MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation Ying Yang a,1 , Marco Marcello b,1 , Volker Endris a , Rainer Saffrich c , Roger Fischer b , Michael F. Trendelenburg b , Rolf Sprengel d , Gudrun Rappold a,⁎ a

Institute of Human Genetics, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany Biomedical structure analysis group, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany c Department of Medicine V, University of Heidelberg, Heidelberg, Germany d Department of Molecular Neurobiology, Max-Planck-Institute for Medical Research, 69120 Heidelberg, Germany b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Over the past several years, it has become clear that the Rho family of GTPases plays an

Received 16 November 2005

important role in various aspects of neuronal development including cytoskeleton

Revised version received

dynamics and cell adhesion processes. We have analysed the role of MEGAP, a GTPase-

22 March 2006

activating protein that acts towards Rac1 and Cdc42 in vitro and in vivo, with respect to its

Accepted 4 April 2006

putative regulation of cytoskeleton dynamics and cell migration. To investigate the effects

Available online 19 April 2006

of MEGAP on these cellular processes, we have established an inducible cell culture model consisting of a stably transfected neuroblastoma SHSY-5Y cell line that endogenously

Keywords:

expresses MEGAP albeit at low levels. We can show that the induced expression of MEGAP

GTPase-activating protein

leads to the loss of filopodia and lamellipodia protrusions, whereas constitutively activated

MEGAP/srGAP3

Rac1 and Cdc42 can rescue the formation of these structures. We have also established

Cell migration

quantitative assays for evaluating actin dynamics and cellular migration. By time-lapse

Actin/microtubule dynamics

microscopy, we show that induced MEGAP expression reduces cell migration by 3.8-fold and

Focal complex

protrusion formation by 9-fold. MEGAP expressing cells also showed impeded microtubule

Video microscopy

dynamics as demonstrated in the TC-7 3x-GFP epithelial kidney cells. In contrast to the wild type, overexpression of MEGAP harbouring an artificially introduced missense mutation R542I within the functionally important GAP domain did not exert a visible effect on actin and microtubule cytoskeleton remodelling. These data suggest that MEGAP negatively regulates cell migration by perturbing the actin and microtubule cytoskeleton and by hindering the formation of focal complexes. © 2006 Elsevier Inc. All rights reserved.

Introduction Cell migration plays a central role in a variety of fundamental processes including embryogenesis, immune response, tissue repair, tumorigenesis, as well as congenital developmental

brain defects. Cell migration is a rather complex process, requiring the coordinated activity of the actin and microtubule cytoskeleton and the adhesion system. It can be described as a multistep cycle comprising of the extension of protrusions at the cell front, formation of stable attachments near the

⁎ Corresponding author. Fax: +49 6221 56 5155. E-mail address: [email protected] (G. Rappold). 1 The first two authors contributed equally to the manuscript. 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.04.001

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protrusions, translocation of the cell body forward, and release of adhesions and retraction at the rear end of the cell [1]. The membrane extension consists of both lamellipodia and filopodia, and it takes place primarily around the cell front. Lamellipodia are broad, veil-like structures, whereas filopodia are thin, cylindrical, needle-like projections. The formation of these highly dynamic protrusive structures at the leading edge of moving cells requires force generated by actin polymerisation. Actin monomers polymerise only onto the existing barbed end of the actin array, so that the actin filaments are elongated towards the cell periphery [2]. The formation of a protrusion initiates the migration cycle process, but in order for the cell to move, the protrusions have to be stabilised to the substratum. These adhesion sites serve as traction points for impellent forces that push the cell moving forward. Migration needs the continuous coordinated formation and disassembly of adhesions [3]. The concerted interaction of microtubules and actin is essential for maintaining cell motility. Dynamic instability of microtubules is required to keep the polarised actin cytoskeleton-based protrusions in the cell-leading edge [4,5]. Microtubules promote the release of substrate adhesions by targeting them at the cell front and rear with different frequencies [6]. This multistep migration process is tightly controlled by the regulated interaction of numerous proteins and the activation of specific signalling pathways. Among intracellular signalling molecules, small GTP-binding proteins of the Rho subfamily, in particular Cdc42, Rac1, and RhoA, have been shown to play important roles in the control of this process through regulating actin dynamics, microtubule cytoskeleton, and the assembly of integrin-mediated focal complexes [7–10]. We have found that haploinsufficiency of a RhoGAP gene, MEGAP, is associated with mental retardation in several patients. MEGAP, also known as WRP [11], represents a functional GTPase-activating (GAP) protein as demonstrated by an in vitro GAP assay leading to an inactivation of the Rac1 and Cdc42-mediated signal transduction processes [12]. To study the presumed role of MEGAP in actin organisation and cell migration, we have established an inducible cell model system that expresses MEGAP under the control of a doxycycline-inducible promoter. The ability of Rho family GTPases in signalling events is dependent on the fraction of GTP/GDP-bound forms in the cell. The switch of GTPases between active GTP-bound states and inactive GDP-bound states is regulated by two divergent factors: guanine nucleotide exchange factors (GEFs) that enhance the exchange of GDP for GTP [13,14] and the GTPase-activating proteins (GAPs) that augment hydrolysis of bound GTP [15]. In addition, GTPases are regulated by guanine nucleotide dissociation inhibitors, which are thought to block the GTPase cycle by sequestering the GDPbound form [16]. Even though a substantial amount of work has been focused on the characterisation of various GAPs [15], little is known to quantitatively define the function of a GAP regarding the direct influence on cytoskeleton dynamics in vivo. We report here the effect of MEGAP towards cellular morphology and cell migration in SHMEGAP cells, which are derived from a SHSY-5Y neuroblastoma line. We demonstrate that expression of MEGAP can downregulate Cdc42 and Rac1

activity and attenuate random migration of the cells by impairing actin and microtubule dynamics, formation of protrusions, and focal complexes.

