Matrin-3 is essential for fibroblast growth factor 2-dependent maintenance of neural stem cells

Abstract

To investigate the mechanisms underlying the maintenance of neural stem cells, we performed two-dimensional fluorescence-difference gel electrophoresis (2D-DIGE) targeting the nuclear phosphorylated proteins. Nuclear phosphorylated protein Matrin-3 was identified in neural stem cells (NSCs) after stimulation using fibroblast growth factor 2 (FGF2). Matrin-3 was expressed in the mouse embryonic subventricular and ventricular zones. Small interfering RNA (siRNA)-mediated knockdown of Matrin-3 caused neuronal differentiation of NSCs in vitro, and altered the cerebral layer structure of foetal brain in vivo. Transfection of Matrin-3 plasmids in which the serine 208 residue was point-mutated to alanine (Ser208Ala mutant Matrin3) and inhibition of Ataxia telangiectasia mutated kinase (ATM kinase), which phosphorylates Matrin-3 Ser208 residue, caused neuronal differentiation and decreased the proliferation of neurosphere-forming stem cells. Thus, our proteomic approach revealed that Matrin-3 phosphorylation was essential for FGF2-dependent maintenance of NSCs in vitro and in vivo.

What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

“…As an example, a high molecular weight protein such as matrin 3 contains many charged amino acids. Consequently, a PTM will lead to a minor spot spacing, so that the modified forms of the protein will appear as an easily recognizable train of spots [ 108 ]. Conversely, a lower molecular weight protein such as triosephosphate isomerase contains a lower number of charged amino acids, so that a single modification brings a much larger spacing on the 2D gels [ 109 ], which is much more difficult to take into account.…”

“…Consequently, a change in the structure of the actin cytoskeleton can be expected, and probed by confocal microscopy, as described in [ 130 , 131 , 132 ]. Detailed analysis of target proteins is also an interesting option [ 108 , 133 ]. Metabolic activity can also be a very good indirect validation (e.g., in [ 111 , 134 , 135 ]), and helps indeed in solving issues that are undecidable through proteomics and even enzyme activities.…”

“…In this strategy, the name of the game is to separate a modified protein form, then to search for the modification(s) that may explain the observed change in the biochemical coordinates (most often the pI). It is perfectly possible to identify phosphorylations at this stage (e.g., in [ 108 , 120 , 145 , 146 , 147 ]), and sometimes phosphorylations that have been undetected by targeted phosphoproteomic approaches can be identified by this approach (e.g., in [ 147 ]). Of course deamidation can also be identified (e.g., in [ 148 ]) but less classical modifications can be detected too.…”

How Do the Different Proteomic Strategies Cope with the Complexity of Biological Regulations in a Multi-Omic World? Critical Appraisal and Suggestions for Improvements

“…Calculations made from the theoretical and observed pI render possible the determination of the number of modifications, but any combination of them in the list of detected modifications may occur. This obviously shows that the separation afforded by 2D gels is clearly suboptimal, but even though, the degree of separation possible and the ability to take into account modifications, even without knowing their mapping, can be very valuable, biologically speaking (e.g., in references [51][52][53]). However it is our opinion that many users of 2DGE proteomics do not use fully this ability of the setup, and tend to confuse the part (a change in one form of the protein) for the whole (a change in the total amount of the protein).…”

Tsukushi is essential for proper maintenance and terminal differentiation of mouse hippocampal neural stem cells

“…patterning and neural plate formation of chicken and Xenopus, where TSK function is mediated by BMP, Vg1, Notch and Xnr2 interactions (Acharjee et al, 2015;Kuriyama et al, 2006;Morris, Almeida, Tanaka, Ohta, & Ohnuma, 2007;Ohta et al, 2006). TSK is involved in the regulation of hair cycle, wound healing and bone formation as well (Niimori et al, 2012;Niimori, Kawano, Niimori-Kita, Ihn, & Ohta, 2014;Yano et al, 2017). It also binds in vitro to connective tissue growth factor (CCN2/CTGF) which plays a regulatory role during skeletal development, growth and regeneration by interacting with BMP and TGF-β (Ohta et al, 2019;Takigawa, 2018).…”