Wangsun Choi

A contractile actomyosin network linked to adherens junctions by Canoe/afadin helps drive convergent extension

Coordination of adhesion and the actin cytoskeleton is critical in morphogenesis. Drosophila germband extension is a model for convergent extension. Canoe/afadin is found to have a novel role in this process. It helps to coordinate a contractile apical actomyosin network with cell shape change and regulates apical polarity protein localization.

Remodeling the zonula adherens in response to tension and the role of afadin in this response

During development, epithelial cells must generate and respond to tension without disrupting epithelial barrier function. The authors use superresolution microscopy in MDCK cells to examine how the zonula adherens (ZA) is remodeled in response to elevated contractility while maintain tissue integrity. They define key roles for zonula occludens family proteins in regulating contractility and for the scaffolding protein afadin in maintaining ZA architecture at tricellular junctions.

Rap1 and Canoe/afadin are essential for establishment of apical–basal polarity in the Drosophila embryo

The small GTPase Rap1 and the actin-junctional linker protein Canoe/afadin are essential for the initial establishment of polarity in Drosophila, acting upstream of Bazooka/Par3 and the adherens junctions. However, feedback and cross-regulation occur, so polarity establishment is regulated by a network of proteins rather than a linear pathway.

The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/Afadin and Enabled.

Analysis of the function of the fly ZO-1 homologue Polychaetoid shows that it is not essential for junctional assembly or maintenance but does play an important role in embryonic morphogenesis. The data suggest that it works with Canoe/afadin and the actin regulator Enabled to regulate actin anchoring at junctions.

Primary platelet signaling cascades and integrin-mediated signaling control ADP-ribosylation factor (Arf) 6-GTP levels during platelet activation and aggregation.

Previous studies showed that ADP-ribosylation factor 6 (Arf6) is important for platelet function; however, little is known about which signaling events regulate this small GTP-binding protein. Arf6-GTP was monitored in platelets stimulated with a number of agonists (TRAP, thrombin, convulxin, collagen, PMA, thapsigargin, or A23187) and all led to a time-dependent decrease in Arf6-GTP. ADP and U46619 were without effect. Using inhibitors, it was shown that the decrease of Arf6-GTP is a direct consequence of known signaling cascades. Upon stimulation via PAR receptors, Arf6-GTP loss could be blocked by treatment with U-73122, BAPTA/AM, Ro-31-8220, or Gö6976, indicating requirements for phospholipase C, calcium, and protein kinase C (PKC) α/β, respectively. The Arf6-GTP decrease in convulxin-stimulated platelets showed similar requirements and was also sensitive to piceatannol, wortmannin, and LY294002, indicating additional requirements for Syk and phosphatidylinositol 3-kinase. The convulxin-induced decrease was sensitive to both PKCα/β and δ inhibitors. Outside-in signaling, potentially via integrin engagement, caused a second wave of signaling that affected Arf6. Inclusion of RGDS peptides or EGTA, during activation, led to a biphasic response; Arf6-GTP levels partially recovered upon continued incubation. A similar response was seen in β3 integrin-null platelets. These data show that Arf6-GTP decreases in response to known signaling pathways associated with PAR and GPVI. They further reveal a second, aggregation-dependent, process that dampens Arf6-GTP recovery. This study demonstrates that the nucleotide state of Arf6 in platelets is regulated during the initial phases of activation and during the later stages of aggregation.

Protein expression in platelets from six species that differ in their open canalicular system.

Platelets contain an invaginated, tubular membranous structure called the surface-connected open canalicular system (SCCS or OCS), which is contiguous with the plasma membrane and serves as a site for granule fusion and as a reservoir of membrane for platelet spreading. According to ultrastructural studies, platelets from some species lack OCS. In an attempt to correlate biochemical and functional attributes with the presence of an OCS, platelets from human, mouse and dog (OCS+), and from cow, camel and horse (OCS−) were analysed for differential protein expression and aggregation in response to thrombin. Among the 18 different cytoskeletal and regulatory proteins examined, five (Rac1, RhoA, Ras, calmodulin and Src) were expressed at higher levels in OCS+ platelets (p < 0.05). Given the role of Arf6 in the formation of tubular invaginations in nucleated cells, the levels of Arf6-GTP were analysed in OCS+ and OCS− platelets. There was no significant correlation between the presence of OCS and total Arf6 or Arf6-GTP levels. Comparison of platelet aggregation between different species suggests that OCS− platelets have delayed responses. This comparison of platelets from six different species, which differ in their OCS, shows the differential expression of known signaling components and foreshadows future studies focusing on OCS formation and function.

Platelets from Munc18c heterozygous mice exhibit normal stimulus-induced release

A critical aspect of hemostasis is the release of clot-forming components from the three intra-platelet stores: dense core granules, alpha-granules and lysosomes. Exocytosis from these granules is mediated by soluble (SNAPs and NSF) and integralmembrane proteins (v- and t-SNAREs). Three SM (Sec1/Munc18) proteins are present in mouse platelets (Munc18a, 18b and 18c) and each potentially regulates exocytosis via modulation of their cognate syntaxin binding partner. To define the molecular machinery required for platelet exocytosis, we analyzed platelets from Munc18c heterozygous knockout mice. These platelets show a decrease in Munc18c but no apparent reduction in other secretory machinery components. No differences in the rates of aggregation or of secretion of [(3)H]-5HT (dense core granules), platelet factor 4 (alpha-granules), or hexosaminidase (lysosomes) were detected between platelets from Munc18c heterozygous knockout or wild-type mice. The platelets also show normal morphology. Contrary to a predicted requirement for Munc18c in platelet secretion, data reported here show that reducing Munc18c levels does not substantially alter platelet function. These data show that despite Munc18cs role in platelet secretion, the lack of a secretion defect may be attributed to compensation by other Munc18 isoforms or that one allele is sufficient to maintain secretion under standard conditions.

Arranging a Cellular Checkerboard

Heterotypic cell adhesion mediated by nectins directs the complex cellular architecture of the inner ear. Biological patterns are all around us, ranging from the complex patterns on butterfly wings to the baroque architecture of kidney tubules. These patterns reflect the organization of millions of cell types in intricate yet reproducible ways to build tissues and organs. Even more remarkable, these patterns often arise through the self-assembly of undifferentiated cells. Understanding how this occurs is a key challenge for biologists. On page 1144 in this issue, Togashi et al. (1) implicate the interactions between nectin adhesion molecules in this process.