Rho-dependent regulation of cell spreading by the tetraspan membrane protein Gas3/PMP22

Abstract

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid-induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.

Figure 1

bcl-2 counteracts apoptosis induced by gas3/PMP22. (A) bcl-2, crmA, gas2, and hPLAP were coexpressed with h-TR in NIH3T3 cells. Six hours after microinjection apoptosis was induced by serum deprivation. Sixteen hours later cells were fixed and processed for immunofluorescence to detect h-TR. Survival was scored as described in the text. Data represent arithmetic means ± SD for five independent experiments (p < 0.001). (B) bcl-2, crmA, gas2, and hPLAP were coexpressed with gas3/PMP22 in NIH3T3 cells. After 24 h from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22. Survival was scored as described in the text. Data represent arithmetic means ± SD for five independent experiments (p < 0.001). (C) gas3/PMP22 and bax killer activity. Different amounts of pGDSV7S containing gas3/PMP22, bax, or gas2 were comicroinjected with pGDSV7h-TR (25 ng/μl) in the nuclei of NIH3T3 cells. After 24 h from microinjection cells were fixed and processed for immunofluorescence to detect h-TR. Survival was scored as described in the text. Datarepresent arithmetic means ± SD for three independent experiments. (D) gas3/PMP22-dependent morphological changes: confocal generated overlay showing cellular and nuclear phenotypes in NIH3T3 cells coexpressing Gas3/PMP22 and Bcl-2 (A) or h-Tr and Bcl-2 (B) or P₀ (C). NIH3T3 cells 24 h after seeding were microinjected with pGDSV3-hTR (100 ng/μl) and pGDSV7-bcl-2 (50 ng/μl), with pGDSV7-gas3/PMP22 (100 ng/μl) and pGDSV7-bcl-2 (50 ng/μl), or with pEXV-P₀ (100 ng/μl). After 24 h cells were fixed and processed for immunofluorescence analysis to visualize Gas3/PMP22 (dA), h-TR (dB), or P₀ (dC) (green) using the specific antibodies. Propidium iodide was used to visualize nuclei (red). Images were overlayered using a Zeiss confocal microscope and are displayed in pseudocolor. Bar, 5 μm.

Figure 2

gas3/PMP22-dependent biological activities in Schwann cells. (A) Confocal generated overlay showing nuclear morphology in rat Schwann cells overexpressing Gas3/PMP22 or h-TR. Schwann cells 24 h after seeding were microinjected with pGDSV7-gas3/PMP22 (100 ng/μl) or with pGDSV3-hTR (100 ng/μl); after 24 h cells were fixed and processed for immunofluorescence analysis to visualize Gas3/PMP22 or h-TR using the specific antibodies (green). Propidium iodide was used to visualize nuclei (red). Images were overlayered using a Zeiss confocal microscope and are displayed in pseudocolor. The arrow indicates a Gas3/PMP22-overexpressing cell. Bar, 5 μm. (B) bcl-2 and h-TR were coexpressed with gas3/PMP22 in Schwann cells. After 24 h from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22. Survival was scored as described in the text. Data represent arithmetic means ± SD for five independent experiments (p < 0.001). (C) Confocal generated overlay showing actin architecture and cell morphology in Schwann cells cells coexpressing Gas3/PMP22 Bcl-2 and Gas2 or h-Tr Bcl-2 and Gas2. Schwann cells 24 h after seeding were microinjected with pGDSV3-hTR (50 ng/μl), pGDSV7-bcl-2 (50 ng/μl), and pGDSV7-gas2 (25 ng/μl) or with pGDSV7-gas3/PMP22 (50 ng/μl), pGDSV7-bcl-2 (50 ng/μl), and pGDSV7-gas2 (25 ng/μl). After 24 h cells were fixed and processed for immunofluorescence analysis to visualize Gas2 (green), using the specific antibody and actin filaments (red) and rhodamine-phalloidin. Images were overlayered using a Zeiss confocal microscope and are displayed in pseudocolor. Bar, 15 μm.

Figure 3

Time-lapse images of a NIH3T3 cell overexpressing gas3/PMP22. Representative cell (arrow) injected with pGDSV7-gas3/PMP22 (100 ng/μl). Pictures at selected times after microinjection (as indicated) show the morphological changes from 17 h and the appearance of membrane blebbing at 28 h. Bar, 20 μm.

Figure 4

Time-lapse images of an NIH3T3 cell overexpressing bax. Representative cell (arrow) injected with pGDSV7-bax (50 ng/μl). Pictures at selected times after microinjection (as indicated) show the appearance of membrane blebbing at 19 h. Bar 20 μm.

Figure 5

Coexpression of Gas3/PMP22 and RhoA-V14 in NIH3T3 cells. (A) AK and rhoA-V14 were coexpressed with gas3/PMP22 in NIH3T3 cells. After 24 h from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22FLAG. Survival and morphological changes were scored as described in the text. Data represent arithmetic means ± SD for seven independent experiments (survival rate, p = 0.225; altered shape, p < 0.001). (B) Immunofluorescence analysis of NIH3T3 cells coexpressing gas3/PMP22 and AK or gas3/PMP22 and rhoA-V14 or gas3/PMP22. NIH3T3 cells 24 h after seeding were microinjected with pEXV-gas3/PMP22FLAG (100 ng/μl) and pEXV-AK (25 ng/μl) or with pEXV-gas3/PMP22FLAG (100 ng/μl) and pEXV-RhoA-V14 (25 ng/μl). After 24 h cells were fixed and processed for immunofluorescence to visualize Gas3/PMP22FLAG. Bar, 5 μm.

