Abstract
BackgroundLymphocyte migration and chemotaxis are essential for effective immune surveillance. A critical aspect of migration is cell polarization and the extension of pseudopodia in the direction of movement. However, our knowledge of the underlying molecular mechanisms responsible for these events is incomplete. Proteomic analysis of the isolated leading edges of CXCL12 stimulated human T cell lines was used to identify glia maturation factor gamma (GMFG) as a component of the pseudopodia. This protein is predominantly expressed in hematopoietic cells and it has been shown to regulate cytoskeletal branching. The present studies were undertaken to examine the role of GMFG in lymphocyte migration.ResultsMicroscopic analysis of migrating T-cells demonstrated that GMFG was distributed along the axis of movement with enrichment in the leading edge and behind the nucleus of these cells. Inhibition of GMFG expression in T cell lines and IL-2 dependent human peripheral blood T cells with shRNAmir reduced cellular basal and chemokine induced migration responses. The failure of the cells with reduced GMFG to migrate was associated with an apparent inability to detach from the substrates that they were moving on. It was also noted that these cells had an increased adherence to extracellular matrix proteins such as fibronectin. These changes in adherence were associated with altered patterns of β1 integrin expression and increased levels of activated integrins as detected with the activation specific antibody HUTS4. GMFG loss was also shown to increase the expression of the β2 integrin LFA-1 and to increase the adhesion of these cells to ICAM-1.ConclusionsThe present studies demonstrate that GMFG is a component of human T cell pseudopodia required for migration. The reduction in migration and increased adherence properties associated with inhibition of GMFG expression suggest that GMFG activity influences the regulation of integrin mediated adhesion.
Highlights
Lymphocyte migration and chemotaxis are essential for effective immune surveillance
glia maturation factor gamma (GMFG) is present in the pseudopodia of SDF1 treated T-lymphocytes The intent of our studies was to examine the roles of proteins found in the pseudopodia of chemotactically stimulated lymphocytes
The presence of GMFG was potentially masked by the presence of higher abundance proteins that were reduced in the pseudopodia preparations
Summary
Lymphocyte migration and chemotaxis are essential for effective immune surveillance. A critical aspect of migration is cell polarization and the extension of pseudopodia in the direction of movement. Proteomic analysis of the isolated leading edges of CXCL12 stimulated human T cell lines was used to identify glia maturation factor gamma (GMFG) as a component of the pseudopodia. This protein is predominantly expressed in hematopoietic cells and it has been shown to regulate cytoskeletal branching. Several groups have shown that GMFG and its homologs interact with purified Arp2/3 complex [7,9,10] and interfere with Arp2/3 induced daughter filament growth They cause removal of actin branches from the parent actin filament (i.e. pruning). These observations suggested that GMFG may be involved in cell movement, possibly by defining the location of Arp2/3 dependent actin branching required for membrane extension or by providing a mechanism for the regeneration of the actin monomer pool required for continued cell pseudopodia extension
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