Human adipose cells in vitro are either refractory or responsive to insulin, reflecting host metabolic state.

While intercellular communication processes are frequently characterized by switch-like transitions, the endocrine system, including the adipose tissue response to insulin, has been characterized by graded responses. Yet here individual cells from adipose tissue biopsies are best described by a switch-like transition between the basal and insulin-stimulated states for the trafficking of the glucose transporter GLUT4. Two statistically-defined populations best describe the observed cellular heterogeneity, representing the fractions of refractive and responsive adipose cells. Furthermore, subjects exhibiting high systemic insulin sensitivity indices (SI) have high fractions of responsive adipose cells in vitro, while subjects exhibiting decreasing SI have increasing fractions of refractory cells in vitro. Thus, a two-component model best describes the relationship between cellular refractory fraction and subject SI. Since isolated cells exhibit these different response characteristics in the presence of constant culture conditions and milieu, we suggest that a physiological switching mechanism at the adipose cellular level ultimately drives systemic SI.

Trapping by clusters of channels, receptors, and transporters: quantitative description.

Various membrane functional units such as receptors, transporters, and channels, whose action necessarily involves capturing diffusing molecules, are often organized into multimeric complexes forming clusters on the cell and organelle membranes. These functional units themselves are usually oligomers of several integral proteins, which have their own symmetry. Depending on the symmetry, they form clusters on different packing lattices. Moreover, local membrane inhomogeneities, e.g., the so-called membrane domains, rafts, stalks, etc., lead to different patterns even within the structures on the same packing lattice. Units in the cluster compete for diffusing molecules and screen each other. Here we propose a general approach that allows one to quantify the screening effects. The approach is used to derive simple approximate formulas giving the trapping rates of diffusing molecules by clusters of absorbers on lattices of different packing symmetries. The obtained results describe smooth variation of the trapping rate from the sum of the rates of individual absorbers forming the cluster to the effective collective rate. The latter shows how the trapping efficiency of an individual absorber decreases as the number of absorbers in the cluster increases and/or the inter-absorber distance decreases. Numerical tests demonstrate good agreement between the rates predicted by the theory and obtained from Brownian dynamics simulations for clusters of different shapes and sizes.

Insulin stimulates translocation of human GLUT4 to the membrane in fat bodies of transgenic Drosophila melanogaster.

The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in 'diabetic' flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin-signaling pathways in Drosophila, demonstrating the utility of TIRFM of tagged sugar transporters to monitor signaling pathways in insects.

Impaired tethering and fusion of GLUT4 vesicles in insulin-resistant human adipose cells.

Systemic glucose homeostasis is profoundly influenced by adipose cell function. Here we investigated GLUT4 dynamics in living adipose cells from human subjects with varying BMI and insulin sensitivity index (Si) values. Cells were transfected with hemagglutinin (HA)-GLUT4-green fluorescent protein (GFP)/mCherry (red fluorescence), and were imaged live using total internal reflection fluorescence and confocal microscopy. HA-GLUT4-GFP redistribution to the plasma membrane (PM) was quantified by surface-exposed HA epitope. In the basal state, GLUT4 storage vesicle (GSV) trafficking to and fusion with the PM were invariant with donor subject Si, as was total cell-surface GLUT4. In cells from insulin-sensitive subjects, insulin augmented GSV tethering and fusion approximately threefold, resulting in a corresponding increase in total PM GLUT4. However, with decreasing Si, these effects diminished progressively. All insulin-induced effects on GLUT4 redistribution and trafficking correlated strongly with Si and only weakly with BMI. Thus, while basal GLUT4 dynamics and total cell-surface GLUT4 are intact in human adipose cells, independent of donor Si, cells from insulin-resistant donors show markedly impaired GSV tethering and fusion responses to insulin, even after overnight culture. This altered insulin responsiveness is consistent with the hypothesis that adipose cellular dysfunction is a primary contributor to systemic metabolic dysfunction.

Insulin regulates Glut4 confinement in plasma membrane clusters in adipose cells.

Insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) is the hallmark of glucose metabolism. In this study we examined insulin's effects on GLUT4 organization in PM of adipose cells by direct microscopic observation of single monomers tagged with photoswitchable fluorescent protein. In the basal state, after exocytotic delivery only a fraction of GLUT4 is dispersed into the PM as monomers, while most of the GLUT4 stays at the site of fusion and forms elongated clusters (60-240 nm). GLUT4 monomers outside clusters diffuse freely and do not aggregate with other monomers. In contrast, GLUT4 molecule collision with an existing cluster can lead to immediate confinement and association with that cluster. Insulin has three effects: it shifts the fraction of dispersed GLUT4 upon delivery, it augments the dissociation of GLUT4 monomers from clusters ∼3-fold and it decreases the rate of endocytic uptake. All together these three effects of insulin shift most of the PM GLUT4 from clustered to dispersed states. GLUT4 confinement in clusters represents a novel kinetic mechanism for insulin regulation of glucose homeostasis.

Trapping of diffusing particles by clusters of absorbing disks on a reflecting wall with disk centers on sites of a square lattice.

A simple approximate formula is derived for the rate constant that describes steady-state flux of diffusing particles through a cluster of perfectly absorbing disks on the otherwise reflecting flat wall, assuming that the disk centers occupy neighboring sites of a square lattice. A distinctive feature of trapping by a disk cluster is that disks located at the cluster periphery shield the disks in the center of the cluster. This competition of the disks for diffusing particles makes it impossible to find an exact analytical solution for the rate constant in the general case. To derive the approximate formula, we use a recently suggested approach [A. M. Berezhkovskii, L. Dagdug, V. A. Lizunov, J. Zimmerberg, and S. M. Bezrukov, J. Chem. Phys. 136, 211102 (2012)], which is based on the replacement of the disk cluster by an effective uniform partially absorbing spot. The formula shows how the rate constant depends on the size and shape of the cluster. To check the accuracy of the formula, we compare its predictions with the values of the rate constant obtained from Brownian dynamics simulations. The comparison made for 18 clusters of various shapes and sizes shows good agreement between the theoretical predictions and numerical results.

Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes.

BACKGROUND: Egress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites. METHODS: Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ homeostasis on the egress programme. RESULTS: A steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER) Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. CONCLUSIONS: The parasite egress programme requires intracellular free Ca2+ for egress initiation, vacuole swelling, and host cell cytoskeleton digestion. The evidence that parasitophorous vacuole swelling, a stage of unaffected egress, is dependent upon a rise in intracellular Ca2+ suggests a mechanism for ionophore-inducible egress and a new target for Ca2+ in the programme liberating parasites from the host cell. A regulatory pathway for egress that depends upon increases in intracellular free Ca2+ is proposed.

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts.

Sphingolipids play important roles in plasma membrane structure and cell signaling. However, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of (15)N-enriched ions from metabolically labeled (15)N-sphingolipids in the plasma membrane, using high-resolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids--both in living cells and during fixation of living cells--exclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous (15)N-sphingolipid microdomains with mean diameters of ∼200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and long-range organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.

Extracellular annexins and dynamin are important for sequential steps in myoblast fusion.

Myoblast fusion into multinucleated myotubes is a crucial step in skeletal muscle development and regeneration. Here, we accumulated murine myoblasts at the ready-to-fuse stage by blocking formation of early fusion intermediates with lysophosphatidylcholine. Lifting the block allowed us to explore a largely synchronized fusion. We found that initial merger of two cell membranes detected as lipid mixing involved extracellular annexins A1 and A5 acting in a functionally redundant manner. Subsequent stages of myoblast fusion depended on dynamin activity, phosphatidylinositol(4,5)bisphosphate content, and cell metabolism. Uncoupling fusion from preceding stages of myogenesis will help in the analysis of the interplay between protein machines that initiate and complete cell unification and in the identification of additional protein players controlling different fusion stages.

Communication: Clusters of absorbing disks on a reflecting wall: competition for diffusing particles.

