Polarity Defects Research Articles

Arabidopsis mTERF6 is required for leaf patterning

To enhance our understanding of the roles of mitochondrial transcription termination factors (mTERFs) in plants, we have taken a reverse genetic approach in Arabidopsis thaliana. One of the mutants isolated carried a novel allele of the mTERF6 gene, which we named mterf6-5. mTERF6 is a chloroplast and mitochondrial localised protein required for the maturation of chloroplast isoleucine tRNA. The mterf6-5 plants are pale and exhibit markedly reduced growth, and altered leaf and chloroplast development. Our qRT-PCR analyses revealed mis-expression of several plastid, mitochondrial and nuclear genes in mterf6-5 plants. Synergistic phenotypes were observed in double mutant combinations of mterf6-5 with alleles of other mTERF genes as well as with scabra3-2, affected in the plastid RpoTp RNA polymerase; these observations suggest a functional relationship between mTERF6, other mTERFs and SCA3. The mterf6-5 mutation also enhanced the leaf dorsoventral polarity defects of the asymmetric leaves1-1 (as1-1) mutant, which resulted in radial leaves. This interaction seemed specific of the impaired mTERF6 function because mutations in the mTERF genes MDA1 or TWR-1/mTERF9 did not result in radialised leaves. Furthermore, the mterf6-5 mutation dramatically increased the leaf phenotype of as2-1 and caused lethality early in vegetative development. Our results uncover a new role for mTERF6 in leaf patterning and highlight the importance of mTERFs in plant development.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as amodel for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism's genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

Prickle1 regulates neurite outgrowth of apical spiral ganglion neurons but not hair cell polarity in the murine cochlea.

In the mammalian organ of Corti (OC), the stereocilia on the apical surface of hair cells (HCs) are uniformly organized in a neural to abneural axis (or medial-laterally). This organization is regulated by planar cell polarity (PCP) signaling. Mutations of PCP genes, such as Vangl2, Dvl1/2, Celsr1, and Fzd3/6, affect the formation of HC orientation to varying degrees. Prickle1 is a PCP signaling gene that belongs to the prickle / espinas / testin family. Prickle1 protein is shown to be asymmetrically localized in the HCs of the OC, and this asymmetric localization is associated with loss of PCP in Smurf mutants, implying that Prickle1 is involved in HC PCP development in the OC. A follow-up study found no PCP polarity defects after loss of Prickle1 (Prickle1-/-) in the cochlea. We show here strong Prickle1 mRNA expression in the spiral ganglion by in situ hybridization and β-Gal staining, and weak expression in the OC by β-Gal staining. Consistent with this limited expression in the OC, cochlear HC PCP is unaffected in either Prickle1C251X/C251X mice or Prickle1f/f; Pax2-cre conditional null mice. Meanwhile, type II afferents of apical spiral ganglion neurons (SGN) innervating outer hair cells (OHC) have unusual neurite growth. In addition, afferents from the apex show unusual collaterals in the cochlear nuclei that overlap with basal turn afferents. Our findings argue against the role of Prickle1 in regulating hair cell polarity in the cochlea. Instead, Prickle1 regulates the polarity-related growth of distal and central processes of apical SGNs.

Domain structures in circular ferroelectric nano-islands with charged defects: A Monte Carlo simulation

The pattern evolution of striped and vortex domain structures in circular ferroelectric nano-islands with in-plane polarization and charged defects is investigated using the Monte Carlo simulation based on the Landau-Devonshire phenomenological theory. The domain structures of islands undergoing different annealing processes are compared. Given embedded charge carriers at the center of islands, the domain patterns would be markedly affected as a result of the competition and balance between the electrostatic charge energy and other free energy terms in the Landau-Devonshire phenomenological theory. The symmetry of islands with different sizes and charge quantities is also analyzed. The present work provides a simple explanation of a variety of ferroelectric nano-islands and proposes an alternative promising approach to tune the domain structures and symmetry for the applications of nano-sized ferroelectric devices.

Astrocytes follow ganglion cell axons to establish an angiogenic template during retinal development.

