Biology subjects

Polka, J. K.

Publications and source records attributed to Polka, J. K..

3 recordsLinked to original sources

bioRxiv2017

Cryo-EM structure of the bacterial actin AlfA reveals unique assembly and ATP-binding interactions and the absence of a conserved subdomain

Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial plasmids and function to partition plasmids into daughter cells. The bacterial actin AlfA segregates plasmids by a mechanism distinct from other partition systems, dependent on its unique dynamic properties. Here, we report the near-atomic resolution cryo-EM structure of the AlfA filament, which reveals a strikingly divergent filament architecture resulting from the loss of a subdomain conserved in all other actins and a novel mode of ATP binding. Its unusual assembly interfaces and nucleotide interactions provide insight into AlfA dynamics, and expand the range of evolutionary variation accessible to actin quaternary structure.\n\nSignificance StatementActin filaments are dynamic cytoskeletal elements that assemble upon ATP binding. Actin homologs are present in all domains of life, and all share a similar three-dimensional structure of the assembling subunit, but evolutionary changes to subunit have generated many different actin filament structures. The filament structure of the bacterial actin AlfA, which positions plasmids - small, circular DNA molecules that encode important genes - to ensure that each daughter cell receives a copy at cell division. AlfA is different from all other actins in two critical ways: it binds to ATP in a unique way, and it is missing a quarter of the conserved structural core. These differences explain unusual AlfA assembly dynamics that underlie its ability to move plasmids.

biophysics↗

bioRxiv2017

Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

Bacteria of the genus Prosthecobacter express homologs of eukaryotic -and {beta}-tubulin, called BtubA and BtubB, that have been observed to assemble into bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may derive from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is GTP-dependent and reversible and that BtubA/B folding does not require chaperones. To better understand bMT behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro bMT assembly using a combination of polymerization kinetics assays, and microscopy. Like eukaryotic microtubules, bMTs exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by bMT polymerization drives a stochastic mechanism of bMT disassembly that occurs via polymer breakage. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of bMT fragments. Unlike MTs, polymerization of bMTs requires KCl, which reduces the critical concentration for BtubA/B assembly and induces bMTs to form stable mixed-orientation bundles in the absence of any additional bMT-binding proteins. Our results suggest that at potassium concentrations resembling that inside the cytoplasm of Prosthecobacter, bMT stabilization through self-association may be a default behavior. The complex dynamics we observe in both stabilized and unstabilized bMTs may reflect common properties of an ancestral eukaryotic tubulin polymer.\n\nImportanceMicrotubules are polymers within all eukaryotic cells that perform critical functions: they segregate chromosomes in cell division, organize intracellular transport by serving as tracks for molecular motors, and support the flagella that allow sperm to swim. These functions rely on microtubules remarkable range of tunable dynamic behaviors. Recently discovered bacterial microtubules composed of an evolutionarily related protein are evolved from a missing link in microtubule evolution, the ancestral eukaryotic tubulin polymer. Using microscopy and biochemical approaches to characterize bacterial microtubules, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules, but differ in how they self-associate into bundles and become destabilized. Our results demonstrate the diversity of mechanisms that microtubule-like filaments employ to promote filament dynamics and monomer turnover.

microbiology↗

bioRxiv2016

Super-resolution microscopy of the β-carboxysome reveals a homogenous matrix

Carbon fixation in cyanobacteria makes a major contribution to the global carbon cycle. The cyanobacterial carboxysome is a proteinaceous microcompartment that protects and concentrates the carbon-fixing enzyme RuBisCO in a paracrystalline lattice, making it possible for these organisms to fix CO2 from the atmosphere. The protein responsible for the organization of this lattice in beta-type carboxysomes of the freshwater cyanobacterium Synechococcus elongatus, CcmM, occurs in two isoforms thought to localize differentially within the carboxysome matrix. Here we use widefield timelapse and 3D-structured illumination microscopy (3D-SIM) to study the recruitment and localization of these two isoforms. We demonstrate that this super-resolution technique is capable of successfully resolving the outer protein shell of the carboxysome from its internal cargo. We develop an automated analysis pipeline to analyze and quantify 3D-SIM images and generate a population level description of carboxysome shell protein, RuBisCO, and CcmM isoform localization. We find that both CcmM isoforms colocalize in space and time, prompting a revised model of the internal arrangement of the beta carboxysome.

cell biology↗