Trapping of diffusing particles by a cluster of absorbing disks on the otherwise reflecting wall is a manifestly many-body problem because of the disk competition for the particles. By replacing the cluster with an effective uniformly absorbing spot, we derive a simple formula for the rate constant that characterizes the trapping. The formula shows how the rate constant depends on the size and shape of the cluster.

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice.

Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and T-tubules and analyzed insulin-stimulated exocytosis at the single vesicle level by total internal reflection fluorescence and confocal microscopy. We found that only 10% of the intracellular GLUT4 pool comprised mobile vesicles, whereas most of the GLUT4 structures remained stationary or tethered at the sarcolemma or T-tubules. In fact, most of the insulin-stimulated exocytosis emanated from pretethered vesicles, whereas the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action but rather locally distributed. Most likely, pretethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis.

Long, saturated chains: tasty domains for kinases of insulin resistance.

The mechanistic basis of how cells respond to increased fatty acids (FAs) is murky but potentially involves receptor-mediated activation or inhibition by different FA classes. Holzer et al. (2011) recently propose in Cell that expansion of intracellular membrane microdomains induced by saturated FA recruit and activate c-Src for JNK activation.

Functional role for transporter isoforms in optimizing membrane transport.

Quantitative analysis of carrier parameters demonstrates that with decreasing substrate concentration the optimal strength of substrate-carrier interaction which maximizes the flux across the membrane increases and requires less fine-tuning than at higher concentrations of the substrate.

Insulin controls the spatial distribution of GLUT4 on the cell surface through regulation of its postfusion dispersal.

While the glucose transporter-4 (GLUT4) is fundamental to insulin-regulated glucose metabolism, its dynamic spatial organization in the plasma membrane (PM) is unclear. Here, using multicolor TIRF microscopy in transfected adipose cells, we demonstrate that insulin regulates not only the exocytosis of GLUT4 storage vesicles but also PM distribution of GLUT4 itself. In the basal state, domains (clusters) of GLUT4 molecules in PM are created by an exocytosis that retains GLUT4 at the fusion site. Surprisingly, when insulin induces a burst of GLUT4 exocytosis, it does not merely accelerate this basal exocytosis but rather stimulates approximately 60-fold another mode of exocytosis that disperses GLUT4 into PM. In contradistinction, internalization of most GLUT4, regardless of insulin, occurs from pre-existing clusters via the subsequent recruitment of clathrin. The data fit a new kinetic model that features multifunctional clusters as intermediates of exocytosis and endocytosis.

A common trafficking route for GLUT4 in cardiomyocytes in response to insulin, contraction and energy-status signalling.

A new mouse model has been developed to study the localisation and trafficking of the glucose transporter GLUT4 in muscle. The mouse line has specific expression of a GFP and HA-epitope-tagged version of GLUT4 under the control of a muscle-specific promoter. The exofacial HA-tag has enabled fluorescent labelling of only the GLUT4 exposed at the external surface. A distinction between sarcolemma labelling and transverse-tubule labelling has also been possible because the former compartment is much more accessible to intact anti-HA antibody. By contrast, the Fab fragment of the anti-HA antibody could readily detect GLUT4 at the surface of both the sarcolemma and transverse tubules. Here, we have used this mouse model to examine the route taken by cardiomyocyte GLUT4 as it moves to the limiting external membrane surface of sarcolemma and transverse-tubules in response to insulin, contraction or activators of energy-status signalling, including hypoxia. HA-GLUT4-GFP is largely excluded from the sarcolemma and transverse-tubule membrane of cardiomyocytes under basal conditions, but is similarly trafficked to these membrane surfaces after stimulation with insulin, contraction or hypoxia. Internalisation of sarcolemma GLUT4 has been investigated by pulse-labelling surface GLUT4 with intact anti-HA antibody. At early stages of internalisation, HA-tagged GLUT4 colocalises with clathrin at puncta at the sarcolemma, indicating that in cells returning to a basal state, GLUT4 is removed from external membranes by a clathrin-mediated route. We also observed colocalisation of GLUT4 with clathrin under basal conditions. At later stages of internalisation and at steady state, anti-HA antibody labeled-GLUT4 originating from the sarcolemma was predominantly detected in a peri-nuclear compartment, indistinguishable among the specific initial stimuli. These results taken together imply a common pathway for internalisation of GLUT4, independent of the initial stimulus.