Materials and methods Reagents, antibodies, DNA constructs, and cell lines DMEM low glucose, HEPES medium, and tetracycline-free fetal calf serum for cell culture were from PAA Laboratories (Linz, Austria). Fugene 6 (Roche Diagnostics, Mannheim, Germany) and Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) were applied for transfection of SHSY-5Y cell line and the line SHMEGAP (derived from SHSY-5Y, stably transfected with MEGAP) with a DNA: reagent ratio of 1:6 and 4:10, respectively. Both blasticidin and zeocin from Invitrogen were used for MEGAP-positive clone selection. Doxycycline (dox) from Invitrogen was used for the induction of MEGAP expression. Bradykinin, PDGF (BB), and TRITCconjugated phalloidin were purchased from Sigma (Munich, Germany). Rhodamine-conjugated dextran, used as a microinjection indicator, was from Sigma as well as poly-lysine; fibronectin was from ICN (ICN Biomedicals Inc. Aurora, Ohio). Poly-lysine and fibronectine were used for coverslips' coating. The following antibodies were used for immunofluorescence, Western blots, and GST-pulldown assay: polyclonal rabbit anti-MEGAP (generated by Pineda, Berlin, Germany); mouse anti-Rac1 (Upstate, Dundee, UK); Cdc42 (BD Transduction Laboratories, Heidelberg, Germany); focal adhesion kinase, FITC-conjugated goat anti-mouse and anti-rabbit (Sigma); and horseradish peroxidase-coupled goat antimouse and goat anti-rabbit (Sigma) secondary antibodies. The pcDNA™6/TR vector (blasticidin selective; Invitrogen) containing the tetracycline repressor was used for establishing the line SHSY-5Y TR1. The mRNA of the MEGAP protein contains three alternatively spliced forms differing in the use of exon 12. The longest form (8327 bp, MEGAP12a) consists of 22 exons with an open reading frame of 3299 bp, encoding a putative protein of 1099 amino acids and including an FCH, GAP, and SH3 domain. The MEGAP12a (MEGAP) cDNA bases from 1 to 3062 were cloned into the pcDNA4/TO/myc–His vector (zeocin selective; Invitrogen) resulting in C-terminal 6× His/myc-tagged plasmid pMEGAP. The MEGAP mutant R542I (pMEGAP R542I) was introduced by PCR-mediated mutagenesis using a primer containing a G to T exchange at position 1727 of the MEGAP cDNA sequences. To generate GFP-Rac1 and GFP-Cdc42 constitutively active (pGFP-Rac1G12V and pGFP-Cdc42G12V) constructs, we cloned GFP cDNA sequence into vector pcDNA3.1+ containing constitutively active Rac1 (pRac1G12V) and Cdc42 sequences (Guthrie cDNA Resource Center, Sayre, USA) at sites NheI/KpnI. Rat γ-actin sequence was cloned from plasmid pβactin-eGFP gamma cyto (kind gift of Prof. Andrew Matus) into plasmid GFP-Cdc42G12V (Cdc42G12V sequence was replaced by Rat γ-actin) at the HindIII/XbaI sites (pGFP-actin). Because MEGAP shows an expression pattern almost entirely restricted to brain, a human neuronal-like neuroblastoma cell line SHSY-5Y (kind gift of Prof. Klaus Unsicker) was

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chosen for the study. TC-7 3x-GFP, a green monkey kidney cell line that stably expresses GFP and E-MAP-115 (ensconsin) microtubule-binding domain (EMTB) fusion protein, was a kind gift of Prof. Chloë Bulinski.

Generation of a stable MEGAP-inducible cell line The neuronal cell line, SHSY-5Y TR1 (TR1), expressing the tetrepressor, was generated by transfection of pcDNA™6/TR vector into the human neuroblastoma cell line SHSY-5Y using the FUGENE 6 reagent. The clones were maintained in DMEM low glucose medium with 20% of tetracycline-free fetal calf serum and were selected by adding 10 mg/ml of blasticidin. The MEGAP-expressing line was established by transfecting pMEGAP into the TR1 line and was double selected by adding 10 mg/ml of blasticidin and 400 mg/ml of zeocin. Clone 16 was used for this study (and is later referred to as SHMEGAP). MEGAP expression was induced by adding 0.5–1 μg/ml doxycycline (dox) for more than 16 h in different clones and could be further defined by Western blot and immunofluorescence.

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Rho GTPase activation assay The levels of the active forms of Rac1 and Cdc42 in induced and uninduced SHMEGAP cells were examined by a Rho GTPase activation assay (GST-pulldown assay). Briefly, the dox-treated and dox-untreated cells in 10-cm dishes were lysed using lysis buffer (50 mM Tris/HCL pH 7.5, 200 mM NaCl, 10 mM MgCL2, 1% NP-40, 5% Glycerol, 1 mM DTT, and 1 mM PMSF). The centrifuge-clarified cell lysates (130 μg proteins for each sample) were incubated for 45 min at 4°C with 20 μg GSTPBD fusion proteins (bound to glutathione–agarose beads; Sigma). The incubated products were washed 3 times with lysis buffer. GTP-bound Rac1 and Cdc42 were detected using (1:1000) monoclonal anti-Rac and Cdc42 antibodies by 12% SDS–PAGE (see Western blot). For the measurement of RhoA activity in the induced and uninduced SHMEGAP line and the parental TR1 line, cells were plated at subconfluency in 10-cm dishes. MEGAP expression was induced by adding doxycyclin 72 h before cell lysis. Equal amounts of cell lysates 50–100 μg (2 mg/ml) were subjected to the luminescence-based G-LISA Rho activation assay (Cytoskeleton Inc., Denver, USA). The