Immature astrocytes and blood vessels enter the developing mammalian retina at the optic nerve head and migrate peripherally to colonize the entire retinal nerve fiber layer (RNFL). Retinal vascularization is arrested in retinopathy of prematurity (ROP), a major cause of bilateral blindness in children. Despite their importance in normal development and ROP, the factors that control vascularization of the retina remain poorly understood. Because astrocytes form a reticular network that appears to provide a substrate for migrating endothelial cells, they have long been proposed to guide angiogenesis. However, whether astrocytes do in fact impose a spatial pattern on developing vessels remains unclear, and how astrocytes themselves are guided is unknown. Here we explore the cellular mechanisms that ensure complete retinal coverage by astrocytes and blood vessels in mouse. We find that migrating astrocytes associate closely with the axons of retinal ganglion cells (RGCs), their neighbors in the RNFL. Analysis of Robo1; Robo2 mutants, in which RGC axon guidance is disrupted, and Math5 (Atoh7) mutants, which lack RGCs, reveals that RGCs provide directional information to migrating astrocytes that sets them on a centrifugal trajectory. Without this guidance, astrocytes exhibit polarization defects, fail to colonize the peripheral retina, and display abnormal fine-scale spatial patterning. Furthermore, using cell type-specific chemical-genetic tools to selectively ablate astrocytes, we show that the astrocyte template is required for angiogenesis and vessel patterning. Our results are consistent with a model whereby RGC axons guide formation of an astrocytic network that subsequently directs vessel development.

Inflammatory cell infiltration and resolution of kidney inflammation is orchestrated by the cold-shock protein Y-box binding protein-1

Tubular cells recruit monocytic cells in inflammatory tubulointerstitial kidney diseases. The cell-cell communication that establishes pro- or anti-inflammatory activities is mainly influenced by cytokines, reactive oxygen species, nitric oxide, and phagocytosis. Key proteins orchestrating these processes such as cold-shock proteins linked with chemoattraction and cell maturation have been identified. The prototypic member of the cold-shock protein family, Y-box binding protein (YB)-1, governs specific phenotypic alterations in monocytic cells and was explored in the present study. Following tubulointerstitial injury by unilateral ureteral obstruction, increased inflammatory cell infiltration and tubular cell CCL5 expression was found in conditional Ybx1 knockout animalswith specific depletion in monocytes/macrophages (YB-1ΔLysM). Furthermore, YB-1ΔLysM mice exhibit enhanced tissue damage, myofibroblast activation, and fibrosis. To investigate relevant molecular mechanism(s), we utilized bone marrow-derived macrophage cultures and found that YB-1-deficient macrophages display defects in cell polarization and function, including reduced proliferation and nitric oxide production, loss of phagocytic activity, and failure to upregulate IL-10 and CCL5 expression in response to inflammatory stimuli. Co-culture with primary tubular cells confirmed these findings. Thus, monocytic YB-1 has prominent and distinct roles for cellular feed-forward crosstalk and resolution of inflammatory processes by its ability to regulate cell differentiation and cytokine/chemokine synthesis.

The Interplay between Trap Density and Hysteresis in Planar Heterojunction Perovskite Solar Cells.

Anomalous current-voltage (J-V) hysteresis in perovskite (PSK) solar cell is open to dispute, where hysteresis is argued to be due to electrode polarization, dipolar polarization, and/or native defects. However, a correlation between those factors and J-V hysteresis is hard to be directly evaluated because they usually coexist and are significantly varied depending on morphology and crystallinity of the PSK layer, selective contacts, and device architecture. In this study, without changing morphology and crystallinity of PSK layer in a planar heterojunction structure employing FA0.9Cs0.1PbI3, a correlation between J-V hysteresis and trap density is directly evaluated by means of thermally induced PbI2 regulating trap density. Increase in thermal annealing time at a given temperature of 150 °C induces growth of PbI2 on the PSK grain surface, which results in significant reduction of nonradiative recombination. Hysteresis index is reduced from 0.384 to 0.146 as the annealing time is increased from 5 to 100 min due to decrease in the amplitude of trap-mediated recombination. Reduction of hysteresis by minimizing trap density via controlling thermal annealing time leads to the stabilized PCE of 18.84% from the normal planar structured FA0.9Cs0.1PbI3 PSK solar cell.

