Presynaptic protein kinase C controls maturation and branch dynamics of developing retinotectal arbors: Possible role in activity‐driven sharpening

Visual activity refines developing retinotectal maps and shapes individual retinal arbors via an NMDA receptor‐dependent mechanism. As retinal axons grow into tectum, they slow markedly and emit many transient side branches behind the tip, assuming a “bottlebrush” morphology. Some branches are stabilized and branch further, giving rise to a compact arbor. The dynamic rate of branch addition and deletion is increased twofold when MK801 is used to block NMDA receptors, as if this prevents release of a stabilizing signal such as arachidonic acid (AA) from the postsynaptic neuron. In optic tract, AA mediates NCAM and L1 stimulation of axon growth by activating presynaptic protein kinase C (PKC) to phosphorylate GAP‐43 and stabilize F‐actin, and, if present in tectum, this growth control pathway could be modulated by postsynaptic activation. To test for the effects on arbor morphology of blocking PKC or AA release, we examined DiO‐labeled retinal axons of larval zebrafish with time‐lapse videomicroscopy. Bath application of the selective PKC inhibitor bisindolylmaleimide from 2 or 3 days onward doubled the rate at which side branches were added and deleted, as seen with MK801, and also prevented maturation of the arbor so that it retained a “bottlebrush” morphology. In order to selectively block the PKC being transported to retinal terminals, we injected the irreversible inhibitor calphostin C into the eye from which the ganglion cells were labeled, and this produced both effects seen with bath application. In contrast, there were no effects of control injections, which included Ringers into the same eye and the same dose into the opposite eye (actually much closer to the tectum of interest), to rule out the possibility that the inhibitor leaked from the eye to act on tectal cells. For comparison, we examined arbors treated with the NMDA blocker MK801 at half‐hour time‐lapse intervals, and detected the twofold rise in rates of branch addition and deletion previously reported in Xenopus larvae, but not the structural effect seen with the PKC inhibitors. In addition, we could produce both effects seen with PKC inhibitors by using RHC80267 to block AA release from DAG lipase, indicating that AA is the main drive for PKC activation. Thus, the results show a distinct role of AA and presynaptic PKC in both maturation of arbor structure and in the dynamic control of branching. The effects on branch dynamics were present regardless of the level of maturity of arbor structure. The fact that they mimicked those of MK801 suggests that presynaptic PKC may be involved in the NMDA receptor‐driven stabilization of developing retinal arbors. © 2003 Wiley Periodicals, Inc. J Neurobiol 58: 328–340, 2004     

Presynaptic protein kinase C controls maturation and branch dynamics of developing retinotectal arbors: possible role in activity-driven sharpening

Visual activity refines developing retinotectal maps and shapes individual retinal arbors via an NMDA receptor-dependent mechanism. As retinal axons grow into tectum, they slow markedly and emit many transient side branches behind the tip, assuming a "bottlebrush" morphology. Some branches are stabilized and branch further, giving rise to a compact arbor. The dynamic rate of branch addition and deletion is increased twofold when MK801 is used to block NMDA receptors, as if this prevents release of a stabilizing signal such as arachidonic acid (AA) from the postsynaptic neuron. In optic tract, AA mediates NCAM and L1 stimulation of axon growth by activating presynaptic protein kinase C (PKC) to phosphorylate GAP-43 and stabilize F-actin, and, if present in tectum, this growth control pathway could be modulated by postsynaptic activation. To test for the effects on arbor morphology of blocking PKC or AA release, we examined DiO-labeled retinal axons of larval zebrafish with time-lapse videomicroscopy. Bath application of the selective PKC inhibitor bisindolylmaleimide from 2 or 3 days onward doubled the rate at which side branches were added and deleted, as seen with MK801, and also prevented maturation of the arbor so that it retained a "bottlebrush" morphology. In order to selectively block the PKC being transported to retinal terminals, we injected the irreversible inhibitor calphostin C into the eye from which the ganglion cells were labeled, and this produced both effects seen with bath application. In contrast, there were no effects of control injections, which included Ringers into the same eye and the same dose into the opposite eye (actually much closer to the tectum of interest), to rule out the possibility that the inhibitor leaked from the eye to act on tectal cells. For comparison, we examined arbors treated with the NMDA blocker MK801 at half-hour time-lapse intervals, and detected the twofold rise in rates of branch addition and deletion previously reported in Xenopus larvae, but not the structural effect seen with the PKC inhibitors. In addition, we could produce both effects seen with PKC inhibitors by using RHC80267 to block AA release from DAG lipase, indicating that AA is the main drive for PKC activation. Thus, the results show a distinct role of AA and presynaptic PKC in both maturation of arbor structure and in the dynamic control of branching. The effects on branch dynamics were present regardless of the level of maturity of arbor structure. The fact that they mimicked those of MK801 suggests that presynaptic PKC may be involved in the NMDA receptor-driven stabilization of developing retinal arbors.     