Fig. 1 – Inducible expression of MEGAP in SHMEGAP line reduces GTP-bound Cdc42 and Rac1, but not GTP-bound RhoA. (A) SHMEGAP cells were treated with dox for 24 h; Western blot was carried out to detect MEGAP protein with polyclonal anti-MEGAP antibody. Lane 1 indicates the undetectable endogenous MEGAP and lane 2 indicates the ectopically expressed MEGAP protein. (B) FITC-conjugated goat anti-rabbit second antibody was used for immunofluorescence against the first anti-MEGAP antibody. Immunostaining confirmed the expression of MEGAP in the cells and its cytoplasmic location, scale bar 40 μm. (C and D) The same amount of proteins from dox-untreated and dox-treated cell lysates were incubated with GST-PBD fusion protein. In lanes C1 and D1, cells were not treated with dox, the GTP-bound Cdc42 and Rac1 could be detected using anti-Cdc42 and anti-Rac antibodies, respectively. However, in lanes C2 and D2, GTP-bound Cdc42 and Rac1 were not detectable due to the expression of MEGAP. In the bottom panel, lanes 1 and 2 (C, D) are control experiments demonstrating the levels of total endogenous Cdc42 and Rac1 expressed in dox-untreated and dox-treated SHMEGAP cells. (E) Active RhoA levels in dox-induced and dox-uninduced SHMEGAP cells were measured with a luminescence-based G-LISA Rho activation assay. On the y axis, relative light units (RLU) are displayed. Columns 1–4 represent GTP-bound RhoA levels of MEGAP-induced cell lysates (1) (2 mg/ml), MEGAP-uninduced cell lysates (2), positive control (3) (5 ng of active RhoA protein), and negative control (4) (lysis buffer), respectively. Three independent experiments were carried out and each measurement was performed in triplicate. The data were analysed with Student's t test and the results showed that there was no statistically significant difference (P = 0.82) in the RhoA levels between dox-induced and dox-uninduced SHMEGAP cells (E).

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assay was performed according to the manufacturers protocol. Luminescence was read on a Lucy II luminometer Anthos (Mikrosysteme, Krefeld, Germany). For each probe, triple measurements were performed and the results from three independent experiments pooled.

Immunofluorescence, focal complexes detection, and Western blot For immunofluorescence, around 5 × 104 of dox-treated and dox-untreated SHMEGAP and TR1 cells were starved in 1% serum for 24 h and plated on 1.8-cm diameter coverslips coated with 0.5 mg/ml poly-lysine in 6-well plates. Cells were fixed immediately for 15 min in 3.7% formaldehyde and permeabilised for 5 min in 0.4% Triton X-100. Polyclonal antibody (1:500) against-MEGAP was used for detecting exogenously expressed MEGAP protein for 1 h, followed by incubation with FITC-conjugated secondary goat antirabbit antibody (1:1000) on 37°C for 45 min. DAPI (4′-6Diamidino-2-phenylindole) (Sigma) dye was used for nuclei staining. To detect the focal complexes, dox-treated and doxuntreated SHMEGAP cells were serum starved for 24 h and plated on 10 μg/ml fibronectin-coated coverslips, mouse antiFAK antibody (1:700) was used, followed by (1:1000) FITCconjugated goat anti-mouse secondary antibody. F-actin was stained by adding TRITC-conjugated phalloidin (stock, 20 μg/ ml, 1:300 dilution) with secondary antibody simultaneously.

Images were obtained on an Axiophot microscope (Zeiss, Germany) equipped with a camera (JAI CV M-300, Yokohama, Japan). Western blot was used for checking the inducible expression of MEGAP in SHMEGAP cells. Of a 6-well plate of dox-treated and dox-untreated cells, one well was lysed using 100 μl lysis buffer; 20 μl of cell lysate was used for 8% SDS–PAGE analysis. Proteins were transferred onto HybondP membrane (Amersham Pharmacia Biotech, Freiburg, Germany) and blocked for more than 2 h or overnight at 4°C in 1× PBS, 0.05% Tween-20, and 3% nonfat dry milk. The filters were then incubated with MEGAP antibody (1:700) followed by horseradish peroxidase-coupled goat anti-rabbit secondary antibody (1:15000). After each incubation with antibodies, the filters were washed with 1× PBS, 0.05% Tween-20 for 7 min, 3 times. The expression of MEGAP was visualised by the ECL detection kit (Amersham Pharmacia Biotech).

Documentation of cell morphology alteration, cell spreading To monitor the morphological changes induced by MEGAP, the SHMEGAP cells were plated on poly-lysine-coated CELLocates (Eppendorf, Hamburg, Germany). These sterile coverslips come with a microgrid pattern that allows the relocation of the target cells even after a period of incubation. The TR1 cells were used as control. The same field of cells was photographed on an Axiovert25 (Carl Zeiss, Göttingen, Germany)

Fig. 2 – Cell rounding and loss of actin filament protrusions induced by expression of MEGAP. (A) The dox-untreated cells showed well-pronounced protrusions. (B) In contrast, after 24 h of dox treatment, cells became rounded and most of the cells lost their ability to form protrusions upon the expression of MEGAP. The dox-treated and dox-untreated SHMEGAP cells were plated on poly-lysine-coated coverslips for 2 h and stained with TRITC-conjugated phalloidin specific for F-actin in the presence of 100 ng/ml bradykinin and 5 ng/ml PDGF BB. (C) The untreated cells formed lamellipodia-like structures interspersed with filopodia-like protrusions in the cell leading edge. (D) The dox-treated cells showed a rounded shape; there were no actin filaments visible in the cells. Scale bar, 20 μm.

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microscope, coupled with a Leica DFO 320 camera (Leica, Heerbrugg, Germany), before and after 24 h of treatment with dox. For cell spreading, dox-treated and dox-untreated cells were starved 24 h in 1% FCS medium and seeded on polylysine-coated coverslips for 2 h. To induce filopodia and lamellipodia formation, we incubated the cells with bradykinin 100 ng/ml for 10 min and PDGF (BB) 5 ng/ml for 15 min, then fixed immediately for 15 min in 3.7% formaldehyde and permeabilised for 5 min in 0.4% Triton X-100. The F-actin was stained by TRITC-conjugated phalloidin (20 μg/ml with a 1:300 dilution). The cells were visualised by the same microscope as in the immunofluorescence analysis. To determine whether constitutively active Cdc42 and Rac1 can rescue MEGAPinduced effects on actin cytoskeleton, we transfected SHMEGAP cells with plasmids GFP-Rac1 G12V and GFP-Cdc42 G12V for 24 h. Three independent experiments were performed and in total 20 cells for each transfection were examined. The remaining steps were as for cell spreading.

