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The Biophysics of Insulin-Glucose Effects in Muscle, Lipid Droplet Kinase Signalling, Calcium Control by Secretory Vesicles, Clustering Theories, Blast-Induced Traumatic Brain Injury, and of Dysferlinopathic Muscular Dystrophies

Joshua Zimmerberg, MD, PhD, Head, Section on Cellular and Membrane Biophysics
Paul S. Blank, PhD, Staff Scientist
Svetlana Glushakova, PhD, Staff Scientist
Vladimir A. Lizunov, MS, Research Fellow
Petr Chlanda, PhD, Visiting Fellow
Glen Humphrey, PhD, Guest Researcher
Ludmila Bezrukov, MS, Chemist
Hang Waters, MS, Biologist
Jane E. Farrington, MS, Contractor
Elena Mekhedov, MA, Contractor
Rea Ravin, PhD, Contractor
Mariam Ghochani, MS, Graduate Student

We study membrane mechanics, intracellular molecules, membranes, viruses, organelles, and cells in order to understand viral and parasite infection, exocytosis, apoptosis, the mechanism of immune protection by stem cells and their cytotoxic potential, and immune dysfunction in microgravity. The overall goals are to understand the mechanisms of cellular secretion and membrane remodeling at the physical, biophysical, and chemical levels. Protein secretion is the climax of the secretory pathway and operates in both constitutive and triggered ways and, to retrieve membrane components, endocytosis is equally important. These processes underlie many pathological processes, such as E. coli toxicosis, malarial infection, viral infections, and the failure of apoptosis in cancer.

This year, we focused on the following six topics. (i) We investigated the mechanism by which insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments; we introduced a muscle-specific transgenic mouse model, in which HA-GLUT4-GFP is expressed under the control of the MCK promoter, and found that HA-GLUT4-GFP translocates to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. (ii) We demonstrated that Ca²⁺ oscillations occur in sea urchin secretory vesicles, the oscillations have super-Poisson noise properties, and the super-Poisson component depends on the magnitude of the Ca²⁺ signal and p-type (Cav2.1) Ca²⁺ channel activity. (iii) We put forward a new hypothesis linking fatty acid–induced activation of c-Src and JNK to insulin resistance and inflammatory response. (iv) We 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. (v) To facilitate analysis of dysferlin, a large membrane-anchored protein required for maintenance of plasmalemmal integrity in muscle, we established a dysferlin-deficient myogenic cell line as a genetic model for dysferlinopathies. (vi) We developed a pneumatic device that delivers shock waves, similar to those known to induce blast-induced traumatic brain injury, within a chamber optimal for fluorescence microscopy.

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of mice.

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, relatively few studies have focused on GLUT4 trafficking in primary skeletal muscle cells.

We expressed recombinant GLUT4 reporters carrying extracellular epitope tags (e.g., myc, HA) and/or fluorescent markers (e.g. GFP) ectopically and analyzed them by fluorescence microscopy. Using multicolor total internal reflection fluorescence (TIRF) microscopy, we previously demonstrated that insulin can regulate the tethering and fusion of GLUT4 storage vesicles, as well as the post-fusion redistribution of GLUT4 molecules to the plasma membrane in adipose cells. However, in muscles cells, it still remains unclear which of these steps are regulated by insulin. Among the most widely used clonal muscle cell lines are rat L6 cells and mouse C2C12 cells, which can both differentiate from myoblasts into insulin-sensitive myotubes. L6 cells, in particular, have been extremely useful in studying the mechanism of insulin action and GLUT4 recycling. We introduced a transgenic mouse expressing the human GLUT4 glucose transporter engineered to encode both an HA tag in the extracellular domain and a fluorescent protein (GFP) at the C-terminus on the cytoplasmic surface of muscle cells. Using this GLUT4 model, we could use the GFP tag to monitor GLUT4 trafficking in live muscle fibers and the HA tag to detect the insertion and exposure of GLUT4 at the cell surface. Using confocal and TIRF microscopy, we dissected the effect of insulin on GLUT4 trafficking and fusion. In contrast to adipose cells, insulin has little effect 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 pre-tethered GLUT4 vesicles, both at the sarcolemma and t-tubules. Taken together, the data suggest that, in skeletal muscles, insulin affects GLUT4 vesicle fusion and has little or no effect on the tethering of GLUT4 vesicles; the data also highlight the differences in insulin regulation of GLUT4 exocytosis between adipose cells and skeletal muscles.

