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ASCB ABSTRACTS: 2004| 2005 | 2006 | 2007 2004 Abstracts Intermediate Filament Dynamics Revealed by Fluorescence Speckle Microscopy L. Chang,1,2 R. D. Goldman,1,2 D. Foethke,3,2 C. M. Waterman-Storer,4,2 T. Wittmann4,2 ; 1 Cell & Molecular Biology, Northwestern University Medical School, Chicago, IL, 2 Physiology Course 2004, MBL, Woods Hole, MA, 3 EMBL, Heidelberg, Germany, 4 Cell Biology, The Scripps Research Institute, La Jolla, CA Presentation Number: 1641 Poster Board Number: B303 Using a stably transfected Ptk1 cell line expressing GFP-vimentin, we have observed, for the first time, intermediate filament (IF) dynamics by fluorescence speckle microscopy (FSM). Under low levels of GFP-vimentin expression, long filaments appear speckled in high resolution fluorescence images. These speckles display a variety of motile properties including tandem movements of linear speckle arrays and also independent movements of speckles towards or away from each other, possibly within a single IF filament or filament bundle. The various speckle movements may represent whole filament movement, subunit movement within a filament, filaments sliding across one another, and non-filamentous precursor particles moving along side an existing filament on a common MT. These various possibilities are being addressed by Hi-Lo-FSM, in which high concentrations of rhodamine labeled vimentin is microinjected into cells expressing low levels of GFP-vimentin, and by multi-spectral FSM using GFP-vimentin and rhodamine-labeled tubulin. Interestingly, IF speckles appear more distinct than MT or actin speckles. This may be due to the unique subunit exchange properties of IF. Subunit exchange along the filament is thought to occur in the form of tetramers. Previous work (see Chang, Singer, Shenoy and Goldman, ASCB 2004 abstract) has suggested that IF precursor structures are co-translationally assembled which may increase the likelihood of a tetrameric unit consisting of more than one GFP-labeled vimentin. Exchange of such structures enriched with GFP, along the filament would result in brighter, more distinguishable speckles. RDG and CW-S are supported by NIGMS. Determinants of mitotic spindle length in Drosophila S2 cells G. Goshima, R. Wollman, O. L. George, P. S. Kunwar, J. M. Scholey, R. D. Vale; Physiology Course 2004, Marine Biological Laboratory, Woods Hole, MA Presentation Number: 2195 Poster Board Number: B190 The length of the metaphase spindle is fairly uniform in a given cell type. Several factors have been proposed to govern spindle length, such as sliding forces generated between antiparallel microtubules and microtubule length governed by microtubule dynamics. To assess the contribution of the two factors to spindle length, we performed RNAi-knockdown and overexpression of force-generating motors and microtubule dynamics regulators in Drosophila S2 cells and examined the effects on spindle length using high-throughput automated microscopy and image analysis. We found that depletion of microtubule depolymerizing kinesins, Klp10A [Kin I] or Klp67A [Kip3], caused a 50% expansion of metaphase spindle compared to wild-type, whereas overexpression of Klp10A caused 20% shortening. Surprisingly, overexpression of force-generating kinesins, Klp61F [BimC/Eg5] or Ncd [KinC], and also RNAi-knockdown of Ncd or dynein had little effect on bipolar spindle length. However, overexpression of Ncd caused an increased frequency of spindle collapse to a monopolar state. Our results indicate that regulation of microtubule dynamics is the major determinant of metaphase spindle length, and that the mitotic spindle size is robust to a wide expression range of force-generating motor proteins in this cell line. We will present a mathematical model that explains these observations, and discuss its generality for other cell types. Clip-170 Cap-Gly Domains: Structural Aspects of In Vivo Plus-End Tracking and In Vitro Microtubule Nucleation K. C. Slep,1 P. Niethammer,2 M. Nonaka,2 R. D. Vale1,3 ; 1 Cellular & Molecular Pharmacology, University of California San Francisco, San Francisco, CA, 2 Physiology Course 2004, Marine Biological Laboratory, Woods Hole, MA, 3 Howard Hughes Medical Institute, San Francisco, CA Presentation Number: L120 Poster Board Number: L120 Clip-170 family members are defined by tandem N-terminal Cap-Gly domains that confer microtubule plus-end tracking activity. The Cap-Gly-based mechanism for plus-end tracking remains to be determined. Here, we present the crystal structure of Clip-170’s Cap-Gly domain I. The structure reveals a unique beta-sheet fold with conserved glycine residues positioned to afford turns between strands. The concave structure exhibits a conserved solvent exposed region, likely to facilitate tubulin binding. We have developed both in vivo and in vitro assays to probe the structural determinants that confer tubulin binding. In vivo, GFP-tagged Clip-170 constructs were analyzed for plus-end tracking capability. To date, deletion constructs reveal that both Cap-Gly domains are required for plus-end tracking while constructs embodying single Cap-Gly domains fail to plus-end track. In vitro, Clip-170 constructs were utilized to promote microtubule nucleation at sub-critical tubulin concentrations. We observed potent nucleation from a Clip-170 construct that embodied both Cap-Gly domains. In contrast, constructs comprised of solitary Cap-Gly domains, while capable of binding tubulin, failed to promote microtubule polymerization. In vivo and in vitro microtubule assays reveal a common mechanism requiring multivalent tubulin interactions to afford both plus-end tracking and microtubule nucleation. A stable chemosensory machine in Escherichia coli revealed by FRAP M. A. DePristo, L. Chang, K. Lipkow, S. Khan, R. D. Vale; Marine Biological Laboratory Physiology Course 2004, Woods Hole, MA Presentation Number: L138 Poster Board Number: L138 Escherichia coli bacteria undergo chemotaxis in response to extracellular concentration gradients of attractive and repulsive molecules. Recent microscopic studies have shown that the chemoreceptors and cytoplasmic chemotaxis proteins are concentrated into clusters at the poles of the cell. In silico models suggest that higher-order spatial organization increases the gain, or sensitivity, of the signaling system, allowing bacteria to respond to minute concentration gradients of attractants or repellents. Despite their importance, the dynamics of molecules in these signaling clusters has not yet been fully explored. Using single-cell fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) in E.coli, we show that the cytosolic pools of the secondary messenger CheY-YFP and the phosphatase CheZ-GFP are highly mobile in Escherichia coli, consistent with their being freely diffusible single molecules. While exchange between polarly localized and cytosolic CheY is also rapid, the polarly localized CheZ population is effectively immobile. This immobility indicates that contacts form between chemoreceptors and the cytoplasmic kinase, CheA, as well as the phosphatase CheZ with nanomolar or greater effective affinity. This estimate is substantially greater than the micromolar affinity measured in vitro for receptor-CheA dimers. Our results demonstrate that individual chemotactic proteins exhibit dramatically different dynamics in the polar cluster. Further, the immobility of CheZ implies that the chemotactic proteins form a highly stable sensory apparatus composed of a network of interacting proteins. |
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