title: 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 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 immunoelectron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labeling 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 the PLCη2 EF-hand domain and articulation of PLCη2 with LIMK-1 in regulating neuritogenesis.
authors.affiliation: School of Medicine, Medical and Biological Sciences Building, North Haugh, University of St Andrews, St Andrews, Fife, KY16 9TF, UK.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Arastoo
authors.affiliation: School of Medicine, Medical and Biological Sciences Building, North Haugh, University of St Andrews, St Andrews, Fife, KY16 9TF, UK.|Bioimaging Centre, Geoffrey Pope Building, College of Life and Environmental Sciences, University of Exeter, Exeter, EX4 4QD, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Hacker
authors.affiliation: School of Medicine, Medical and Biological Sciences Building, North Haugh, University of St Andrews, St Andrews, Fife, KY16 9TF, UK.|Veterans Affairs Medical Center, Miami, FL, 33125, USA.
authors.forename: Petra
authors.initials: P
authors.lastname: Popovics
authors.affiliation: School of Medicine, Medical and Biological Sciences Building, North Haugh, University of St Andrews, St Andrews, Fife, KY16 9TF, UK.
authors.forename: John M
authors.initials: JM
authors.lastname: Lucocq
authors.affiliation: School of Medicine, Medical and Biological Sciences Building, North Haugh, University of St Andrews, St Andrews, Fife, KY16 9TF, UK. ajs21@st-andrews.ac.uk.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Stewart
mesh_terms: D000818:Animals; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D054461:Lim Kinases / Q000378:metabolism*; D051379:Mice; D016501:Neurites / Q000187:drug effects*; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D014212:Tretinoin; D054461:Lim Kinases; C515893:Limk1 protein, mouse; D054801:Phosphoinositide Phospholipase C; C503884:Plch2 protein, mouse
keywords: Calcium signaling; Cell differentiation; Electron microscopy; Neuritogenesis; Protein–protein interaction