Abstract
Movement of hematopoietic stem/progenitor cells into (engraftment) and out of (mobilization) the bone marrow involves actin cytoskeleton and chemotaxis. Members of the Rho GTPase family have been well known for their critical roles in morphogenesis and cell migration via regulating actin assembly. Loss of Rac1 and Rac2 alleles leads to defective engraftment and massive mobilization of hematopoietic progenitor cells (HPCs), which are associated with impaired chemotaxis and cortical filamentous (F)-actin polymerization (Gu et al., Science 302: 445–449). RhoH, a hematopoietic-specific member of the RhoE subfamily, negatively regulates HPC engraftment, chemotaxis, F-actin polymerization and Rac activities (Gu et al., Blood 105: 1467–1475). These findings suggest that RhoH may antagonize Rac function in regulating these cellular processes. However, molecular mechanism of the cross-talk between these Rho GTPases is not defined. In this study, we examined the role of RhoH in actin cytoskeleton organization, chemotaxis and Rac membrane translocation in response to stromal-derived factor 1α (SDF-1α) using RhoH-deficient HPCs and retrovirus-mediated expression of EGFP-fusion proteins.
RhoH−/− HPCs exhibit increased migration in response to SDF-1α, especially at low concentration, as compared with wild-type (WT) cells [10ng/ml SDF-1α: 3.5 +/− 0.9 vs. 12.3 +/− 1.8; 100ng/ml SDF-1α: 21.4 +/− 1.7 vs. 32.3 +/− 3.4, migrated cells (%), WT vs. RhoH−/−, n=3, p< 0.01]. Migration without SDF-1α stimulation of RhoH−/− cells is also enhanced. RhoH−/− HPCs assemble cortical F-actin without SDF-1α stimulation, under conditions in which WT cells do not show F-actin polymerization [cells with F-actin (%): 8.9 +/− 0.9 vs. 72.8 +/− 4, WT vs. RhoH−/−, n=6, p<0.001]. Additionally, RhoH−/− HPCs exhibit increased active, GTP-bound Rac GTPases. PAK, a known downstream effector of Rac in regulating actin cytoskeleton, also shows hyperphosphorylation in RhoH-/− HPCs, suggesting that RhoH may regulate actin assembly and cell migration through Rac-mediated pathway. In support of this, expression of a dominant negative Rac1N17 mutant blocks cortical F-actin assembly in RhoH−/− cells [cells with F-actin (%): 60 +/− 1 vs. 19 +/− 7, EGFP-Rac1 vs. Rac1N17, n=2].
To further address the mechanism by which RhoH cross-talks to affect Rac signaling, we examine the role of RhoH in subcellular localization of EGFP-Rac proteins. SDF-1α induces activation of Rac, leading to translocation to the cell membrane where it co-localizes with lipid rafts and mediates cortical F-actin assembly in HPCs. In contrast, the dominant negative Rac1N17 does not localize to the cell membrane after SDF-1α stimulation. In RhoH−/− HPCs, EGFP-Rac protein presents at the cell membrane in the absence of SDF-1α [cells with membrane-localized EGFP-Rac1 (%): 7.5 +/− 3.9 vs. 44.5 +/− 6.4, WT vs. RhoH−/−, n=2]. In contrast, overexpression of RhoH in HPCs blocks translocation to the cell membrane after SDF-1α stimulation of Rac1, Rac2 and active Rac1V12. Finally, we found that RhoH, a constitutively active, GTP-bound protein, preferentially localizes to the cell membrane even in the absence of SDF-1α. This localization is dependent upon the prenylation site and the c-terminal domains of RhoH. Lack of membrane localization is associated with defective biological function. Together, our data suggest that RhoH is essential for proper cortical F-actin assembly and chemotaxis of HPCs via regulating Rac activation and membrane localization, and implicates a functional cross-talk between RhoH and Rac.
Acrosomal reaction of the Thyone sperm. III. The relationship between actin assembly and water influx during the extension of the acrosomal process.
