PubMed Health⌕ Search

Biomedical subjects

M P Sheetz

Publications and source records attributed to M P Sheetz.

At least 91 records · Page 5Linked to original sources
PubMed1993

Concomitant changes in mitochondria and intermediate filaments during heat shock and recovery of chicken embryo fibroblasts.

Utilizing video-enhanced differential interference contrast microscopy of chicken embryo fibroblasts, we observed dramatic changes in the localization and morphology of mitochondria shortly after cells were subjected to a mild heat shock. At normal temperatures mitochondria were distributed in the cell cytoplasm as elongated, tubular, and dynamic organelles but upon heat shock they moved to the perinuclear region and formed a tight ring of short swollen and--in some cases--fused vesicles. Vital dye staining of mitochondria with rhodamine 123 and indirect immunofluorescence staining with antibodies against the mitochondrial-matrix protein, HSP 60, confirmed these results. Using cells double labeled with antibodies to vimentin and the HSP 60 protein, we found that the changes in mitochondria were accompanied by perturbations of the intermediate filament network that we and others have reported previously for heat shocked cells. Microtubules remained largely unaltered by our heat shock treatment and the redistribution of intermediate filaments and mitochondria occurred even in the presence of taxol, a microtubule stabilizing drug. The effects of heat shock on mitochondria were reversed when cells were returned to normal temperatures and their recovery to their normal state coincided with return of normal intermediate filament morphology. This recovery was blocked in cells treated with actinomycin D during heat shock, a result indicating that a heat shock protein may be required for recovery. These data are consistent with previously published observations that mitochondria are associated with the intermediate filament network but they extend this interaction to a cell system responding to a physiological stress normally experienced by the intact organism.

Animals↗
PubMed1993

Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated.

We have used laser optical trapping and nanometer-level motion analysis to investigate the cytoskeletal associations and surface dynamics of beta 1 integrin, a cell-substrate adhesion molecule, on the dorsal surfaces of migrating fibroblast cells. A single-beam optical gradient trap (laser tweezers) was used to restrain polystyrene beads conjugated with anti-beta 1 integrin mAbs and place them at desired locations on the cell exterior. This technique was used to demonstrate a spatial difference in integrin-cytoskeleton interactions in migrating cells. We found a distinct increase in the stable attachment of beads, and subsequent rearward flow, on the lamellipodia of locomoting cells compared with the retracting portions. Complementary to the enhanced linkage of integrin at the cell lamellipodium, the membrane was more deformable at the rear versus the front of moving cells while nonmotile cells did not exhibit this asymmetry in membrane architecture. Video microscopy and nanometer-precision tracking routines were used to study the surface dynamics of integrin on the lamellipodia of migrating cells by monitoring the displacements of colloidal gold particles coated with anti-beta 1 integrin mAbs. Small gold aggregates were rapidly transported preferentially to the leading edge of the lamellipod where they resumed diffusion restricted along the edge. This fast transport was characterized by brief periods of directed movement ("jumps") having an instantaneous velocity of 37 +/- 15 microns/min (SD), separated by periods of diffusion. In contrast, larger aggregates of gold particles and the large latex beads underwent slow, steady rearward movement (0.85 +/- 0.44 micron/min) (SD) at a rate similar to that reported for other capping events and for migration of these cells. Cell lines containing mutated beta 1 integrins were used to show that the cytoplasmic domain is essential for an asymmetry in attachment of integrin to the underlying cytoskeletal network and is also necessary for rapid, intermittent transport. However, enhanced membrane deformability at the cell rear does not require integrin-cytoskeletal interactions. We also demonstrated that posttranslational modifications of integrin could potentially play a role in these phenomena. These results suggest a scheme for the role of dynamic integrin-mediated adhesive interactions in cell migration. Integrins are transported preferentially to the cell front where they form nascent adhesions. These adhesive structures grow in size and associate with the cytoskeleton that exerts a rearward force on them. Dorsal aggregates more rearward while those on the ventral side remain fixed to the substrate allowing the cell body to move forward. Detachment of the cell rear occurs by at least two modes: (a) weakened integrin-cytoskeleton interactions, potentially mediated by local modifications of linkage proteins, which lead to weakened cell-substratum interactions and (b) ripping of integrins and the highly deformable membrane from the cell body.

3T3 Cells↗
PubMed1993

Glycoprotein motility and dynamic domains in fluid plasma membranes.

The diffusion measurements of glycoproteins have further supported a fluid mosaic model of membrane structure, but the basis of the lower apparent diffusion coefficients in biological membranes remains incompletely understood. In the specific case of glycoproteins with a single alpha-helix spanning the membrane, studies indicate that the major frictional drag is in the external protein layer and not the bilayer. Only in the erythrocyte membrane does the internal protein layer clearly control the lateral diffusion coefficient of a glycoprotein with a large cytoplasmic domain. In cultured cells, the barriers to lateral displacements over long distances are primarily on the cytoplasmic surface and not in the external matrix. Active movements of individual or small groups of glycoproteins both forward and rearward on cells appear to result from the interactions with moving cytoskeletal structures. Membrane turnover as well as transient attachment to the cytoskeleton can produce dynamic domains in the membrane that would depend on motile activity. Recent technological advances enable simultaneous monitoring of specific cell functions and glycoprotein motility, making it possible to correlate membrane fluidity and active glycoprotein movements with cell function.

