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Emergent complexity in myosin V-based organelle inheritance.
F. D. Mast1, R. A. Rachubinski1, J. B. Dacks1; 1Cell Biology, Univ Alberta, Edmonton, AB, Canada
How is adaptability generated in complex biological systems composed of interacting cellular machineries, each with individual, separate and functionally critical jobs to perform? Organelle inheritance is one such system that requires the coordination of several robust and ancient cellular modules, including the cell cycle, cytoskeleton and organelle biogenesis/identity. Budding yeasts have emerged as powerful models to study these processes as organelles compete for access to myosin V motors that travel along polarized actin cables to vectorially deliver bound cargo to the bud. Under the direction of the cell cycle, myosin V motors are recruited to organelles by specific interactions between their carboxyl-terminal globular tail domain and organelle-specific receptors. We used comparative genomics, phylogenetics and secondary structural modeling to characterize the evolutionary history of these organelle- specific receptors. We find that while some receptors are retained widely across the animals and fungi, others are limited primarily to the Saccharomycetaceae family of budding yeast, with the emergent pattern of a conserved biogenic and inheritance factor often paired with an evolutionarily novel inheritance adaptor. We propose an evolutionary model whereby the emergence of myosin V-based organelle inheritance has utilized mechanisms of paralogy, the exploration of sequence space and the appearance of pliable, evolutionarily novel adaptor proteins. Our findings suggest an overarching evolutionary mechanism for how diverse cargoes compete for a single myosin V motor in organelle transport and detail one system’s solution to obtaining evolutionary adaptability amongst constrained cellular modules. We are continuing to explore the consequences of the model with functional studies on the regulation of myosin V- based organelle inheritance.
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Comprehensive analysis reveals dynamic and evolutionary plasticity of Rab GTPases and membrane traffic in Tetrahymena thermophila.
A. Turkewitz1, L. Bright1; 1Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL
Cellular sophistication is not exclusive to multicellular organisms, and unicellular eukaryotes can resemble differentiated animal cells in their complex network of membrane-bound structures. These comparisons can be illuminated by genome-wide surveys of key gene families. We report a systematic analysis of Rabs in a complex unicellular Ciliate, including gene prediction and phylogenetic clustering, expression profiling based on public data, and Green Fluorescent Protein (GFP) tagging. Rabs are monomeric GTPases that regulate membrane traffic. Because Rabs act as compartment-specific determinants, the number of Rabs in an organism reflects intracellular complexity. The Tetrahymena Rab family is similar in size to that in humans and includes both expansions in conserved Rab clades as well as many divergent Rabs. Importantly, more than 90% of Rabs are expressed concurrently in growing cells, while only a small subset appears specialized for other conditions. By localizing most Rabs in living cells, we could assign the majority to specific compartments. These results validated most phylogenetic assignments, but also indicated that some sequence-conserved Rabs were co-opted for novel functions. Our survey uncovered a rare example of a nuclear Rab and substantiated the
existence of a previously unrecognized core Rab clade in eukaryotes. Strikingly, several functionally conserved pathways or structures were found to be associated entirely with divergent Rabs. These pathways may have permitted rapid evolution of the associated Rabs or may have arisen independently in diverse lineages and then converged. Thus, characterizing entire gene families can provide insight into the evolutionary flexibility of fundamental cellular pathways.
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Functional Genomics of Cell Regeneration in the Giant Ciliate Stentor coeruleus. M. Slabodnick1, W. Marshall1; 1Univ California-San Francisco, San Francisco, CA
The mechanisms that specify cell shape and organization are not currently understood. Ciliates provide ideal model systems to tackle problems of cell morphology due to their complex cell organization and unique patterning. With current advances in technology along with work done in Paramecium tetraurelia and Tetrahymena thermophila, it is now possible to apply these tools to studying other organisms. Stentor coeruleus is a large, ~1mm long, single cell with a highly patterned cell cortex and the ability to regenerate and reorganize after surgical or chemical manipulations. The ease of surgical manipulations gives Stentor significant advantages over other ciliate models. Using the surgical techniques unique to Stentor as well as modern RNA interference (RNAi) methods, visualization techniques, and genomic sequencing I will revive Stentor as a model for studying cell polarity and organization. With the current state of Next Generation Sequencing it has become feasible for a lab to sequence the genome of a Eukaryotic organism. We have begun our own sequencing effort for Stentor’s macronuclear genome in order to facilitate the development of a better experimental toolbox. We have been able to repeat many of the surgical experiments performed by Vance Tartar, Noël de Terra and others. Using data obtained from preliminary sequences I constructed RNAi vectors that target endogenous Stentor genes and here I provide evidence that methodology developed for other ciliates can function in Stentor as well. Results for RNAi of Alpha-Tubulin and Mob1 result in dramatic changes in cell polarity and organization of the cortex and provide strong evidence that studies in Stentor can yield exciting and useful results. Knocking down Alpha-Tubulin, a key structural component in the cortex, results in clear cortical defects and problems with cell regeneration. This is very different from the Mob1 knockdown, which results in the drastic elongation of cells and other cortical aberrations. Using RNAi in conjunction with the unique microsurgical methods available in Stentor, it should be possible to restore this classical system to its previous status as a central model for addressing many important questions, including centriole structure, cell polarity, biological pattern formation, and cellular regeneration.