Insulin regulates fusion of GLUT4 vesicles independent of Exo70-mediated tethering.

Insulin regulates cellular glucose uptake by changing the amount of glucose transporter-4 (GLUT4) in the plasma membrane through stimulation of GLUT4 exocytosis. However, how the particular trafficking, tethering, and fusion steps are regulated by insulin is still debated. In a 3T3-L1 adipocyte cell line, the Exocyst complex and its Exo70 subunit were shown to critically affect GLUT4 exocytosis. Here we investigated the effects of Exo70 on tethering and fusion of GLUT4 vesicles in primary isolated rat adipose cells. We found that Exo70 wild type was sequestered away from the plasma membrane in non-stimulated cells, and its overexpression had no effect on GLUT4 trafficking. The addition of insulin increased the amount of Exo70 in the vicinity of the plasma membrane and stimulated the tethering and fusion of GLUT4 vesicles, but the rates of fusion and GLUT4 exposure were not affected by overexpression of Exo70. Surprisingly, the Exo70-N mutant induced insulin-independent tethering of GLUT4 vesicles, which, however, did not lead to fusion and exposure of GLUT4 at the plasma membrane. Upon insulin stimulation, the stationary pretethered GLUT4 vesicles in Exo70-N mutant cells underwent fusion without relocation. Taken together, our data suggest that fusion of GLUT4 vesicles is the rate-limiting step regulated by insulin downstream of Exo70-mediated tethering.

Cellular biophysics: bacterial endospore, membranes and random fluctuation.

Purposeful motion of biological processes can be driven by Brownian motion of macromolecular complexes with one-sided binding biasing movement in one direction: a Brownian ratchet, now proposed to explain membrane motion during a phagocytosis-like process in bacteria.

Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.

Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

Shape control through molecular segregation in giant surfactant aggregates.

Mixtures of cationic and anionic surfactants crystallized at various ratios in the absence of added salt form micrometer-sized colloids. Here, we propose and test a general mechanism explaining how this ratio controls the shape of the resulting colloidal structure, which can vary from nanodiscs to punctured planes; during cocrystallization, excess (nonstoichiometric) surfactant accumulates on edges or pores rather than being incorporated into crystalline bilayers. Molecular segregation then produces a sequence of shapes controlled by the initial mole ratio only. Using freeze-fracture electron microscopy, we identified three of these states and their corresponding coexistence regimes. Fluorescence confocal microscopy directly showed the segregation of anionic and cationic components within the aggregate. The observed shapes are consistently reproduced upon thermal cycling, demonstrating that the icosahedral shape corresponds to the existence of a local minimum of bending energy for facetted icosahedra when the optimal amount of excess segregated material is present.

Shape bistability of a membrane neck: a toggle switch to control vesicle content release.

Shape dynamics and permeability of a membrane neck connecting a vesicle and plasma membrane are considered. The neck is modeled by a lipid membrane tubule extended between two parallel axisymmetric rings. Within a range of lengths, defined by system geometry and mechanical properties of the membrane, the tubule has two stable shapes: catenoidal microtubule and cylindrical nanotubule. The permeabilities of these two shapes, measured as ionic conductivity of the tubule interior, differ by up to four orders of magnitude. Near the critical length the transitions between the shapes occur within less than a millisecond. Theoretical estimates show that the shape switching is controlled by a single parameter, the tubule length. Thus the tubule connection can operate as a conductivity microswitch, toggling the release of vesicle content in such cellular processes as "kiss-and-run" exocytosis. In support of this notion, bistable behavior of membrane connections between vesicles and the cell plasma membrane in macrophages is demonstrated.