Biology subjects

Quintanilla, M. A.

Publications and source records attributed to Quintanilla, M. A..

4 recordsLinked to original sources

bioRxiv2024

Non-muscle myosin 2 can incorporate into established filaments in cells without an assembly competence domain

Myosin 2 dynamically assembles into filaments that exert force on the actin cytoskeleton. To form filaments, myosin 2 monomers transition between folded and unfolded states. Monomer unfolding exposes an extended coiled-coil that interacts with other monomers in parallel and antiparallel fashions, enabling bipolar filament formation. A C-terminal domain of the coiled-coil, termed assembly competence domain (ACD), has been repeatedly identified as necessary for filament assembly. Here, we revisit ACD contribution when full-length filaments are present. Non-muscle myosin 2A lacking the ACD ({Delta}ACD) initially appears diffuse, but triton extraction of cytosolic fraction reveals cytoskeletal association. Disruption of the folded monomer enhances the cytoskeletal fraction, while inhibition of endogenous filament assembly appears to reduce it. Finally, high resolution imaging of endogenous and exogenous bipolar filamentous structures reveals highly coincident signal, suggesting {Delta}ACD constructs co-assemble with endogenous myosin 2A filaments. Our data demonstrate that while the ACD is required for de novo filament assembly, it is not required for monomers to recognize and associate with established filaments in cells. More broadly, this highlights the existence of distinct mechanisms governing myosin 2 monomer assembly into nascent filaments, and monomer recognition and association with established filaments to maintain steady-state contractile networks.

cell biology↗

bioRxiv2024

The PCM scaffold enables RNA localization to centrosomes

As microtubule-organizing centers, centrosomes direct assembly of the bipolar mitotic spindle required for chromosome segregation and genome stability. Centrosome activity requires the dynamic assembly of pericentriolar material (PCM), the composition and organization of which changes throughout the cell cycle. Recent studies highlight the conserved localization of several mRNAs encoded from centrosome-associated genes enriched at centrosomes, including Pericentrin-like protein (Plp) mRNA. However, relatively little is known about how RNAs localize to centrosomes and influence centrosome function. Here, we examine mechanisms underlying the subcellular localization of Plp mRNA. We find that Plp mRNA localization is puromycin-sensitive, and the Plp coding sequence is both necessary and sufficient for RNA localization, consistent with a co-translational transport mechanism. We identify regions within the Plp coding sequence that regulate Plp mRNA localization. Finally, we show that protein-protein interactions critical for elaboration of the PCM scaffold permit RNA localization to centrosomes. Taken together, these findings inform the mechanistic basis of Plp mRNA localization and lend insight into the oscillatory enrichment of RNA at centrosomes.

cell biology↗

bioRxiv2023

Local Monomer Levels and Established Filaments Potentiate Non-Muscle Myosin 2 Assembly

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet the biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the actin architecture plays a minimal direct role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes sub-resolution filament stacks prior to partitioning into clusters that feed higher-order networks. Together these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

cell biology↗

bioRxiv2023

Non-muscle myosin 2 filaments are processive in cells

Directed transport of cellular components is often dependent on the processive movements of cytoskeletal motors. Myosin 2 motors predominantly engage actin filaments of opposing orientation to drive contractile events, and are therefore not traditionally viewed as processive. However, recent in vitro experiments with purified non-muscle myosin 2 (NM2) demonstrated myosin 2 filaments could move processively. Here, we establish processivity as a cellular property of NM2. Processive runs in central nervous system-derived CAD cells are most apparent as processive movements on bundled actin in protrusions that terminate at the leading edge. We find that processive velocities in vivo are consistent with in vitro measurements. NM2 makes these processive runs in its filamentous form against lamellipodia retrograde flow, though anterograde movement can still occur in the absence of actin dynamics. Comparing the processivity of NM2 isoforms, we find that NM2A moves slightly faster than NM2B. Finally, we demonstrate that this is not a cell-specific property, as we observe processive-like movements of NM2 in the lamella and subnuclear stress fibers of fibroblasts. Collectively, these observations further broaden NM2 functionality and the biological processes in which the already ubiquitous motor can contribute.

cell biology↗