Molecular counting of myosin force generators in growing filopodia.

ABSTRACT

Animal cells build actin-based surface protrusions to enable biological activities ranging from cell motility to mechanosensation to solute uptake. Long-standing models of protrusion growth suggest that actin filament polymerization provides the primary mechanical force for "pushing" the plasma membrane outward at the distal tip. Expanding on these actin-centric models, our recent studies used a chemically inducible system to establish that plasma membrane-bound myosin motors, which are abundant in protrusions and accumulate at the distal tips, can also power robust filopodial growth. How protrusion resident myosins coordinate with actin polymerization to drive elongation remains unclear, in part because the number of force generators and thus, the scale of their mechanical contributions remain undefined. To address this gap, we leveraged the SunTag system to count membrane-bound myosin motors in actively growing filopodia. Using this approach, we found that the number of myosins is log-normally distributed with a mean of 12.0 ± 2.5 motors [GeoMean ± GeoSD] per filopodium. Together with unitary force values and duty ratio estimates derived from biophysical studies for the motor used in these experiments, we calculate that a distal tip population of myosins could generate a time averaged force of ∼tens of pN to elongate filopodia. This range is comparable to the expected force production of actin polymerization in this system, a point that necessitates revision of popular physical models for protrusion growth. SIGNIFICANCE STATEMENT This study describes the results of in-cell molecular counting experiments to define the number of myosin motors that are mechanically active in growing filopodia. This data should be used to constrain future physical models of the formation of actin-based protrusions.