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ASCB ABSTRACTS: 2004| 2005 | 2006 | 2007 2005 Abstracts: Elucidating the Role of Myosin II Contraction in Adhesion Protein Dynamics and Maturation. A. S. Kirby,1,2 Y. Zilberman,1,3 D. B. Weibel,1,4 L. C. Kapitein,1,5 C. Waterman-Storer1,6; 1Physiology Course 2005, Marine Biological Laboratory, Woods Hole, MA, 2Cell Biology, Duke University, Durham, NC, 3Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel, 4Chemistry and Chemical Biology, Harvard University, Cambridge, MA, 5Physics and Astronomy Laser Center, Vrije University, Amsterdam, The Netherlands, 6Cell Biology, Scripps Research Institute, La Jolla, CA Presentation Number: 334 Poster Board Number: B289 Cells move by coupling forces generated in the actin cytoskeleton to the extracellular matrix via trans-membrane focal adhesions. RhoGTPase-regulated myosinII contraction of the cytoskeleton linked to adhesion induces integrin clustering and focal adhesion “maturation.” We hypothesize that tension-induced maturation of focal adhesions results in changes in protein composition and dynamics that may influence adhesion morphology, strength, and signaling. We first aimed to elucidate the relationship between contractility, maturation and protein binding/dissociation at focal adhesions. We performed FRAP of GFP-conjugated integin_5, FAK, paxillin, zyxin, talin, and vinculin expressed in mouse embryo fibroblasts. Protein dynamics fell into two categories, those with low fluorescence-recovery halftimes (t1/2) (<25s; talin, paxillin FAK and zyxin), and those more stably bound in adhesion sites (integrin_5, t1/2~100s; vinculin, t1/2~250s). Cells were treated with myosinII inhibitors to reduce contractility or transfected with activated Rac1 to induce immature focal complexes, and FRAP was performed on integrin_5, talin, and vinculin. Talin and integrin dynamics were relatively insensitive to these treatments, while t1/2 of GFP-vinculin was greatly reduced by both activated Rac1 (~80s) and myosinII inhibition (<50s). We next sought to determine how induction of myosinII contraction promotes organization of actin bundles and maturation of adhesions. Cells co-expressing cherry-actin and GFP-paxillin were treated with myosin inhibitors to disassemble focal adhesions. Contraction was induced by drug washout and cells were analyzed by time-lapse fluorescence microscopy. During contraction, no de-novo adhesion formation was seen, rather focal complexes matured into focal adhesions by fusion of small preexisting clusters that increased GFP-paxillin fluorescence over time. Nascent actin bundles appeared to grow from fusing focal complexes, as opposed to being recruited from preexisting f-actin. These results indicate that myosinII contraction promotes maturation of adhesions by induction of actin filament growth and tight vinculin binding. Interaction Between Prokaryotic Actin ParM and the R1-plasmid Kinetechore Complex ParR/parC: Force Generation, Polarity, and Insertional Polymerization. E. C. Garner,1 H. G. Garcia,2 L. Coin,2 L. Trichet,2 D. R. Mullins1; 1Biochemistry, UCSF, San Francisco, CA, 2Physiology course, Marine Biological Labs, Woods Hole, MA Presentation Number: 922 Poster Board Number: B146 The R1 par operon is thought to construct a minimal DNA-segregating spindle from three components, the centromeric DNA sequence parC, the DNA binding protein ParR, and the actin homolog ParM. In vivo, the ParR/C complex is thought to couple ParM polymerization to plasmid segregation. In vitro, ParM filaments exhibit rapid spontaneous nucleation, symmetrical elongation, and dynamic instability. Using Total Internal Reflection Fluorescence (TIRF) microscopy, we show that the ParR/parC complex stabilizes individual ParM filaments against catastrophic depolymerization. ParR/parC-mediated stabilization produces a population of long filaments that grow asymmetrically with one fast-growing and one slow-growing end. The fast-growing end elongates by insertion of ParM monomers at the interface with ParR/parC. To demonstrate that the connection between ParM and the ParR/parC complex is capable of generating force, we coupled ParR/parC complexes to micron-sized polystyrene spheres and incubated them with ParM in the presence of non-hydrolyzable nucleotides. Assembly of ParM filaments occurs preferentially at the surface of the microsphere and filaments are rapidly organized into an asymmetrical bundle. Elongation of filaments at the interface with ParR/parC generates force propelling the microshpere through the medium. By polarization microscopy the filaments in the bundle are aligned parallel to the direction of motion. By confocal fluorescence microscopy the bundle has a hollow core. Based on these results, we propose a model for assembly and function of a DNA-segregating ParM spindle. Mechanisms for Mitotic Spindle Pole Focusing in the Absence of Functional Centrosomes G. Goshima, A. Kirby, H. Barak, L. Krueger, S. Sivaramakrishnan, R. D. Vale; Physiology Course 2005, Marine Biological Laboratory, Woods Hole, MA Presentation Number: 994 Poster Board Number: B224 The centrosome is the dominant microtubule (MT) nucleation site during mitotic