Biomedical subjects

D F Kucik

Publications and source records attributed to D F Kucik.

10 recordsLinked to original sources

PubMed2001

The microtubule cytoskeleton participates in control of beta2 integrin avidity.

Leukocyte avidity is regulated by cytoskeletal constraints, which keep beta(2) integrins in an inactive mode. Releasing these constraints results in increased lateral mobility and clustering of integrins, effectively activating adhesion. At least part of the constraint on beta(2) integrins is due to actin; whether other cytoskeletal components are involved has not previously been investigated. Microtubules are a candidate for control of integrin rearrangement, because they modulate focal adhesions, which are sites of interaction between integrins and the cytoskeleton. Here we report that both depolymerization of microtubules by colchicine or nocodazole and stabilization of microtubules by taxol increased the lateral mobility of beta(2) integrins, activating adhesion. Increased integrin mobility was accompanied by an increase in tyrosine phosphorylation of paxillin, a biochemical event associated with activation of beta(2) integrins. Further, C3 exoenzyme, an inhibitor of Rho, blocked induction of integrin mobility by nocodazole, but not by taxol, suggesting that there are multiple microtubule-dependent pathways to integrin rearrangement, only some of which require Rho activity. Taken together, our data suggest that a dynamic microtubule system is required to regulate integrin-cytoskeleton interactions. Furthermore, these data demonstrate that microtubules participate in control of integrin rearrangement, one of the earliest steps in activation of integrin-mediated adhesion.

Animals↗

PubMed2001

Identification and kinetic analysis of the interaction between Nck-2 and DOCK180.

Nck-2 is a newly identified adapter protein comprising three N-terminal SH3 domains and one C-terminal SH2 domain. We have identified in a yeast two-hybrid screen DOCK180, a signaling protein implicated in the regulation of membrane ruffling and migration, as a binding protein for Nck-2. Surface plasmon resonance analyses reveal that the second and the third SH3 domains interact with the C-terminal region of DOCK180. The interactions mediated by the individual SH3 domains, however, are much weaker than that of the full length Nck-2. Furthermore, a point mutation that inactivates the second or the third SH3 domain dramatically reduced the interaction of Nck-2 with DOCK180, suggesting that both SH3 domains contribute to the DOCK180 binding. A major Nck-2 binding site, which is recognized primarily by the third SH3 domain, has been mapped to residues 1819-1836 of DOCK180. Two additional, albeit much weaker, Nck-2 SH3 binding sites are located to DOCK180 residues 1793-1810 and 1835-1852 respectively. Consistent with the mutational studies, kinetic analyses by surface plasmon resonance suggest that two binding events with equilibrium dissociation constants of 4.15+/-1.9x10(-7) M and 3.24+/-1.9x10(-9) M mediate the binding of GST-Nck-2 to GST fusion protein containing the C-terminal region of DOCK180. These studies identify a novel interaction between Nck-2 and DOCK180. Furthermore, they provide a detailed analysis of a protein complex formation mediated by multiple SH3 domains revealing that tandem SH3 domains significantly enhance the weak interactions mediated by each individual SH3 domain.

Amino Acid Sequence↗

PubMed2001

Activation-enhanced alpha(IIb)beta(3)-integrin-cytoskeleton interactions outside of focal contacts require the alpha-subunit.

Integrins link the cell's cytoskeleton to the extracellular matrix, as well as to receptors on other cells. These links occur not only at focal contacts but also at smaller integrin-containing protein complexes outside of focal contacts. We previously demonstrated the importance of focal contact-independent integrin-cytoskeleton interactions of beta(2) integrins: activation of adhesion resulted from a release of integrins from cytoskeletal constraints. To determine whether changes in integrin-cytoskeleton interactions were related to activation of the integrin, we used single particle tracking to examine focal contact-independent cytoskeletal associations of alpha(IIb)beta(3)-integrin, in which activation results in a large conformational change. Direct activation of alpha(IIb)beta(3) by mutation did not mimic activation of lymphocytes with phorbol ester, because it enhanced integrin-cytoskeleton interactions, whereas activation of lymphocytes decreased them. Using additional integrin mutants, we found that both alpha- and beta-cytoplasmic domains were required for these links. This suggests that 1) both beta(2)- and beta(3)-integrins interact with the cytoskeleton outside of focal contacts; 2) activation of a cell and activation of an integrin are distinct processes, and both can affect integrin-cytoskeleton interactions; and 3) the role of the alpha-subunit in integrin-cytoskeleton interactions in at least some circumstances is more direct than generally supposed.

Amino Acid Motifs↗

PubMed1999

Weak dependence of mobility of membrane protein aggregates on aggregate size supports a viscous model of retardation of diffusion.

Proteins in plasma membranes diffuse more slowly than proteins inserted into artificial lipid bilayers. On a long-range scale (>250 nm), submembrane barriers, or skeleton fences that hinder long-range diffusion and create confinement zones, have been described. Even within such confinement zones, however, diffusion of proteins is much slower than predicted by the viscosity of the lipid. The cause of this slowing of diffusion on the micro scale has not been determined and is the focus of this paper. One way to approach this question is to determine the dependence of particle motion on particle size. Some current models predict that the diffusion coefficient of a membrane protein aggregate will depend strongly on its size, while others do not. We have measured the diffusion coefficients of membrane glycoprotein aggregates linked together by concanavalin A molecules bound to beads of various sizes, and also the diffusion coefficients of individual concanavalin A binding proteins. The measurements demonstrate at most a weak dependence of diffusion coefficient on aggregate size. This finding supports retardation by viscous effects, and is not consistent with models involving direct interaction of diffusing proteins with cytoskeletal elements.

Animals↗

PubMed1997

Blood substitutes: evolution and future applications.