Thus, while in adipose cells the main effect of insulin is to stimulate tethering/docking of GLUT4 vesicles to the plasma membrane, in muscles cells the majority of insulin-stimulated GLUT4 exocytosis occurs from pre-tethered/docked vesicles, with little effect on the mobile GLUT4 pool. This suggests that the major site of action of insulin in muscles is activation of the GLUT4 vesicle fusion machinery at the sarcolemma and T-tubular region rather than the release of GLUT4 from the intracellular retention cycle, as proposed for 3T3-L1 adipocytes. The low GLUT4 traffic intensity we observed in isolated FDB (flexor digitorum brevis) and soleus muscles, together with data from others, suggests that an overall redistribution of GLUT4 in response to insulin in skeletal muscle does not require significant relocation of GLUT4 vesicles from distant intracellular pools. Also, the rather long delay between recruitment of new GLUT4 vesicles and their fusion contrasts with GLUT4 translocation in adipose cells, in which the time of tethering preceding fusion is relatively short for the majority of GLUT4 fusion events. Together with the abundance of relatively stationary GLUT4 structures near the sarcolemma, the data suggest that the bulk of the insulin response may be achieved by local exocytosis of pre-tethered GLUT4 vesicles.

We propose that HA-GLUT4-GFP will be a useful model to study specific defects in GLUT4 translocation in muscle associated with metabolic disorders such as insulin resistance and type 2 diabetes.

Calcium oscillations in intracellular granules

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 on the amount of Ca²⁺ transported through the plasma membrane, Ca²⁺ released from intracellular organelles, and available endogenous buffering mechanisms. In addition to the established roles of the endoplasmic reticulum and mitochondria in regulating intracellular Ca²⁺, the involvement of the secretory vesicle has received increasing attention, in part, because the Ca²⁺ content of vesicles is high. The high intra-vesicular Ca²⁺ content could affect the local intracellular environment surrounding the vesicle because vesicles contain Ca²⁺ channels and other Ca²⁺ transport mechanisms—by supplying Ca²⁺ to critical sites close to the release machinery; even small changes in 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 affect exocytotic release. Thus, intra-vesicular Ca²⁺ dynamics may be an inherent vesicle property that is important for secretion and signaling.

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 between the Ca²⁺ dependence of their release and the release of synaptic and other secretory vesicles. We evaluated Ca²⁺ dynamics in fluorescently labeled sea urchin secretory vesicles using confocal microscopy. 71% of the vesicles examined exhibited one or more transient increases in the fluorescence signal, which were damped in time. The detection of transient increases in signal depended on the affinity of the fluorescence indicator; we estimated the free Ca²⁺ concentration in the secretory vesicles to be in the range of 10 to 100 μM. Non-linear stochastic analysis revealed extra variance in the Ca²⁺-dependent fluorescence signal. This noise process increased linearly with the amplitude of the Ca²⁺ signal. Both the magnitude and spatial properties of the noise process depended on the activity of the vesicles' p-type (Cav2.1) Ca²⁺ channels. Blocking the p-type Ca²⁺ channels with agatoxin reduced signal variance and altered the spatial noise pattern within the vesicle. The fluorescence signal properties are consistent with the vesicles' Ca²⁺ dynamics and not simply a result of obvious physical properties such as gross movement artifacts or pH–driven changes in Ca²⁺ indicator fluorescence. The results suggest that the free Ca²⁺ content of cortical secretory vesicles is dynamic, a property that may modulate the exocytotic fusion process and may play 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, plays a role in Ca²⁺ homeostasis and signaling.

Lipid droplets as signalling hubs

In this era of unprecedented caloric excess, we face an increased incidence of obesity, metabolic syndrome, and diabetes mellitus; natural selection has left us ill equipped for unrestricted food intake. The first adverse sign is insulin resistance/decreased 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, also known as 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 harbor the putative signaling domains accumulate intracellularly, like endosomes. We proposed that they included lipid droplets (LDs)—organelles composed of neutral lipids and covered with a phospholipid monolayer. LDs are induced upon FA uptake by cells with similar timescales. Dietary fat can induce new lipid droplets. Rich in saturated chains, these new monolayer surfaces could induce the accumulation of dually myristoylated and palmitoylated proteins via interaction of their saturated alkyl chains. Long-chain saturated fatty acids may also overload the sphingomyelin synthesis pathway and lead to accumulation of ceramides—lipid signaling molecules also capable of inducing insulin resistance. 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 signaling hub. It is conceivable that LDs enriched in saturated FAs possess biophysical properties positive for c-Src selectivity in a manner similar to that 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.

Cluster functions in diffusion

Trapping of diffusing particles by a cluster of absorbing disks on an otherwise reflecting wall is a manifestly many-body problem because of the disk competition for the particles. The key idea of our approach is to replace the cluster by an effective uniform spot, which is partially absorbing, and then to use a Collins-Kimball-like formula to find the rate constant. The formula shows how the rate constant depends on the size and shape of the cluster. The effective trapping rate of the spot, obtained by boundary homogenization, accounts for the many-body effects owing to the competition of the disks for diffusing particles.