Microinjection One day prior to the injection, cells were seeded on the small glass coverslips CELLocate. Injection capillaries were pulled with an automatic puller (P-87 Brown Flaming Micropipette Puller, Sutter Instrument Company, Novato CA, USA). In each experiment, 100–200 cells were usually injected within 20– 30 min. The plasmids pMEGAP and pRac1G12V were diluted in PBS at a concentration of 20 ng/ml and coinjected into TC-7 3x-

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GFP cells with sterile filtered rhodamine B isothiocyanatedextran (R9379; Sigma ChemicalCo., Munich, Germany) at concentrations of 1.5%. Plasmids pMEGAP (50 ng/ml) and pMEGAP R542I (50 ng/ml) were also coinjected with pGFP-actin (60 ng/ml) into TR1 cells. The probes were centrifuged before injection for 10 min at 20,000×g. To minimise the cytotoxical effects of the modified values of temperature and CO2 during the injection, the cells were always kept in an incubator, overlaid with 2 ml medium, and injected as quickly as possible. Microinjection was performed with an AIS injection system (Zeiss, Jena, Germany).

Video microscopy and imaging analysis TC-7 3x-GFP, SHMEGAP, and TR1 cells plated on glass coverslips were imaged always 24 h after transfection or microinjection. For microscopic analysis, each coverslip was placed in a custom-designed chamber, medium with HEPES was introduced, and the chamber was placed on a heating stage to minimise the cytotoxical effects of the modified values of temperature and CO2 during the imaging. Epifluorescence microscopy was performed with an Axioplan microscope (Carl Zeiss, Jena, Germany) operating with a Planapochromat 63× lens and the appropriate filter sets for the used fluorescence stainings. Images and sequences were taken with a Hamamatsu C7780 Peltier cooled 3-chip CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan). Camera exposure and shutter controls were under the control of Simple PCI software (COMPIX, Pittsburgh, USA).

Fig. 3 – Constitutively active Cdc42 and Rac1 rescue filopodia and lamellipodia formation in MEGAP-expressing SHMEGAP cells. Constitutively active Cdc42- and Rac1-GFP fusion proteins were transfected into SHMEGAP line for 24 h and stained with TRITC-conjugated phalloidin. Three independent experiments were performed and for each experiment 20 of the successfully transfected cells were examined. In all transfected cells, MEGAP expression induced by dox treatment could no longer suppress the formation of filopodia (A, B) and lamellipodia (C, D) mediated by constitutively active Cdc42G12V and Rac1G12V, respectively. The untransfected cells in panels A–D still showed a rounded morphology. Scale bar, 20 μm.

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Fig. 4 – Loss of focal complexes in MEGAP-expressing cells. Cells were serum starved for 24 h. The dox-treated and dox-untreated cells were replated on 10 μg/ml fibronectin-coated coverslips and stained with focal adhesion kinase (FAK) antibody for 2 h, followed by FITC-conjugated anti-mouse second antibody. (A–C) Dox-untreated cells produced dot-like and oblong-shaped focal complexes (green) on a lamellipodia-like protrusion in panel A; (B) a TRITC-conjugated phalloidin staining for F-actin of panel A; and (C) the merged picture of panels A and B. (D–F) In panel D, Dox-treated cells could not assemble focal complexes and showed a rounded shape; panel E was TRITC-conjugated phalloidin staining of panel D; and panel F was the merged picture of panels D and E. Scale bar, 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Two types of time-lapse images were collected: a short time-lapse for the calculation of area variation, and a long one for the analysis of motility and cellular protrusions. Short time-lapses were collected at intervals of 5–15 s, the whole duration of the movie being between 5 and 10 min. Up to 1-s exposures were used for both red and green images. To examine the morphometric variations in the cell during time, colour-encoded overlays were created and areas were quantified with the Simple PCI software; the resulting data were directly logged to Microsoft Excel for analysis. The quantitative analysis of unprocessed images was always found to be in agreement with the visual analysis of morphological criteria in processed colour overlays, indicating that the image-processing regime did not degrade the positional information in the images. Long time-lapses were collected in bright field at intervals of 5 min, with a microscope Olympus IX70 microscope (Olympus Optical, Hamburg, Germany) used with an incubation chamber to maintain cell culture conditions at 37°C and 5% CO2. Images were acquired every 5 min with a colorview-12 camera (SIS, Münster, Germany) using a 20× objective, during a period from 24 to 48 h and stored on a PC workstation for further analysis. Image acquisition and processing was controlled by SIS AnalySIS 3.2 software. Long time-lapses were analysed with Image J (National Institute of Mental Health, Bethesda, Maryland, USA) and the resulting data were directly logged to Microsoft Excel for analysis. The following criteria were applied to cell motility measurements: (1) the cell had to remain discernable throughout the whole

Fig. 5 – Reduction of migration speed in MEGAP-expressing cells. The average length of tracks (in μm/h, y axis) from 10 dox-treated and 10 dox-untreated SHMEGAP cells as well as 10 dox-treated and 10 dox-untreated TR1 cells is shown over a period of more than 14 h. TR1 cells represent the parental neuronal cell line SHSYTR1 expressing the tet-repressor. The untreated SHMEGAP cells traveled a distance more than 3.8 times longer than the SHMEGAP-treated cells, the P value was <7.884e−05. ‘X’ indicates a significant difference between dox-untreated and dox-treated cells. The P values calculated between dox-untreated and dox-treated TR1 cells and between dox-treated TR1 and dox-untreated SHMEGAP cells were not significant, as <0.8528 and <0.8404, respectively. See also movie 1 (A–D).

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Fig. 6 – MEGAP-expressing cells lost the ability to form protrusions. The dox-untreated and dox-treated cells were analysed and compared for their capacity of forming protrusions by counting the number of protrusions through 12 h. Panels A and B are example montages taken from dox-untreated and dox-treated cells showing time intervals of 50 min (see also movie 2). (A) The untreated cells were forming longer and dynamic protrusions throughout the movie. (B) Dox-treated cells either did not produce or produced very short and relatively static protrusions during 250 min. The untreated cells produced 9-fold more protrusions than the treated cells. The statistical analysis of 10 untreated and treated cells revealed a significant difference with the P value <9.698e−08. Scale bar, 10 μm.

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movie; (2) the displacements of the centre of the cell were recorded every 10 min and all the contributions were added up at the end and converted in micron; and (3) in case of mitosis, just one of the daughter cells was traced. The following criteria were applied to analysis of cellular

protrusions: (1) the cell had to remain discernable throughout the whole movie; (2) the cellular protrusions were counted as any lamellipodium or filopodium that lasted at least 10 min; (3) in case of mitosis, the protrusions of just one of the daughter cells were counted; and (4) only the

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appearance of a protrusion was recorded as an event, not its disappearance.

dox-induced and dox-uninduced SHMEGAP cells, suggesting that RhoA activity is not regulated by MEGAP (Fig. 1E).

