McQueen, P. G. | Search bioRxiv

bioRxiv2022

Enabled primarily controls filopodial morphology, not actin organization, in the TSM1 growth cone in Drosophila

Ena/VASP proteins are processive actin polymerases that are required throughout animal phylogeny for many morphogenetic processes, including axon growth and guidance. Here we use live imaging of morphology and actin organization in the TSM1 axon of the Drosophila wing to dissect the mechanism of Ena action. We find that altering Ena activity has a substantial impact on filopodial morphology in this growth cone, but exerts only modest effects on actin organization. This is in contrast to the main regulator of Ena, Abl tyrosine kinase, which has profound effects on actin and only mild effects on TSM1 growth cone morphology. These data suggest that the primary role of Ena in this axon may be to link actin to morphogenetic processes of the plasma membrane, rather than regulating actin organization itself. These data also suggest that a key role of Ena, acting downstream of Abl, may be to maintain a constant filopodial organization of the growth cone, even as Abl activity varies in response to guidance cues in the environment. Summary statementWe dissect the function of the actin polymerase, Enabled, in axon growth by live-imaging of actin dynamics and axon morphology of the TSM1 neuron in its native environment in vivo.

neuroscience↗

bioRxiv2021

Computational simulations reveal that Abl activity controls cohesiveness of actin networks in growth cones

Extensive studies of growing axons have revealed many individual components and protein interactions that guide neuronal morphogenesis. Despite this, however, we lack any clear picture of the emergent mechanism by which this nanometer-scale biochemistry generates the multi-micron scale morphology and cell biology of axon growth and guidance in vivo. To address this, we studied the downstream effects of the Abl signaling pathway using a computer simulation software (MEDYAN) that accounts for mechanochemical dynamics of active polymers. Previous studies implicate two Abl effectors, Arp2/3 and Enabled, in Abl-dependent axon guidance decisions. We now find that Abl alters actin architecture primarily by activating Arp2/3, while Enabled plays a more limited role. Our simulations show that simulations mimicking modest levels of Abl activity bear striking similarity to actin profiles obtained experimentally from live-imaging of actin in wild type axons in vivo. Using a graph-theoretical filament-filament contact analysis, moreover, we find that networks mimicking hyperactivity of Abl (enhanced Arp2/3) are fragmented into smaller domains of actin that interact weakly with each other, consistent with the pattern of actin fragmentation observed upon Abl overexpression in vivo. Two perturbative simulations further confirm that high Arp2/3 actin networks are mechanically disconnected and fail to mount a cohesive response to perturbation. Taken together, these data provide a molecular-level picture of how the large-scale organization of the axonal cytoskeleton arises from the biophysics of actin networks. Highlight summaryHow do single-molecule dynamics produce multi-micron scale changes in actin organization in an extending axon? Comparison of computational simulations to in vivo data suggests that Abl kinase and Arp2/3 expand actomyosin networks by fragmenting into multiple domains, thus toggling the axon between states of local vs global internal connectivity.

cell biology↗