Animal cell cytokinesis: the role of dynamic changes in the plasma membrane proteome and lipidome

In animal cells, cytokinesis is characterised by the formation of the mitotic spindle that signals the assembly of an actomyosin ring between the spindle poles. Contraction of this ring drives ingression of the cleavage furrow, and culminates in the formation of a thin intercellular bridge between the daughter cells. At the centre of this bridge is the midbody, which is thought both to provide a site of attachment for the plasma membrane furrow and act as foci for the spatial and temporal control mechanisms that drive abscission. This review will focus upon recent studies that offer new insight into these events, in particular studies that elaborate on the mechanism of attachment between the furrow plasma membrane and the underlying cytoskeleton, and how dynamic changes in membrane composition might underpin key aspects of cytokinesis

ESCRT function in cytokinesis: location, dynamics and regulation by mitotic kinases

Mammalian cytokinesis proceeds by constriction of an actomyosin ring and furrow ingression, resulting in the formation of the midbody bridge connecting two daughter cells. At the centre of the midbody resides the Flemming body, a dense proteinaceous ring surrounding the interlocking ends of anti-parallel microtubule arrays. Abscission, the terminal step of cytokinesis, occurs near the Flemming body. A series of broad processes govern abscission: the initiation and stabilisation of the abscission zone, followed by microtubule severing and membrane scission—The latter mediated by the endosomal sorting complex required for transport (ESCRT) proteins. A key goal of cell and developmental biologists is to develop a clear understanding of the mechanisms that underpin abscission, and how the spatiotemporal coordination of these events with previous stages in cell division is accomplished. This article will focus on the function and dynamics of the ESCRT proteins in abscission and will review recent work, which has begun to explore how these complex protein assemblies are regulated by the cell cycle machinery

Alternate routes to the cell surface underpin insulin-regulated membrane trafficking of GLUT4

Insulin-stimulated delivery of glucose transporters (GLUT4) from specialized intracellular GLUT4 storage vesicles (GSVs) to the surface of fat and muscle cells is central to whole-body glucose. This translocation and subsequent internalization of GLUT4 back into intracellular stores transits numerous small membrane-bound compartments (internal GLUT4-containing vesicles; IGVs) including GSVs, but the function of these different compartments is not clear. Cellugyrin and sortilin define distinct populations of IGV; sortilin-positive IGVs represent GSVs, but the function of cellugyrin-containing IGVs is unknown. Here we demonstrate a role for cellugyrin in intracellular sequestration of GLUT4 in HeLa cells and have used a proximity ligation assay to follow changes in pairwise associations between cellugyrin, sortilin, GLUT4 and membrane trafficking machinery following insulin-stimulation of 3T3-L1 adipoctyes. Our data suggest that insulin stimulates traffic from cellugyrin- to sortilin- membranes, and that cellugyrin-IGVs provide an insulin-sensitive reservoir to replenish GSVs following insulin-stimulated exocytosis of GLUT4. Furthermore, our data support the existence of a pathway from cellugyrin-membranes to the surface of 3T3-L1 adipocytes that bypasses GSVs under basal conditions, and that insulin diverts traffic away from this into GSVs

A complex network of interactions between mitotic kinases, phosphatases and ESCRT proteins regulates septation and membrane trafficking in S. pombe

Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast

Tyrosine phosphorylation of Munc18c on residue 521 abrogates binding to Syntaxin 4

Background: Insulin stimulates exocytosis of GLUT4 from an intracellular store to the cell surface of fat and muscle cells. Fusion of GLUT4-containing vesicles with the plasma membrane requires the SNARE proteins Syntaxin 4, VAMP2 and the regulatory Sec1/Munc18 protein, Munc18c. Syntaxin 4 and Munc18c form a complex that is disrupted upon insulin treatment of adipocytes. Munc18c is tyrosine phosphorylated in response to insulin in these cells. Here, we directly test the hypothesis that tyrosine phosphorylation of Munc18c is responsible for the observed insulin-dependent abrogation of binding between Munc18c and Syntaxin 4. Results: We show that Munc18c is directly phosphorylated by recombinant insulin receptor tyrosine kinase in vitro. Using pull-down assays, we show that phosphorylation abrogates binding of Munc18c to both Syntaxin 4 and the v-SNARE VAMP2, as does the introduction of a phosphomimetic mutation into Munc18c ( Y521E). Conclusion: Our data indicate that insulin-stimulated tyrosine phosphorylation of Munc18c impairs the ability of Munc18c to bind its cognate SNARE proteins, and may therefore represent a regulatory step in GLUT4 traffic

ESCRT function in cytokinesis : location, dynamics and regulation by mitotic kinases

Mammalian cytokinesis proceeds by constriction of an actomyosin ring and furrow ingression, resulting in the formation of the midbody bridge connecting two daughter cells. At the centre of the midbody resides the Flemming body, a dense proteinaceous ring surrounding the interlocking ends of anti-parallel microtubule arrays. Abscission, the terminal step of cytokinesis, occurs near the Flemming body. A series of broad processes govern abscission: the initiation and stabilisation of the abscission zone, followed by microtubule severing and membrane scission—The latter mediated by the endosomal sorting complex required for transport (ESCRT) proteins. A key goal of cell and developmental biologists is to develop a clear understanding of the mechanisms that underpin abscission, and how the spatiotemporal coordination of these events with previous stages in cell division is accomplished. This article will focus on the function and dynamics of the ESCRT proteins in abscission and will review recent work, which has begun to explore how these complex protein assemblies are regulated by the cell cycle machinery

