Hundreds of myosin 10s are pushed to the tips of filopodia and could cause traffic jams on actin

Abstract

Myosin 10 (Myo10) is a vertebrate-specific motor protein well known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the numbers of Myo10 in filopodia. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. Here we combined SDS-PAGE analysis with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it tends to be enriched at opposite ends of the cell. Hundreds of Myo10 are found in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane deformations in addition to the numbers of Myo10 required for filopodia initiation. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.

Molecular counting of myosin force generators in growing filopodia

“…This point further implies that the number of myosins promoting elongation may be set at the initiation of filopodial growth. In contrast to our findings, a recent study in U2OS cells demonstrated that over-expression of Myo10FL leads to filopodial tips that contain hundreds of motors (median of ~360 molecules), with a large range around this value (2-65,000 molecules) (33). Although we employed a direct single molecule visualization strategy, that study used a population level biochemical approach combined with epifluorescence microscopy to approximate the number of motors in individual cells, and in turn, single filopodia.…”

The dynamics of actin protrusions can be controlled by tip-localized myosin motors

“…Myosin motors accumulated at the protrusion tip could contribute to combatting this force, as well as create space for polymerization to occur. Recent data published by Shangguan and Rock ( 62 ) estimates ∼360 MYO10 molecules occupy a single filopodium, distributed mostly at the protrusion tip ( 62 ). Given that a single molecule of MYO10 can generate a maximum force of ∼1pN per single step ( 31 ) with a duty ratio of roughly 60% ( 48 ), an ensemble of ∼100 active MYO10 molecules at the protrusion tip could contribute around 60pN of tip-ward force along the membrane per step.…”