Distinct and redundant functions of Esama and VE-cadherin during vascular morphogenesis

The cardiovascular system forms during early embryogenesis and adapts to embryonic growth by sprouting angiogenesis and vascular remodeling. These processes require fine-tuning of cell-cell adhesion to maintain and re-establish endothelial contacts, while allowing cell motility. We have compared the contribution of two endothelial cell-specific adhesion proteins, VE-cadherin (VE-cad/Cdh5) and Esama (endothelial cell-selective adhesion molecule a), during angiogenic sprouting and blood vessel fusion (anastomosis) in the zebrafish embryo by genetic analyses. Different combinations of mutant alleles can be placed into a phenotypic series with increasing defects in filopodial contact formation. Contact formation in esama mutants appears similar to wild type, whereas esama-/-; ve-cad+/- and ve-cad single mutants exhibit intermediate phenotypes. The lack of both proteins interrupts filopodial interaction completely. Furthermore, double mutants do not form a stable endothelial monolayer, and display intrajunctional gaps, dislocalization of Zo-1 and defects in apical-basal polarization. In summary, VE-cadherin and Esama have distinct and redundant functions during blood vessel morphogenesis, and both adhesion proteins are central to endothelial cell recognition during anastomosis.

Effects of structural defects and polarization charges in InGaN-based double-junction solar cell

The performance of a double heterojunction solar cell based on Indium Gallium Nitride (InGaN) including a tunnel junction was simulated. The most challenging aspects of InGaN solar cells development being the crystal polarization and structural defects detrimental effects, their impact on the solar cell performances has been investigated in detail. The solar cell simulation was performed using physical models and InGaN parameters extracted from experimental measurements. The optimum efficiency of the heterojunction solar cell was obtained using a multivariate optimization method which allows to simultaneously optimize eleven parameters. The optimum defect free efficiency obtained is 24.4% with a short circuit current JSC=12.92mA/cm2, an open circuit voltage VOC=2.29V and a fill factor FF=82.55%. The performances evolution as functions of the polarization and the defects types and parameters was studied from their maximum down to as low as a 2% efficiency.

Phosphorylation potential of Drosophila E-Cadherin intracellular domain is essential for development and adherens junction biosynthetic dynamics regulation

Phosphorylation of a highly conserved serine cluster in the intracellular domain of E-Cadherin is essential for binding to β-Catenin in vitro . In cultured cells, phosphorylation of specific serine residues within the cluster is also required for regulation of adherens junction (AJ) stability and dynamics. However, much less is known about how such phosphorylation of E-Cadherin regulates AJ formation and dynamics in vivo . In this report, we generated an extensive array of Drosophila E-Cadherin (DE-Cad) endogenous knock-in alleles that carry mutations targeting this highly conserved serine cluster. Analyses of these mutations suggest that the overall phosphorylation potential, rather than the potential site-specific phosphorylation, of the serine cluster enhances the recruitment of β-Catenin by DE-Cad in vivo . Moreover, phosphorylation potential of the serine cluster only moderately increases the level of β-Catenin in AJs and is in fact dispensable for AJ formation in vivo . Nonetheless, phosphorylation-dependent recruitment of β-Catenin is essential for development, probably by enhancing the interactions between DE-Cad and α-Catenin. In addition, several phospho-mutations dramatically reduced the biosynthetic turnover rate of DE-Cad during apical-basal polarization, and such biosynthetically stable DE-Cad mutants specifically rescued the polarity defects in embryonic epithelia lacking the polarity proteins Stardust and Crumbs.

Sequence Polymorphism and Intrinsic Structural Disorder as Related to Pathobiological Performance of the Helicobacter pylori CagA Oncoprotein.

CagA, an oncogenic virulence factor produced by Helicobacter pylori, is causally associated with the development of gastrointestinal diseases such as chronic gastritis, peptic ulcers, and gastric cancer. Upon delivery into gastric epithelial cells via bacterial type IV secretion, CagA interacts with a number of host proteins through the intrinsically disordered C-terminal tail, which contains two repeatable protein-binding motifs, the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif and the CagA multimerization (CM) motif. The EPIYA motif, upon phosphorylation by host kinases, binds and deregulates Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2), a bona fide oncoprotein, inducing pro-oncogenic mitogenic signaling and abnormal cell morphology. Through the CM motif, CagA inhibits the kinase activity of polarity regulator partitioning-defective 1b (PAR1b), causing junctional and polarity defects while inducing actin cytoskeletal rearrangements. The magnitude of the pathobiological action of individual CagA has been linked to the tandem repeat polymorphisms of these two binding motifs, yet the molecular mechanisms by which they affect disease outcome remain unclear. Recent studies using quantitative techniques have provided new insights into how the sequence polymorphisms in the structurally disordered C-terminal region determine the degree of pro-oncogenic action of CagA in the gastric epithelium.

