Comparative proteomics of primitive hematopoietic cell populations reveals differences in expression of proteins regulating motility

Abstract

Lineage-marker depleted (Lin ؊ ) murine bone marrow cells expressing stem cell antigen 1 (Sca-1) were sorted on the basis of stem cell factor receptor (c-kit) expression to obtain Lin ؊ Sca ؉ Kit ؉ or Lin ؊ Sca ؉ Kit ؊ cells. Lin ؊ Sca ؉ Kit ؊ cells have a markedly greater chemotactic response to stromal derived factor-1 (SDF-1). Using a novel fluorescent stain, we show that both populations generate similar levels of a key messenger, phosphatidylinositol 3,4,5 trisphosphate (PIP 3 ), in response to SDF-1. Differences in motile behavior may therefore lie downstream of phosphatidylinositol 3-kinase (PI3-kinase) activation at the level of cytoskeleton regulation. The 2 cell populations were compared using 2-dimensional difference gel electrophoresis (2D-DIGE), with a maleimide CyDye fluorescent protein labeling technique that has enhanced sensitivity for low abundance samples. Comparative proteomic analysis of Cy3-and Cy5-labeled protein samples allows relative quantification of protein spots present in both cell populations; of these, 73% were common. Key protein differences were adseverin and gelsolin, actin microfilament splicing proteins, regulated by Rac, downstream of PI3-kinase activation. Adseverin was shown to be acetylated, a novel modification for this protein. Differences in major regulators of cell shape and motility between the 2 populations can explain the differential response to SDF-1. IntroductionHematopoietic stem cells have the capacity to self-renew and also to differentiate to form a number of mature cell types with distinct morphologies and functional activities. The distinctive features of the stem cell that give it longevity and the ability to balance its self-renewal and differentiation to the required degree in any circumstance remain unclear. These biologic properties are encoded in the transcriptional profile, or transcriptome, of stem cells. The properties of a stem cell can therefore be defined in terms of the gene expression profiles of stem cells. Retention of stem cell characteristics will rely in part on the cohort of genes specifically expressed in these primitive cells, but not in their maturing progeny. This axiom has led to transcriptional analysis of long-and short-term repopulating stem cells and other primitive populations (defined via expression of specific markers) to define the genes expressed for self-renewal and pluripotency. 1,2 The generation of these stem cell "blueprints" then offers the opportunity to investigate the functional activity of the proteins produced as a consequence of gene expression and to systematically study gene networks controlling stem cell biology.The relationship between messenger RNA production and protein synthesis is not, however, strictly correlative. 3 In a range of different organisms it has become clear that changes in the expression of a specific protein do not necessarily relate directly to altered transcription. Recent developments in protein separation and relative quantification allow populations of cells to be compared at the pr...

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