The development of oxygen-carrying blood substitutes has progressed significantly in the last decade with phase I and phase II clinical trials of both hemoglobin-based and perfluorocarbon-based oxygen carriers nearing completion. As these products approach clinical use it is important for the laboratory medicine community to be aware of their effects on routine laboratory testing and the settings in which they might be used. Here we review the forces driving the development of oxygen-carrying blood substitutes, the clinical settings in which they might be used, the major categories of oxygen carriers in clinical trials, and the challenges faced by these products as they approach clinical use.

Animals↗

PubMed1996

Adhesion-activating phorbol ester increases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes.

Lymphocytes activate adhesion to intracellular adhesion mlecule 1 (ICAM-1) via leukocyte function associated antigen 1 (LFA-1), their major beta 2 integrin, in response to PMA (phorbol 12-myristate 13-acetate) without an increase in the number of receptors expressed. The molecular details of the mechanism are unknown. To determine the effect of PMA activation on LFA-1 movement within the plasma membrane, we used the single particle tracking technique to measure the diffusion rate of LFA-1 molecules on EBV-transformed B cells before and after PMA activation. Diffusion of LFA-1 on unactivated cells was restricted compared to CR1 (CD35), another transmembrane protein of equivalent size. PMA caused a 10-fold increase in the diffusion rate of LFA-1 without any effect on CD35. The increased LFA-1 motion induced by PMA was random, not directed, indicating that it was due to a release of constraints rather than the application of forces. The diffusion rates of LFA-1 are consistent with cytoskeletal attachment before and free diffusion after PMA. Cytochalasin D led to an equivalent increase in mobility and, at low doses, stimulated adhesion, implying that the nonadhesive state of LFA-1 is actively maintained by the lymphocyte cytoskeleton.

Biological Transport↗

PubMed1991

Actin tracks.

Explore the source record for details and available documents.

Actin Cytoskeleton↗

PubMed1991

Preferential attachment of membrane glycoproteins to the cytoskeleton at the leading edge of lamella.

The active forward movement of cells is often associated with the rearward transport of particles over the surfaces of their lamellae. Unlike the rest of the lamella, we found that the leading edge (within 0.5 microns of the cell boundary) is specialized for rearward transport of membrane-bound particles, such as Con A-coated latex microspheres. Using a single-beam optical gradient trap (optical tweezers) to apply restraining forces to particles, we can capture, move and release particles at will. When first bound on the central lamellar surface, Con A-coated particles would diffuse randomly; when such bound particles were brought to the leading edge of the lamella with the optical tweezers, they were often transported rearward. As in our previous studies, particle transport occurred with a concurrent decrease in apparent diffusion coefficient, consistent with attachment to the cytoskeleton. For particles at the leading edge of the lamella, weak attachment to the cytoskeleton and transport occurred with a half-time of 3 s; equivalent particles elsewhere on the lamella showed no detectable attachment when monitored for several minutes. Particles held on the cell surface by the laser trap attached more strongly to the cytoskeleton with time. These particles could escape a trapping force of 0.7 X 10(-6) dyne after 18 +/- 14 (sd) s at the leading edge, and after 64 +/- 34 (SD) s elsewhere on the lamella. Fluorescent succinylated Con A staining showed no corresponding concentration of general glycoproteins at the leading edge, but cytochalasin D-resistant filamentous actin was found at the leading edge. Our results have implications for cell motility: if the forces used for rearward particle transport were applied to a rigid substratum, cells would move forward. Such a mechanism would be most efficient if the leading edge of the cell contained preferential sites for attachment and transport.

Animals↗

PubMed1990

Cell migration does not produce membrane flow.

We have previously reported that rearward migration of surface particles on slowly moving cells is not driven by membrane flow (Sheetz, M. P., S. Turney, H. Qian, and E. L. Elson. 1989. Nature (Lond.). 340:284-288) and recent photobleaching measurements have ruled out any rapid rearward lipid flow (Lee, J., M. Gustafsson, D. E. Magnussen, and K. Jacobson. 1990. Science (Wash. DC.) 247:1229-1233). It was not possible, however, to conclude from those studies that a slower or tank-tread membrane lipid flow does not occur. Therefore, we have used the technology of single particle tracking to examine the movements of diffusing particles on rapidly locomoting fish keratocytes where the membrane current is likely to be greatest. The keratocytes had a smooth lamellipodial surface on which bound Con A-coated gold particles were observed either to track toward the nuclear region (velocity of 0.35 +/- 0.15 micron/s) or to diffuse randomly (apparent diffusion coefficient of [3.5 +/- 2.0] x 10(-10) cm2/s). We detected no systematic drift relative to the cell edge of particles undergoing random diffusion even after the cell had moved many micrometers. The average net particle displacement was 0.01 +/- 2.7% of the cell displacement. These results strongly suggest that neither the motions of membrane proteins driven by the cytoskeleton nor other possible factors produce a bulk flow of membrane lipid.

Animals↗

PubMed1989

Forward transport of glycoproteins on leading lamellipodia in locomoting cells.

In several types of locomoting cells, active rearward transport of particles on the cell surface has been observed and correlated with motility. No forward transport of particles has previously been reported, however. Here we report rapid forward transport of concanavalin A-coated gold particles on the dorsal surfaces of lamellipodia of fish epidermal keratocytes. These movements are active, not diffusive, and more rapid than either rearward particle transport or the rate of cell locomotion. We observed forward transport in migrating, but not in stationary cells, and could block the movement by treatment with cytochalasin D. These studies demonstrate for the first time that small numbers of glycoproteins can be actively transported on the surface of the cell to the front of the lamellipodium. We suggest that this mechanism transports proteins involved in cell locomotion, such as proteins necessary for adhesion, and could also produce an extensile force.

Animals↗