Cul3, a Cullin family scaffold protein, is thought to mediate the assembly of a large number of SCF (Skp1-Cullin1-F-box protein)-like ubiquitin ligase complexes through BTB domain substrate-recruiting adaptors. Cul3 controls early embryonic development in several genetic models through mechanisms not understood. Very few functional substrate/adaptor pairs for Cul3 ubiquitin ligases have been identified. Here, we show that Cul3 knockdown in human cells results in abnormal actin stress fibers and distorted cell morphology, owing to impaired ubiquitination and degradation of small GTPase RhoA. We identify a family of RhoA-binding BTB domain adaptors conserved from insects to mammals, designated BACURDs. BACURDs form ubiquitin ligase complexes, which selectively ubiquitinate RhoA, with Cul3. Dysfunction of the Cul3/BACURD complex decreases cell migration potential and impairs RhoA-mediated convergent extension movements during Xenopus gastrulation. Our studies reveal a previously unknown mechanism for controlling RhoA degradation and regulating RhoA function in various biological contexts, which involves a Cul3/BACURD ubiquitin ligase complex.
The Brownian motion of molecules at thermal equilibrium usually has a finite correlation time and will eventually be randomized after a long delay time, so that their displacement follows the Gaussian statistics. This is true even when the molecules have experienced a complex environment with a finite correlation time. Here, we report that the lateral motion of the acetylcholine receptors on live muscle cell membranes does not follow the Gaussian statistics for normal Brownian diffusion. From a careful analysis of a large volume of the protein trajectories obtained over a wide range of sampling rates and long durations, we find that the normalized histogram of the protein displacements shows an exponential tail, which is robust and universal for cells under different conditions. The experiment indicates that the observed non-Gaussian statistics and dynamic heterogeneity are inherently linked to the slow-active remodelling of the underlying cortical actin network.
N-syndecan (syndecan-3) was previously isolated as a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and suggested to mediate the neurite growth-promoting signal from cell matrixbound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. In the present study, we have transfected N18 neuroblastoma cells with a rat N-syndecan cDNA and show that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinaseactive fractions that bind to the cytosolic moiety of Nsyndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. We suggest that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway. HB-GAM1 was initially isolated from neonatal rat brain as a neurite outgrowth-promoting protein, the expression of which in brain corresponds to the stage of rapid axonal growth (1). Molecular cloning of full-length cDNA identified a novel secretory sequence (2). The same cDNA sequence was reported for pleiotrophin, a protein suggested to be mitogen for fibroblastic cells (3, 4). The HB-GAM/pleiotrophin sequence shares approximately 50% homology with the midkine protein involved in retinoic acid-induced cell differentiation (5-7).The expression of HB-GAM in the axon pathways of the brain and in the basement membranes outside of brain (8 -9) and the neurite outgrowth-promoting property of HB-GAM in vitro (see Refs. 1 and 4) have suggested interaction of matrixassociated HB-GAM with a cell surface receptor. Furthermore, HB-GAM is expressed at the surface of developing muscle cells and is suggested to play a role in the development of nerve/ muscle contacts (10 -12). N-syndecan (syndecan-3) has recently been isolated from detergent extracts of perinatal rat brain as a receptor or coreceptor for HB-GAM using recombinant HB-GAM as an affinity matrix (13). N-syndecan is localized at the surface of neurites and their growth cones in rat primary neurons growing on HB-GAM-coated matrix in vitro (13). Furthermore, HB-GAM and N-syndecan are spatiotemporally co-expressed in developing rat brain (14).The cell surface N-syndecan interacts with HB-GAM through its heparan sulfate chains (15). This interaction is enhanced by assembly of the heparan sulfat...
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