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Adaptation of cell shape and polarization through the formation and retraction of cellular protrusions requires balancing of endocytosis and exocytosis combined with fine-tuning of the local activity of small GTPases like Rab8. Here, we show that endocytic turnover of the plasma membrane at protrusions is directly coupled to surface removal and inactivation of Rab8. Removal is induced by reduced membrane tension and mediated by the GTPase regulator associated with focal adhesion kinase-1 (GRAF1, also known as ARHGAP26), a regulator of clathrin-independent endocytosis. GRAF1-depleted cells were deficient in multi-directional spreading and displayed elevated levels of GTP-loaded Rab8, which was accumulated at the tips of static protrusions. Furthermore, GRAF1 depletion impaired lumen formation and spindle orientation in a 3D cell culture system, indicating that GRAF1 activity regulates polarity establishment. Our data suggest that GRAF1-mediated removal of Rab8 from the cell surface restricts its activity during protrusion formation, thereby facilitating dynamic adjustment of the polarity axis. [ABSTRACT FROM AUTHOR]

The apicobasal polarity of enterocytes determines where the brush border membrane (apical membrane) will form, but how this apical membrane faces the lumen is not well understood. The electrical signal across the epithelium could serve as a coordinating cue, orienting and polarizing enterocytes. Here, we show that applying a physiological electric field to intestinal epithelial cells, to mimic the natural electric field created by the transepithelial potential difference, polarized phosphorylation of the actin-binding protein ezrin, increased expression of intestinal alkaline phosphatase (ALPI, a differentiation marker) and remodeled the actin cytoskeleton selectively on the cathode side. In addition, an applied electric field also activated ERK1/2 and LKB1 (also known as STK11), key molecules in apical membrane formation. Disruption of the tyrosine protein kinase transmembrane receptor Ror2 suppressed activation of ERK1/2 and LKB1 significantly, and subsequently inhibited apical membrane formation in enterocytes. Our findings indicate that the endogenous electric field created by the transepithelial potential difference might act as an essential coordinating signal for apical membrane formation at a tissue level, through activation of LKB1 mediated by Ror2 -- ERK signaling. [ABSTRACT FROM AUTHOR]

Cell polarization relies on small GTPases, such as Cdc42, which can break symmetry through self-organizing principles, and landmarks that define the axis of polarity. In fission yeast, microtubules deliver the Tea1-Tea4 complex to mark cell poles for growth, but how this complex activates Cdc42 is unknown. Here, we show that ectopic targeting of Tea4 to cell sides promotes the local activation of Cdc42 and cell growth. This activity requires that Tea4 binds the type I phosphatase (PP1) catalytic subunit Dis2 or Sds21, and ectopic targeting of either catalytic subunit is similarly instructive for growth. The Cdc42 guanine-nucleotide-exchange factor Gef1 and the GTPase-activating protein Rga4 are required for Tea4-PP1-dependent ectopic growth. Gef1 is recruited to ectopic Tea4 and Dis2 locations to promote Cdc42 activation. By contrast, Rga4 is locally excluded by Tea4, and its forced colocalization with Tea4 blocks ectopic growth, indicating that Rga4 must be present, but at sites distinct from Tea4. Thus, a Tea4-PP1 landmark promotes local Cdc42 activation and growth both through Cdc42 GEF recruitment and by creating a local trough in a Cdc42 GAP. [ABSTRACT FROM AUTHOR]

Adhesive interactions of cadherins induce crosstalk between adhesion complexes and the actin cytoskeleton, allowing strengthening of adhesions and cytoskeletal organization. The underlying mechanisms are not completely understood, and microtubules (MTs) might be involved, as for integrin-mediated cell -- extracellular-matrix adhesions. Therefore, we investigated the relationship between N-cadherin and MTs by analyzing the influence of N-cadherin engagement on MT distribution and dynamics. MTs progressed less, with a lower elongation rate, towards cadherin adhesions than towards focal adhesions. Increased actin treadmilling and the presence of an actomyosin contractile belt, suggested that actin relays inhibitory signals from cadherin adhesions to MTs. The reduced rate of MT elongation, associated with reduced recruitment of end-binding (EB) proteins to plus ends, was alleviated by expression of truncated N-cadherin, but was only moderately affected when actomyosin was disrupted. By contrast, destabilizing actomyosin fibers allowed MTs to enter the adhesion area, suggesting that tangential actin bundles impede MT growth independently of MT dynamics. Blocking MT penetration into the adhesion area strengthened cadherin adhesions. Taken together, these results establish a crosstalk between N-cadherin, F-actin and MTs. The opposing effects of cadherin and integrin engagement on actin organization and MT distribution might induce bias of the MT network during cell polarization. [ABSTRACT FROM AUTHOR]

