calyculin-a and jasplakinolide

We report advances in quantitative fluorescent speckle microscopy to generate simultaneous maps of cytoskeleton flow and rates of net assembly and disassembly in living cells. We apply this tool to analyze the filamentous actin (F-actin) dynamics at the front of migrating cells. F-actin turnover and flow are both known to be factors of cell locomotion. However, how they are orchestrated to produce directed cell movements is poorly understood. Our approach to data analysis allows us to examine their interdependence. Our maps confirm the previously described organization of flow into a lamellipodium and a lamellum, both exhibiting retrograde flow; and a convergence zone, where lamellum retrograde flow meets with slow anterograde flow of cortical F-actin at the ventral side of the cell body. The turnover maps show the well known actin polymerization at the leading edge, but also indicate that approximately 90% of the polymer disassembles at the lamellipodium-lamellum junction. Strong depolymerization is also found in the convergence zone, where meshwork contraction is prominent. To determine whether contraction and depolymerization are coupled events, we have treated cells with calyculin A, which is known to promote myosin activity. Stimulated contraction was accompanied by accelerated retrograde flow and increased depolymerization throughout the lamellum, whereas disassembly at the lamellipodium-lamellum junction remained unaffected. There appear to be two distinct depolymerization mechanisms, of which one depends directly on meshwork contraction.

Topics: Actins; Animals; Biopolymers; Cell Movement; Cells, Cultured; Cytoplasmic Structures; Cytoskeleton; Cytotoxins; Depsipeptides; Epithelial Cells; Lung; Marine Toxins; Microscopy, Fluorescence; Oxazoles; Peptides, Cyclic; Perfusion; Pseudopodia; Reproducibility of Results; Salamandridae; Time Factors

Na(+)/Ca(2+) exchange activity was studied in transfected Chinese hamster ovary (CHO) cells expressing the wild-type cardiac exchanger (NCX1.1) or mutants created by site-directed mutagenesis. The activity of the wild-type exchanger, but not exchanger mutants deficient in Ca(2+)-dependent activation, was inhibited by sphingolipids such as ceramide and sphingosine. We propose that sphingolipids interfere with the regulatory activation of exchange activity by Ca(2+) and suggest that this interaction provides a means for monitoring and regulating diastolic Ca(2+) levels in beating cardiac myocytes. Exchange activity in CHO cells was also linked, through a poorly understood feedback mechanism, to Ca(2+) accumulation within internal stores such as the endoplasmic reticulum and the mitochondria. Finally, the F-actin cytoskeleton was shown to modulate exchange activity through interactions involving the exchanger's central hydrophilic domain. We conclude that regulation of exchange activity in intact cells involves multiple interactions with various lipid species, cytosolic Ca(2+), organellar Ca(2+) stores, and the cytoskeleton.

Topics: Animals; Axons; Calcium; Calcium Channels; CHO Cells; Cricetinae; Cytosol; Decapodiformes; Depsipeptides; Heart; Marine Toxins; Mitochondria; Mutagenesis, Site-Directed; Myocardial Contraction; Oxazoles; Peptides, Cyclic; Recombinant Proteins; Sodium; Sodium Channels; Sodium-Calcium Exchanger; Sphingosine; Transfection

We previously reported that glucose can be released from GLUT2-null hepatocytes through a membrane traffic-based pathway issued from the endoplasmic reticulum. Here, we further characterized this glucose release mechanism using biosynthetic labeling protocols. In continuous pulse-labeling experiments, we determined that glucose secretion proceeded linearly and with the same kinetics in control and GLUT2-null hepatocytes. In GLUT2-deficient hepatocytes, however, a fraction of newly synthesized glucose accumulated intracellularly. The linear accumulation of glucose in the medium was inhibited in mutant, but not in control, hepatocytes by progesterone and low temperature, as previously reported, but, importantly, also by microtubule disruption. The intracellular pool of glucose was shown to be present in the cytosol, and, in pulse-chase experiments, it was shown to be released at a relatively slow rate. Release was not inhibited by S-4048 (an inhibitor of glucose-6-phosphate translocase), cytochalasin B, or progesterone. It was inhibited by phloretin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and low temperature. We conclude that the major release pathway segregates glucose away from the cytosol by use of a membrane traffic-based, microtubule-dependent mechanism and that the release of the cytosolic pool of newly synthesized glucose, through an as yet unidentified plasma membrane transport system, cannot account for the bulk of glucose release.