spindle formation. However, animal somatic cells also can generate MTs near chromosomes independently of centrosome function. In Drosophila S2 cells, RNAi of centrosomin (CNN) leads to loss of functional centrosomes, and spindles MTs are generated and organized into a bipolar array via chromatin-dependent pathway, as occurs during meiosis. Despite the absence of functional centrosomes, the spindle MTs, including kinetochore microtubule bundles (K-fiber), are reasonably well focused at their minus-ends in CNN RNAi cells. To understand the mechanism of K-fiber focusing in the absence of centrosomes, we performed double RNAi screening of CNN with ~200 known mitotic genes, and identified Ncd (a minus-end-directed kinesin) and Asp (a putative homolog of vertebrate NuMA) as essential factors for K-fiber focusing in CNN spindle. Interestingly, unlike what has been described for meiotic spindles reconstituted using Xenopus egg extracts, RNAi knockdown of dynein/dynactin subunits did not affect the pole focusing of acentrosomal spindles in S2 cells. We suggest that pole focusing by dynein requires a centrosome and involves to transport K-fibers along centrosome MTs. Ncd and Asp, on the other hand, can crossbridge minus ends of K-fibers and contribute to pole focusing in the absence of centrosomes. Organization and Force Generation of Interphase Microtubules in Fission Yeast L. Krueger,1 C. Pantoga,1 D. Bhatt,2 D. Foethke,3,1 M. Dogterom,4,1 F. Nedelec,3,1 P. Tran,2 M. Janson2,1; 1Physiology Course, Marine Biological Laboratory, Woods Hole, MA, 2Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, 3Cell Biology and Biophysics Program, EMBL, Heidelberg, Germany, 4FOM Institute AMOLF, Amsterdam, The Netherlands Presentation Number: 2456 Poster Board Number: B228 Interphase fission yeast cells organize 2 to 5 microtubule bundles that run along the long axis of cells. Each bundle contains two or more antiparallel microtubules that are overlapped and bound to the nuclear envelope at their minus ends, while the plus ends grow toward the cell ends. At the cell poles polymerization creates pushing forces that position the nucleus towards the cell middle. Microtubule bundles also deliver polarity factors to the cell cortex and thereby restrict cell growth to the cell poles. To perform these symmetry-maintaining roles it seems important that multiple bundles are well dispersed over the nuclear envelope and that the growth dynamics of microtubules is regulated at the cell cortex. This summer at the Woods Hole physiology course we investigated possible regulatory mechanisms. First, we observed that bundles in wild type cells are more regularly spaced over the nuclear envelope than would be expected by random placement. We used the software Cytosim to simulate the process of bundle formation and organization. We investigated whether a factor that selectively bundles anti-parallel microtubules could explain the observed bundle distribution. Second, we investigated how microtubule growth dynamics responds to forces generated in contact with the cell wall. We inferred growth velocities and forces from the buckling of single microtubules in contact with cell walls. For practical purposes we used round mutant cells (mor2-phenotype) that lacked the microtubule bundling protein ase1p. Our analysis shows that microtubule growth in yeast may be optimized for force generation as the growth velocity was decreased less by force than previously observed for microtubules polymerized from pure tubulin. Clustering of Chemotactic Receptors in E. coli with Altered Morphologies D. Weibel, I. Schneider, E. Garner, R. D. Vale, S. Khan; The Physiology Course, Marine Biological Laboratory, Woods Hole, MA Presentation Number: 2632 Poster Board Number: B413 E. coli possess a sophisticated signal transduction machinery that involves receptor-mediated binding of extracellular attractants and repellants and an intracellular relay mechanism that control the rotation of the flagellar motor. The signal transduction components are concentrated at one pole of the bacteria. This high degree of spatial order is thought to be important producing a high degree of cooperativity that is necessary for sensing shallow gradients of attractants/repellants. However, the mechanism that produces this pole clustering is unknown. Here, we have visualized a receptor-GFP fusion protein (TAR-GFP) and examined conditions that might affect its clustering. We chemically depolymerized the bacterial actin MreB, which produces spherical instead of rod-shaped cells, and found that receptors remained clustered. We also produced long filamentous bacteria by inhibiting septation, confirming that additional receptor clusters formed along the length of the rods, and these nonpolar clusters often appeared to have regular spacing. Finally, we observed small microclusters of TAR-GFP that diffused rapidly in the membrane. We propose these microclusters might be precursors of larger receptor clusters and that clustering reflects a self-assembly process that does not require the bacterial cytoskeleton and occurs in cells with dramatically different shapes. |
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