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

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.

Seeing is believing: Current methods to observe sperm acrosomal exocytosis in real time

“…These limitations impeded the use of this probe to study the actin cytoskeleton in real time using live cells. However, a novel membrane permeable fluorescent probe, called SiR‐actin, that binds to actin filaments in vivo (Lukinavičius et al, 2014; Magliocca et al, 2017; Yamazaki et al, 2018) allowed to image actin dynamics.…”

Mitotic Centromere-Associated Kinesin (MCAK/KIF2C) Regulates Cell Migration and Invasion by Modulating Microtubule Dynamics and Focal Adhesion Turnover

“…After 60 min release, the cells started to equalize their α-tubulin content in HeLa CRISPRi/a cells, though with big standard derivations (Figure 5F,G, 7th and 8th graph). These data could explain the impaired actin dynamics observed in CRISPRi/a cells released from latrunculin B treatment (Figure 5B,D,E), since the actin-and MT-network are functionally coupled by various cross-linking proteins, and MT organization can restrict F-actin alignment [47,48], further enhancing the indirect regulation of cell motility by MCAK.…”

Interrogating Synaptic Architecture: Approaches for Labeling Organelles and Cytoskeleton Components

“…SiR-actin has been used in a number of studies focused on super resolution imaging of actin cytoskeleton in neurons (D’Este et al, 2015, 2016; Gokhin et al, 2015; Qu et al, 2016; Hoyle et al, 2017) and appears to be the easiest tool to label actin so far: its usage does not require transfection, cell membrane permeabilization or other manipulations to deliver the probe in the cell. A conjugate of silicon-rhodamine and microtubule-stabilizing drug docetaxel, named SiR-tubulin (Lukinavičius et al, 2014), can be used for visualization of microtubules (Robison et al, 2016; Dmitrieff et al, 2017; Magliocca et al, 2017; Larsson et al, 2018; Paknikar et al, 2019). Other similar fluorogenic probes based on STED-compatible dyes (such as 510R, 580CP, GeR) and tubulin-binding drugs cabazitaxel and larotaxel have been also developed recently (Lukinavičius et al, 2018).…”