Attenuation of N-glycosylation causes polarity and adhesion defects in the C. elegans embryo

The Caenorhabditiselegans early embryo is highly polarized, requiring sequestration of cytoplasmic polarity factors at the plasma membrane. This compartmentalization aids asymmetric distribution of lipids and proteins, which is partially responsible for the fates of the daughter cells. Since most plasma membrane proteins are glycosylated, we determined the effect of attenuation of N-glycosylation on cell polarity. While polarity establishment was not perturbed, the size difference between the two cells formed in first cell division (AB and P1) was more variable in embryos with reduced N-glycosylation than in the mock-treated embryos. In addition, among other deficiencies, we observed spindle orientation defects in two-cell embryos. Moreover, cell-cell adhesion was specifically lost at the two-cell stage when N-glycosylation was reduced. This loss-of-adhesion phenotype was rescued by interfering with polarity establishment, indicating that polarity establishment enforces plasma membrane compartmentalization. Consistent with this idea, the decreased plasma membrane levels of the adhesion proteins E-cadherin and MAGI-1 in ribo-1(RNAi) embryos were restored in the absence of functional PAR-2. Our data suggest a general role for N-glycosylation in plasma membrane compartmentalization and cell polarity.

The Yeast Claudin Dcv1 Is Essential for the Maintenance of Distinct Membrane Domains and Polarized Mating Functions

Chemotropic and chemotactic cells exhibit a remarkable ability to interpret chemical gradients and sense direction. The mating response of the budding yeast S. cerevisiae is a model chemotropic system. The two haploid mating types, MATa and MATα, sense the pheromone secreted by cells of the opposite type, polarize their growth to form a mating projection towards the closest mating partner, and fuse to form diploids. In MATa cells, the Ste2 pheromone receptor is the primary gradient sensor. Ste2 is uniformly distributed on the plasma membrane (PM) in vegetative cells, but upon ligand binding, it is rapidly internalized. It then reappears as a polarized crescent that coincides with the incipient mating projection site. In mating mixtures, the newly emerged receptor crescents orient towards the closest mating partner. Although actin‐dependent directed secretion stabilizes and amplifies receptor polarity, we have shown that receptor polarization precedes the polarization of actin cables and occurs in the absence of actin‐directed secretion. In contrast, internalization of the receptor is essential for its polarization. How is receptor polarity established upstream of actin‐directed secretion and what are the key players?In a directed genetic screen, we found that deletion of the yeast claudin homolog, DCV1, conferred a significant defect in receptor polarization without affecting actin‐directed secretion. Unlike wild type (WT) cells, dcv1Δ cells were unable to establish a receptor polarization site that could be amplified independently of f‐actin. Based on its primary structure, Dcv1 was predicted to be an integral membrane protein. Immuno‐fluorescence (IF) microscopy in cells expressing Dcv1‐HA suggested that the protein localizes uniformly to the PM in vegetative cells and away from the mating projection in pheromone‐treated cells. Studies of cells expressing fluorescently‐tagged Dcv1 confirmed the IF results. Furthermore, Dcv1 appeared to localize as a ring at the base of newly formed mating projections. Cells co‐expressing fluorescently‐tagged forms of Dcv1 and the receptor exhibited inverse localization of these proteins concurrent with morphogenesis. This result is consistent with the observation that claudins promote the formation of membrane domains and act as membrane barriers. dcv1Δ cells also exhibited abnormal localization of various lipids in the PM. These include phosphotidylinositol, phosphotidylserine, and sterols. Preliminary lipidomic studies using mass spectrometry suggest differences in the phospholipid composition of the PM in WT and dcv1Δ cells. Consistent with its effect on receptor polarization, dcv1Δ conferred a defect in pheromone‐gradient sensing. Time‐lapse imaging of mating mixtures also revealed that Dcv1 is required for a variety of polarized mating functions in addition to receptor redistribution, and for efficient mating. In summary, our data implicate a gene of previously unknown function in the polarization of proteins and plasma membrane lipids that contribute to efficient chemotropism and mating. We propose that the yeast claudin Dcv1 plays a role in organizing mating‐specific membrane domains essential for the polarization of the receptor and other mating‐specific proteins.Support or Funding InformationNational Science Foundation

