Biomedical Sciences ETDs

Publication Date

7-1-2012

Abstract

The spatial organization and dynamics of proteins and lipids within the cell membrane is important for the regulation of cell signaling, adhesion, and cell communication. Within the bone marrow niche, communication between hematopoietic stem/progenitor cells (HSPCs) and niche cells is essential for regulating their proliferation, differentiation, and survival. Our previous work has ascertained that HSPCs utilize a polarized domain on the plasma membrane that serves as the contact site with osteoblasts, which are important members of the bone marrow niche. Using human primary CD34+ stem/progenitor cells and the progenitor-like KG1a cell line, we found this domain to be enriched in the specific tetraspanin proteins, CD63, CD81, and CD82. Tetraspanins are multi-spanning membrane proteins that act as scaffolds for the organization of membrane domains important for regulating adhesion and signaling. CD82 is of particular interest, as it is highly expressed on HSPCs and downregulated during HSPC differentiation. Our characterization of CD82 function using CD82-blocking antibodies revealed a significant decrease in adhesion of HSPCs to niche cells as well as in the in vivo homing and engraftment capabilities of these cells. To determine the molecular mechanisms of CD82s role in adhesion, we have generated CD82 overexpression and knockdown cell lines using the KG1a background. Our data indicate that the level of CD82 expression positively correlates with the extent of adhesion to fibronectin and osteoblasts but has no effect on binding to collagen I or laminin. The increase in adhesion we observed with CD82 overexpression was inhibited by the VLA-4-specific peptide, LDV, indicating a potential role for the VLA-4 (α4β1) integrin. Investigations into potential CD82-mediated mechanisms of VLA-4 regulation have revealed that CD82 regulates both the expression and avidity of VLA-4 but does not regulate its affinity. Taken together, the VLA-4 expression and avidity changes could account for the observed adhesion changes with differing CD82 expression levels. Finally, assessment of CD82 palmitoylation using KG1a CD82 palmitoylation mutant cells revealed that palmitoylation may be required for the CD82-induced changes in adhesion.

Keywords

Hematopoietic Stem Cell, Bone Marrow, Niche, Osteoblast, Fibronectin, Adhesion, Integrin, ╬▒4╬▓1, VLA-4, Tetraspanin, CD82

Document Type

Thesis

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Masters

Department Name

Biomedical Sciences Graduate Program

First Advisor

Gillette, Jennifer

First Committee Member (Chair)

Buranda, Tione

Second Committee Member

Cannon, Judy

Third Committee Member

Hartley, Rebecca

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