Biology subjects

Mullins, R. D.

Publications and source records attributed to Mullins, R. D..

4 recordsLinked to original sources

bioRxiv2018

LC3 and STRAP regulate actin filament assembly by JMY during autophagosome formation

During autophagy actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in non-starved cells, cytoplasmic JMY co-localizes with STRAP, a regulator of JMYs nuclear functions, on non-motile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMYs de novo actin nucleation activity via a cryptic actin-binding sequence near JMYs N-terminus, and STRAP inhibits JMYs ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3- and JMY-dependent actin network formation on membranes, and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMYs actin assembly activities in trans during autophagy.\n\neTOC BlurbThe actin regulator JMY creates filament networks that move membranes during autophagy. We find that, in unstarved cells, JMY is inhibited by interaction with the STRAP protein, but upon starvation JMY is recruited away from STRAP and activated by LC3.

cell biology↗

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

Three-dimensional actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 m), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

cell biology↗

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↗