MK801 increases retinotectal arbor size in developing zebrafish without affecting kinetics of branch elimination and addition

Visual activity refines the retinotopic map formed on tectum during regeneration and development in goldfish through an N-methyl-D-aspartate (NMDA) receptor-mediated mechanism. Retinal arbors are enlarged in fish with unrefined maps. Here, we examined the effect of NMDA receptor blockers on the development of retinotectal arbors in zebrafish. Since visual behaviors begin 68-79 h postfertilization, we blocked NMDA receptors by immersion of larvae in MK801, AP5, or CPP starting at either 48 or 72 h. We then labeled axons with DiI at 72 or 96 h and examined them 5-9 h later. Arbors at 101-105 h (31 cases) were larger than at 77-79 h (11 cases): The average number of branches increased from 4.0 to 7.6 and the area (convex polygon method) increased by 42%. Blocking NMDA receptors with MK801 from 72 to 101-105 h significantly enlarged arbor size, but the number of branches remained roughly the same. The length and area of the arbors were both significantly increased (21% and 36%), whereas the width increased by a smaller amount (6%). This increase was reflected in longer distances between branches within the arbor (interbranch segments, +13%) as well as in the summed length of all branches (+28%). This selective effect on the extent but not number of branches is in agreement with our previous report of strobe effects in both developing and regenerating projections in goldfish, and supports the role of NMDA receptors in the first 24 h of synaptic transmission. We also used DiO to label arbors in time-lapse images taken at hourly intervals from 77 to 112 h. These sequences confirmed that individual arbors grew during this time, but showed that rates of branch addition and deletion and branch lifetimes were unaltered by the MK801 treatment. This is consistent with a simple model of random insertion of new branches and selective activity-driven elimination of those at the periphery to keep the normal arbor focused. Blocking NMDA receptors is postulated to randomize the elimination allowing the periphery to expand, thus accounting for the enlarged areas, without change in branch numbers or branch dynamics.     

Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607--3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95--106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na(+),K(+)-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.     

KIBRA suppresses apical exocytosis through inhibition of aPKC kinase activity in epithelial cells

Epithelial cells possess apical-basolateral polarity and form tight junctions (TJs) at the apical-lateral border, separating apical and basolateral membrane domains. The PAR3-aPKC-PAR6 complex plays a central role in TJ formation and apical domain development during tissue morphogenesis. Inactivation and overactivation of aPKC kinase activity disrupts membrane polarity. The mechanism that suppresses active aPKC is unknown. KIBRA, an upstream regulator of the Hippo pathway, regulates tissue size in Drosophila and can bind to aPKC. However, the relationship between KIBRA and the PAR3-aPKC-PAR6 complex remains unknown. We report that KIBRA binds to the PAR3-aPKC-PAR6 complex and localizes at TJs and apical domains in epithelial tissues and cells. The knockdown of KIBRA causes expansion of the apical domain in MDCK three-dimensional cysts and suppresses the formation of apical-containing vacuoles through enhanced de novo apical exocytosis. These phenotypes are restored by inhibition of aPKC. In addition, KIBRA directly inhibits the kinase activity of aPKC in vitro. These results strongly support the notion that KIBRA regulates epithelial cell polarity by suppressing apical exocytosis through direct inhibition of aPKC kinase activity in the PAR3-aPKC-PAR6 complex.     