Results

Expression of MEGAP leads to cell rounding and loss of protrusions in SHMEGAP cells

Inducible expression of MEGAP in SHMEGAP cells reduces GTP-bound Cdc42 and Rac1 We have previously reported that MEGAP is predominantly expressed in fetal and adult brain tissues and that the GAP domain of MEGAP acts towards the GTPases Rac1 and Cdc42 but not RhoA in vitro [12]. Rac1 and Cdc42 have been implicated in the regulation of cytoskeleton-related cellular processes such as cell polarisation, migration, neurite outgrowth, and phagocytosis [17,18]. To evaluate the function of MEGAP in the control of these GTPases and to analyse the cytoskeleton modulation specifically induced by ectopically expressed MEGAP, we generated a MEGAP-inducible cell line termed SHMEGAP. SHMEGAP is derived from the neuroblastoma cell line SHSY-5Y TR1 (containing the tet-repressor) and expresses endogenous MEGAP albeit at low amounts, which is generally not detectable on a Western blot. In SHMEGAP cells, expression of MEGAP can be controlled by a Tet-on mammalian expression system by applying doxycycline (dox). Fig. 1A, lane 2, shows the expression of MEGAP protein when SHMEGAP cells were treated with 1 μg/ml dox for 24 h compared to the untreated situation (lane 1). Immunofluorescence staining of the same cell line with an anti-MEGAP antibody revealed that nearly all treated cells express the given MEGAP protein, which localised to the cytoplasm (Fig. 1B), whereas in untreated cells, endogenous MEGAP was undetectable on a Western blot (Fig. 1A) but detectable from an RT-PCR assay carried out with cell lysates (data not shown). By Rho GTPase activation assay, we could confirm that MEGAP reduces GTP-bound Cdc42 and Rac1 in vivo in SHMEGAP cells (Figs. 1C and D). To assess the influence of MEGAP on the level of active RhoA, we have introduced a more sensitive, luminescencebased G-LISA Rho activation assay, as RhoA levels in SHMEGAP cells were below detection level of conventional RBD assays. The results indicate that there was no statistically significant difference (P = 0.82) in the RhoA levels between

By comparing the dox-untreated and dox-treated cells, the cell morphological changes were analysed. Using CELLocate, we were able to monitor the same group of cells prior and post treatment. We found that expression of MEGAP led to cell rounding and loss of filopodia and lamellipodia structures (Fig. 2B). This effect was predominant regardless of the presence of extracellular agonists like bradykinin and PDGF. The effect was reproducible using different MEGAP-expressing clones. Doxuntreated SHMEGAP cells were able to produce protrusions (Fig. 2A). As control, the parental cell line TR1 maintained the ability to form protrusions independently from dox treatment (data not shown). To answer the question whether the abovementioned changes were derived from MEGAP-mediated actin cytoskeleton remodelling, we replated the dox-treated and doxuntreated cells to poly-lysine-coated coverslips for 2 h and stained them with TRITC-conjugated phalloidin. It was obvious that the dox-treated cells could not properly spread and form actin-based structures on coated coverslips. The Cdc42-mediated and bradykinin-induced filopodia and the Rac1-mediated and PDGF-induced lamellipodia were not observed in most cells (Fig. 2D). These phenomenons were reversible after dox was removed from the culture medium. Moreover, the treated cells lost RhoA-associated stress fibers as well. In contrast, filopodia and lamellipodia structures were clearly visible in untreated cells (Fig. 2C). In the control TR1 line, both dox-treated and dox-untreated cells displayed the specific actin-related structures (data not shown). These data suggest that expression of MEGAP has an influence on cell shape, causing a general rounding up and loss of protrusions.

MEGAP exerts its function upstream of Cdc42 and Rac1 in the signal transduction pathways Previous assays have shown that MEGAP exerts an effect on the cytoskeleton structure. Microinjection and transfection of

Fig. 7 – Loss of actin dynamics in the cell leading edge upon MEGAP expression. SHMEGAP cells transfected with the GFP-actin construct were placed 24 h later in a custom-designed chamber and mounted on a heating stage in the presence of agonists (5 ng/ml of PDGF BB and 100 ng/ml of bradykinin). The time-lapse video microscopy was carried out using Axioplan (Zeiss) microscope and Hamamatsu C7780 Peltier cooled 3 CCD camera with a time interval of 10 s for more than 5 min. (A) Dox-untreated cell and (C) MEGAP R542I mutant injected cell showed pronounced actin lamellipodia-like protrusions and membrane ruffles in the cell leading edge. (B) cells treated with dox and (D) cells injected with MEGAP showed the typical MEGAP-expressing morphology with a rounded and contracted cell body, without actin-based protrusions. Scale bar, 5 μm. Actin dynamics were further quantitatively analysed by taking single protrusions and calculating perimeter and area variation of the protrusions between subsequent frames for the entire time-lapse duration. Panels E and F are montages with frames selected every 100 s. (E) Dox-untreated cells exhibited marked alterations of perimeter and area. (F) Dox-treated cells remained nearly unchanged. Panels G and H illustrated the typical pattern of the perimeter and area variations under different regimens (untreated: O, dox treated: ο). (I and J) Mean variation (perimeter and area) every 10 s over the whole duration of the time-lapse. The black (1), gray (2), and white (3) columns represent the dox-untreated, the dox-treated, and MEGAP R542I mutant injected cells, respectively. “X” indicates a significant difference between dox-untreated and dox-treated cells (P < 0.03 and <0.0028, respectively, for perimeter and area). The variations of perimeter and area were not significant between dox-untreated and mutant injected cells (P < 0.3251 and P < 0.847).

