizing these contacts remains elusive. Research has demonstrated that the Rho GTPase, Rac1 is activated upon E-cadherin adhesion and localized to sites of initial cell-cell contacts. These levels subsequently decrease as cell-cell contacts become more stable. Previous work on Rac1/E-cadherin at localized areas of cell-cell contact indicate that Rac1 is localized to sites of cell contact, but may or may not affect E-cadherin accumulation at cell-cell contacts. My project involves further examination of the molecular and cellular mechanisms governing activation and inactivation of Rac1 at de novo sites of cell adhesion and the potential role of E-cadherin during this process.
|Crosstalk between Cell-Matrix and Cell-Cell Adhesion
The balance between cell-matrix and cell-cell adhesions is of foremost importance for tissue development and homeostasis. The deregulation of the integration of either or both of these adhesion cues can lead to pathological cell behaviours such as metastatic cell migration in cancer. To understand the mechanisms underlying this balance, we study cell behaviours as a function of controlled changes in cell-cell and/or cell-matrix adhesion cues. We use an original surface functionalization approach to expose isolated epithelial cells to micro patterns of cell-matrix or cell-cell adhesion proteins (collagen and E-cadherin, respectively) in close apposition. We show that cellular E-cadherins and focal adhesion proteins segregate between cadherin and collagen patterns, respectively. Simultaneously, cell motility is biased by pattern shape and lamellipodial activity dampened on cadherin patterns. This system provides the basis for a systematic analysis of signalling proteins involved in cell-cell and cell-matrix adhesion crosstalk.
|- Nicolas Borghi
|Allosteric regulation of alpha-catenin
|The adherens junction is dependent upon homophilic binding between cadherins from opposing cells and is mediated intracellularly through binding to b-catenin, which in turn binds a-catenin. While it was previously believed that cadherins are directly linked to the actin cytoskeleton through a-catenin, recent data from our lab show that a-catenin cannot simultaneously bind b-catenin and actin filaments in vitro; a-catenin monomer preferentially binds the cadherin-b-catenin complex whereas the homodimer (likely formed during cell-cell adhesion) preferentially binds actin filaments. However, the existence of a monomer-dimer transition and its effects on actin dynamics have not been demonstrated in vivo. I aim to investigate the allosteric regulation of a-catenin by inducing a-catenin dimerization and testing the effects on membrane dynamics, migration, and adhesion. To induce a-catenin dimerization independent of cell-cell adhesion, we recruit a-catenin to the membrane to mimic the increased local concentration of a-catenin during cell-cell adhesion.
|- Jacqueline Benjamin
|APC at mother centriole
|Centrosomes nucleate and organize MTs into arrays important for different stages of the cell cycle. The mother centriole anchors and focuses MTs at the different stages of the cell cycle. Recently, proteins have been found that specifically localize to the mother centriole throughout the cell cycle. It is believed that these proteins function in centriole duplication and microtubule organization. We have discovered that localization of APC and EB1 at centrosomes is specific to the mother centriole and is independent of cytoplasmic microtubules (movie). We will do molecular biological and cell biological studies to further investigate the function of APC and EB1 at the mother centriole.
|- Shirin Bahmanyar
Mechanisms of initial cell polarization and TGN protein sorting
Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We are studying when and how initial cell polarization occurs in MDCK cells and found that polarization occurs shortly after initial cell-cell contact: The lateral targeting patch (microtubules, Exocyst, SNAREs) is established rapidly following adhesion and newly synthesized basolateral proteins are directly targeted from the Trans-Golgi Network to the site of initial cell-cell adhesion. We are currently interested in how the lateral targeting patch is assembled and how basolateral proteins are sorted into transport vesicles at the TGN and how they arrive at the lateral targeting patch.
|- Lene Nejsum
Regulative properties of Alpha-catenin in zebrafish development
Adherens junctions (AJ) play key roles in cell-cell adhesion, cell migration and cell signaling in a wide variety of biological processes. AJs are dynamic structures composed of cadherin, catenins and other proteins that locally regulate the actin cytoskeleton. An important unanswered question is how these structures are regulated during complex cell rearrangements when cell-cell contacts are maintained as cells migrate. We combine classical zebrafish methodologies with cell biochemical approaches and high resolution live cell imaging to define mechanisms underlying epithelial cell rearrangements driven by remodeling and stabilization of AJs in gastrulation. Two components of the AJ, E-cadherin and the beta- catenin, are required, but the function of alpha-catenin is not known. Significantly, the Nelson’s laboratory identified new functions for alpha-catenin that may be particularly important during cell rearrangements, and we seek to test these functions in vivo using zebrafish gastrulation as a model of dynamic cell adhesion and migration.
