Classical cadherins accumulate at cell-cell contacts as a characteristic response to productive adhesive ligation. Such local accumulation of cadherins is a developmentally regulated process that supports cell adhesiveness and cell-cell cohesion. Yet the molecular effectors responsible for cadherin accumulation remain incompletely understood. We now report that Myosin 2 is critical for cells to concentrate E-cadherin at cell-cell contacts. Myosin 2 is found at cadherin-based cell-cell contacts and its recruitment requires E-cadherin activity. Indeed, both Myosin 2 recruitment and its activation were stimulated by E-cadherin homophilic ligation alone. Inhibition of Myosin 2 activity by blebbistatin or ML-7 rapidly impaired the ability of cells to concentrate E-cadherin at adhesive contacts, accompanied by decreased cadherin-based cell adhesiveness. The total surface expression of cadherins was unaffected, suggesting that Myosin 2 principally regulates the regional distribution of cadherins at the cell surface. The recruitment of Myosin 2 to cadherin contacts, and its activation, required Rho kinase; furthermore, inhibition of Rho kinase signaling effectively phenocopied the effects of Myosin 2 inhibition. We propose that Myosin 2 is a key effector of Rho-Rho kinase signaling that regulates cell-cell adhesion by determining the ability of cells to concentrate cadherins at contacts in response to homophilic ligation.

Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen 1 protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of the trans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process. INTRODUCTIONInsulin stimulates glucose uptake in muscle and fat cells by triggering translocation of the glucose transporter GLUT4 from an intracellular compartment to the cell surface (Bryant et al., 2002). The intracellular localization of GLUT4 in adipocytes includes the endosomal system, trans-Golgi network (TGN), cytoplasmic tubulovesicular elements and the cell surface, suggesting a complex intracellular trafficking itinerary (Slot et al., 1991b;Martin et al., 2000a). Although previous studies have indicated a role for endosomes in GLUT4 trafficking (Slot et al., 1991b;Livingstone et al., 1996) the precise role of the TGN is not clear. Several observations suggest an important role for the TGN in GLUT4 trafficking. First, there is a significant amount of GLUT4 in the TGN area in insulin-responsive cells (Slot et al., 1991a,b;Ralston and Ploug, 1996;Wang et al., 1996;Slot et al., 1997;Ploug et al., 1998;Martin et al., 2000a). Second, ϳ60% of the entire GLUT4 pool is localized to atrial natriuretic factorcontaining secretory granules in atrial cardiomyocytes and this seems to be due to recycling of GLUT4 through the TGN area (Slot et al., 1997). Third, there is significant overlap between GLUT4 and proteins known to traffic between the TGN and endosomes, including the cation-dependent mannose 6-phosphate receptor (Martin et al., 2000a), the cation-independent mannose 6-phosphate receptor (Kandror and Pilch, 1996), and adaptor-related protein complex-1 (Gillingham et al., 1999;Martin et al., 2000b These data suggest that the TGN contributes to the trafficking of GLUT4, adding a further layer of complexity to understanding the insulin-regulated movement ...

In contrast to the well-established relationship between cadherins and the actin cytoskeleton, the potential link between cadherins and microtubules (MTs) has been less extensively investigated. We now identify a pool of MTs that extend radially into cell-cell contacts and are inhibited by manoeuvres that block the dynamic activity of MT plus-ends (e.g. in the presence of low concentrations of nocodazole and following expression of a CLIP-170 mutant). Blocking dynamic MTs perturbed the ability of cells to concentrate and accumulate E-cadherin at cell-cell contacts, as assessed both by quantitative immunofluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis, but did not affect either transport of E-cadherin to the plasma membrane or the amount of E-cadherin expressed at the cell surface. This indicated that dynamic MTs allow cells to concentrate E-cadherin at cell-cell contacts by regulating the regional distribution of E-cadherin once it reaches the cell surface. Importantly, dynamic MTs were necessary for myosin II to accumulate and be activated at cadherin adhesive contacts, a mechanism that supports the focal accumulation of E-cadherin. We propose that this population of MTs represents a novel form of cadherin-MT cooperation, where cadherin adhesions recruit dynamic MTs that, in turn, support the local concentration of cadherin molecules by regulating myosin II activity at cell-cell contacts.

Inositol phospholipids have been implicated in almost all aspects of cellular physiology including spatiotemporal regulation of cellular signaling, acquisition of cellular polarity, specification of membrane identity, cytoskeletal dynamics, and regulation of cellular adhesion, motility, and cytokinesis. In this review, we examine the critical role phosphoinositides play in these processes to execute the establishment and maintenance of cellular architecture. Epithelial tissues perform essential barrier and transport functions in almost all major organs. Key to their development and function is the establishment of epithelial cell polarity. We place a special emphasis on highlighting recent studies demonstrating phosphoinositide regulation of epithelial cell polarity and how individual cells use phosphoinositides to further organize into epithelial tissues.P hosphoinositides (PIs) are essential components of cellular membranes in eukaryotes. Though these specialized lipids comprise less than 1% of the cellular lipid cohort, they play key roles in many fundamental biological processes (Di Paolo and De Camilli 2006;Saarikangas et al. 2010). PIs possess such far ranging roles by serving as specialized membrane docking sites for effectors of numerous cellular signal transduction cascades. PIs also serve as precursors of lipid second messengers. They are concentrated on the cytosolic face of cellular membranes (Fig. 1A) and rapidly diffuse within the plane of the membrane. Reversible phosphorylation of the myo-inositol head group of phosphatidylinositol (PtdIns) at positions 3, 4, and 5 (Fig. 1B) gives rise to the seven PI isoforms identified in eukaryotic cells. PtdIns(4)P and PtdIns(4,5)P 2 are constitutively present in membranes and comprise the largest pool of cellular PIs, whereas PtdIns(3,4,5)P 3 is essentially undetectable in most types of unstimulated cells (Lemmon and Ferguson 2000;Saarikangas et al. 2010).The spatiotemporally regulated production and turnover of phosphoinositides is crucial for localized PI signaling and function. Numerous phosphatidylinositol kinases and phosphatases are involved in regulating the metabolism of the various PI isoforms (Fig. 1)

The structural organisation of pancreatic β-cells in the islets of Langerhans is relatively unknown. Here, using three-dimensional (3D) two-photon, 3D confocal and 3D block-face serial electron microscopy, we demonstrate a consistent in situ polarisation of β-cells and define three distinct cell surface domains. An apical domain located at the vascular apogee of β-cells, defined by the location of PAR-3 (also known as PARD3) and ZO-1 (also known as TJP1), delineates an extracellular space into which adjacent β-cells project their primary cilia. A separate lateral domain, is enriched in scribble and Dlg, and colocalises with E-cadherin and GLUT2 (also known as SLC2A2). Finally, a distinct basal domain, where the β-cells contact the islet vasculature, is enriched in synaptic scaffold proteins such as liprin. This 3D analysis of β-cells within intact islets, and the definition of distinct domains, provides new insights into understanding β-cell structure and function.