GLUT4 expression and glucose transport in human induced pluripotent stem cell-derived cardiomyocytes

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) have the potential to transform regenerative cardiac medicine and the modelling of cardiac disease. This is of particular importance in the context of diabetic cardiomyopathy where diabetic individuals exhibit reduced cardiac diastolic contractile performance in the absence of vascular disease, significantly contributing towards high cardiovascular morbidity. In this study, the capacity of iPSC-CM to act as a novel cellular model of cardiomyocytes was assessed. The diabetic phenotype is characterised by insulin resistance, therefore there was a specific focus upon metabolic parameters. Despite expressing crucial insulin signalling intermediates and relevant trafficking proteins, it was identified that iPSC-CM do not exhibit insulin-stimulated glucose uptake. iPSC-CM are spontaneously contractile however contraction mediated uptake was not found to mask any insulin response. The fundamental limitation identified in these cells was a critical lack of expression of the insulin sensitive glucose transporter GLUT4. Using comparative immunoblot analysis and the GLUT-selective inhibitor BAY-876 to quantify expression of these transporters, we show that iPSC-CM express high levels of GLUT1 and low levels of GLUT4 compared to primary cardiomyocytes and cultured adipocytes. Interventions to overcome this limitation were unsuccessful. We suggest that the utility of iPSC-CMs to study cardiac metabolic disorders may be limited by their apparent foetal-like phenotype

Insulin-stimulated GLUT4 translocation – size is not everything!

Insulin-regulated trafficking of the facilitative glucose transporter GLUT4 has been studied in many cell types. The translocation of GLUT4 from intracellular membranes to the cell surface is often described as a highly specialised form of membrane traffic restricted to certain cell types such as fat and muscle, which are the major storage depots for insulin-stimulated glucose uptake. Here, we discuss evidence that favours the argument that rather than being restricted to specialised cell types, the machinery through which insulin regulates GLUT4 traffic is present in all cell types. This is an important point as it provides confidence in the use of experimentally tractable model systems to interrogate the trafficking itinerary of GLUT4

The Glucose Transporter GLUT4 and the Aminopeptidase vp165 Colocalise in Tubulo-Vesicular Elements in Adipocytes and Cardiomyocytes

The aminopeptidase vp165 is one of the major polypeptides enriched in GLUT4-containing vesicles immuno-isolated from adipocytes. In the present study we have confirmed and quantified the high degree of colocalisation between GLUT4 and vp165 using double label immuno-electron microscopy on vesicles isolated from adipocytes and heart. The percentage of vp165-containing vesicles that also contained GLUT4 was 91%, 76%, and 86% in rat adipocytes, 3T3-L1 adipocytes, and rat heart, respectively. Internalisation of a transferrin/HRP (Tf/HRP) conjugate by 3T3-L1 adipocytes, followed by diaminobenzidine treatment in intact cells, resulted in ablation of only 41% and 45% of GLUT4 and vp165, respectively, whereas endosomal markers are almost quantitatively ablated. Using immuno-electron microscopy on cryosections it was determined that in atrial cardiomyocytes GLUT4 and vp165 colocalised in a population of tubulo-vesicular (T-V) elements that were often found close to the plasma membrane. Double label immunocytochemistry indicated a high degree of overlap in these T-V elements between GLUT4 and vp165. However, in atrial cardiomyocytes a large proportion of GLUT4 was also present in secretory granules containing atrial natriuretic factor (ANF). In contrast, very little vp165 was detected in ANF granules. These data indicate that GLUT4 and vp165 are colocalised in an intracellular, post-endocytic, tubulo-vesicular compartment in adipocytes and cardiomyocytes suggesting that both proteins are sorted in a similar manner in these cells. However, GLUT4 but not vp165 is additionally localised in the regulated secretory pathway in atrial cardiomyocytes

SNARE phosphorylation: a control mechanism for insulin-stimulated glucose transport and other regulated exocytic events

Trafficking within eukaryotic cells is a complex and highly regulated process; events such as recycling of plasma membrane receptors, formation of multivesicular bodies, regulated release of hormones and delivery of proteins to membranes all require directionality and specificity. The underpinning processes, including cargo selection, membrane fusion, trafficking flow and timing, are controlled by a variety of molecular mechanisms and engage multiple families of lipids and proteins. Here, we will focus on control of trafficking processes via the action of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, in particular their regulation by phosphorylation. We will describe how these proteins are controlled in a range of regulated trafficking events, with particular emphasis on the insulin-stimulated delivery of glucose transporters to the surface of adipose and muscle cells. Here, we focus on a few examples of SNARE phosphorylation which exemplify distinct ways in which SNARE machinery phosphorylation may regulate membrane fusion