Cell line for research on dysferlinopathic muscular dystrophies

Limb girdle muscular dystrophy 2b (LGMD2b) and Myoshi myopathy (MM) are late-onset muscular dystrophies caused by point mutations and deletions resulting in reduced levels, or absence, of the protein dysferlin. In dysferlinopathy patients, the sarcolemma displays characteristic abnormalities, including 0.1–2.0 μm discontinuities, thickened basal lamina, and accumulations of small vesicles at the sarcolemma, features suggesting that dysferlin is required for maintenance of sarcolemmal integrity. Dysferlin is a large (200 kDa) membrane-anchored protein with six C2 domains, with sequence similarity to synaptotagmins. By analogy to synaptotagmin's function as a possible calcium sensor in exocytosis, it has been proposed that dysferlin serves as a calcium sensor for membrane repair. Isolated wild-type mouse muscle fibers can reseal sarcolemmal wounds in the presence of 1 mM Ca²⁺, but fibers from a dysferlin knock-out mouse are defective in resealing, based on the unimpeded uptake of FM143 fluorescent dye following laser wounding.

Studies of muscle damage and repair in vivo suggest that dysferlin has other functions in addition to membrane resealing. The A/J mouse strain has a spontaneous dysferlin mutation, attributable to a retrotransposon insertion in intron 4 of the dysf gene, and no detectable dysferlin protein expression. A/J mice exhibit a progressive muscular dystrophy, appearing at two months, in the proximal muscles and spreading to the distal muscles by five months. A/J mice exhibit a defect in recovery from muscle injury caused by a single large strain-lengthening contraction. Large strain-lengthening contractions produce microtears in muscle fibers, which spontaneously reseal, based on retention of fluorescent dextran. A/J muscle fibers appear to reseal normally following injury, but become necrotic a few days later and must be replaced by new myogenesis.

To study membrane resealing in a model cell system, we developed a dysferlin-deficient cell line (GREG cells) from the A/J mouse strain. GREG cells have no detectable dysferlin expression but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. Thus, dysferlin is not required for either myoblast proliferation or fusion into myotubes in this cell line. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM143 dye. Under such wounding conditions, the majority (66%) of GREG myotubes lack the capacity to repair membranes, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. The extent of the membrane repair defect in GREG myotubes is variable; approximately 34% of the GREG myotubes exhibit membrane repair, compared with 100% of dysferlin-positive C2C12 myotubes. The heterogeneous membrane-resealing deficiency suggests that myotubes possess both dysferlin-dependent and -independent modes of membrane repair. Dysferlin-independent membrane repair could represent a genetic compensatory process operant in the presence of dysferlin deficiency.

Tramatic brain injury: a new model for microscopic examination of human brain cultures in blast-like pressure waves

Traumatic brain injury (TBI) is a major public health problem. Since 2001, over 150,000 US military personnel have been diagnosed with a mild form of TBI, often after exposure to an explosive blast (bTBI), with a spectrum of neurological and psychological deficits resulting from changes in brain function. The mechanism responsible for primary bTBI following a blast shock wave remains unknown. The mechanisms of the primary injury phase, a direct result of the shock wave generated by an explosion, are the least understood. The blast shock wave (BSW) of primary bTBI is a transient, solitary supersonic pressure wave with a rapid (sub-msec) increase in pressure (i.e., compression) followed by a more slowly developing (msec) rarefaction phase of low pressure (i.e., tension). In the majority of bTBI, the peak pressure is low; exposure to blasts estimated to create 10 atm peak pressure in the skull for a few milliseconds can result in death for unprotected persons. We asked the central question whether the BSW itself acts directly on human brain cells or indirectly through secondary shear stresses.

We attached a pneumatic device to one of 96 wells positioned on a microscope and varied the amplitude of the pressure transient with an adjustable quick-release plug. The characteristics of the wave-form are comparable with those recorded in open field blasts; the pressure wave-form profile closely resembles a classic Friedlander curve. We generated simulated blasts with rise times in the 0.1 msec range and a two-component falling phase: a fast component dropping below ambient pressure within 0.5 msec and a slower component returning to ambient pressure within 2 msecs; we examined pressures 5–15 atm above ambient pressure.

