Exploring the mechanisms behind filopodia formation

Abstract

Filopodia are actin-filled membrane projections used by cells to explore their surroundings. Their structure is well understood, however the mechanisms behind filopodia formation are unclear. For example, mammalian myosin 10 (Myo10) and its evolutionarily distant, functional homologue amoeboid Myo7 (DdMyo7) are both essential for filopodia formation in their respective lineage, yet the role they play in filopodia formation remains unclear. To help elucidate mechanisms involved in filopodia formation, filopodia proteins are often manipulated and the resulting impact on filopodia formation measured, which relies on visualizing them with an actin stain, a filopodia tip marker, or a membrane marker. However, the diverse array of cell types that extend filopodia, from amoeboid to mammalian cells, and different visualization methods can make it challenging to find a reliable filopodia analysis workflow. Chapter 2 contained within this thesis describes the development of filoVision, a novel filopodia analysis workflow that uses deep learning to enable automated, rapid analysis of tip-marked filopodia and can be easily tailored to new cell types and imaging conditions. Myo10 is a fast motor optimized for movement on parallel bundles of actin and thought to transport actin polymerases to filopodia tips, but it’s not known if the DdMyo7 motor operates similarly. Chapter 3 describes steps taken to characterize a forced dimer of the amoeboid filopodial myosin motor, DdMyo7, using single molecule motility assays to determine if it is capable of transporting polymerases from the base of a filopodium to its tip, as proposed for the fast mammalian filopodial myosin motor, Myo10, or if its motor activity is more aligned with its other slow Myo7 family members. Single molecule TIRF assays of the dimerized DdMyo7 motor revealed that it is a slow, processive motor that moves along actin at ∼ 40 nm/sec. The activity of the motor is significantly reduced in the presence of Ca2+. The slow speed of DdMyo7 is like that of other Myo7 family motors, but is at least 10 times slower than its functional counterpart Myo10 and of amoeboid filopodia extension. The slow velocity establishes that the DdMyo7 motor is unlikely to serve as a transporter but rather is better-suited to serve as a cross-linker, bringing actin filaments together to form nascent parallel bundles of actin during filopodia initiation as well as provide force at the tips of filopodia during ongoing elongation. This demonstrates that the DdMyo7 and Myo10 motors likely play different roles during filopodia formation, despite both being critical for filopodia formation in amoeboa (DdMyo7) and metazoan (Myo10) cells.