Identifying the dynamics of actin and tubulin polymerization in iPSCs and in iPSC-derived neurons.

Valentina Magliocca,Stefania Petrini,Rossella Borghi,Zeinab Abbaszadeh,Enrico Bertini,Alessia Niceforo,Tiziana Franchin,Claudia Compagnucci

doi:10.18632/oncotarget.22571

Abstract

The development of the nervous system requires cytoskeleton-mediated processes coordinating self-renewal, migration, and differentiation of neurons. It is not surprising that many neurodevelopmental problems and neurodegenerative disorders are caused by deficiencies in cytoskeleton-related genes. For this reason, we focus on the cytoskeletal dynamics in proliferating iPSCs and in iPSC-derived neurons to better characterize the underpinnings of cytoskeletal organization looking at actin and tubulin repolymerization studies using the cell permeable probes SiR-Actin and SiR-Tubulin. During neurogenesis, each neuron extends an axon in a complex and changing environment to reach its final target. The dynamic behavior of the growth cone and its capacity to respond to multiple spatial information allows it to find its correct target. We decided to characterize various parameters of the actin filaments and microtubules. Our results suggest that a rapid re-organization of the cytoskeleton occurs 45 minutes after treatments with de-polymerizing agents in iPSCs and 60 minutes in iPSC-derived neurons in both actin filaments and microtubules. The quantitative data confirm that the actin filaments have a primary role in the re-organization of the cytoskeleton soon after de-polymerization, while microtubules have a major function following cytoskeletal stabilization. In conclusion, we investigate the possibility that de-polymerization of the actin filaments may have an impact on microtubules organization and that de-polymerization of the microtubules may affect the stability of the actin filaments. Our results suggest that a reciprocal influence of the actin filaments occurs over the microtubules and vice versa in both in iPSCs and iPSC-derived neurons.

Highlights

Genetic investigation in humans have revealed that several mutations occurring in genes involved in the development of a functional cytoskeleton are fundamental for the correct neurologic development of the nervous system
We focus on the cytoskeletal dynamics in proliferating induced pluripotent stem cells (iPSCs) and in iPSC-derived neurons to better characterize the underpinnings of cytoskeletal organization looking at actin and tubulin repolymerization studies using the cell permeable probes SiR-Actin and SiR-Tubulin
Our results show that the quantitative parameters (Filament Mean Length, the Filament Mean Diameter, the Filament Mean Volume, Dendrite Mean Length, the Dendrite Mean Diameter, the Dendrite Mean Volume and the no. of Sholl Intersections) recorded for the re-polymerization of F-actin and MTs are significantly different both during proliferation of iPSCs and in differentiated neurons derived from iPSCs

Summary

Introduction

Genetic investigation in humans have revealed that several mutations occurring in genes involved in the development of a functional cytoskeleton are fundamental for the correct neurologic development of the nervous system. The cytoskeleton is involved in a broad series of events regulating neurogenesis and maintenance of the neuronal function, alterations of genes controlling cytoskeletal dynamics lead to severe www.impactjournals.com/oncotarget neurological diseases. In addition to defects in the development of the nervous system, alterations of the cytoskeleton can affect the maintenance of a functional neuronal network, and leading to neurodegenerative disorders. The exact sequence of events leading to neuronal death as well as the molecular determinants for the “dying back” type of axonopathy (where progressive axonal degeneration begins distally and spreads proximally to the cell body), is still obscure and no therapy currently exists to treat the neurodegenerative progression

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