Podosomes are actin-rich adhesion and invasion structures. Especially in macrophages, podosomes exist in two subpopulations, large precursors at the cell periphery and smaller podosomes (successors) in the cell interior. To date, the mechanisms that differentially regulate these subpopulations are largely unknown. Here, we show that the membrane-associated protein supervillin localizes preferentially to successor podosomes and becomes enriched at precursors immediately before their dissolution. Consistently, podosome numbers are inversely correlated with supervillin protein levels. Using deletion constructs, we find that the myosin II regulatory Nterminus of supervillin [SV(1-174)] is crucial for these effects. Phosphorylated myosin light chain (pMLC) localizes at supervillinpositive podosomes, and time-lapse analyses show that enrichment of GFP-supervillin at podosomes coincides with their coupling to contractile myosin-IIA-positive cables. We also show that supervillin binds only to activated myosin IIA, and a dysregulated N-terminal construct [SV(1-830)] enhances pMLC levels at podosomes. Thus, preferential recruitment of supervillin to podosome subpopulations might both require and induce actomyosin contractility. Using siRNA and pharmacological inhibition, we demonstrate that supervillin and myosin IIA cooperate to regulate podosome lifetime, podosomal matrix degradation and cell polarization. In sum, we show here that podosome subpopulations differ in their molecular composition and identify supervillin, in cooperation with myosin IIA, as a crucial factor in the regulation of podosome turnover and function. [ABSTRACT FROM AUTHOR]

The position of the nucleus is regulated in different developmental stages and cellular events. During polarization, the nucleus moves away from the future leading edge and this movement is required for proper cell migration. Nuclear movement requires the LINC complex components nesprin-2G and SUN2, which form transmembrane actin-associated nuclear (TAN) lines at the nuclear envelope. Here we show that the nuclear envelope protein Samp1 (NET5) is involved in nuclear movement during fibroblast polarization and migration. Moreover, we demonstrate that Samp1 is a component of TAN lines that contain nesprin-2G and SUN2. Finally, Samp1 associates with SUN2 and lamin A/C, and the presence of Samp1 at the nuclear envelope requires lamin A/C. These results support a role for Samp1 in the association between the LINC complex and lamins during nuclear movement. [ABSTRACT FROM AUTHOR]

Rac proteins are the only canonical Rho family GTPases in Dictyostelium, where they act as key regulators of the actin cytoskeleton. To monitor the dynamics of activated Rac1 in Dictyostelium cells, a fluorescent probe was developed that specifically binds to the GTPbound form of Rac1. The probe is based on the GTPase-binding domain (GBD) from PAK1 kinase, and was selected on the basis of yeast two-hybrid, GST pull-down and fluorescence resonance energy transfer assays. The PAK1 GBD localizes to leading edges of migrating cells and to endocytotic cups. Similarly to its role in vertebrates, activated Rac1 therefore appears to control de novo actin polymerization at protruding regions of the Dictyostelium cell. Additionally, we found that the IQGAP-related protein DGAP1, which sequesters active Rac1 into a quaternary complex with actin-binding proteins cortexillin I and cortexillin II, localizes to the trailing regions of migrating cells. Notably, PAK1 GBD and DGAP1, which both bind to Rac1-GTP, display mutually exclusive localizations in cell migration, phagocytosis and cytokinesis, and opposite dynamics of recruitment to the cell cortex upon stimulation with chemoattractants. Moreover, cortical localization of the PAK1 GBD depends on the integrity of the actin cytoskeleton, whereas cortical localization of DGAP1 does not. Taken together, these results imply that Rac1 GTPases play a dual role in regulation of cell motility and polarity in Dictyostelium. [ABSTRACT FROM AUTHOR]

