Research
So far, despite the profound impact that GIMAP5 deficiency is known to have in terms of both lymphoid deficiency and autoimmunity (i.e. the type 1 diabetic BB rat strain as well as a rat model of eosinophilic bowel disease – see Cousins et al. 2006), very little is understood of the cellular mechanisms by which GIMAP proteins produce their effects on lymphocyte development and survival. We are using different experimental approaches to uncover the function and mechanisms of action of the GIMAP family.
Genetic manipulation of GIMAP genes in mice.
We are creating conditional gene knockouts for some of the GIMAP genes of the mouse in order to identify essential requirements for them in selected tissues. In a recent publication (Saunders et al., 2010), we have described how deletion of the gene for GIMAP1 early in lymphoid development leads to a severe deficit of mature cells in both the T and B cell lineages, although the precursor lymphocytes in the bone marrow and thymus are scarcely affected.
The effects of this mutation on the T cell lineage have close similarities to what has been seen in mice and rats deficient in GIMAP5 but the additional severe effects on B cells are a novel feature.
These findings establish GIMAP1 as an important pro-survival factor for mature T and B lymphocytes. Analysis of the full ‘germline’ deletion of GIMAP1 is in progress in order to identify any role this gene may play in non-lymphoid tissues.
Cellular and sub-cellular expression of the GIMAPs.
The natural distribution of GIMAP proteins in different tissues, and within cells, has not been described in much detail. In order to help establish basic facts we have been developing panels of polyclonal and monoclonal antibodies with which to examine the endogenous expression of GIMAPs. Our collection of antibodies now identifies many of the GIMAPs in mouse, rat and human cells. We have made use of these antibodies to examine, for instance, the interesting changes in GIMAP protein expression that take place through the different stages of B and T cell development in the bone marrow and thymus of mice (Saunders et al., 2010).
We have also been keen to learn more about the intracellular location of GIMAP proteins. Some of them (notably GIMAP1 and GIMAP5) have protein regions which anchor them to membranes. Identifying in which sub-cellular membranes they are to be found could give important clues about their function. From detailed studies we have drawn the unexpected conclusion that GIMAP5 is a lysosomal protein (Wong et al., 2010). By contrast, we found that GIMAP1 is located in the Golgi system of mouse lymphoid cells. In the future, we will be interested to look for dynamic changes in the intracellular expression of GIMAP proteins during cellular responses to stimuli such as antigen-receptor activation, apoptosis induction and others.
GIMAP1 expressed in a mouse NKT cell line [C1498] is located in the Golgi apparatus
Panel A – DAPI stain for DNA;
Panel B – detection of giantin, a standard marker for the Golgi; Panel C – detection of endogenous GIMAP1 using a specific monoclonal antibody; Panel D – overlay of Panels A-C demonstrates substantial co-localisation of GIMAP1 with giantin.
Myc-tagged human GIMAP5 in stably transfected HEK293 cells detected in vesicular structures including lysosomes.
Protein-protein interactions of the GIMAP GTPases.
As stated above, very little is known as yet about the biochemical mechanisms by which GIMAP GTPases exert their functional effects. In an attempt to address this knowledge gap directly, we have embarked on experiments to identify protein binding partners of the GIMAPs. Different approaches that we are trying include direct immunoprecipitation of GIMAP-protein complexes (using our highly specific antibodies), affinity purification using GST-GIMAP fusion protein pull-downs from cell or tissue extracts and tag-mediated affinity purification using engineered GIMAP constructs.
Updated 22 August, 2011