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constitutively active or dominant-negative RhoA, Rac1, and Cdc42 GTPases into several cell lines were previously applied for evaluating the function of GTPases in the regulation of cell polarity, membrane-ruffling, and other membrane structures [19]. To examine whether the role of MEGAP in the inhibition of actin filament formation is carried out through the GTPase pathway, with particular regard to Rac1 and Cdc42, GTP constitutively positive Cdc42 and Rac1-GFP fusion constructs were transfected into the SHMEGAP line for 24 h. Three independent experiments were performed and the successfully transfected cells were examined. In all transfected cells, the immunofluorescence data indicate that expression of MEGAP can no longer block the filopodia and lamellipodia formation in GTP constitutively positive Cdc42G12V (Figs. 3A and B) and Rac1G12V (Figs. 3C and D) transfected cells. The untransfected surrounding cells were, however, exhibiting rounding and loss of protrusions. These results demonstrate that MEGAP exerts its function upstream of Cdc42 and Rac1 in the signal transduction pathways.

Expression of MEGAP inhibits the formation of focal complexes Formation of stable attachments near the leading edge of the protrusion is one of the indispensable steps in the cell migration cycle. In migrating cells, the Rho family of small GTPases is an important regulator of adhesion dynamics [3]. Rac1 is required for the formation of new adhesions at the cell front and may regulate their turnover through downstream effectors or by antagonising Rho activity [20–22]. Integrins functionally link the extracellular matrix such as fibronectin to the actin cytoskeleton and are involved in this process [23]. Because MEGAP downregulates Rac1 and Cdc42 activities and leads to cell rounding and to the loss of protrusions, we speculated that expression of MEGAP may also influence the formation of focal complexes. To prove this, the dox-treated SHMEGAP cells were serum starved, replated onto fibronectin-coated coverslips, and immunostained using focal adhesion kinase (one of the components of focal contacts) antibody. We found that indeed dox-untreated cells showed normal formation of focal complexes (Figs. 4A– C), whereas dox-treated SHMEGAP cells could not form focal complexes (Figs. 4D–F). In addition, the loss of focal adhesions was also observed in the dox-treated SHMEGAP cells (data not shown).

Reduction of migration and protrusion formation upon MEGAP induction GTPases play an important role in the regulation of cell migration through remodelling the cytoskeleton [9]. Because MEGAP reduces GTP-bound Cdc42 and Rac1 and affects the formation of filopodia and lamellipodia, it may therefore also influence the migration of the cells. To determine this function of MEGAP, we have examined the random migration of SHMEGAP cells by real-time video-microscopy. The doxtreated and dox-untreated cells were monitored by time-lapse with 5 min interval, for more than 15 h. The collected total tracks of 10 treated and 10 untreated cells from 150 frames were analysed. The results showed that untreated cells moved 3.8-fold quicker (movie 1A) than the treated cells (P < 7.884e

−05) (movie 1B; Fig. 5). In a comparison between the doxtreated and dox-untreated TR1 line (movies 1C and D), the dox-treated TR1 and the dox-untreated SHMEGAP line showed no significant difference with P values of <0.8528 and <0.8404, respectively. To be able to move forward on a two-dimensional substrate, cells need to form protrusions consisting of filopodia and lamellipodia at the cell periphery. The lamellipodia structures are dominating on the leading edge and often move backwards to form ruffles of the membrane [2]. To determine the influence of MEGAP on SHMEGAP cell motility, the ability of protrusion formation in MEGAP-induced SHMEGAP cells was evaluated. We counted the appearance of filopodia and lamellipodia over a period of 12 h for 10 doxtreated and 10 dox-untreated cells. The results show a clear difference in the frequency of protrusion formation between the dox-untreated cells (Fig. 6A; movie 2A) and dox-treated cells (Fig. 6B; movie 2B). The formation of protrusions in untreated SHMEGAP cells was 9 times higher than in treated cells (P < 9.698e−08). The frequency of protrusion formation between dox-treated and dox-untreated TR1 cells, untreated SHMEGAP, and treated TR1 cells was, however, not significantly altered, with P values of <0.6672 and <0.8593, respectively.

Loss of actin dynamics in the cell leading edge upon MEGAP induction Previous analysis has indicated that MEGAP-expressing cells have a reduced ability to form protrusions. To investigate how MEGAP affects this actin-related process, we transfected GFPactin into the SHMEGAP cell line. At 24 h after transfection, short-range time-lapse fluorescent video microscopy analysis revealed in dox-untreated SHMEGAP cells (Fig. 7A; movie 3A) a clear cell polarisation and a strong membrane ruffling in the region of the cellular leading edge. In contrast, a consistent diminution in actin dynamics was observed in dox-treated SHMEGAP cells. Most of the cells presented a rounded form with a complete absence of protrusions and appeared highly contracted (Fig. 7B; movie 3B). To determine whether the treatment with dox may represent a cytotoxic effect on the cells, the parental cell line TR1 transfected with the GFP-actin construct was also treated with dox. The results clearly indicated that TR1 cells are unaffected by the dox treatment (data not shown). In order to assess whether the GAP domain of MEGAP is required for MEGAP functions, we replaced the conserved amino acid Arg542 with isoleucine in the GAP domain. The GFP-actin and the mutant MEGAP R542I were coinjected into the parental cell line TR1. The presence of MEGAP R542I in the cells was confirmed by immunofluorescence. Cell polarisation and clear membrane ruffling could be observed at the cellular leading edge (Fig. 7C; movie 3C). To investigate how MEGAP affects this actin-related process, we coinjected GFP-actin and the plasmid pMEGAP into the parental cell line TR1. At 24 h after injection, short-range time-lapse fluorescent video microscopy analysis revealed a consistent diminution in actin dynamics. Most of the cells presented a rounded form with a complete absence of protrusions and appeared highly contracted (Fig. 7D; movie 3D).