Animals↗
PubMed1993

Lytic granules from cytotoxic T cells exhibit kinesin-dependent motility on microtubules in vitro.

One major mechanism of cell-mediated cytolysis is the polarized secretion of lytic granules, a process which is highly dependent on microtubules. We isolated lytic granules from murine cytotoxic T cells and tested their ability to bind to and move along microtubules in vitro. In the presence of a motor-containing supernatant, the granules bound to the microtubules and moved along them at an average maximal rate of 1 microns/second. Virtually every granule could bind to microtubules, and about half translocated within a few seconds of binding. Motility required exogenous cytosolic motors, hydrolyzable nucleotides, and an intact granule membrane. Although the motor preparation used to support granule movement contains both plus- and minus-end-directed motor proteins, granule movement was strongly biased toward microtubule plus-ends. Inactivation of cytoplasmic dynein had little effect on granule binding and movement, but immuno-depletion of kinesin from the motor preparation inhibited granule binding by 50%. These results indicate that most granule movement in this assay is mediated by kinesin. The speed and direction of granule movement in vitro are sufficient to account for the release of lytic granules in the intact T cell. This model system should be valuable for studying the interactions of secretory granules with microtubules, and for identifying the regulatory factors involved.

Animals↗
PubMed1993

Regulation of vesicle transport in CV-1 cells and extracts.

To investigate the regulation of microtubule-dependent vesicle motility, we have studied the effects of pharmacological agents on the frequency and velocity of small vesicle movements in intact CV-1 cells. Nocodazole, but not cytochalasin B or D, abolished vesicle movements, indicating that these movements were microtubule and not actin-dependent. The frequency of vesicle movements was stimulated maximally sixfold by okadaic acid from a resting value of 1.6 movements/min per microns2 in serum-starved CV-1 cells. Other activators in decreasing order of effectiveness are fetal calf serum, dibutyryl cAMP, cholera toxin, genistein, A23187, and trental. On the other side, taxol inhibited vesicle movements by a factor of four. The activators, okadaic acid, fetal calf serum, and dibutyryl cAMP, also increased vesicle velocity and run length, while taxol decreased vesicle velocity. Although modulation of the frequency of vesicle movements over a > 20-fold range was observed, under all conditions the fraction of vesicles moving inward versus outward did not significantly change. Only in the case of taxol was the distribution of microtubules altered within this same time period. Both inward and outward microtubule-dependent vesicle movements therefore appear to be coordinately regulated. The enhanced vesicle motility elicited by fetal calf serum in intact cells correlated with in vitro measurements of vesicle motility and velocity on purified microtubules using microtubule affinity-purified motors and carbonate-washed vesicles from cells treated with fetal calf serum. This suggests that the amount of vesicular intracellular membrane traffic is coordinately regulated with microtubule-dependent motor activity.

Adenylyl Cyclases↗
PubMed1992

Kinesin and cytoplasmic dynein binding to brain microsomes.

Movement of cellular organelles in a directional manner along polar microtubules is driven by the motor proteins, kinesin and cytoplasmic dynein. The binding of these proteins to a microsomal fraction from embryonic chicken brain is investigated here. Both motors exhibit saturation binding to the vesicles, and proteolysis of vesicle membrane proteins abolishes binding. The maximal binding for kinesin is 12 +/- 1.7 and 43 +/- 2 pmol per mg of vesicle protein with or without 1 mM ATP, respectively. The maximal binding for cytoplasmic dynein is 55 +/- 3.8 and 73 +/- 3.7 pmol per mg of vesicle protein with or without ATP, respectively. These values correspond to 1-6 sites per vesicle of 100-nm diameter. The nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate (AMP-PNP), inhibited kinesin binding to vesicles but increased kinesin binding to microtubules. An antibody to the kinesin light chain also inhibited vesicle binding to kinesin. In the absence but not presence of ATP, competition between the two motors for binding was observed. We suggest that there are two distinguishable binding sites for kinesin and cytoplasmic dynein on these organelles in the presence of ATP and a shared site in the absence of ATP.

Adenosine Triphosphate↗
PubMed1992

Extension of filopodia by motor-dependent actin assembly.

A variety of mechanisms have been proposed to explain the forward extension of cytoplasm in advancing cells and axonal growth cones, including actin polymerization and osmotic swelling. Based on our observations of the filopodia of cultured neuronal growth cones, we propose a mechanism involving motor-induced extension and retraction. We observed that filopodia (actin-based protrusions 0.2-0.5 mu in diameter) extend and retract from growth cone lamellae at the same rate. Further, force is generated at the tips of filopodia which is sufficient to produce compressive buckling of the proximal portion of the filopodium. From our analysis of these movements we suggest that a motor protein powers both the extension and retraction of filopodia.

Actins↗
PubMed1992

Kinectin, a major kinesin-binding protein on ER.

