Radial somatic F-actin organization affects growth cone dynamics during early neuronal development.

Durga Praveen Meka,Irina Schaefer,Oliver Kobler,Birgit Schwanke,Dennis Eggert,Marina Mikhaylova,Sergei Klykov,Robin Scharrenberg,Michael R Kreutz,Carlos G Dotti,Sabine Windhorst,Bing Zhao,Melanie Richter,Theresa König,Froylan Calderon De Anda

doi:10.15252/embr.201947743

Abstract

The centrosome is thought to be the major neuronal microtubule‐organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin‐organizing center, raising the possibility that neuronal development may, in addition, require a centrosome‐based actin radial organization. Here, we report, using super‐resolution microscopy and live‐cell imaging of cultured rodent neurons, F‐actin organization around the centrosome with dynamic F‐actin aster‐like structures with F‐actin fibers extending and retracting actively. Photoactivation/photoconversion experiments and molecular manipulations of F‐actin stability reveal a robust flux of somatic F‐actin toward the cell periphery. Finally, we show that somatic F‐actin intermingles with centrosomal PCM‐1 (pericentriolar material 1 protein) satellites. Knockdown of PCM‐1 and disruption of centrosomal activity not only affect F‐actin dynamics near the centrosome but also in distal growth cones. Collectively, the data show a radial F‐actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers, hence sustaining initial neuronal development.

Highlights

The centrosome is thought to be the major neuronal microtubuleorganizing center (MTOC) in early neuronal development, producing microtubules with a radial organization
We found a preferential localization of cytosolic F-actin puncta near the centrosome in cultured rat hippocampal neurons labeled with phalloidin and in neurons in the developing mouse cortex labeled with Lifeact-GFP
We used single molecule localization microscopy (SMLM/STORM) to image Phalloidin-Alexa647 and corroborated that F-actin is organized around the centrosome in a pocket-like structure, where several F-actin puncta surrounded the centrosome with individual puncta exhibiting an aster-like organization (Appendix Fig S2)

Summary

Introduction

The centrosome is thought to be the major neuronal microtubuleorganizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. Albeit in vitro, recent work showed that isolated centrosomes could serve as an actin-organizing center, raising the possibility that neuronal development may, in addition, require a centrosome-based actin radial organization. We report, using super-resolution microscopy and live-cell imaging of cultured rodent neurons, F-actin organization around the centrosome with dynamic F-actin asterlike structures with F-actin fibers extending and retracting actively. We show that somatic F-actin intermingles with centrosomal PCM-1 (pericentriolar material 1 protein) satellites. Knockdown of PCM-1 and disruption of centrosomal activity affect F-actin dynamics near the centrosome and in distal growth cones. The data show a radial F-actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers, sustaining initial neuronal development

Methods

Results

Conclusion