Our findings show that human brain cells in culture are indifferent to blast-induced fast transient BSW consisting of sub-msec rise time, positive peaks of up to 15 atm, followed by tensions of 0.2 atm, of msec total duration. Furthermore, we showed that the cells only respond with global elevations in intracellular free Ca²⁺ when sufficient shear forces are simultaneously induced with the pressure profiles. The results make it unlikely that the primary effect of a BSW on brain cells in vivo is a direct effect of the compression and tension forces created by the pressure transient. While the pressure transient created in our system is very similar to the classic Friedlander curve, it is possible that significant differences exist between the nature of the shear forces created by our system and those induced during a blast in vivo. Furthermore, we do not know the magnitudes of the low-pressure components that develop at brain cells during an actual blast. However, the observed correlations between cellular response and shear forces, and the lack of correlation with pressure, suggest that shear forces are likely involved in the primary injury phase of bTBI (see below).

Calcium has been implicated in the induction of neuronal death during TBI and stroke; calcium is elevated for long periods (days in cells surviving TBI and stroke). In our experiments, calcium is elevated transiently for short periods (seconds to a few minutes), and cell death does not occur even after 20 hours following this excitation. Thus, the mechanism of mild bTBI may differ from that in TBI and stroke injuries, which do lead to cell death. Brain cells exposed to blast wave profiles lacking shear forces had no calcium response, even at peak pressures up to 15 atm and trough pressures of 0.2 atm, suggesting that a shear-dependent mechanism of primary bTBI may involve mechano-sensitive channels, lipidic pores, or uniquely vulnerable regions of the neuronal plasma membrane, leading to activation of a small population of cells and subsequent amplification through cell-cell signaling. The high curvature stress at the necks of pre-synaptic and post-synaptic boutons or fine processes of astrocytes may be an example of vulnerable regions given that the curvature stress would add to the shear stress at those points, known to disassemble during homogenization.

The influence of a controlled shear stress on cells in general and neurons in particular has been investigated in a variety of model systems including a rotating cone, linear actuator, and micro-fluidic vacuum transfection. Using primary human brain cell cultures at the level of single and small networks of cells, we found that shear forces acting at cellular length scales, rather than changes in pressure, control intracellular calcium, the major activation parameter of CNS–derived cell culture. Rapid compression and positive tension alone have been ruled out as the origin of calcium-dependent cell-cell signaling following a BSW. It is now possible to evaluate both the pharmacology of the propagated calcium response associated with a blast in the presence of shear forces and the behavior of other cellular markers during varied blast conditions.

Additional Funding

Jain Foundation
Center for Neuroscience & Regenerative Medicine (CNRM) Award
Bench-to-Bedside Award
Supplementary Award for Dietary Supplements

Publications

Ravin R, Blank PS, Steinkamp A, Rappaport SM, Ravin N, Bezrukov L, Guerrero-Cazares H, Quinones-Hinojosa A, Bezrukov SM, Zimmerberg J. Shear forces during blast, not abrupt changes in pressure alone, generate calcium activity in human brain cells. PLoS One 2012;7:e39421.
Humphrey GW, Mekhedov E, Blank PS, de Morree A, Pekkurnaz G, Nagaraju K, Zimmerberg J. GREG cells, a dysferlin-deficient myogenic mouse cell line. Exp Cell Res 2012;318:127-135.
Berezhkovskii AM, Dagdug L, Lizunov VA, Zimmerberg J, Bezrukov SM. Communication: Clusters of absorbing disks on a reflecting wall: competition for diffusing particles. J Chem Phys 2012;136:211102.
Lizunov VA, Stenkula KG, Lisinski I, Gavrilova O, Yver DR, Chadt A, Al-Hasani H, Zimmerberg J, Cushman SW. Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice. Am J Physiol Endocrinol Metab 2012;302:E950-E960.
Raveh A, Valitsky M, Shani L, Coorssen JR, Blank PS, Zimmerberg J, Rahamimoff R. Observations of calcium dynamics in cortical secretory vesicles. Cell Calcium 2012;52:217-225.

Collaborators

Jens R. Coorssen, PhD, University of Western Sydney, Penrith, Australia
Samuel W. Cushman, PhD, Diabetes Branch, NIDDK, Bethesda, MD
Klaus Gawrisch, PhD, Laboratory of Membrane Biochemistry and Biophysics, NIAAA, Bethesda, MD
Hugo Guerrero-Cazares, MD, The Johns Hopkins University, Baltimore, MD
Samuel T. Hess, PhD, University of Maine, Orono, ME
Mary Kraft, PhD, University of Illinois at Urbana-Champaign, Urbana, IL
Kanneboyina Nagaraju, MD, Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC
Alfredo Quinones-Hinojosa, MD, The Johns Hopkins University, Baltimore, MD
Adi Raveh, Hebrew University-Hadassah Medical School, Jerusalem, Israel
Thomas S. Reese, MD, Laboratory of Neurobiology, NINDS, Bethesda, MD
Karin G. Stenkula, PhD, Diabetes Branch, NIDDK, Bethesda, MD

Contact

For more information, email zimmerbj@mail.nih.gov.

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