A unique role for clathrin light chain A in cell spreading and migration.

Clathrin heavy chain is the structural component of the clathrin triskelion, but unique functions for the two distinct and highly conserved clathrin light chains (CLCa and CLCb, also known as CLTA and CLTB, respectively) have been elusive. Here, we show that following detachment and replating, CLCa is uniquely responsible for promoting efficient cell spreading and migration. Selective depletion of CLCa, but not of CLCb, reduced the initial phase of isotropic spreading of HeLa, H1299 and HEK293 cells by 60-80% compared to siRNA controls, and wound closure and motility by ∼50%. Surface levels of β1-integrins were unaffected by CLCa depletion. However, CLCa was required for effective targeting of FAK (also known as PTK2) and paxillin to the adherent surface of spreading cells, for integrin-mediated activation of Src, FAK and paxillin, and for maturation of focal adhesions, but not their microtubule-based turnover. Depletion of CLCa also blocked the interaction of clathrin with the nucleation-promoting factor WAVE complex, and altered actin distribution. Furthermore, preferential recruitment of CLCa to budding protrusions was also observed. These results comprise the first identification of CLCa-specific functions, with implications for normal and neoplastic integrin-based signaling and cell migration

G protein–coupled receptor/arrestin3 modulation of the endocytic machinery

Nonvisual arrestins (arr) modulate G protein–coupled receptor (GPCR) desensitization and internalization and bind to both clathrin (CL) and AP-2 components of the endocytic coated pit (CP). This raises the possibility that endocytosis of some GPCRs may be a consequence of arr-induced de novo CP formation. To directly test this hypothesis, we examined the behavior of green fluorescent protein (GFP)-arr3 in live cells expressing β2-adrenergic receptors and fluorescent CL. After agonist stimulation, the diffuse GFP-arr3 signal rapidly became punctate and colocalized virtually completely with preexisting CP spots, demonstrating that activated complexes accumulate in previously formed CPs rather than nucleating new CP formation. After arr3 recruitment, CP appeared larger: electron microscopy analysis revealed an increase in both CP number and in the occurrence of clustered CPs. Mutant arr3 proteins with impaired binding to CL or AP-2 displayed reduced recruitment to CPs, but were still capable of inducing CP clustering. In contrast, though constitutively present in CPs, the COOH-terminal moiety of arr3, which contains CP binding sites but lacks receptor binding, did not induce CP clustering. Together, these results indicate that recruitment of functional arr3–GPCR complexes to CP is necessary to induce clustering. Latrunculin B or 16°C blocked CP rearrangements without affecting arr3 recruitment to CP. These results and earlier studies suggest that discrete CP zones exist on cell surfaces, each capable of supporting adjacent CPs, and that the cortical actin membrane skeleton is intimately involved with both the maintenance of existing CPs and the generation of new structures

Exosome-mediated Transfer of αvβ3 Integrin from Tumorigenic to Nontumorigenic Cells Promotes a Migratory Phenotype.

The αvβ3 integrin is known to be highly upregulated during cancer progression and promotes a migratory and metastatic phenotype in many types of tumors. We hypothesized that the αvβ3 integrin is transferred through exosomes and, upon transfer, has the ability to support functional aberrations in recipient cells. Here, for the first time, it is demonstrated that αvβ3 is present in exosomes released from metastatic PC3 and CWR22Pc prostate cancer cells. Exosomal β3 is transferred as a protein from donor to nontumorigenic and tumorigenic cells as β3 protein or mRNA levels remain unaffected upon transcription or translation inhibition in recipient cells. Furthermore, it is shown that upon exosome uptake, de novo expression of an αvβ3 increases adhesion and migration of recipient cells on an αvβ3 ligand, vitronectin. To evaluate the relevance of these findings, exosomes were purified from the blood of TRAMP mice carrying tumors where the expression of αvβ3 is found higher than in exosomes from wild-type mice. In addition, it is demonstrated that αvβ3 is coexpressed with synaptophysin, a biomarker for aggressive neuroendocrine prostate cancer. IMPLICATIONS: Overall this study reveals that the αvβ3 integrin is transferred from tumorigenic to nontumorigenic cells via exosomes, and its de novo expression in recipient cells promotes cell migration on its ligand. The increased expression of αvβ3 in exosomes from mice bearing tumors points to its clinical relevance and potential use as a biomarker. Mol Cancer Res; 14(11); 1136-46. ©2016 AACR

The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids

Panoramic Views of the Cygnus Loop

We present a complete atlas of the Cygnus Loop supernova remnant in the light of [O III] (5007), H alpha, and [S II] (6717, 6731). Despite its shell-like appearance, the Cygnus Loop is not a current example of a Sedov-Taylor blast wave. Rather, the optical emission traces interactions of the supernova blast wave with clumps of gas. The surrounding interstellar medium forms the walls of a cavity through which the blast wave now propagates, including a nearly complete shell in which non-radiative filaments are detected. The Cygnus Loop blast wave is not breaking out of a dense cloud, but is instead running into confining walls. The interstellar medium dominates not only the appearance of the Cygnus Loop but also the continued evolution of the blast wave. If this is a typical example of a supernova remnant, then global models of the interstellar medium must account for such significant blast wave deceleration.Comment: 28 pages AAS Latex, 28 black+white figures, 6 color figures. To be published in The Astrophysical Journal Supplement Serie

Design, Implementation and First Measurements with the Medipix Neutron Camera in CMS

The Medipix detector is the first device dedicated to measuring mixed-field radiation in the CMS cavern and able to distinguish between different particle types. Medipix2-MXR chips bump bonded to silicon sensors with various neutron conversion layers developed by the IEAP CTU in Prague were successfully installed for the 2008 LHC start-up in the CMS experimental and services caverns to measure the flux of various particle types, in particular neutrons. They have operated almost continuously during the 2010 run period, and the results shown here are from the proton run between the beginning of July and the end of October 2010. Clear signals are seen and different particle types have been observed during regular LHC luminosity running, and an agreement in the measured flux rate is found with the simulations. These initial results are promising, and indicate that these devices have the potential for further and future LHC and high energy physics applications as radiation monitoring devices for mixed field environments, including neutron flux monitoring. Further extensions are foreseen in the near future to increase the performance of the detector and its coverage for monitoring in CMS.Comment: 15 pages, 16 figures, submitted to JINS

Negative regulation of syntaxin4/SNAP-23/VAMP2-mediated membrane fusion by Munc18c <i>In Vitro</i>

Background: Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes. Methodology/Principal Findings Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex. Conclusion/Significance Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion

Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

Following antigen recognition, B cell receptor (BCR)-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2) is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs). Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system