Mutation of Kinesin-6 Kif20b causes defects in cortical neuron polarization and morphogenesis

BackgroundHow neurons change their cytoskeleton to adopt their complex polarized morphology is still not understood. Growing evidence suggests that proteins that help build microtubule structures during cell division are also involved in building and remodeling the complex cytoskeletons of neurons. Kif20b (previously called MPP1 or Mphosph1) is the most divergent member of the Kinesin-6 family of “mitotic” kinesins that also includes Kif23/MKLP1 and Kif20a/MKLP2. We previously isolated a loss-of-function mouse mutant of Kif20b and showed that it had a thalamocortical axon guidance defect and microcephaly.MethodsWe demonstrate here, using the mouse mutant, that Kif20b is required for neuron morphogenesis in the embryonic neocortex. In vivo and in vitro cortical neurons were labeled and imaged to analyze various aspects of morphogenesis.ResultsLoss of Kif20b disrupts polarization as well as neurite outgrowth, branching and caliber. In vivo, mutant cortical neurons show defects in orientation, and have shorter thinner apical dendrites that branch closer to the cell body. In vitro, without external polarity cues, Kif20b mutant neurons show a strong polarization defect. This may be due in part to loss of the polarity protein Shootin1 from the axonal growth cone. Those mutant neurons that do succeed in polarizing have shorter axons with more branches, and longer minor neurites. These changes in shape are not due to alterations in cell fate or neuron layer type. Surprisingly, both axons and minor neurites of mutant neurons have increased widths and longer growth cone filopodia, which correlate with abnormal microtubule organization. Live analysis of axon extension shows that Kif20b mutant axons display more variable growth with increased retraction.ConclusionsThese results demonstrate that Kif20b is required cell-autonomously for proper morphogenesis of cortical pyramidal neurons. Kif20b regulates neuron polarization, and axon and dendrite branching, outgrowth, and caliber. Kif20b protein may act by bundling microtubules into tight arrays and by localizing effectors such as Shootin1. Thus it may help shape neurites, sustain consistent axon growth, and inhibit branching. This work advances our understanding of how neurons regulate their cytoskeleton to build their elaborate shapes. Finally, it suggests that neuronal connectivity defects may be present in some types of microcephaly.

Atrophin controls developmental signaling pathways via interactions with Trithorax-like.

Mutations in human Atrophin1, a transcriptional corepressor, cause dentatorubral-pallidoluysian atrophy, a neurodegenerative disease. Drosophila Atrophin (Atro) mutants display many phenotypes, including neurodegeneration, segmentation, patterning and planar polarity defects. Despite Atro's critical role in development and disease, relatively little is known about Atro's binding partners and downstream targets. We present the first genomic analysis of Atro using ChIP-seq against endogenous Atro. ChIP-seq identified 1300 potential direct targets of Atro including engrailed, and components of the Dpp and Notch signaling pathways. We show that Atro regulates Dpp and Notch signaling in larval imaginal discs, at least partially via regulation of thickveins and fringe. In addition, bioinformatics analyses, sequential ChIP and coimmunoprecipitation experiments reveal that Atro interacts with the Drosophila GAGA Factor, Trithorax-like (Trl), and they bind to the same loci simultaneously. Phenotypic analyses of Trl and Atro clones suggest that Atro is required to modulate the transcription activation by Trl in larval imaginal discs. Taken together, these data indicate that Atro is a major Trl cofactor that functions to moderate developmental gene transcription.

MTCL1 plays an essential role in maintaining Purkinje neuron axon initial segment.