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Systems-level analysis of multicellular microbial community structure.
A. M. Valm1,2, J. L. Mark Welch1, R. Oldenbourg1,2, G. G. Borisy1; 1Marine Biological Laboratory, Woods Hole, MA, 2Brown University, Providence, RI
Just as the phenotypically different cells that make up multicellular organisms are distributed in tissues with structures that embody specific functions, microbial cells with different metabolic functions form unique spatial structures and coordinate their activities as multicellular units, e.g. biofilms. Fundamentally, microbial communities differ from eukaryotic tissues because their cellular constituents may be genetically distinct; in fact, up to hundreds of different species may be present in a single biofilm. Any number of probes may be designed to identify the different species present in a community; however, the ability to unambiguously distinguish more than a few different labels in a single fluorescence image has been severely hampered by the
excitation cross-talk and signal bleed-through of fluorophores with highly overlapping excitation and emission spectra.
We recently developed a fluorescence labeling, imaging, and analysis method to greatly expand the number of identifiable labels in a single image, which we call Combinatorial Labeling and Spectral Imaging (CLASI). Application of our CLASI technique to human dental plaque using fluorescence in situ hybridization (FISH) enabled the first quantitative analysis of the spatial distribution of 15 different taxa of microbes in a biofilm. Proximity analysis was used to determine the frequency of inter- and intrataxon cell-to-cell associations, which revealed statistically significant intertaxon pairings. Cells of the genera Prevotella and Actinomyces showed the most interspecies associations, suggesting a central role for these genera in establishing and maintaining biofilm complexity. In a proof-of-principle experiment, we further demonstrate that we can distinguish 120 differently labeled E. coli in a mixture labeled with binary combinations of 16 fluorophores using a novel linear unmixing algorithm constrained to identify specific combinations of fluorophores. Our results provide an initial systems-level structural analysis of biofilm organization. We believe that the CLASI approach will be useful for a systems level analysis of many complex molecular structures within cells. Supported by the Sloan Foundation and NIH Grants F31DE019576 and RC1DE20630.
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Electrical Spiking in Escherichia coli Probed with a Fluorescent Voltage Indicating Protein.
J. Kralj1, D. Hochbaum2, A. Douglass3, A. Cohen1; 1Chemistry, Harvard University, Cambridge, MA, 2Applied Physics, Harvard University, Cambridge, MA, 3Biology, Harvard University, Cambridge, MA
Bacteria have many voltage- and ligand-gated ion channels, and population-level measurements indicate that membrane potential is important for bacterial survival. However, it has not been possible to probe voltage dynamics in an intact bacterium. Here we developed a method to measure membrane potential on a single cell which revealed electrical spiking in Escherichia coli. To probe bacterial membrane potential we engineered a voltage-sensitive fluorescent protein based on green-absorbing proteorhodopsin. Expression of the Proteorhodopsin Optical Proton Sensor (PROPS) in E. coli revealed electrical spiking at up to 1 Hz. Within a nominally homogeneous population of bacteria, cells showed a variety of voltage dynamics. Spiking was sensitive to chemical and physical perturbations, and coincided with rapid efflux of a small-molecule fluorophore, suggesting that bacterial efflux machinery may be electrically regulated.
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Control of cell differentiation by the regulatory molecules ppGpp and inorganic polyphosphate.
C. Boutte1, J. Henry2, S. Crosson1; 1Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 2Committee on Microbiology, University of Chicago, Chicago, IL
The molecules guanosine tetraphosphate (ppGpp) and inorganic polyphosphate (PolyP) function as global regulators of cell physiology in both prokaryotes and eukaryotes. These molecules have the capacity to directly bind and regulate the activity of a range of proteins in the cell including RNA polymerase and proteases. Our recent studies on bacterial cells have focused on understanding the molecular logic underlying regulated synthesis of ppGpp and PolyP, and the downstream control of cell development by these important signaling molecules.
We have defined the input logic and direct transcriptional targets of ppGpp in the oligotrophic bacterium, Caulobacter crescentus. The sole ppGpp synthase, SpoTCC, binds to and is
regulated by the ribosome, and exhibits AND-type control logic. We further demonstrate a regulatory link between the synthesis of ppGpp and PolyP and demonstrate that both molecules function to control of the timing of Caulobacter cell differentiation. Specifically, Caulobacter differentiates from a motile, foraging swarmer cell into sessile, replication-competent stalked cell during its cell cycle, akin to the G1->S transition in eukaryotic cells. This developmental
transition is inhibited by nutrient deprivation to favor the motile swarmer state. Both ppGpp and PolyP inhibit this transition in rich and glucose-depleted medium. Upon exhaustion of available carbon, swarmer cells lacking the ability to synthesize ppGpp or PolyP erroneously initiate chromosome replication, proteolyze the replication inhibitor CtrA, localize the cell-fate determinant DivJ, and develop polar stalks. These results provide evidence that ppGpp and polyP function as cell-type specific developmental regulators.