Neuronal filopodium formation induced by the membrane glycoprotein M6a (Gpm6a) is facilitated by coronin‐1a, Rac1, and p21‐activated kinase 1 (Pak1)

Abstract

Stress-responsive neuronal membrane glycoprotein M6a (Gpm6a) functions in neurite extension, filopodium and spine formation and synaptogenesis. The mechanisms of Gpm6a action in these processes are incompletely understood. Previously, we identified the actin regulator coronin-1a (Coro1a) as a putative Gpm6a interacting partner. Here, we used co-immunoprecipitation assays with the anti-Coro1a antibody to show that Coro1a associates with Gpm6a in rat hippocampal neurons. By immunofluorescence microscopy, we demonstrated that in hippocampal neurons Coro1a localizes in F-actin-enriched regions and some of Coro1a spots co-localize with Gpm6a labeling. Notably, the over-expression of a dominant-negative form of Coro1a as well as its downregulation by siRNA interfered with Gpm6a-induced filopodium formation. Coro1a is known to regulate the plasma membrane translocation and activation of small GTPase Rac1. We show that Coro1a co-immunoprecipitates with Rac1 together with Gpm6a. Pharmacological inhibition of Rac1 resulted in a significant decrease in filopodium formation by Gpm6a. The same was observed upon the co-expression of Gpm6a with the inactive GDP-bound form of Rac1. In this case, the elevated membrane recruitment of GDP-bound Rac1 was detected as well. Moreover, the kinase activity of the p21-activated kinase 1 (Pak1), a main downstream effector of Rac1 that acts downstream of Coro1a, was required for Gpm6a-induced filopodium formation. Taken together, our results provide evidence that a signaling pathway including Coro1a, Rac1, and Pak1 facilitates Gpm6a-induced filopodium formation. Keywords: Coronin-1a, filopodium, hippocampal neuron, membrane glycoprotein M6a, Ras-related C3 botulinum toxin substrate 1 (Rac1), rat. Abbreviations used: CA, constitutively activate; Coro1a, coronin-1a; DN, dominant negative; ECL, enhanced chemiluminescence; EGFP, enhanced green fluorescent protein; GAPs, GTPase activating proteins; GDIs, GDP dissociation inhibitors; GEFs, guanine nucleotide exchange factors; Gpm6a, neuronal membrane glycoprotein M6a; N2a, mouse neuro-2a neuroblastoma cell line; Pak1, p21-activated kinase 1; PBS, phosphate-buffered saline; Rac1, Ras-related C3 botulinum toxin substrate 1; RFP, red fluorescent protein; ROI, region of interest; SDS, sodium dodecyl sulfate; TBS, tris-buffered saline; wt, wild type. 46

Gene expression profiling of fast- and slow-growing non-functioning gonadotroph pituitary adenomas

“…Two other genes that definitely need further characterization are GPM6A (the highest positive fold of change on RNA-seq) and PCDH18 (the highest association with TVDT). GPM6A is highly expressed in brain tissue and is involved in neuronal differentiation, including differentiation and migration of neuronal stem cells, neuronal plasticity and filopodia outgrowth and motility and probably synapse formation (42). PCDH18 belongs to the protocadherin gene family and is thought to play a role in the establishment and function of specific cell-cell connections in the brain (43).…”

Alanine Scanning Mutagenesis of the C-Terminal Cytosolic End of Gpm6a Identifies Key Residues Essential for the Formation of Filopodia

“…Each experiment was scored blind. Previously we have shown that all filopodial protrusions induced by Gpm6a overexpression are labeled by phalloidin ( Alvarez Julia et al, 2016 ) and that quantification of phalloidin-labeled filopodia coincided with quantification when EGFP is used to detect filopodia in transfected neurons ( Alvarez Julia et al, 2016 ).…”

“…Dissociated neuronal cultures were prepared from hippocampi of embryonic day 19 Sprague–Dawley rats obtained from the Faculty of Veterinary Sciences (Buenos Aires, Argentina), as described previously ( Alvarez Julia et al, 2016 ). Briefly, hippocampal tissue was treated with 0.25% (wt/vol) trypsin in HBSGK buffer (4.8 g/l HEPES, 8.7 g/l NaCl, 0.22 g/l KCl, 0.36 g/l glucose, pH 7.4) for 15 min at 37°C.…”

“…In primary hippocampal neurons, filopodium density (the number of filopodia per 45 μm neurite length) was quantified as described previously ( Alvarez Julia et al, 2016 ) in 35–55 neurites of 10–20 different neurons from each transfection condition done in duplicates in at least two independent experiments. Each experiment was scored blind.…”

“…The localization of Gpm6a in lipid microdomains as well as Src kinases and MAPK activity have been reported to participate ( Scorticati et al, 2011 ). More recently, it has been shown that the actin regulator Coronin 1a associates with Gpm6a in rat hippocampal neurons and facilitates the formation of filopodia by Gpm6a and Rac1/Pak1 signaling pathway has been shown to be involved ( Fuchsova et al, 2015 ; Alvarez Julia et al, 2016 ).…”