A novel function for the PAR complex in subcellular morphogenesis of tracheal terminal cells in Drosophila melanogaster

The processes that generate cellular morphology are not well understood. To investigate this problem, we use Drosophila melanogaster tracheal terminal cells, which undergo two distinct morphogenetic processes: subcellular branching morphogenesis and subcellular apical lumen formation. Here we show these processes are regulated by components of the PAR-polarity complex. This complex, composed of the proteins Par-6, Bazooka (Par-3), aPKC, and Cdc42, is best known for roles in asymmetric cell division and apical/basal polarity. We find Par-6, Bazooka, and aPKC, as well as known interactions between them, are required for subcellular branch initiation, but not for branch outgrowth. By analysis of single and double mutants, and isolation of two novel alleles of Par-6, one of which specifically truncates the Par-6 PDZ domain, we conclude that dynamic interactions between apical PAR-complex members control the branching pattern of terminal cells. These data suggest that canonical apical PAR-complex activity is required for subcellular branching morphogenesis. In addition, we find the PAR proteins are downstream of the FGF pathway that controls terminal cell branching. In contrast, we find that while Par-6 and aPKC are both required for subcellular lumen formation, neither Bazooka nor a direct interaction between Par-6 and aPKC is needed for this process. Thus a novel, noncanonical role for the polarity proteins Par-6 and aPKC is used in formation of this subcellular apical compartment. Our results demonstrate that proteins from the PAR complex can be deployed independently within a single cell to control two different morphogenetic processes.     

In vivo observations of timecourse and distribution of morphological dynamics in Xenopus retinotectal axon arbors

Changes in neuronal structure can contribute to the plasticity of neuronal connections in the developing and mature nervous system. However, the expectation that they would occur slowly precluded many from considering structural changes as a mechanism underlying synaptic plasticity that occurs over a period of minutes to hours. We took time-lapse confocal images of retinotectal axon arbors to determine the timecourse, magnitude, and distribution of changes in axon arbor structure within living Xenopus tadpoles. Images of axons were collected at intervals of 3 min, 30 min, and 2 h over total observation periods up to 8 h. Branch additions and retractions in arbors imaged at 3- or 30-min intervals were confined to shorter branches. Sites of additions and retractions were distributed throughout the arbor. The average lifetime of branches was about 10 min. Branches of up to 10 μm could be added to the arbor within a single 3-min observation interval. Observations of arbors at 3-min intervals showed rapid changes in the structure of branchtips, including transitions from lamellar growth cones to more streamlined tips, growth cone collapse, and re-extension. Simple branchtips were motile and appeared capable of exploratory behavior when viewed in time-lapse movies. In arbors imaged at 2-h intervals over a total of 8 h, morphological changes included longer branches, tens of microns in length. An average of 50% of the total branch length in the arbor was remodeled within 8 h. The data indicate that the elaboration of the arbor occurs by the random addition of branches throughout the arbor, followed by the selective stabilization of a small fraction of the new branches and the retraction of the majority of branches. Stabilized branches can then elongate and support the addition of more branches. These data show that structural changes in presynaptic axons can occur very rapidly even in complex arbors and can therefore play a role in forms of neuronal plasticity that operate on a timescale of minutes. © 1996 John Wiley & Sons, Inc.     

A distinct PAR complex associates physically with VE-cadherin in vertebrate endothelial cells

A cell polarity complex consisting of partitioning defective 3 (PAR-3), atypical protein kinase C (aPKC) and PAR-6 has a central role in the development of cell polarity in epithelial cells. In vertebrate epithelial cells, this complex localizes to tight junctions. Here, we provide evidence for the existence of a distinct PAR protein complex in endothelial cells. Both PAR-3 and PAR-6 associate directly with the adherens junction protein vascular endothelial cadherin (VE-cadherin). This association is direct and mediated through non-overlapping domains in VE-cadherin. PAR-3 and PAR-6 are recruited independently to cell-cell contacts. Surprisingly, the VE-cadherin-associated PAR protein complex lacks aPKC. Ectopic expression of VE-cadherin in epithelial cells affects tight junction formation. Our findings suggest that in endothelial cells, another PAR protein complex exists that localizes to adherens junctions and does not promote cellular polarization through aPKC activity. They also point to a direct role of a cadherin in the regulation of cell polarity in vertebrates.     

Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling

Cell polarity in Drosophila epithelia, oocytes and neuroblasts is controlled by the evolutionarily conserved PAR/aPKC complex, which consists of the serine-threonine protein kinase aPKC and the PDZ-domain proteins Bazooka (Baz) and PAR-6. The PAR/aPKC complex is required for the separation of apical and basolateral plasma membrane domains, for the asymmetric localization of cell fate determinants and for the proper orientation of the mitotic spindle. How the complex exerts these different functions is not known. We show that the lipid phosphatase PTEN directly binds to Baz in vitro and in vivo, and colocalizes with Baz in the apical cortex of epithelia and neuroblasts. PTEN is an important regulator of phosphoinositide turnover that antagonizes the activity of PI3-kinase. We show that Pten mutant ovaries and embryos lacking maternal and zygotic Pten function display phenotypes consistent with a function for PTEN in the organization of the actin cytoskeleton. In freshly laid eggs, the germ plasm determinants oskar mRNA and Vasa are not localized properly to the posterior cytocortex and pole cells do not form. In addition, the actin-dependent posterior movement of nuclei during early cleavage divisions does not occur and the synchrony of nuclear divisions at syncytial blastoderm stages is lost. Pten mutant embryos also show severe defects during cellularization. Our data provide evidence for a link between the PAR/aPKC complex, the actin cytoskeleton and PI3-kinase signaling mediated by PTEN.     

GAP43 phosphorylation is critical for growth and branching of retinotectal arbors in zebrafish

Visual activity acts via NMDA Receptors to refine developing retinotectal maps by shaping retinal arbors. Retinal axons add and delete transient branches, and the dynamic rates increase when MK801 blocks NMDARs, as if this prevents release of a stabilizing signal. Ca⁺⁺ entry through NMDARs activates phospholipase A2 (cPLA2) to release arachidonic acid (AA), which taps into a presynaptic growth control mechanism. NCAM, L1, N‐cadherin, and FGF all stimulate axon growth via AA activation of protein kinase C to phosphorylate GAP43 and polymerize/stabilize F‐actin. Our previous results show that blocking cPLA2 mimics NMDAR blockers, whereas exogenous AA reverses the increased dynamics, and PKC inhibitors also arrest growth. To test whether this activity‐driven F‐actin control mechanism shapes retinotectal arbors in zebrafish, we used the alpha‐1‐tubulin promoter to express GAP43‐GFP fusion proteins in retinal ganglion cells, and imaged arbors in time‐lapse to test for effects of GAP43 levels and its phosphorylation. Overexpressing wildtype GAP43 gave faster growth and larger arbors (#branches, spatial extent, total length of branches) at three days and especially four days. Surprisingly, the N‐terminal 20 amino acid segment alone caused the same increase in branching, but no increase in growth. Earlier studies implicate this region in activating G_o resulting in collapse of growth cones, which is now known to precede branch initiation. In contrast, GAP43 with ser41 mutated to ala (S41A) to prevent phosphorylation did not increase either branching or growth but resulted in immature, elongated arbors even at four to five days. In support of this atrophic effect, only half of brain/spinal neurons expressing S41A successfully initiated axonal outgrowth (vs. nearly 100% for wtGAP43). These results suggest that the region around the ser41 phosphorylation site, which binds CaM and PIP2, promotes growth only when phosphorylated, and also activates the branching control region in the first 10–20 amino acids. Whereas phosphorylation introduces a bulky negative charge group, mutation of serine to arginine introduces a bulky positive charge. But this also produced the same growth and branching as phosphorylation, suggesting that the effect of phosphorylation is through hydrophilic bulk rather than negative charge, in agreement with other IQ motifs. The results implicate the cPLA2‐AA‐PKC‐GAP43 pathway as part of an F‐actin based mechanism that both stabilizes new synapses and initiates new branches near effective synapses. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 897–911, 2010     

PAR3beta,a novel homologue of the cell polarity protein PAR3, localizes to tight junctions

The cell polarity protein PAR3, conserved from the nematode to the vertebrate, forms a complex with PAR6 and atypical protein kinase C (aPKC), and the protein complex occurs at the tight junctions in mammalian epithelial cells. Here we have cloned human cDNA for a novel PAR3 homologue, designated PAR3beta, whose messages are present in a variety of tissues and most abundantly expressed in the adult and fetal kidneys. The encoded protein of 1,205 amino acids contains a region homologous to the aPKC-binding domain of PAR3alpha, another human homologue previously identified, and three PDZ domains; the first PDZ domain of PAR3alpha is considered to interact with PAR6. Unexpectedly, in contrast to other PAR3s found in various species, PAR3beta is incapable of binding to any isotypes of PAR6 or aPKC. Nevertheless PAR3beta, expressed intrinsically or extrinsically, localizes to the tight junctions, indicating that the localization does not require the ternary complex formation.     