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Fig. 8 – Reduction of microtubule dynamics in MEGAP-injected TC-7 3x-GFP cells. The microtubules are visualised in the cells as green bundles in the stably expressed 3x-GFP-EMTB. The TC-7 3x-GFP cells were plated on CELLocate and 24–48 h later rhodamine-conjugated dextran was injected into the nuclei of the cells. The microtubule dynamics were visualised (24 h later) by placing the CELLocate in a custom-designed chamber and mounted on a heating stage. Time-lapses were collected with the time interval of 10 s for 5 min. (A) The red staining of the nucleus indicated a successful injection of rhodamine-conjugated dextran. The cell displays a normal microtubular array, with free extremities. (B) The montage from the selected inset in panel A showed that the ends of microtubules in the cell cortical region present high dynamic instability. The “−” and “+” symbols indicated the changes of the microtubules (shortening and extending) between selected frames. (C) The MEGAP-injected cells displayed a rounded shape and the ends of microtubules were not distinguishable. (D) The montage from the selected inset in panel C indicated that the changes of the microtubules between the frames were quite small. The ends of the microtubules were intertwined and not able to move due to end buckling. (E) The cells injected with pMEGAP and pRac1G12V (both 20 ng/μl) displayed a normal microtubular array with free ends. (F) A montage from the selected inset (E) shows the microtubules' dynamic instability. The “−” symbol indicates the shortening of two microtubules between selected frames. The montages B, D, and F display frames recorded every 20 s. Scale bar, 5 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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We further quantified the effect of MEGAP on actin dynamics at the protrusions in more than 30 cells over a period of 300 s using time-lapse with an interval of 10 s. The pixels of each fluorescently labelled lamellipodia picture were counted under the various treatment regimens, and morphometric cellular parameters that were changing during the time-lapse, such as area or perimeter of the protrusion, were analysed. The montages of protrusions (Fig. 7E) displayed clear variations in perimeter and area. The montages of protrusions (Fig. 7F) showed small changes during the whole period of time-lapse. To have a visualisation of these variations, we plotted the pixel number against time (see Materials and methods). Figs. 7G–H indicate the typical patterns of variations in perimeter and area for doxuntreated and dox-treated cells. The relative variations of both perimeter and area between two consequent frames in dox-treated cells were around 3-fold smaller than in the untreated cells. A statistical analysis relative to the variations of these morphometric parameters was carried out by Student's t test. In Figs. 7I–J, the black (1), gray (2), and white (3) columns represent the dox-untreated, the dox-treated, and MEGAP R542I mutant injected cells, respectively. Although the values for perimeter and area relative to the mutant are always somewhat smaller than the ones of uninduced cells, the difference was not significant (P < 0.3251 and P < 0.84789). However, the differences between the dox-untreated and doxtreated cells were significantly higher (P values <0.03 and <0.0028, respectively, for perimeter and area). Taken together, these results indicate that cells expressing MEGAP showed significantly reduced dynamic properties in the cell leading edge area.

MEGAP-injected TC-7 3x-GFP cells reduce dynamics of microtubules The dynamic remodelling of the actin and the microtubule cytoskeleton and the reciprocal cross-link between these two systems is essential for cell migration. It has been reported that the polymerisation of microtubules led to activation of Rac1, inducing actin polymerisation [24,25] and that the destruction of microtubules inhibited the formation of lamellipodia protrusions [26]. It is therefore interesting to investigate whether the downregulation of the GTPase Rac1 by MEGAP can affect microtubule dynamics. For this purpose, we have used the TC-7 3x-GFP cell line that stably expresses GFP-EMTB, a GFP fusion construct containing the microtubule-binding domain of a microtubule associated protein [27]. This cell line provides a straightforward way to analyse the effect of MEGAP on microtubule dynamics using real time fluorescent microscopy. MEGAP and the mutant MEGAP R542I with rhodamine-conjugated dextran as indicator were coinjected into TC-7 3x-GFP cells. In 80% of successfully injected nuclei of TC-7 3x-GFP cells (injected either with the indicator or with MEGAP R542I), microtubules had normal dynamics as described by Faire and coworkers [27]. Fig. 8A shows an example of an entire TC-7 3x-GFP cell with normal microtubule pattern and Fig. 8B (montage of inset from Fig. 8A) indicates some microtubule instability in the cell periphery. A dramatic reduction of microtubule dynamic

instability and diminished microtubule motility was, however, detected in the cell cortical region of MEGAP-injected TC-7 3x-GFP cells (Figs. 8C and D, montage of inset from Fig. 8C). The cells rounded up and the ends of the microtubules were entwined together along the periphery of the cells, thereby reducing their overall motility, a phenomenon already described as “microtubule-end buckling” [28]. Our hypothesis is that the downregulation of Rac1 by MEGAP can cause the reduction in microtubules dynamics. To test this hypothesis, we coinjected pRac1G12V and pMEGAP into TC-7 3x-GFP cells. Eighty percent of the successfully injected cells showed normal microtubules dynamics, suggesting that the constitutive active Rac1 was able to override the effect of MEGAP by rescuing the physiological movements of the microtubules (Figs. 8E and F).

Discussion Over the past several years, it has become clear that the Rho family of GTPases plays an important role in different aspects of neuronal development and neuronal morphogenesis including neurite outgrowth and differentiation, axonal pathfinding, and dendrite spine formation and maintenance. Among the non-specific mental retardation genes (including MEGAP) identified so far, about half belong to Rho-linked genes involved in Rho-GTPase [29]. Patients with syndromic and nonsyndromic mental retardation share as common feature a structural and quantitative abnormality of dendritic spines in the brain [30]. These highly dynamic structures are composed of actin and are important for the connectivity and information processing in brain. The remodelling of the dendritic spines is controlled by the active forms of Rho-GTPases [31]. Cell migration plays an important role in axon outgrowth, dendrite branching and dendritic spine morphogenesis [32]. In order to understand the function of MEGAP in the development of mental retardation, it is therefore important to analyse the role of MEGAP on migration. To investigate the presumed role of MEGAP in actin organisation and cell migration, we used a tetracyclineregulated system in which the expression of MEGAP was controlled by a doxycycline-inducible promoter. The expression of the full-length MEGAP, a Cdc42/Rac1 GAP predominantly expressed in brain, results in cell rounding and loss of protrusions at the cell leading edge of SHMEGAP cells. We have also shown that constitutively active Cdc42 and Rac1 can rescue these events. MEGAP expression also inhibits the formation of focal complexes and attenuates cell migration as shown by time-lapse microscopy. For all the functions of actin and microtubule dynamics, an intact RhoGAP domain with a conserved arginine is required. The MEGAP mutant (R542I)-injected cells demolishes all these effects and maintained similar dynamics as the MEGAP-uninduced cells, strongly suggesting that the GAP domain is required for the MEGAP activity. Disrupting effects on the cytoskeleton have been shown for other GAPs as well [33]. Microinjection of CdGAP, a Cdc42/ Rac GAP, into serum-starved fibroblasts inhibited both the Rac1- and Cdc42-mediated filopodia and lamellipodia