Topics: Animals; Antiporters; Calcium; Carbon Radioisotopes; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Cholesterol; Culture Media; Cytochalasin B; Depsipeptides; Endoplasmic Reticulum; Energy Metabolism; Glucose; Glucose Transporter Type 2; Glucose-6-Phosphate; Hepatocytes; Kinetics; Marine Toxins; Mice; Mice, Knockout; Monosaccharide Transport Proteins; Nocodazole; Oxazoles; Peptides, Cyclic; Phloretin; Phosphotransferases; Progesterone; Uncoupling Agents

One popular model for the activation of store-operated Ca2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP3) receptors on the stores to bind to, and thus activate, store-operated Ca2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I(CRAC), the most widely distributed and best characterised store-operated Ca2+ current, in a model system, the RBL-1 rat basophilic cell line. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I(CRAC). Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I(CRAC) was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores. Pre-treatment with the membrane-permeable InsP3 receptor antagonist 2-APB blocked the activation of I(CRAC). However, intracellular dialysis with 2-APB failed to prevent I(CRAC) from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I(CRAC) had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La3+. Studies with the more conventional InsP3 receptor antagonist heparin revealed that occupation of the intracellular InsP3-sensitive receptors was not necessary for the activation or maintenance of I(CRAC). Similarly, the InsP3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I(CRAC). Exposure to Li+, which reduces InsP3 levels by interfering with inositol monophosphatase, also failed to alter I(CRAC). Caffeine and Li+ did not affect the size of the intracellular Ca2+ signal that arose when external Ca2+ was re-admitted to cells

Topics: Animals; Basophils; Boron Compounds; Caffeine; Calcium; Calcium Channels; Calcium Signaling; Cell Line; Cell Size; Cytochalasin D; Cytoskeleton; Depsipeptides; Enzyme Inhibitors; Heparin; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Lithium; Marine Toxins; Microscopy, Fluorescence; Nocodazole; Oxazoles; Patch-Clamp Techniques; Peptides, Cyclic; Rats; Receptors, Cytoplasmic and Nuclear; Thapsigargin; Time Factors

The nature of the mechanism underlying store-mediated Ca(2+) entry has been investigated in human platelets through a combination of cytoskeletal modifications. Inhibition of actin polymerization by cytochalasin D or latrunculin A had a biphasic time-dependent effect on Ca(2+) entry, showing an initial potentiation followed by inhibition of Ca(2+) entry. Moreover, addition of these agents after induction of store-mediated Ca(2+) entry inhibited the Ca(2+) influx mechanism. Jasplakinolide, which reorganizes actin filaments into a tight cortical layer adjacent to the plasma membrane, prevented activation of store-mediated Ca(2+) entry but did not modify this process after its activation. In addition, jasplakinolide prevented cytochalasin D-induced inhibition of store-mediated Ca(2+) entry. Calyculin A, an inhibitor of protein serine/threonine phosphatases 1 and 2 which activates translocation of existing F-actin to the cell periphery without inducing actin polymerization, also prevented activation of store-mediated Ca(2+) entry. Finally, inhibition of vesicular transport with brefeldin A inhibited activation of store-mediated Ca(2+) entry but did not alter this mechanism once initiated. These data suggest that store-mediated Ca(2+) entry in platelets may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, which shows close parallels to the events mediating secretion.

Topics: Actins; Biological Transport; Blood Platelets; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cell Membrane; Cytochalasin D; Cytoskeleton; Depsipeptides; Endoplasmic Reticulum; Humans; Inositol 1,4,5-Trisphosphate; Marine Toxins; Models, Biological; Oxazoles; Peptides, Cyclic; Thiazoles; Thiazolidines