Research ArticleMEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation
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 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], [8], [9], [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 GDP-bound 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.
Section snippets
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
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
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
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).
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The first two authors contributed equally to the manuscript.