Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth
Abstract
Neurite growth is central to the formation and differentiation of functional neurons and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for calcium-dependent PLCη2 activation. To do this we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCη2 by comparing immuno-electron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labelling for PLCη2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCη2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of PLCη2 and its articulation with LIMK-1 in regulating neuritogenesis.
Citation
Arastoo , M , Hacker , C , Popovics , P , Lucocq , J M & Stewart , A J 2016 , ' Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth ' , Histochemistry and Cell Biology , vol. 145 , no. 2 , pp. 163-173 . https://doi.org/10.1007/s00418-015-1390-7
Publication
Histochemistry and Cell Biology
Status
Peer reviewed
ISSN
0948-6143Type
Journal article
Rights
© The Author(s) 2015 This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Description
We gratefully acknowledge financial support from the Wellcome Trust (Grant No. WT089803MA to J.M.L.) and the School of Medicine, University of St Andrews.Collections
Items in the St Andrews Research Repository are protected by copyright, with all rights reserved, unless otherwise indicated.