Microtubules induce self-organization of polarized PAR domains in Caenorhabditis elegans zygotes

A hallmark of polarized cells is the segregation of the PAR polarity regulators into asymmetric domains at the cell cortex. Antagonistic interactions involving two conserved kinases, atypical protein kinase C (aPKC) and PAR-1, have been implicated in polarity maintenance, but the mechanisms that initiate the formation of asymmetric PAR domains are not understood. Here, we describe one pathway used by the sperm-donated centrosome to polarize the PAR proteins in Caenorhabditis elegans zygotes. Before polarization, cortical aPKC excludes PAR-1 kinase and its binding partner PAR-2 by phosphorylation. During symmetry breaking, microtubules nucleated by the centrosome locally protect PAR-2 from phosphorylation by aPKC, allowing PAR-2 and PAR-1 to access the cortex nearest the centrosome. Cortical PAR-1 phosphorylates PAR-3, causing the PAR-3-aPKC complex to leave the cortex. Our findings illustrate how microtubules, independently of actin dynamics, stimulate the self-organization of PAR proteins by providing local protection against a global barrier imposed by aPKC.     

Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation

A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.     

Hippocampal neuronal polarity specified by spatially localized mPar3/mPar6 and PI 3-kinase activity

How a neuron becomes polarized remains an outstanding question. Here, we report that selection of the future axon among neurites of a cultured hippocampal neuron requires the activity of growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-kinase), as well as atypical protein kinase C (aPKC). The PI 3-kinase activity, highly localized to the tip of the newly specified axon of stage 3 neurons, is essential for the proper subcellular localization of mPar3, the mammalian homolog of C. elegans polarity protein Par3. Polarized distribution of not only mPar3 but also mPar6 is important for axon formation; ectopic expression of mPar6 or mPar3, or just the N terminus of mPar3, leaves neurons with no axon specified. Thus, neuronal polarity is likely to be controlled by the mPar3/mPar6/aPKC complex and the PI 3-kinase signaling pathway, both serving evolutionarily conserved roles in specifying cell polarity.     

Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis

BACKGROUND: The Par-3/Par-6/aPKC complex is a key regulator of cell polarity in a number of systems. In Drosophila, this complex acts at the zonula adherens (adherens junctions) to establish epithelial polarity and helps to orient the mitotic spindle during asymmetric neuroblast divisions. In MDCKII cells, this complex localizes to the zonula occludens (tight junctions) and appears to regulate epithelial polarity. However, the in vivo role of this complex during vertebrate embryogenesis is not known, due to the lack of relevant mutations. RESULTS: We have positionally cloned the zebrafish heart and soul (has) mutation, which affects the morphogenesis of several embryonic tissues, and show that it encodes atypical protein kinase C lambda (aPKC lambda). We find that loss of aPKC lambda affects the formation and maintenance of the zonula adherens in the polarized epithelia of the retina, neural tube, and digestive tract, leading to novel phenotypes, such as the formation of multiple lumens in the developing intestine. In addition, has mutants display defects in gut looping and endodermal organ morphogenesis that appear to be independent of the defects in epithelial polarity. Finally, we show that loss of aPKC lambda leads to defects in spindle orientation during progenitor cell divisions in the neural retina. CONCLUSIONS: Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.     

