Actin is an integral component of epithelial apical junctions, yet the interactions of branched actin regulators with apical junction components are still not clear. Biochemical data have shown that α-catenin inhibits Arp2/3-dependent branched actin. These results suggested that branched actin is only needed at earliest stages of apical junction development. We use live imaging in developing C. elegans embryos to test models for how WAVE-induced branched actin collaborates with other apical junction proteins during the essential process of junction formation and maturation. We uncover both early and late essential roles for WAVE in apical junction formation. Early, as the C. elegans intestinal epithelium becomes polarized, we find that WAVE components become enriched concurrently with the Cadherin components and before the DLG-1 apical accumulation. Live imaging of F-actin accumulation in polarizing intestine supports that the Cadherin complex components and branched actin regulators work together for apical actin enrichment. Later in junction development, the apical accumulation of WAVE and Cadherin components is shown to be interdependent: Cadherin complex loss alters WAVE accumulation, and WAVE complex loss increases Cadherin accumulation. To determine why Cadherin levels rise when WVE-1 is depleted, we use FRAP to analyze Cadherin dynamics and find that loss of WAVE as well as of the trafficking protein EHD-1/RME-1 increases Cadherin dynamics. EM studies in adults depleted of branched actin regulators support that WVE-1 maintains established junctions, presumably through its trafficking effect on Cadherin. Thus we propose a developmental model for junction formation where branched actin regulators are tightly interconnected with Cadherin junctions through their previously unappreciated role in Cadherin transport.

The newer member of the tubulin superfamily, gamma-tubulin, is known to mediate microtubule nucleation from the centrosome of eukaryotic cells with the aid of some other proteins. The major amount of gamma-tubulin is believed to be located in the centrosome before the onset of mitotic division. However, a considerable amount has been found in the cytoplasm in the form of a complex whose function is not well known. Microtubules are most abundant in brain tissues and brain microtubules have been extensively used in many in vitro studies. Thus, it is relevant to use brain tissue to characterize cytoplasmic gamma-tubulin complex. Here we show that cytoplasmic gamma-tubulin in brain tissues exists as a ring complex as in other tissues. Interestingly, along with the common members of the gamma-TuRC reported from several tissues and species, the purified brain cytoplasmic complex contains some high molecular weight proteins including alpha and beta nonerythroid spectrin which are not found in other tissues. Immunohistochemical studies of brain tissue sections also show the co-localization of gamma-tubulin and spectrin. The possible implications have been discussed.

Gamma-tubulin is the major protein involved in the nucleation of microtubules from centrosomes in eukaryotic cells. It is present in both cytoplasm and centrosome. However, before centrosome maturation prior to mitosis, gamma-tubulin concentration increases dramatically in the centrosome, the mechanism of which is not known. Earlier it was reported that cytoplasmic gamma-tubulin complex isolated from goat brain contains non-erythroid spectrin/fodrin. The major role of erythroid spectrin is to help in the membrane organisation and integrity. However, fodrin or non-erythroid spectrin has a distinct pattern of localisation in brain cells and evidently some special functions over its erythroid counterpart. In this study, we show that fodrin and γ-tubulin are present together in both the cytoplasm and centrosomes in all brain cells except differentiated neurons and astrocytes. Immunoprecipitation studies in purified centrosomes from brain tissue and brain cell lines confirm that fodrin and γ-tubulin interact with each other in centrosomes. Fodrin dissociates from centrosome just after the onset of mitosis, when the concentration of γ-tubulin attains a maximum at centrosomes. Further it is observed that the interaction between fodrin and γ-tubulin in the centrosome is dependent on actin as depolymerisation of microfilaments stops fodrin localization. Image analysis revealed that γ-tubulin concentration also decreased drastically in the centrosome under this condition. This indicates towards a role of fodrin as a regulatory transporter of γ-tubulin to the centrosomes for normal progression of mitosis.

