Gosain, H. | bioRxiv Science

bioRxiv2024

TC10 differently controls the dynamics of Exo70 in growth cones of cortical and hippocampal neurons

The exocyst is an octameric protein complex that acts as a tether for GOLGI-derived vesicles at the plasma membrane during exocytosis. It is involved in membrane expansion during axonal outgrowth. Exo70 is a major subunit of the exocyst complex and is controlled by TC10, a Rho family GTPase. How TC10 affects the dynamics of Exo70 at the plasma membrane is not well understood. There is also evidence that TC10 controls Exo70 dynamics differently in nonpolar cells and axons. To address this, we used super-resolution microscopy to study the spatially resolved effects of TC10 on Exo70 dynamics in HeLa cells and the growth cone of cortical and hippocampal neurons. We generated single particle localization and trajectory maps and extracted mean square displacements, diffusion coefficients, and alpha coefficients to characterize Exo70 diffusion. We found that the diffusivity of Exo70 was different in nonpolar cells and in the growth cone of neurons. TC10 stimulated the mobility of Exo70 in HeLa cells, but decreased the diffusion of Exo70 in the growth cone of cortical neurons. In contrast to cortical neurons, TC10 overexpression did not affect the mobility of Exo70 in the axonal growth cone of hippocampal neurons. These data suggest that mainly exocyst tethering in cortical neurons was under the control of TC10.

biophysics↗

bioRxiv2024

Tagging of Exo70 at the N-terminus compromises its assembly with the exocyst complex and changes its spatiotemporal behavior at the plasma membrane

The vesicle-tethering exocyst complex is a key regulator of cell polarity. The subunit Exo70 is required for the targeting of the exocyst complex to the plasma membrane. While the N-terminus of Exo70 is important for its regulation by GTPases, the C-terminus binds to PI(4,5)P2 and Arp2/3. Here, we compare N- and C-terminal tagged Exo70 with respect to subcellular localization, dynamics and function in cell membrane expansion. Using high-resolution imaging, we determined the spatial distribution and dynamics in different sub-compartments of un-polarized and polarized cells. With lattice light-sheet microscopy, we show that HaloTag-Exo70, but not Exo70-HaloTag, promotes the outgrowth of filopodia-like structures from the axon of hippocampal neurons. Fluorescence lifetime imaging of sfGFP-Exo70 and molecular modeling results suggest that the assembly of sfGFP-Exo70 with the exocyst complex is reduced. This is supported by single particle tracking data showing higher mobility of N- than C-terminal tagged Exo70 at the plasma membrane. The distinct spatiotemporal properties of N-terminal tagged Exo70 were correlated with pronounced filopodia formation in unpolarized cells and neurons, a process that is less reliant on exocyst complex formation. We therefore propose that N-terminal tagging of Exo70 shifts its activity to processes that are less exocyst-dependent. Why it mattersIn the life sciences, the high-resolution visualization of processes in living cells is of great interest. To tag proteins, they are fused with fluorescent proteins, usually at the N- or C-terminus of the amino acid sequence. We show here that the position of the tag alters the function and interaction of Exo70, a polypeptide involved in vesicle fusion, but also in membrane bending and expansion. Using state-of-the-art microscopic techniques, single particle localization and tracking, fluorescence lifetime imaging microscopy and co-localization in combination with modeling, we conclude that N-terminal tagging of Exo70 impairs its assembly with the exocyst complex and instead promotes the interaction of free Exo70 with the actin skeleton, which favors controlled membrane expansion into filipodia.

biophysics↗