by Hee-Don Chae, Katherine E. Lee, David A. Williams, and Yi Gu Cross-talk between RhoH and Rac1 in regulation of actin cytoskeleton and chemotaxis of hematopoietic progenitor cells by Hee-Don Chae, Katherine E. Lee, David A. Williams, and Yi Gu Blood Volume 111(5):2597-2605 March 1, 2008 ©2008 by American Society of Hematology

Enhanced chemotaxis, F-actin assembly, and Rac activity in Rhoh−/− HPCs. (A) In vitro migration of Lin−/c-kit+ cells in a transwell chamber assay in response to increasing concentration of SDF-1α for 4 hours. Enhanced chemotaxis, F-actin assembly, and Rac activity in Rhoh−/− HPCs. (A) In vitro migration of Lin−/c-kit+ cells in a transwell chamber assay in response to increasing concentration of SDF-1α for 4 hours. (B) Time course of migration of Lin−/c-kit+ cells in response to SDF-1α (100 ng/mL). (C) In vitro migration of Lin−/c-kit+ cells through FN-CH296–coated filters in a transwell chamber assay in response to SDF-1α for 4 hours. (D) Chemokinesis of Lin−/c-kit+ cells in uniform concentration of SDF-1α (100 ng/mL; 4 hours). Data represent the percentage of the migrated cells as the mean plus or minus SD; n = 3. (E) F-actin assembly. Lin−/c-kit+ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds before being stained with rhodamine-labeled phalloidin and 4′,6-diamidino-2-phenylindole. The percentage of cells with cortical F-actin is shown as the mean plus or minus SD; n = 3. Hee-Don Chae et al. Blood 2008;111:2597-2605 ©2008 by American Society of Hematology

Rac-dependent RhoH effects on cortical F-actin assembly and chemotaxis. Rac-dependent RhoH effects on cortical F-actin assembly and chemotaxis. (A,B) Levels of the active, GTP-bound Rac proteins of Lin−/c-kit+ cells after stimulation with 100 ng/mL SDF-1α. The levels of GTP-bound Rac and total Rac proteins in whole-cell lysates were examined in parallel. Ratio of GTP-bound Rac GTPases was quantified by densitometry measurements. The relative ratio is shown as the mean plus or minus SD (n > 3) except WT 5-minute data (average±range; n = 2). (C) Expression of EGFP–Rac1N17 inhibited chemotaxis induced by SDF-1α. WT, Rhoh−/− LDBM cells were transduced with retroviral viruses expressing EGFP-tagged the dominant-negative Rac1 mutant Rac1N17 (EGFP–Rac1N17) or vector control. The percentage of sorted EGFP+/c-kit+ cells migrated toward SDF-1α in a transwell chamber for 4 hours was calculated. Data represent the mean plus or minus SD; n = 3. (D) Inhibitory effect of EGFP–Rac1N17 on cortical F-actin assembly. Sorted EGFP+/c-Kit+ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds and stained with rhodamine-labeled phalloidin and 4′,6-diamidino-2-phenylindole (DAPI). A total of 200 cells were counted under fluorescent microscope. Data represent the percentage of cells with cortical F-actin as the mean plus or minus SD; n = 3. Hee-Don Chae et al. Blood 2008;111:2597-2605 ©2008 by American Society of Hematology

Activity-dependent localization of Rac1. Activity-dependent localization of Rac1. (A,B) WT LDBM cells were transduced with EGFP–Rac1, EGFP–Rac1V12, or EGFP–Rac1N17 retroviral vectors. Sorted EGFP+/c-Kit+ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds, fixed, and stained with rhodamine-labeled phalloidin (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). The percentage of cells with membrane bound Rac1 is shown as the mean plus or minus SD; n = 3. (C) Rac1 localizes to lipid rafts in response to SDF-1α treatment. WT LDBM cells were transduced with EGFP-Rac1 retroviral vector. Sorted EGFP+/c-Kit+ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds, fixed, and then stained with Alexa Fluor 550–labeled CTxB (red). (D) Negative effect of MβCD on Rac1 localization and F-actin polymerization. WT LDBM cells were transduced with EGFP-Rac1. Sorted EGFP+/c-Kit+ cells were treated with 5 mM MβCD for 30 minutes to deplete cholesterol followed by the addition of SDF-1α (100 ng/mL) for 30 seconds. The cells were fixed and stained with rhodamine-labeled phalloidin or Alexa Fluor 550–labeled CTxB. The representative images from 3 independent experiments are shown. Hee-Don Chae et al. Blood 2008;111:2597-2605 ©2008 by American Society of Hematology