CDC-42 regulation of non-muscle myosin/NMY-2 is required for polarity maintenance in the one-cell embryo of Caenorhabditis elegans. CDC-42 and NMY-2 regulate polarity throughout embryogenesis, but their contribution to later events of morphogenesis are less understood. We have shown that epidermal enclosure requires the GTPase CED-10/Rac1 and WAVE/Scar complex, its effector, to promote protrusions that drive enclosure through the branch actin regulator Arp2/3. Our analysis here of RGA-8, a homolog of SH3BP1/Rich1/ARHGAP17/Nadrin, with BAR and RhoGAP motifs, suggests it regulates CDC-42, so that actin and myosin/NMY-2 promote ventral enclosure during embryonic morphogenesis. Genetic and molecular data suggest RGA-8 regulates CDC-42, and phenocopies the CDC-42 pathway regulators WASP-1/WSP-1 and the F-BAR proteins TOCA-1 and TOCA-2. Live imaging shows RGA-8 and WSP-1 enrich myosin and regulate F-actin in migrating epidermal cells during ventral enclosure. Loss of RGA-8 alters membrane recruitment of active CDC-42. We propose TOCA proteins and RGA-8 use BAR domains to localize and regenerate CDC-42 activity, thus regulating F-actin levels, through the branched actin regulator WSP-1, and myosin enrichment. RhoGAP RGA-8 thus polarizes epithelia, to promote cell migrations and cell shape changes of embryonic morphogenesis.

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RGA-8, a protein with membrane binding and actin regulatory motifs, promotes embryonic morphogenesis by localizing active CDC-42 in developing epithelia, thus controlling actin and actin motors during cell movements. Abstract -CDC-42 regulation of non-muscle myosin/ NMY-2 is required for polarity maintenance in the one-cell embryo of C. elegans. CDC-42 and NMY-2 regulate polarity throughout embryogenesis, but their contribution to later events of morphogenesis are less understood.We have shown that epidermal enclosure requires the GTPase CED-10/Rac1 and WAVE/Scar complex, its effector, to promote protrusions that drive enclosure through the branch actin regulator Arp2/3. Our analysis here of RGA-8, a homolog of SH3BP1/Rich1/ARHGAP17/Nadrin, with BAR and RhoGAP motifs, suggests it regulates CDC-42, so that NMY-2 promotes two events of epidermal morphogenesis: ventral enclosure and elongation. Genetic and molecular data suggest RGA-8 regulates CDC-42, and the CDC-42 effectors WSP-1 and MRCK-1, in parallel to F-BAR proteins TOCA-1 and TOCA-2.The RGA-8-CDC-42-WSP-1 pathway enriches myosin in migrating epidermal cells during ventral enclosure. We propose TOCA proteins and RGA-8 use BAR domains to localize and regenerate CDC-42 activity, thus regulating F-actin levels, through the branched actin regulator WSP-1, and myosin polarity through the myosin kinase MRCK-1. Regulated CDC-42 thus polarizes epithelia, to control cell migrations and cell shape changes of embryonic morphogenesis.

Cadherin dynamics drive morphogenesis, while defects in Cadherin polarity contribute to diseases, including cancers. However, the forces polarizing Cadherin membrane distribution are not well understood. We previously showed that WAVE-dependent branched actin polarizes Cadherin distribution, and suggested one mechanism is protein transport. While previous studies suggested that WAVE is enriched at various endocytic organelles, the role of WAVE in protein traffic is understudied. Here we test the model that WAVE regulates Cadherin by polarizing its transport. In support of this model we show that: 1) endogenously tagged WAVE accumulates in vivo at several endocytic organelles, including recycling endosomes and at the Golgi; 2) likewise, Cadherin protein accumulates at recycling endosomes and Golgi; 3) loss of WAVE components reduces Cadherin accumulation at apically directed RAB-11-positive recycling endosomes and increases accumulation at Golgi. In addition, live imaging illustrates that dynamics and velocity of recycling endosomes enriched for RAB-11::GFP and RFP::RME-1 are reduced in animals depleted of WAVE components, and RAB-11::GFP movements are misdirected, suggesting WAVE powers and directs their movements. This in vivo study demonstrates the importance of WAVE in promoting polarized transport in epithelia, and supports a model that WAVE promotes cell-cell adhesion and polarity by promoting Cadherin transport. [Media: see text] [Media: see text] [Media: see text]