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formation, respectively [34]. However, the in vivo functions of GAPs are not always identical to their GAP activities in vitro. In contrast to the effects of its GAP domain in vitro, CRGAP-72, for example, induces membrane ruffling and lamellipodia and filopodia formation in vivo [35]. These functions likely involve binding of the GAP to activated Cdc42 and Rac1, targeting them to certain subcellular sites and triggering local morphological changes rather than its GAP activity. In response to integrin engagement with the extracellular matrix, cells form focal contacts (consisting of focal complexes and focal adhesions) thought to serve as sites for coordination between cell adhesion and cell motility [36,37]. After integrin engagement, several downstream tyrosine kinases, such as FAK and Src, are activated and phosphorylate substrates including GEFs and other adaptor proteins, leading to the activation of Cdc42 and Rac1, which are required for focal complexes formation [10,38]. In our study, MEGAP-expressing cells were not able to form integrin-mediated focal complexes, probably due to the interruption of the integrin-mediated pathway as a consequence of the downregulation of Rac1 and Cdc42. In a recent study, a Cdc42/Rac1 GAP protein, CR-GAP72, was described to colocalise with focal adhesions and stress fibers via its C-terminal region and to reduce the formation of these structures [35]. Due to the low level of expression of endogenous MEGAP in SHMEGAP cells, we were not able to determine an association among MEGAP, focal contacts, and the actin filaments. Yet the induction of MEGAP overexpression by dox caused a general disruption of focal complexes and the actin cytoskeleton. In addition, the loss of focal adhesions and stress fibers, phenomena typically related to a downregulation of RhoA, were also observed in MEGAPexpressing cells. Under the chosen experimental conditions, however, RhoA was not downregulated, as previously described [11]. The reason for the disappearance of stress fibers, therefore, still remains enigmatic. We speculate that these phenomena may likely not relate to the GAP activity of MEGAP. In this study, we have focused on the effect of MEGAP in the regulation of cytoskeleton dynamics and cell migration. Green fluorescent protein (GFP) tagging combined with realtime fluorescent microscopy [39,40] allowed an optimal and direct visualisation of the dynamic changes of the cytoskeleton induced by MEGAP in living cells. Statistical analysis of our data from real-time video microscopy indicated that MEGAP reduces migration speed of SHMEGAP cells by 4-fold and protrusion formation rate by 9-fold. In contrast to Cdc42/Rac GAPs, RhoGAPs have been shown to promote cell migration. Arthur and Burridge [41], for example, demonstrated that expression of the wild-type p190 RhoGAP decreased transient integrin signalling-mediated RhoA activity, promoting the formation of membrane protrusions, and enhancing motility of the cells. BPGAP1, a RhoA GAP, was reported recently to augment the migration rate of 293T cells by 2-fold through binding with cortactin and facilitating its translocation and the assembly of actin to the cell periphery, leading to enhanced cell motility [42,43]. The harmonisation of the actin/microtubule systems is required for cell migration and regulated through the Rho

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family of GTPases [44,45]. The assembly of microtubules activates Rac1 and promotes in turn actin-based protrusions [25,46]. Activation of Rac1 and Cdc42 enhances microtubule growth through their effector p65PAK, probably by regulating the microtubule destabilising protein, Op18/ stathmin [47,48]. We therefore examined a possible role of MEGAP in the regulation of microtubule cytoskeleton in TC7 3x-GFP living cells that were microinjected either with wild-type or mutant (R542I) MEGAP. We observed a reduction in the movement of microtubules and a loss of microtubule dynamic instability in the cell periphery (end “buckling”) with wild-type MEGAP. In contrast, the cells injected with the mutant (R542I) exhibited similar microtubule instability as the control injected only with indicator dye. To further test how MEGAP affects microtubules dynamics in the presence of constitutive active Rac1, we injected TC-7 3x-GFP cells with pMEGAP and pRac1G12V. Microtubules in the injected cells displayed normal dynamics. These results suggest that MEGAP may reduce microtubules dynamics through the downregulation of Rac1. This may indicate that Rac1 can rescue downstream signalling, formerly blocked by MEGAP, possibly by restoring the physiological inactivation of the Op18 gene [48]. The exact mechanism by which MEGAP regulates microtubule instability is still unknown. MEGAP belongs to a subfamily of GAPs consisting of srGAP1, srGAP2, and MEGAP/srGAP3 [49]. They all contain three functional domains: an N-terminal FCH (Fes/CIP4 homology) domain, a central RhoGAP domain, and a Cterminal SH3 domain [33]. Besides the GAP domain required for the GAP activity, the SH3 domain was reported to interact with WAVE1, attenuating Rac-mediated signalling [35]. The function of the FCH domain of this subfamily of GTPases has not been described so far in respect to cytoskeleton regulation. There are reports, however, concerning a putative regulatory function of the microtubule cytoskeleton in different FCH domain-containing proteins. Rapostlin, the first identified effector of Rnd2 GTPase, directly binds to microtubules through its FCH domain and this interaction is essential for the neurite branching in P12 cells [50]. The FCH-containing Fes/Fps (Fes) tyrosine kinase is involved in Semaphorin3A-mediated signalling and plays a critical role in microtubule nucleation and bundling through its FCH domain in Cos-7 cells [51]. Therefore, further investigation of the association between the FCH domain of MEGAP and microtubules may help to understand the effect of MEGAP on microtubule dynamics. The rather specific expression of MEGAP mRNA in fetal and adult mouse and human brain ([12], unpublished data) suggests an involvement of MEGAP in neuronal development and neuronal-related processes. Indeed, preliminary data from our laboratory showed that expression of MEGAP induces retraction of nerve growth factor-induced neurites in SHMEGAP cells. This retraction could lead to aberrant neuronal morphogenesis, dendritogenesis, and connectivity between neuronal cells. One of our further interests therefore is to analyse MEGAP function in the regulation of neurogenesis in SHMEGAP cells and neuronal primary culture cells as well as in the living animals.

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Acknowledgments Y. Y. and V. E. were supported by the BMBF grant GS0117 and GS0167 to G. R.; M. M. by a joint collaboration Hamamatsu/ DKFZ (project PA 11631).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2006.04.001.

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