Dishevelled promotes axon differentiation by regulating atypical protein kinase C

The atypical protein kinase C (aPKC) in complex with PAR3 and PAR6 is required for axon-dendrite differentiation, but the upstream factors responsible for regulating its activity are largely unknown. Here, we report that in cultured hippocampal neurons aPKC is directly regulated by Dishevelled (Dvl), an immediate downstream effector of Wnt. We found that downregulation of Dvl abrogated axon differentiation, whereas Dvl overexpression resulted in multiple axon formation. Interestingly, Dvl was associated with aPKC and this interaction resulted in aPKC stabilization and activation. Furthermore, the multiple axon formation resulting from Dvl overexpression was attenuated by expressing a dominant-negative aPKC in these neurons and overexpression of aPKC prevented the loss of axon caused by Dvl downregulation. Finally, Wnt5a, a noncanonical Wnt, activated aPKC and promoted axon differentiation. The Wnt5a effect on axon differentiation was attenuated by downregulating Dvl or inhibiting aPKC. Thus, Dvl-aPKC interaction can promote axon differentiation mediated by the PAR3-PAR6-aPKC complex.     

Blood Vessel Epicardial Substance (Bves) Regulates Epidermal Tight Junction Integrity through Atypical Protein Kinase C

Bves is widely observed in the cell junction of the skin, epicardium, intestine, and cornea of both developmental embryos and mature adults. However, it is not clear how Bves confers its role in intercellular adhesion. Here, we identified the zebrafish bves (zBves) and found that the epidermal barrier function could be disrupted after knockdown of Bves, and these zBves morphants were sensitive to osmotic stress. A loss of zBves would affect the partitioning defective protein (PAR) junctional complex identified by the rescue experiment with tjp-2/ZO-2 or the PAR complex (par-3, par-6, and prkci/atypical (a)PKC) mRNAs, in which the survival rate of embryos increased 11, 24, 25, and 28%, respectively, after injection with junctional components; the tjp-2 and aPKC mRNA-rescued embryos also had 24 and 45% decreases in the defective rate. Immunofluorescent studies demonstrated that the aggregation of aPKC around the cell junctions had disintegrated in zBves morphants. However, the expression and assembly of zBves were not influenced by aPKC-MO. These results indicate that a loss of zBves affects the proteins involved in the pathway of the PAR junctional complex, especially aPKC, and both aPKC and Bves are indispensable to claudin expression.     

PAR‐3 controls endothelial planar polarity and vascular inflammation under laminar flow

Impaired cell polarity is a hallmark of diseased tissue. In the cardiovascular system, laminar blood flow induces endothelial planar cell polarity, represented by elongated cell shape and asymmetric distribution of intracellular organelles along the axis of blood flow. Disrupted endothelial planar polarity is considered to be pro‐inflammatory, suggesting that the establishment of endothelial polarity elicits an anti‐inflammatory response. However, a causative relationship between polarity and inflammatory responses has not been firmly established. Here, we find that a cell polarity protein, PAR‐3, is an essential gatekeeper of GSK3β activity in response to laminar blood flow. We show that flow‐induced spatial distribution of PAR‐3/aPKCλ and aPKCλ/GSK3β complexes controls local GSK3β activity and thereby regulates endothelial planar polarity. The spatial information for GSK3β activation is essential for flow‐dependent polarity to the flow axis, but is not necessary for flow‐induced anti‐inflammatory response. Our results shed light on a novel relationship between endothelial polarity and vascular homeostasis highlighting avenues for novel therapeutic strategies.     

Atypical protein kinase C controls sea urchin ciliogenesis

The atypical protein kinase C (aPKC) is part of the conserved aPKC/PAR6/PAR3 protein complex, which regulates many cell polarity events, including the formation of a primary cilium at the apical surface of epithelial cells. Cilia are highly organized, conserved, microtubule-based structures involved in motility, sensory processes, signaling, and cell polarity. We examined the distribution and function of aPKC in the sea urchin embryo, which forms a swimming blastula covered with motile cilia. We found that in the early embryo aPKC is uniformly cortical and becomes excluded from the vegetal pole during unequal cleavages at the 8- to 64-cell stages. During the blastula and gastrula stages the kinase localizes at the base of cilia, forming a ring at the transition zone between the basal body and the elongating axoneme. A dose-dependent and reversible inhibition of aPKC results in mislocalization of the kinase, defective ciliogenesis, and lack of swimming. Thus, as in the primary cilium of differentiated mammalian cells, aPKC controls the growth of motile cilia in invertebrate embryos. We suggest that aPKC might function to phosphorylate kinesin and so activate the transport of intraflagellar vesicles.