Previous studies have shown that microtubule-based organelle transport requires a membrane receptor but no kinesin-binding membrane proteins have been isolated. Chick embryo brain microsomes have kinesin bound to their surface, and after detergent solubilization, a matrix with an antibody to the kinesin head domain (SUK-4) (Ingold et al., 1988) bound the solubilized kinesin and retained an equal amount of a microsome protein of 160-kD. Similarly, velocity sedimentation of solubilized membranes showed that kinesin and the 160-kD polypeptide cosedimented at 13S. After alkaline treatment to remove kinesin from the microsomes, the same 160-kD polypeptide doublet bound to a kinesin affinity resin and not to other proteins tested. Biochemical characterization localized this protein to the cytoplasmic face of brain microsomes and indicated that it was an integral membrane protein since it was resistant to alkaline washing. mAbs raised to chick 160-kD protein demonstrated that it was absent in the supernatant and concentrated in the dense microsome fraction. The dense microsome fraction also had the greatest amount of microtubule-dependent motility. With immunofluorescence, the antibodies labeled the ER in chick embryo fibroblasts (similar to the pattern of bound kinesin staining in the same cells) (Hollenbeck, P. J. 1989. J. Cell Biol. 108:2335-2342), astroglia, Schwann cells and dorsal root ganglion cells but staining was much less in the Golgi regions of these cells. Because this protein is a major kinesin-binding protein of motile vesicles and would be expected to bind kinesin to the organelle membrane, we have chosen the name, kinectin, for this protein.

Animals↗
PubMed1992

Can indirect cost funding be improved?

Like the tax laws many possible loopholes are in these proposed changes and therefore they have to be considered very carefully. If there is such a thing as a collective wisdom, it should be brought to bear on this problem. With strong and well-considered actions, which would be implemented in a controlled way, the mechanisms for funding research could be improved to encourage efficient use of funds.

Costs and Cost Analysis↗
PubMed1991

Lateral movements of membrane glycoproteins restricted by dynamic cytoplasmic barriers.

Cell membranes often are patchy, composed of lateral domains. These domains may be formed by barriers within or on either side of the membrane bilayer. Major histocompatibility complex (MHC) class 1 molecules that were either transmembrane- (H-2Db) or glycosylphosphatidylinositol (GPI)-anchored (Qa2) were labeled with antibody-coated gold particles and moved across the cell surface with a laser optical tweezers until they encountered a barrier, the barrier-free path length (BFP). At room temperature, the BFPs of Qa2 and H-2Db were 1.7 +/- 0.2 and 0.6 +/- 0.1 (micrometers +/- SEM), respectively. Barriers persisted at 34 degrees C, although the BFP for both MHC molecules was fivefold greater at 34 degrees C than at 23 degrees C. This indicates that barriers to lateral movement are primarily on the cytoplasmic half of the membrane and are dynamic.

Animals↗
PubMed1991

Transcription by single molecules of RNA polymerase observed by light microscopy.

The kinetics of transcription by Escherichia coli RNA polymerase relate directly to the regulation of transcription and to the properties of processive enzymes in general, but analysis of RNA polymerase movement along the DNA template has so far been limited to the study of populations of enzyme molecules. The ability to view nanometre-sized particles with the light microscope suggested a method of monitoring transcription by individual RNA polymerase molecules. We describe here the behaviour of 40-nm-diameter particles of colloidal gold attached to the ends of DNA molecules being transcribed by RNA polymerase immobilized on a glass surface. The tethered gold particles are released from the surface at times after addition of nucleoside triphosphates that are consistent with the kinetics of transcription by RNA polymerase in solution. Analysis of the brownian motion of the gold particles enabled us to measure the movement along the template DNA of individual polymerase molecules.

Bacterial Proteins↗
PubMed1991

Axonal transport: beyond kinesin and cytoplasmic dynein.

In vitro and in vivo studies of specific neuronal fast and slow transport components are presently reshaping our understanding of how the processes of vesicular and cytoskeletal transport are regulated in axons and dendrites. Evidence suggests that vesicles possess an inherent directionality, possibly the result of their motor receptor proteins responding to intracellular cues, which then allows movement with either kinesin or cytoplasmic dynein.

Animals↗
PubMed1991

Single particle tracking. Analysis of diffusion and flow in two-dimensional systems.

Analysis of the trajectories of small particles at high spatial and temporal resolution using video enhanced contrast microscopy provides a powerful approach to characterizing the mechanisms of particle motion in living cells and in other systems. We present here the theoretical basis for the analysis of these trajectories for particles undergoing random diffusion and/or systematic transport at uniform velocity in two-dimensional systems. The single particle tracking method, based on observations of the trajectories of individual particles, is compared with methods that characterize the motions of a large collection of particles such as fluorescence photobleaching recovery. Determination of diffusion coefficients or transport velocities either from correlation of positions or of velocities of the particles is discussed. A result of practical importance is an analysis of the dependence of the expected statistical uncertainty of these determinations on the number of position measurements. This provides a way of judging the accuracy of the diffusion coefficients and transport velocities obtained using this approach.

Diffusion↗