Subcellular localization of endogenous Rac1. Subcellular localization of endogenous Rac1. Lin−/c-kit+ cells were stimulated with SDF-1α (100 ng/mL) for indicated times, fixed, and then stained with anti-Rac1 mAb (green), rhodamine-labeled phalloidin (red), and 4′,6-diamidino-2-phenylindole (blue). The leading edges are indicated with a white arrow. The representative images from 3 independent experiments are shown. Hee-Don Chae et al. Blood 2008;111:2597-2605 ©2008 by American Society of Hematology

RhoH regulates subcellular localization of Rac1. RhoH regulates subcellular localization of Rac1. (A) WT LDBM cells were transduced with retroviral vectors expressing HA-tagged RhoH-IRES-YFP (RhoH) or vector control (YFP). Sorted YFP+/c-Kit+ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds, fixed, and stained with anti-Rac1 mAb (green), rhodamine-labeled phalloidin (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Phase contrast images show the cellular morphology. Some cells were polarized after SDF-1α stimulation. The leading edge of polarized cell is indicated with a white arrow. (B) EGFP-Rac1, EGFP-Rac1V12, and EGFP-Rac2 localization in WT and RhoH-overexpressing cells. WT LDBM cells were transduced with retroviral vectors expressing Rac1 (EGFP-Rac1), EGFP-tagged Rac2 (EGFP-Rac2), or constitutive active Rac1 mutant Rac1V12 (EGFP-Rac1V12) with or without HA-tagged RhoH-IRES-YFP (RhoH–YFP). Sorted EGFP+/c-Kit+ or EGFP+/YFP+/c-Kit+ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds, fixed, and stained with rhodamine-labeled phalloidin or Alexa Fluor 550–labeled CTxB (red) and DAPI (blue). The representative images from 3 independent experiments are shown. Hee-Don Chae et al. Blood 2008;111:2597-2605 ©2008 by American Society of Hematology

The C-terminal domains of RhoH are required for its function. The C-terminal domains of RhoH are required for its function. (A) Schematic representation of RhoH mutants. Numbers indicate amino acid position within the sequence. (B) Subcellular localization of RhoH deletion mutants. WT LDBM cells were transduced with retroviral vectors expressing EGFP-tagged WT or C-terminal–deleted RhoH constructs (EGFP-RhoH, EGFP-RhoHΔPR, EGFP-RhoHΔCT). Sorted EGFP+/c-Kit+ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds, fixed, and stained with Alexa Fluor 550–labeled CTxB (red) and 4′,6-diamidino-2-phenylindole (DAPI). (C) Subcellular localization of RhoH deletion mutants in 32D cells. 32D cells were transduced with HA-tagged RhoH-YFP, RhoHÁPR-YFP, or RhoHÁCT-YFP. Sorted YFP+ 32D cells were fractionated into cytosolic fraction (S), detergent-soluble membrane fraction (P), detergent-insoluble cytoskeleton-enriched membrane fraction, and nuclear fraction. RhoH was detected with anti-HA antibody. (D) C-terminal prenylation site and polybasic domain of RhoH are required for its inhibition of cortical F-actin assembly. WT LDBM cells were transduced with retroviral vectors expressing RhoH-YFP, RhoHΔPR-YFP, or RhoHΔCT-YFP. Sorted YFP+/c-Kit+ cells were stimulated with SDF-1α (0 or 100 ng/mL) for 30 seconds before being fixed and stained with rhodamine-labeled phalloidin (red) and DAPI (blue). A total of 200 cells were counted under fluorescent microscope. Data represent the percentage of cells with cortical F-actin as the mean plus or minus SD; n = 3. Hee-Don Chae et al. Blood 2008;111:2597-2605 ©2008 by American Society of Hematology