Biology subjects

Kovar, D. R.

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

5 recordsLinked to original sources

bioRxiv2018

Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia crosslinker fascin

Ena/VASP are tetrameric assembly factors that bind F-actin barbed ends continuously while increasing their elongation rate within dynamic bundled networks such as filopodia. We used single-molecule TIRFM and developed a kinetic model to dissect Ena/VASPs processive mechanism on bundled filaments. Notably, Ena/VASPs processive run length increases with the number of both bundled filaments and Ena arms, revealing avidity facilitates enhanced processivity. Moreover, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Finally, enhanced processivity on trailing barbed ends of bundled filaments is an evolutionarily conserved property of Ena/VASP homologs and is specific to fascin-bundled filaments. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, we discovered a novel regulatory mechanism whereby bundle size and bundling protein specificity control activities of a processive assembly factor.

cell biology↗

bioRxiv2017

Tropomyosin and a-actinin cooperation inhibits fimbrin association with actin filament networks in fission yeast

We previously discovered that competition between fission yeast actin binding proteins (ABPs) for association with F-actin helps facilitate their sorting to different F-actin networks. Specifically, competition between actin patch ABPs fimbrin Fim1 and cofilin Adf1 enhances each others activities, and rapidly displaces tropomyosin Cdc8 from the F-actin network. However, these interactions dont explain how Fim1, a robust competitor, is prevented from associating equally well with other F-actin networks. Here, with a combination of fission yeast genetics, live cell fluorescent imaging, and in vitro TIRF microscopy, we identified the contractile ring ABP -actinin Ain1 as a key sorting factor. Fim1 competes with Ain1 for association with F-actin, which is dependent upon their residence time on F-actin. Remarkably, although Fim1 outcompetes both contractile ring ABPs Ain1 and Cdc8 individually, Cdc8 enhances the bundling activity of Ain1 10-fold, allowing the combination of Ain1 and Cdc8 to inhibit Fim1 association with contractile ring F-actin.

cell biology↗

bioRxiv2017

Cofilin Drives Rapid Turnover and Fluidization of Entangled F-actin

The shape of most animal cells is controlled by the actin cortex, a thin, isotropic network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and turnover: Myosin-II molecular motors drive deformations required for cell division, migration, and tissue morphogenesis, while turnover of the molecular components of the actin cortex relax stress and facilitate network reorganization. While many aspects of F-actin network viscoelasticity are well-characterized in the presence and absence of motor activity, a mechanistic understanding of how non-equilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system wherein the steady-state length and turnover rate of F-actin in entangled solutions are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and nucleate filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. Microrheology measurements demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover, and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number in spite of sustained severing.\n\nSignificance StatementWhen an animal cell moves or divides, a disordered network of actin filaments (F-actin) plays a central role in controlling the resulting changes in cell shape. While it is known that continual turnover of F-actin by cofilin-mediated severing aids in reorganization of the cellular cytoskeleton, it is unclear how the turnover of structural elements alters the mechanical properties of the network. Here we show that severing of F-actin by cofilin results in a stress relaxation mechanism in entangled solutions characterized by a single-timescale set by the severing rate. Additionally, we identify ATP hydrolysis and nucleotide-dependent cofilin binding as sufficient ingredients to generate a non-equilibrium steady-state in which asymmetric F-actin disassembly preserves filament number in spite of sustained severing.

biophysics↗

bioRxiv2017

Partitioning and Enhanced Self-Assembly of Actin in Polypeptide Coacervates

Biomolecules exist and function in cellular micro-environments that control their spatial organization, local concentration and biochemical reactivity. Due to the complexity of native cytoplasm, the development of artificial bioreactors and cellular mimics to compartmentalize, concentrate and control the local physicochemical properties is of great interest. Here, we employ self-assembling polypeptide coacervates to explore the partitioning of the ubiquitous cytoskeletal protein actin into liquid polymer-rich droplets. We find that actin spontaneously partitions into coacervate droplets and is enriched by up to {approx}30-fold. Actin polymerizes into micrometer-long filaments and, in contrast to the globular protein BSA, these filaments localize predominately to the droplet periphery. We observe up to a 50-fold enhancement in the actin filament assembly rate inside coacervate droplets, consistent with the enrichment of actin within the coacervate phase. Together these results suggest that coacervates can serve as a versatile platform in which to localize and enrich biomolecules to study their reactivity in physiological environments.\n\nSIGNIFICANCE STATEMENTLiving cells harbor many protein-rich membrane-less organelles, the biological functions of which are defined by compartment composition and properties. Significant differences between the physico-chemical properties of these crowded compartments and the dilute solutions in which biochemical reactions are traditionally studied pose a major challenge for understanding regulation of organelle composition and component activity. Here, we report the spontaneous partitioning and accelerated polymerization of the cytoskeletal protein actin inside model polypeptide coacervates as a proof-of-concept demonstration of coacervates as bioreactors for studying biomolecular reactions in cell-like environments. Our work introduces exciting avenues for the use of synthetic polymers to control the physical and biological properties of bioreactors in vitro, enabling studies of biochemical reactions in cell-like micro-environments.

biophysics↗

bioRxiv2016

Chlamydomonas reinhardtii formin and profilin are optimized for acute rapid actin filament assembly

The regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in Chlamydomonas is the fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed end elongation at equimolar concentrations to actin. However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-bound actin. PRF1 further favors FOR1-mediated actin assembly by potently inhibiting Arp2/3 complex-mediated actin assembly. Furthermore, for1 and prf1-1 mutants, as well as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 cooperate to selectively and rapidly assemble F-actin at the right time and place.\n\nSUMMARY STATEMENTThe Chlamydomonas reinhardtii formin FOR1 initiates rapid assembly of fertilization tubule actin filaments from monomers associated with the actin-assembly inhibitor profilin PRF1.

cell biology↗