Enucleation, the final step in terminal differentiation of mammalian red blood cells, is an essential process in which the nucleus surrounded by the plasma membrane is budded off from the erythroblast to form a reticulocyte. Most molecular events in enucleation remain unclear. Here we show that enucleation requires establishment of cell polarization that is regulated by the microtubule-dependent local activation of phosphoinositide 3-kinase (PI3K). When the nucleus becomes displaced to one side of the cell, actin becomes restricted to the other side, where dynamic cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a narrow constriction in the cell surface, forming a bud. The PI3K products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are highly localized at the cytoplasmic side of the plasma membrane. PI3K inhibition caused impaired cell polarization, leading to a severe delay in enucleation. Depolymerization of microtubules reduced PI3K activity, resulting in impaired cell polarization and enucleation. We propose that enucleation is regulated by microtubules and PI3K signaling in a manner mechanistically similar to directed cell locomotion. [ABSTRACT FROM AUTHOR]

In several migratory cells, the microtubule-organizing center (MTOC) is repositioned between the leading edge and nucleus, creating a polarized morphology. Although our understanding of polarization has progressed as a result of various scratch-wound and cell migration studies, variations in culture conditions required for such assays have prevented a unified understanding of the intricacies of MTOC and nucleus positioning that result in cell polarization. Here, we employ a new SMRT (for sparse, monolayer, round, triangular) analysis that uses a universal coordinate system based on cell centroid to examine the pathways regulating MTOC and nuclear positions in cells plated in a variety of conditions. We find that MTOC and nucleus positioning are crucially and independently affected by cell shape and confluence; MTOC off-centering correlates with the polarization of single cells; acto-myosin contractility and microtubule dynamics are required for single-cell polarization; and end binding protein 1 and light intermediate chain 1, but not Par3 and light intermediate chain 2, are required for single-cell polarization and directional cell motility. Using various cellular geometries and conditions, we implement a systematic and reproducible approach to identify regulators of MTOC and nucleus positioning that depend on extracellular guidance cues. [ABSTRACT FROM AUTHOR]

The dynamic rearrangement of cell-cell adhesion is one of the major physiological events in tissue development and tumor metastasis. Polarized ceil migration, another key event, is a tightly regulated process that occurs during tissue development, chemotaxis and wound healing. Rho-family small GTPases, especially Rac1 and Cdc42, play pivotal roles in these processes through one of their effectors, IQGAPI. Recent studies reveal that IQGAPI regulates cadherin-mediated cell-cell adhesion both positively and negatively. It captures and stabilizes microtubules through the microtubule-binding protein CLIP-170 near the cell cortex, leading to establishment of polarized cell morphology and directional cell migration. Furthermore, Rac1 and Cdc42 link the adenomatous polyposis coli (APC) protein to actin filaments through IQGAP1 at the leading edge and thereby regulate polarization and directional migration. [ABSTRACT FROM AUTHOR]

Two CEACAM1 isoforms with different cytoplasmic domains, CEACAM1-L and CEACAM1-S, are unequally distributed in polarized epithelial MDCK cells. CEACAM1-S is exclusively apical whereas CEACAM1-L occurs both in apical and lateral cell surfaces. Using confocal microscopy and CEACAM1-L mutants, we identified several amino acids in the cytoplasmic domain that were instrumental for the lateral localization. Tyr515, but not Tyr488, constituted a prominent lateral targeting signal. Pervanadate-stimulated Tyr phosphorylation induced rapid phosphatidylinositol 3-kinase-dependent disappearance of lateral CEACAM1-L, whereas staurosporine, a Ser/Thr kinase inhibitor, resulted in slower phosphatidylinositol 3-kinase-independent disappearance. Both drugs caused accumulation of CEACAM1-L in a late endosome/lysosome compartment. Colocalization studies of occludin, ZO-1, E-cadherin, βcatenin and desmoplakin indicated that laterally localized CEACAM1-L was present in adherens junctions but not in tight junctions or desmosomes. Overexpressed CEACAM1L did not affect the organization of tight junction or adherens junction proteins, but perturbed the arrangement of desmosomes. The abundance of desmosomes in the lateral cell surfaces decreased significantly and the submembraneous cytokeratin filaments became disorganized. The signal for desmosomal perturbance resided within amino acids 484-518 in the C-terminal part of the cytoplasmic domain, among which an intact Tyr515 was indispensable. [ABSTRACT FROM AUTHOR]