Figure 6

Coexpression of Gas3/PMP22, RhoA-V14 and Bcl-2 in NIH3T3 cells. (A) AK, rhoA-V14, and bcl-2 were coexpressed with gas3/PMP22 in NIH3T3 cells. After 24 h from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22FLAG. Survival and morphological changes were scored as described in the text. Data represent arithmetic means ± SD for seven independent experiments (p < 0.001). (B) gas3/PMP22 and rhoA-V14 were coexpressed in NIH3T3NEO and in NIH3T3bcl-2 cells by nuclear microinjection. After 24 h from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22FLAG. Survival and morphological changes were scored as described in the text. Data represent arithmetic means ± SD for three independent experiments.

Figure 7

Gas3/PMP22 effect on cell shape and spreading can be modulated by bacterial toxin regulating Rho activity. (A) gas3/PMP22 FLAG was overexpressed in NIH3T3 cells by nuclear microinjection. After microinjection cells were treated or not with CNF-1 in serum-free medium; 24 h later cells were fixed and processed for immunofluorescence to detect Gas3/PMP22FLAG. Morphological changes were scored as described in the text. Data represent arithmetic means ± SD for four independent experiments (p = 0.002). (B) Immunofluorescence

Figure 8

Overexpression of Gas3/PMP22 CMT1A point mutants in NIH3T3 cells. gas3/PMP22wt-FLAG, gas3/PMP22L16P-FLAG, gas3/PMP22S79C-FLAG, and gas3/PMP22G150D-FLAG were overexpressed in NIH3T3 cells by nuclear microinjection. After 24 h from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22FLAG. Survival and morphological changes were scored as described in the text. Data represent arithmetic means ± SD for five independent experiments (p < 0.001).analysis of NIH3T3 cells expressing gas3/PMP22 in the presence of CNF-1. NIH3T3 cells 24 h after seeding were microinjected with pGDSV7-gas3/PMP22FLAG (100 ng/μl). After microinjection medium was changed to serum free, and cells were treated or not with CNF-1. Twenty-four hours later cells were fixed and processed for immunofluorescence to visualize Gas3/PMP22FLAG. Bar, 25 μm. (C) C3 exoenxyme was coexpressed with gas3/PMP22 or gas2 in REF 52 cells. After 24 h from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22FLAG or Gas2. Morphological changes were scored as described in the text. Data represent arithmetic means ± SD for four independent experiments (p < 0.001). (D) Immunofluorescence analysis of REF 52 cells expressing gas3/PMP22. REF 52 cells 24 h after seeding were microinjected with pGDSV7-gas3/PMP22FLAG (100 ng/μl) and pcDNA3NEO (25 ng/μl), with pGDSV7-gas3/PMP22FLAG (100 ng/μl) and pcDNA3C3 (25 ng/μl), or with pGDSV7-gas2 (100 ng/μl) and pcDNA3C3 (25 ng/μl). Twenty-four hours later cells were fixed and processed for immunofluorescence to visualize Gas3/PMP22FLAG or Gas2. Bar, 25 μm.

Figure 9

Wortmannin (WT) can agument changes of cell shape and spreading induced by Gas3/PMP22. (A) gas3/PMP22 FLAG was overexpressed in NIH3T3 cells by nuclear microinjection. After 6 h cells were treated or not with WT and LY294002; 12 h later cells were fixed and processed for immunofluorescence to detect Gas3/PMP22FLAG. Survival and morphological changes were scored as described in the text. Data represent arithmetic means ± SD for five independent experiments (altered shape, p < 0.001). (B) Immunofluorescence analysis of NIH3T3 cells expressing gas3/PMP22 in the presence of WT. NIH3T3 cells 24 h after seeding were microinjected with pEXV-gas3/PMP22FLAG (50 ng/μl). After 6 h cells were treated or not with WT; 12 h later cells were fixed and processed for immunofluorescence to visualize Gas3/PMP22FLAG. Bar, 25 μm. (C) AK and rhoA-V14 were coexpressed with gas3/PMP22 in NIH3T3 cells. After 6 h cells were treated or not with WT; 12 h later cells were fixed and processed for immunofluorescence to detect Gas3/PMP22FLAG. Survival and morphological changes were scored as described in the text. Data represent arithmetic means ± SD for five independent experiments (altered shape, p < 0.001).

Figure 10

Gas3/PMP22 overexpression and stress fiber formation. Swiss 3T3 grown for 48 h in 0.1% FCS (A) and then treated for 30 min with LPA (B) were stained to visualize microfilaments. Serum-starved Swiss 3T3 cells were microinjected with pEXVhGas3/PMP22 (100 ng/μl). After 15 h from microinjection, LPA was added, and 30 min later cells were fixed and processed for immunofluorescence analysis to visualize actin filaments (D and F), Gas3/PMP22 (C, E, and G) and talin (H). Arrows indicate microinjected cells. Bar, 10 μm.

Figure 11

Schematic representation of Gas3/PMP22 biological activities. Gas3/PMP22 could regulate both susceptibility to apoptosis and morphological changes. Bcl-2 counteracts the apoptotic response, whereas Rho specifically counteracts morphological changes.