The axon initial segment (AIS) is a specialized domain essential for neuronal function, the formation of which begins with localization of an ankyrin-G (AnkG) scaffold. However, the mechanism directing and maintaining AnkG localization is largely unknown. In this study, we demonstrate that invivo knockdown of microtubule cross-linking factor 1 (MTCL1) in cerebellar Purkinje cells causes loss of axonal polarity coupled with AnkG mislocalization. MTCL1 lacking MT-stabilizing activity failed to restore these defects, and stable MT bundles spanning the AIS were disorganized in knockdown cells. Interestingly, during early postnatal development, colocalization of MTCL1 with these stable MT bundles was observed prominently in the axon hillock and proximal axon. These results indicate that MTCL1-mediated formation of stable MT bundles is crucial for maintenance of AnkG localization. We also demonstrate that Mtcl1 gene disruption results in abnormal motor coordination with Purkinje cell degeneration, and provide evidence suggesting possible involvement of MTCL1 dysfunction in the pathogenesis of spinocerebellar ataxia.

P101 Interleukin-13 induces polarity defects in the intestinal epithelia

P101 Interleukin-13 induces polarity defects in the intestinal epithelia

Control of nuclear organization by F-actin binding proteins

ABSTRACTThe regulation of nuclear shape and deformability is a key factor in controlling diverse events from embryonic development to cancer cell metastasis, but the mechanisms governing this process are still unclear. Our recent study demonstrated an unexpected role for the F-actin bundling protein fascin in controlling nuclear plasticity through a direct interaction with Nesprin-2. Nesprin-2 is a component of the LINC complex that is known to couple the F-actin cytoskeleton to the nuclear envelope. We demonstrated that fascin, which is predominantly associated with peripheral F-actin rich filopodia, binds directly to Nesprin-2 at the nuclear envelope in a range of cell types. Depleting fascin or specifically blocking the fascin-Nesprin-2 complex leads to defects in nuclear polarization, movement and cell invasion. These studies reveal a novel role for an F-actin bundling protein in control of nuclear plasticity and underline the importance of defining nuclear-associated roles for F-actin binding proteins in future.

Sustained centrosome-cortical contact ensures robust polarization of the one-cell C. elegans embryo

In C. elegans, the anterior-posterior axis is established at the one-cell stage when the embryo polarizes along its long axis. One model suggests that a cue from the centrosome triggers symmetry breaking and is then dispensable for further steps in the process. In the absence of the initial centrosome cue, a redundant mechanism, reliant on the centrosome's microtubules, can polarize the cell. Despite this model, data from multiple sources suggest that direct centrosome-contact with the cortex may play a role in ensuring robust polarization. Some of this past work includes analysis of pam-1 mutants, which lack a functional puromycin-sensitive aminopeptidase and have aberrant centrosome positioning and variable polarization defects. To better understand the role of centrosome dynamics in polarization, we looked in detail at centrosome behavior in relation to key polarity landmarks in pam-1 mutants as well as those lacking cortical flows. We provide evidence for a model in which sustained direct contact between the centrosome and the cortex acts to reinforce both the actomyosin and the microtubule-dependent pathways. This contact is necessary for polarization when flows are inhibited.

Talin regulates integrin β1-dependent and -independent cell functions in ureteric bud development.

Kidney collecting system development requires integrin-dependent cell-extracellular matrix interactions. Integrins are heterodimeric transmembrane receptors consisting of α and β subunits; crucial integrins in the kidney collecting system express the β1 subunit. The β1 cytoplasmic tail has two NPxY motifs that mediate functions by binding to cytoplasmic signaling and scaffolding molecules. Talins, scaffolding proteins that bind to the membrane proximal NPxY motif, are proposed to activate integrins and to link them to the actin cytoskeleton. We have defined the role of talin binding to the β1 proximal NPxY motif in the developing kidney collecting system in mice that selectively express a Y-to-A mutation in this motif. The mice developed a hypoplastic dysplastic collecting system. Collecting duct cells expressing this mutation had moderate abnormalities in cell adhesion, migration, proliferation and growth factor-dependent signaling. In contrast, mice lacking talins in the developing ureteric bud developed kidney agenesis and collecting duct cells had severe cytoskeletal, adhesion and polarity defects. Thus, talins are essential for kidney collecting duct development through mechanisms that extend beyond those requiring binding to the β1 integrin subunit NPxY motif.