1. In this study, we have introduced a transgenic mouse expressing human GLUT4 engineered to encode both the HA tag in the extracellular domain and a fluorescent protein (GFP) at the COOH terminus on the cytoplasmic surface of muscle cells. Using this GLUT4 model, we were able to use the GFP tag to monitor GLUT4 trafficking in live muscle fibers as well as the HA tag to detect the insertion and exposure of GLUT4 at the cell surface. Using confocal and TIRF microscopy, we have dissected the effect of insulin on GLUT4 trafficking and fusion. In contrast to adipose cells, insulin has little effect of on the recruitment of GLUT4 vesicles from the interior of the skeletal muscle cell;rather, insulin-stimulated GLUT4 translocation was mostly driven by fusion of pretethered GLUT4 vesicles, at both the sarcolemma and T-tubules. All together, these data suggest that in skeletal muscles insulin affects GLUT4 vesicle fusion and has little or no effect on the tethering of GLUT4 vesicles. These data highlight the differences in insulin regulation of GLUT4 exocytosis in adipose cells and skeletal muscles. Muscle is a major direct contributor to mammalian systemic glucose homeostasis. It is now well established that insulin stimulates glucose transport in adipose and muscle cells through the translocation of glucose transporter 4 (GLUT4) from intracellular sites to the plasma membrane. However, whereas the molecular mechanism of GLUT4 translocation has been extensively studied in primary adipose cells and cultured adipocytes during the past years, relatively few studies have focused on GLUT4 trafficking in primary skeletal muscle cells. In part, this is due to the presence of abundant microfibrillar protein and large amounts of nuclei such that most fractionation protocols suffer from poor resolution for the analysis of the subcellular distribution of GLUT4 in skeletal muscle. Likewise, because of technical limitations, morphological analyses of GLUT4 in skeletal muscle by photolabeling techniques, immunofluorescence, and electron microscopy have not provided sufficient information about the kinetics and dynamics of GLUT4 recycling through the multiple intracellular compartments. 2. In this era of unprecedented caloric excess, we face increased incidences of obesity, metabolic syndrome, and diabetes mellitus;natural selection has left us ill equipped for unrestricted food. The first adverse sign is insulin resistancedecreased glucose transport into cells that is matched by an increase in serum insulin at the cost of elevated blood insulin, free fatty acids (FAs), and inflammatory mediators to maintain blood glucose homeostasis. Although the insulin receptor signaling cascade is redundant, with one insulin receptor substrate compensating for the loss of the other's function, c-Jun n-terminal kinase family members 1 and 2 (JNK, aka stress-activated protein kinases, a subset of mitogen-activated protein kinases), when activated, act as intracellular mediators of insulin resistance by disrupting both arms of this cascade. The cellular structures whose membranes harboring the putative signaling domains accumulate intracellularly, like endosomes. Could they include lipid droplets (LDs), organelles composed of neutral lipids and covered with a phospholipid monolayer? LD are induced upon FA uptake by cells with similar timescales. Notably, the LD monolayer also contains the typical raft markers flotilin-1 and caveolin-1, and the LD monolayer may function as a monolayer domain and a signaling hub. It is conceivable that LDs enriched in saturated FA may possess biophysical properties positive for c-Src selectivity in a similar way as suggested for rafts. However, LD monolayers derived from ER may have insufficient anionic lipid, despite sufficient phosphatidyl inositol (PI) for signaling. Nevertheless, saturated FA may differentially induce lipid droplets from membranes of the endosomal system, rich in anionic phospholipids (C. Jackson and K. Soni, personal communication). Thus the special domains for activation may be on endosomal-derived LD. 3. Many cell functions are regulated by the intracellular Ca2+ concentration, among them secretion/exocytosis/endocytosis (i.e. neurotransmitter release), fertilization, programmed cell death, and gene expression. The intracellular Ca2+ concentration, in turn, depends upon the amount of Ca2+ transported through the plasma membrane, the Ca2+ released from intracellular organelles, and the endogenous buffering mechanisms available. Among the intracellular organelles that can store and release Ca2+, the roles of the endoplasmic reticulum and the mitochondria have been established;the involvement of the secretory vesicle in the regulation of intracellular Ca2+ has received increasing attention, in part, because the Ca2+ content of vesicles is high. For example, the total Ca2+ content of the cholinergic synaptic vesicles of the electric ray is ∼120 mM. Of interest is whether this high Ca2+ content affects the local intracellular environment surrounding the vesicle. Intra-vesicular Ca2+ could affect the Ca2+ concentration adjacent to vesicles because vesicles contain Ca2+ channels and other Ca2+ transport mechanisms. These transport mechanisms may contribute to the function of the vesicle by supplying Ca2+ to critical sites close to the release machinery;even small changes of the local Ca2+ concentration can have a profound effect on the release of transmitter. It has been demonstrated that changes in the vesicular Ca2+ concentration can have effects on exocytotic release. Intra-vesicular Ca2+ dynamics may be an inherent vesicle property that is important to secretion and signaling. Some vesicle types have the necessary machinery to support dynamic Ca2+ behavior including Ca2+ oscillations. The dynamic behaviorr can be stimulated by inositol 1,4,5-trisphosphate (InsP3), and is pH and potassium dependent, showing many similarities to other internal storage compartments involved in Ca2+ release and uptake. At the cellular level, Ca2+ oscillations are also observed in both space and time, and the relationship between these dynamic properties, signaling and coupled biochemical pathways, is the subject of both experimental and theoretical study. At the cellular level, Ca2+ dynamics have been evaluated using deterministic, stochastic, and chaotic models. Large docked (i.e. stationary), fusion-ready secretory vesicles, amenable to confocal microscopy, are found in the sea urchin egg;there is also a striking similarity in the Ca2+ dependence of their release and the release of synaptic and other secretory vesicles. In this project we demonstrate that Ca2+ oscillations occur in sea urchin secretory vesicles and these oscillations have super-Poisson noise properties. The super-Poisson component is dependent upon the magnitude of the Ca2+ signal and p-type (Cav2.1) Ca2+ channel activity. These Ca2+ properties may have a role in the regulation of the secretory/exocytotic pathway. The results of these studies suggest an evolutionarily conserved vesicular Ca2+ handling mechanism that, along with those of the endoplasmic reticulum and mitochondria, has a role in Ca2+ homeostasis and signaling. 4. We have proposed a new approach to the calculation of the rate constant that characterizes trapping of diffusing particles by a cluster of identical circular, perfectly absorbing, non-overlapping disks located on the otherwise reflecting flat wall. The key idea of the approach is to replace the cluster by an effective uniform spot, which is partially absorbing, and then to use the Collins-Kimball-like formula, Eq. (2), to find the rate constant. The effective trapping rate of the spot, obtained by boundary homogenization, accounts for the many-body effects due to the competition of the disks for diffusi

Project Start
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Support Year
22
Fiscal Year
2012
Total Cost
$1,519,614
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Epstein, Jonathan A; Blank, Paul S; Searle, Brian C et al. (2016) ProteinProcessor: A probabilistic analysis using mass accuracy and the MS spectrum. Proteomics 16:2480-90
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Cabral, Wayne A; Ishikawa, Masaki; Garten, Matthias et al. (2016) Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta. PLoS Genet 12:e1006156
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Berezhkovskii, Alexander M; Dagdug, Leonardo; Lizunov, Vladimir A et al. (2014) Trapping by clusters of channels, receptors, and transporters: quantitative description. Biophys J 106:500-9

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