The present study demonstrated that GMFG is enriched in the pseudopodia of migrating human T lymphocytes. Interference with GMFG expression markedly inhibited SDF1 stimulated chemotaxis. This effect was associated with reduced migration and a failure to sustain cellular polarity and directed movement. The inability of the cells to move was associated with an increase in total β1 integrin levels which included elevated expression levels of the high-affinity conformation of the β1 subunit. These changes and the increased adhesion of GMFG-KD cells to fibronectin appeared to be predominantly related to increased α5β1 levels. This was further supported by the fact that a synthetic RGD containing peptide and inhibitory antibodies specific for the α5 and β1 subunits reduced the elevated adhesion of the GMFG-KD lines to levels comparable with control cells. The loss of GMFG also seems to impact on the distribution of integrin molecules, as there is a propensity for GMFG-KD cells to accumulate β1 integrins within the uropod. This accumulation, in combination with increased surface expression levels, was consistent with failure of the cells to detach from the substrate. Furthermore the changes in integrin levels and activity were not restricted to the α5β1 integrin suggesting that GMFG activity plays a broader role in control of cellular adhesion.
GMFG is a member of the actin-depolymerizing factor homology (ADF-H) family with actin depolymerizing activity [7
]. Recent studies indicate that GMFG mediated depolymerization differs from that of other ADF-H members such as cofilin [10
]. GMFG does not bind directly to actin, rather it interacts with Arp2/3 complex members which can induce debranching of actin filaments. Interestingly, while GMFG reduces the rate of actin filament assembly, the distribution and absolute amount of F-actin ultimately produced during in vitro assays is unaltered in the absence of GMFG (data not shown). These observations raise the possibility that GMFG may be required for the transition from branched actin networks at the leading edge to more filamentous structures that are at immediately proximal to the leading edge of migrating cells. These filaments may provide the cytoskeletal architecture for interaction with integrins through integrin associated actin binding proteins. Such interactions could also regulate integrin activation and distribution to generate the adhesion required for cell movement.
Several GMFG interaction partners have been identified to date. GMFG was identified in immunoprecipitates of Arp2/3 complexes in Hela cells that were overexpressing GMFG [7
]. Subsequently, several groups confirmed the interaction of murine, yeast or dictyostelium GMFs and the Arp2/3 complex using purified proteins [9
]. WAVE2 was also co-precipitated with Myc tagged GMFG in dHL60 [5
]. However it is unclear if the latter interaction relates to cell motility rather than chemotaxis as these results were obtained with cells that had not been treated with exogenous chemokine.
The present studies demonstrated a role for GMFG in the control of T cell adhesion and its subsequent effects on cell migration in stably transduced lymphocyte lines and short term IL2 dependent peripheral blood T cells. The loss of GMFG has also been shown to decrease the chemotactic responses of both PMN and differentiated HL60 cells [5
]. The phosphorylation levels of p21 activated kinases (PAK 1 or PAK 2) and p38 MAPK were also found to be decreased following fMLF or CXCL8 stimulation of dHL60 cells. PAK1/2 plays multiple roles in the regional regulation of actin dynamics and myosin function [17
]. The strength of focal adhesions can be regulated by Pak1/2 activity as inhibition of either the activity or expression of Pak1/2 results in increased numbers of nascent long lived adhesions. The net effect of these alterations is a reduction in cellular migration. Additionally, the conformation of α5β1 integrin is known to switch from low to high affinity in focal adhesions [18
]. Hence the increased proportion of activated β1 integrin observed on GMFG-KD cells would be consistent with a Pak1/2 mediated increase in the number of focal adhesions. An intriguing possibility is that GMFG alters cytoskeletal organization which impacts on the frequency and properties of integrin contacts with the extracellular matrix. The strength of α5β1 binding can be enhanced by cytoskeletal tension forces generated by myosin II [19
], and it is conceivable that GMFG could be involved in the regulation of force generation by its effects on the cytoskeleton. The observed changes in the integrin expression patterns and activity may be a reflection of architectural changes that accompany the shifts in motility induced by GMFG loss. These are points for future examination which could provide new insights as to the regulation of lymphocyte adhesion.