|- Antonino Schepis
Mechanisms of APC regulation during cell extension
Adenomatous polyposis coli (APC) is a microtubule (MT) binding protein that clusters at sites of MT-cortex interactions during membrane extension in several cell types. This APC-MT interaction is known to be regulated by phosphorylation. However, it is still unclear how APC phosphorylation changes during the cell extension process. We are studying how APC is regulated during cell extension formation in PC12 cells induced by nerve growth factor (NGF), which involves the activation of several protein kinase signaling cascades and MT reorganization. Using a pharmacological approach we are identifying signaling cascades affecting APC clustering and phosphorylation states. Our study also aims to identify important residues on APC that mediate its regulation via these signaling cascades and how APC phosphorylation can affect MT dynamics and cell extension.
|- Hector Y. Caro-Gonzalez
|APC and microtubule dynamics in cell morphogenesis
|We are examining how microtubule-associated APC clusters at the cortex locally regulate cytoskeletal re-organization during membrane outgrowth and contact formation, and how APC clusters are in turn regulated by Wnt signaling and signals from the cadherin complex at the cell surface: 1). Identify and characterize proteins in cortical APC clusters, and determine their role in APC cluster formation; 2). Characterize the function of membrane-attached APC clusters in microtubule assembly and formation of membrane extensions; and 3). Identify and characterize signaling pathways from Wnt and cadherins that regulate APC cluster function. The significance of these proposed studies is they will provide novel understanding of how cytoskeletal restructuring is controlled in locally defined, subcellular domains in order to specify membrane dynamics and cell-cell contacts in response to extracellular signals.
|- Angela Barth and Kathy Siemers
|Cadherins on the Brain
|Synapses are highly specialized, asymmetric cell-cell junctions
that relay information from neurons to target cells. Cadherins
and associated catenins modulate synapse formation, maturation and
function; however, the mechanisms by which they do so remain unclear.
In conjunction with the Garner lab here at Stanford, we are studying
the role of cadherins and catenins in synapse formation and function
in cultured neurons. We
are examining the dynamics of cadherin/catenin synaptic localization using fluorescently-tagged proteins and live cell imaging. We are also investigating molecular events downstream of N-cadherin engagement during
early stages of synapse formation. Finally, we are exploring the role of a-catenin in regulating actin dynamics within dendritic spines.
|- Adam Kwiatkowski
|The role of cadherin complexes in cell adhesion and motility
|During normal development and cancer, epithelia undergo shape transformations that range from cohesive sheet migration, cell position changes within tissues, to individual cell migration after dissociation. Such cell movements require coordinated cross-talk between cadherins, involved in cell-cell interactions, and integrin adhesion receptors, involved in cell migration. How different cadherins contribute to the balance between adhesion and motility of cells within tissues is unknown. We use functionalized micro-patterned surfaces comprised of alternating stripes of extracellular matrix and cadherins to analyze changes in individual cell behaviors. We suggest that desmosomal cadherin adhesion is rigid, resulting in reduced cell migration rate and shear-resistant epithelium. Conversely, E-cadherin junctions provide fluid contacts that can maintain tissue cohesion while allowing movement of cells past each other and en masse sheet migration.
|- Molly Lowndes
|p120-catenin: A master regulator of cell-cell adhesion
|The Adherens Junction is known to initiate the formation of cell-cell contacts and is comprised of the proteins E-cadherin, p120-catenin, β-catenin, and α-catenin. It is proposed that the p120/E-cadherin interaction stabilizes E-cadherin at the plasma membrane. Uncoupling of the interaction results in endocytosis of E-cadherin which may then be degraded or recycled back to the membrane. Hakai, an E3 ligase, has been identified to ubiquitinate E-cadherin resulting in endocytosis. The mechanism in which E-cadherin is degraded still remains to be determined. p120 may also play a role in the dynamics involved in de novo cell-cell contact formation. p120 is known to regulate the actin cytoskeleton in motile cells possibly through interactions with Rho family GTPases. It is not known if these interactions occur during the initial formation of cell contacts. Our lab has characterized the dynamics of the initial cell adhesion process in relation to localized zones of E-cadherin accumulation and localized zones of Rac and Rho activity present in the forming contact. It has yet to be determined how these zones of activity are regulated.
|- Andrea Hartsock
|The cellular slime mold Dictyostelium discoideum exists under normal conditions as single-celled amoebae that prey on bacteria in forest soils. However, when starved, the cells aggregate and undergo a multicellular developmental process involving 10,000 100,000 cells. I have identified a protein in Dictyostelium which appears based on sequence analysis to be a common ancestor of alpha-catenin, which is involved in adherens junction formation, and vinculin, which is present at focal adhesions. I am using biochemistry, genetics, and imaging approaches to characterize this protein and determine whether it plays a role in cell adhesion. I also hope to understand its role in multicellular development. My work will shed light on the evolution of multicellularity and cell adhesion, and also provide insight into the role that cell adhesion plays in morphogenetic processes.
|- Daniel Dickinson