Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network

In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies establish roles for Hip1R and actin in CCV budding from the TGN for lysosome biogenesis

IPIP27 coordinates PtdIns(4,5)P2 homeostasis for successful cytokinesis

During cytokinesis, an actomyosin contractile ring drives the separation of the two daughter cells. A key molecule in this process is the inositol lipid PtdIns(4,5)P 2 , which recruits numerous factors to the equatorial region for contractile ring assembly. Despite the importance of PtdIns(4,5)P 2 in cytokinesis, the regulation of this lipid in cell division remains poorly understood. Here, we identify a role for IPIP27 in mediating cellular PtdIns(4,5)P 2 homeostasis. IPIP27 scaffolds the inositol phosphatase oculocerebrorenal syndrome of Lowe (OCRL) by coupling it to endocytic BAR domain proteins. Loss of IPIP27 causes accumulation of PtdIns(4,5)P 2 on aberrant endomembrane vacuoles, mislocalization of the cytokinetic machinery, and extensive cortical membrane blebbing. This phenotype is observed in Drosophila and human cells and can result in cytokinesis failure. We have therefore identified IPIP27 as a key modulator of cellular PtdIns(4,5)P 2 homeostasis required for normal cytokinesis. The results indicate that scaffolding of inositol phosphatase activity is critical for maintaining PtdIns(4,5)P 2 homeostasis and highlight a critical role for this process in cell division. Carim et al. reveal that the IPIP27 protein, which physically couples the inositol phosphatase OCRL to endocytic BAR domain proteins, is required for cellular phosphoinositide homeostasis and normal cell division. The results show the importance of physically scaffolding inositol phosphatase activity within cells

Three-dimensional printing of shape memory alloys

A novel variant of the three-dimensional printing technique has been developed and used to manufacture NiTi parts. Instead of metal powder the process uses granules, which consist of a mixture of metal powder and organic binder. These granules are spread on a working table. Then a solvent is dispensed with a printer head to consolidate a selected area of the granule bed and the table is moved down. The "solvent on granule" printing process is repeated until a threedimensional green body is obtained. The green part is finally debinded and sintered to obtain a dense and fully metallic part. NiTi parts have been successfully produced by this technique with densities of about 95% of the theoretical density. Detailed information on the microstructure has been obtained by X ray diffraction. Sintered parts exhibit shape memory effect, which has been measured during thermal cycling under tensile stress

Three-dimensional printing of stainless steel parts

Stainless steel parts have been manufactured by two different layer by layer additive processes. The first one is a standard three dimensional process, in which metal powders are bound by selective deposition of binder with a printer head. The second one is a novel process, which is based on the selective deposition of a solvent on metal-polymer granule beds. The microstructures of green and sintered parts are characterized by optical and scanning electron microscopy, and the mechanical properties evaluated by hardness and tensile tests. Solvent on granule printing allows to reach mechanical properties similar to those of metal injection moulding parts

RNAi-mediated Hip1R Silencing Results in Stable Association between the Endocytic Machinery and the Actin Assembly Machinery

Actin filaments transiently associate with the endocytic machinery during clathrin-coated vesicle formation. Although several proteins that might mediate or regulate this association have been identified, in vivo demonstration of such an activity has not been achieved. Huntingtin interacting protein 1R (Hip1R) is a candidate cytoskeletal-endocytic linker or regulator because it binds to clathrin and actin. Here, Hip1R levels were lowered by RNA interference (RNAi). Surprisingly, rather than disrupting the transient association between endocytic and cytoskeletal proteins, clathrin-coated structures (CCSs) and their endocytic cargo became stably associated with dynamin, actin, the Arp2/3 complex, and its activator, cortactin. RNAi double-depletion experiments demonstrated that accumulation of the cortical actin-endocytic complexes depended on cortactin. Fluorescence recovery after photobleaching showed that dynamic actin filament assembly can occur at CCSs. Our results provide evidence that Hip1R helps to make the interaction between actin and the endocytic machinery functional and transient

Programmed death-1 ligands-transfected dendritic cells loaded with glutamic acid decarboxylase 65 (GAD65) inhibit both the alloresponse and the GAD65-reactive lymphocyte response

Type 1 diabetes (T1D) is due to a loss of immune tolerance to islet antigens, such as glutamic acid decarboxylase 65 (GAD65), for which islet transplantation is a promising therapy. Therefore, the generation of tolerance aiming at both alloantigen and GAD65 will help therapeutic intervention greatly in T1D. In this study, we tested the effect of programmed death-1 ligands (PD-L1)-transfected dendritic cells (DC) loaded with GAD65 on the alloresponse and GAD65-reactive lymphocyte response. The DC2·4 cell line was transfected with PD-L1 and co-cultured with GAD65. BALB-c mice were primed, respectively, by intraperitoneal injection with GAD65, PD-L1-transfected- or non-transfected DC (PD-L1/DC or DC), and PD-L1-transfected- or non-transfected DC loaded with GAD65 (PD-L1/DC/GAD65 or DC/GAD65). Splenocytes of treated mice were isolated and restimulated in vitro with GAD65 or the various DC populations above being used as stimulators, respectively. In the mixed lymphocyte reaction, DC/GAD65 were able to stimulate both allogeneic and GAD65-reactive lymphocytes. However, PD-L1/DC/GAD65 were poorer than DC/GAD65 at activating the GAD65-reactive lymphocyte response. Further, although PD-L1/DC could inhibit the alloresponse, PD-L1/DC/GAD65 were more effective at down-regulating the GAD65-reactive lymphocyte response. More importantly, PD-L1/DC/GAD65-primed lymphocytes exhibited the weakest proliferation when again restimulated in vitro by PD-L1/DC/GAD65. Additionally, PD-L1/DC/GAD65 down-regulated interferon-γ and up-regulated interleukin-10 production by activated lymphocytes. Therefore, combined stimulation in vivo and in vitro by PD-L1/DC/GAD65 could inhibit both the alloresponse and the GAD65-reactive lymphocyte response, which may contribute to controlling diabetes and islet transplant rejection