Multi-PDZ (PSD-95/Discs large/Zonula-occludens-1) domain proteins play a crucial role in the establishment and maintenance of cell polarization. The novel multi-PDZ domain protein FRMPD2 is a potential scaffolding protein consisting of an N-terminal KIND domain, a FERM domain and three PDZ domains. Here we show that FRMPD2 is localized in a polarized fashion in epithelial cells at the basolateral membrane and partially colocalizes with the tight-junction marker protein Zonula-occludens-1. Downregulation of FRMPD2 protein in Caco-2 cells is associated with an impairment of tight junction formation. We find that the FERM domain of FRMPD2 binds phosphatidylinositols and is sufficient for membrane localization. Moreover, we demonstrate that recruitment of FRMPD2 to cell-cell junctions is strictly E-cadherin-dependent, which is in line with our identification of catenin family proteins as binding partners for FRMPD2. We demonstrate that the FERM domain and binding of the PDZ2 domain to the armadillo protein p0071 are required for basolateral restriction of FRMPD2. Moreover, the PDZ2 domain of FRMPD2 is sufficient to partially redirect an apically localized protein to the basolateral membrane. Our results provide novel insights into the molecular function of FRMPD2 and into the targeting mechanism of peripheral membrane proteins in polarized epithelial cells. [ABSTRACT FROM AUTHOR]

Although cells migrate in a constrained 3D environment in vivo, in-vitro studies have mainly focused on the analysis of cells moving on 2D substrates. Under such conditions, the Golgi complex is always located towards the leading edge of the cell, suggesting that it is involved in the directional movement. However, several lines of evidence indicate that this location can vary depending on the cell type, the environment or the developmental processes. We have used micro contact printing (μCP) to study the migration of cells that have a geometrically constrained shape within a polarized phenotype. Cells migrating on micropatterned lines of fibronectin are polarized and migrate in the same direction. Under such conditions, the Golgi complex and the centrosome are located behind the nucleus. In addition, the Golgi complex is often displaced several micrometres away from the nucleus. Finally, we used the zebrafish lateral line primordium as an in-vivo model of cells migrating in a constrained environment and observe a similar localization of both the Golgi and the centrosome in the leading cells. We propose that the positioning of the Golgi complex and the centrosome depends on the geometrical constraints applied to the cell rather than on a precise migratory function in the leading region. [ABSTRACT FROM AUTHOR]

Oxysterol-binding protein (OSBP)-related protein 3 (ORP3) is highly expressed in epithelial, neuronal and hematopoietic cells, as well as in certain forms of cancer. We assessed the function of ORP3 in HEK293 cells and in human macrophages. We show that ORP3 interacts with R-Ras, a small GTPase regulating cell adhesion, spreading and migration. Gene silencing of ORP3 in HEK293 cells results in altered organization of the actin cytoskeleton, impaired cell-cell adhesion, enhanced cell spreading and an increase of ß1 integrin activity-effects similar to those of constitutively active R-Ras(38V). Overexpression of ORP3 leads to formation of polarized cell-surface protrusions, impaired cell spreading and decreased ß1 integrin activity. In primary macrophages, overexpression of ORP3 leads to the disappearance of podosomal structures and decreased phagocytotic uptake of latex beads, consistent with a role in actin regulation. ORP3 is phosphorylated when cells lose adhesive contacts, suggesting that it is subject to regulation by outside-in signals mediated by adhesion receptors. The present findings demonstrate a new function of ORP3 as part of the machinery that controls the actin cytoskeleton, cell polarity and cell adhesion. [ABSTRACT FROM AUTHOR]

How formation of the front and rear of a cell are coordinated during cell polarization in migrating cells is not well understood. Time-lapse microscopy of live primary chick embryo heart fibroblasts expressing GFP-actin show that, prior to cell polarization, polymerized actin in the cell body reorganizes to form oriented actin-filament bundles spanning the entire cell body. Within an average of 5 minutes of oriented actin bundles forming, localized cell-edge retraction initiates at either the side or at one end of the newly formed bundles and then elaborates around the nearest end of the bundles to form the cell rear, the first visual break in cell symmetry. Localized net protrusion occurs at the opposing end of the bundles to form the cell front and lags formation of the rear of the cell. Consequently, cells acquire full polarity and start to migrate in the direction of the long axis of the bundles, as previously documented for already migrating cells. When ADF/cofilin family protein activity or actin-filament disassembly is specifically blocked during cell polarization, reorganization of polymerized actin to form oriented actin-filament bundles in the cell body fails, and formation of the cell rear and front is inhibited. We conclude that formation of oriented actin-filament bundles in the cell body requires ADF/cofilin family proteins, and is an early event needed to coordinate the spatial location of the cell rear and front during fibroblast polarization. [ABSTRACT FROM AUTHOR]