Myosin-Induced Actin Filament Bundle Organization – Myo-n-Ease

We characterize each myosin and actin regulatory protein subdomain activity separately, measure the binding affinity between protein partners and then assess the resulting actin dynamics.
Partner #2, an expert on myosin motors, provides structural insights on myosin and Espin proteins in order to design and produce motors for functional studies by Partner #1.
To study the action of proteins on actin assembly dynamics, Partner #1 uses a microfluidics/TIRF experimental approach, capable of addressing actin bundle organization and regulation, with high control over the biochemical conditions.

We found that myosin X is able to remodel a branched actin network in vitro, in the absence of any other actin-binding protein. This would explain the emergence of filopodia in cells, consistent with the convergent elongation model.
Collectively, myosin X can produce a coordinated activity to induce the beating of parallel filament bundles. This activity is only possible for unbranched filaments with one end anchored to a substrate. In the absence of anchoring, myosins tend to separate the bundle into individual filaments. This relative sliding activity of the filaments between each other is a sign of myosin aggregation that is still poorly understood.
We have started to study the activity of myosins in an actin network, in the presence of other regulatory proteins. In the presence of fascin, a protein responsible for grouping filaments into bundles, myosins X are able to bind to the bundles. However, their ability to bend these structures results in erratic brending/beating, and sometimes breakage of the bundles.

Over the next few semesters, we will address the remaining questions in this project, following its progress as planned. In particular, we will seek to determine the architectural details of the branching networks that myosin X are able to remodel. We will then be able to study the growth of actin bundles generated by myosin X, in the presence of regulatory proteins responsible for the elongation of the barbed end of the filaments (formin and/or Ena/VASP). We will then study the transport properties of myosins as a function of the architecture of actin networks.

The results obtained so far have not been published or presented at international conferences. Depending on the progress of the project, a reasonable goal could be to communicate these results to the community by mid-2022.

Filopodia and stereocilia are two typical membrane protrusions, that perform specific functions related to the integration of mechanical cues. Filopodia are specialized dynamic actin-rich protrusions with emerging roles in the probing of cell surroundings (ECM, neighboring cells, ...) and guidance of cell migration. An amazing feature of hair cell stereocilia, essential for their function, is the staircase morphology of the bundles which implies the precise control of the actin-filled protrusion length by the interaction of myosins motors and actin binding regulators. Current models of stereocilia growth highlight the importance of the actin-filament growth control at the tip while the rest of the stereocilia core remains stable.
The marked differences in bundle size, width and actin turnover between filopodia and stereocilia are attributed to the differences in the activity of actin elongators at the tip, but also of the actin bundling proteins and myosin motors involved in building these cellular protrusions, namely Myo10 in filopodia and Myo3 in stereocilia.
Though the role of myosin motors has frequently been limited to cargo tip delivery, there is now evidence that myosins are required to initiate the bundling of parallel actin filaments and play exquisite role in the control of the stereocilia bundles in concert with actin binding proteins. In particular, Myo3 forms a very intriguing ternary complex with espin-1, a protein known to bundle actin filaments. It is thus essential to focus our attention on how myosin and actin regulatory proteins work cooperatively to gain insights into the molecular events controlling filopodia and stereocilia length.
We propose to investigate the similarities and differences of the Myo10 and Myo3 motors, their multiple activities on actin bundle dynamics. The originality of the Myo’N’Ease project relies on the unique combination of structural approaches with the study of actin assembly dynamics using in vitro reconstituted actin networks in a microfluidics system. Insights from structural studies will be tested in precise assays that reconstitute actin structures in physiological biochemical conditions, which is essential to propose a valuable mechanistic description.