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The projects below describe examples of the many kinds of research projects that took place in the course in 2004.
Project by Lynne Chang in Clare Waterman-Storer’s section 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. Multi-spectral FSM using GFP-vimentin and X-rhodamine-labeled tubulin was used to address some of these possibilities. Interestingly, IF speckles appear brighter and 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. Evidence for co-translational assembly of IF precursor structures suggests that a tetrameric unit may consist of more than one GFP-labeled vimentin. Exchange of such structures enriched with GFP, along the filament would result in brighter, more distinguishable speckles. Ongoing studies inspired by these preliminary observations made at the Physiology course include Hi-Lo-FSM, in which high concentrations of X-rhodamine labeled vimentin are microinjected into cells expressing low levels of GFP-vimentin, enabling us to distinguish IF precursor particles from subunits within filaments. Spatial Organization and Dynamics of Molecules Involved in Chemotaxis in Bacteria Project performed with Ron Vale, Dennis Bray and Shahid Khan in the regular research session and in post-course research Bacterial sense extra-cellular concentration gradients of attractive and repulsive molecules using a signaling network of transmembrane and cytosolic proteins in a process called bacterial chemotaxis. The wealth of knowledge about the chemotactic response has enabled a rich interplay between experiment and modeling. One clear outcome is that bacteria are far more sensitive to concentration gradients than modeling predicts, known as the “gain problem.” Recently, microscopic studies have shown that the chemotactic proteins are not distributed uniformly throughout the cytosol, but instead occur in localized sub-cellular structures, suggestive of a higher-order spatial organization. In the course, we examined the intra-cellular dynamics of the chemotactic proteins CheY and CheZ using single-cell fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). Specifically, we show that CheY and CheZ behave like single molecules undergoing free diffusion in the cytosol, while the clustered form is highly static and has limited exchange with the cytosolic pool. However, the CheZ cluster expands rapidly upon exposure to the chemo-attractant aspartate. These results constitute the first evidence that the chemotactic proteins participate in a highly stable macromolecular machine that responds dynamically to chemo-attractants and repellants.
Project by Roy Wollman, Olivia L. George, and Prabhat S. Kunwar in Ron Vale’s section with help from teaching assistant Gohta Goshima 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. Technically, this involved preparing cells in 96 well plate format, staining for tubulin, gamma-tubulin, and DNA, automated imaging using an Axon IMAGE EXPRESS, and then developed Matlab programs (Roy Wollman) to identify mitotic spindles in the images (only ~1% of cells were mitotic). 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. Roy and Gohta are continuing work on this project, including developing a mathematical model that explains these observations. Studying Network Dynamics in Actin Comet Tails using Fluorescent Speckle Microscopy Project by Lucia Sironi and Judit Tóth in Dyche Mullins’ section with help from teaching assistant Orkun Akin The actin cytoskeleton is thought to be the engine of amoeboid motility. In vitro models of actin-based motility, developed originally with the intracellular pathogen Listeria monocytogenes, have been valuable in studying the biophysical mechanism of force generation. Efforts to describe this mechanism are represented by two major models which differ in the length scale of their approach: The Brownian Ratchet model treats a single polymerizing actin filament as the force generating unit; the Elastic Gel (EG) model considers large scale deformations of crosslinked actin networks. Specifically, the latter model proposes that an actin network generated and crosslinked on a closed surface - such as the lateral surface of a motile Listeria monocytogenes - can store elastic energy which can subsequently be released to perform mechanical work and propel that surface forward. Since the length scale of the deformations predicted by the EG model are of the order of the surface curvature of the motile particle, it should be possible to directly observe the expected network dynamics using fluorescent speckle microscopy (FSM). Therefore, we performed time-lapse FSM imaging of actin comet tails generated on ActA-coated 5µm polysyrene beads in an in vitro motility system based on purified protein components. Actin network dynamics were studied with a variety of techniques, including kymograph analysis and speckle tracking. We find that actin polymerized on the lateral surface of beads does not converge toward the central axis of the comet tail, inconsistent with the elastic relaxation predicted by the EG model. While further study is needed, this preliminary observation suggest that the EG model is an incomplete description of actin-based force generation.
Transcriptional Control in Yeast Erin O’Shea’s group Working with Erin O’Shea in the systems biology module, students focused on quantitative understanding of biological behaviors. One project involved dissection of the role of positive and negative feedback in a simple signal transduction pathway. A second project attempted to identify the molecular mechanisms that suppress randomness or noise in the regulation of gene expression. A third tested a control theory model of eukaryotic systems that enable nutrient homeostasis. Small groups of students cooperated to integrate data collection, automated image analysis, and computational modeling. Important results from students’ work included identification of underlying
See also the movie section: Pho84 endocytosis in response to increased intracellular phosphate levels |
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