Identifying the dynamics of actin and tubulin polymerization in iPSCs and in iPSC-derived neurons
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Valentina Magliocca1,*, Stefania Petrini2,*, Tiziana Franchin3, Rossella Borghi1,4, Alessia Niceforo1,4, Zeinab Abbaszadeh2, Enrico Bertini1 and Claudia Compagnucci1
1Department of Neuroscience, Unit of Neuromuscular and Neurodegenerative Diseases, Laboratory of Molecular Medicine, Bambino Gesủ Children’s Research Hospital, IRCCS, Rome 00146, Italy
2Confocal Microscopy Core Facility, Research Laboratories, Bambino Gesủ Children’s Research Hospital, IRCCS, Rome 00146, Italy
3Research Laboratories, Bambino Gesủ Children’s Research Hospital, IRCCS, Rome 00146, Italy
4Department of Science-LIME, University “Roma Tre”, Rome 00146, Italy
*These authors have contributed equally to this work
Keywords: actin filaments; microtubules; 3D image analysis; iPSCs; neuron
Received: August 30, 2017 Accepted: October 28, 2017 Published: November 15, 2017
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.
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