James F. Johnson

Signaling and cytoskeletal requirements in erythroblast enucleation

To understand the role of cytoskeleton and membrane signaling molecules in erythroblast enucleation, we developed a novel analysis protocol of multiparameter high-speed cell imaging in flow. This protocol enabled us to observe F-actin and phosphorylated myosin regulatory light chain (pMRLC) assembled into a contractile actomyosin ring (CAR) between nascent reticulocyte and nucleus, in a population of enucleating erythroblasts. CAR formation and subsequent enucleation were not affected in murine erythroblasts with genetic deletion of Rac1 and Rac2 GTPases because of compensation by Rac3. Pharmacologic inhibition or genetic deletion of all Rac GTPases altered the distribution of F-actin and pMRLC and inhibited enucleation. Erythroblasts treated with NSC23766, cytochalasin-D, colchicine, ML7, or filipin that inhibited Rac activity, actin or tubulin polymerization, MRLC phosphorylation, or lipid raft assembly, respectively, exhibited decreased enucleation efficiency, as quantified by flow cytometry. As assessed by high-speed flow-imaging analysis, colchicine inhibited erythroblast polarization, implicating microtubules during the preparatory stage of enucleation, whereas NSC23766 led to absence of lipid raft assembly in the reticulocyte-pyrenocyte border. In conclusion, enucleation is a multistep process that resembles cytokinesis, requiring establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte.

Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen.

Background The small Rho GTPases Rac1 and Rac2 have both overlapping and distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. The role of these Rac GTPases in erythropoiesis has not yet been fully elucidated. Design and Methods Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background, in mouse hematopoietic cells in vivo. The erythroid progenitors and precursors in the bone marrow and spleen of these genetically engineered animals were evaluated by colony assays and flow cytometry. Apoptosis and proliferation of the different stages of erythroid progenitors and precursors were evaluated by flow cytometry. Results Erythropoiesis in Rac1−/−;Rac2−/− mice is characterized by abnormal burst-forming unit-erythroid colony morphology and decreased numbers of megakaryocyte-erythrocyte progenitors, erythroid colony-forming units, and erythroblasts in the bone marrow. In contrast, splenic erythropoiesis is increased. Combined Rac1 and Rac2 deficiency compromises proliferation of the megakaryocyte-erythrocyte progenitor population in the bone marrow, while it allows increased survival and proliferation of megakaryocyte-erythrocyte progenitors in the spleen. Conclusions These data suggest that Rac1 and Rac2 GTPases are essential for normal bone marrow erythropoiesis but that they are dispensable for erythropoiesis in the spleen, implying different signaling pathways for homeostatic and stress erythropoiesis.

R-Ras and Rac GTPase Cross-talk Regulates Hematopoietic Progenitor Cell Migration, Homing, and Mobilization

Adult hematopoietic progenitor cells (HPCs) are maintained by highly coordinated signals in the bone marrow. The molecular mechanisms linking intracellular signaling network of HPCs with their microenvironment remain poorly defined. The Rho family GTPase Rac1/Rac2 has previously been implicated in cell functions involved in HPC maintenance, including adhesion, migration, homing, and mobilization. In the present studies we have identified R-Ras, a member of the Ras family, as a key signal mediator required for Rac1/Rac2 activation. We found that whereas Rac1 activity is up-regulated upon stem cell factor, integrin, or CXCL12 stimulation, R-Ras activity is inversely up-regulated. Expression of a constitutively active R-Ras mutant resulted in down-regulation of Rac1-activity whereas deletion of R-Ras led to an increase in Rac1/Rac2 activity and signaling. R-Ras−/− HPCs displayed a constitutively assembled cortical actin structure and showed increased directional migration. Rac1/Rac2 inhibition reversed the migration phenotype of R-Ras−/− HPCs, similar to that by expressing an R-Ras active mutant. Furthermore, R-Ras−/− mice showed enhanced responsiveness to G-CSF for HPC mobilization and exhibited decreased bone marrow homing. Transplantation experiments indicate that the R-Ras deficiency-induced HPC mobilization is a HPC intrinsic property. These results indicate that R-Ras is a critical regulator of Rac signaling required for HPC migration, homing, and mobilization.

Rac1 targeting suppresses p53 deficiency–mediated lymphomagenesis

Mutation of the p53 tumor suppressor is associated with disease progression, therapeutic resistance, and poor prognosis in patients with lymphoid malignancies and can occur in approximately 50% of Burkitt lymphomas. Thus, new therapies are needed to specifically target p53-deficient lymphomas with increased efficacy. In the current study, the specific impact of inhibition of the small GTPase Rac1 on p53-deficient B- and T-lymphoma cells was investigated. p53 deficiency resulted in increased Rac1 activity in both B-cell and T-cell lines, and its suppression was able to abrogate p53 deficiency-mediated lymphoma cell proliferation. Further, Rac targeting resulted in increased apoptosis via a p53-independent mechanism. By probing multiple signaling axes and performing rescue studies, we show that the antiproliferative effect of Rac1 targeting in lymphoma cells may involve the PAK and Akt signaling pathway, but not the mitogen-activated protein (MAP) kinase pathway. The effects of inhibition of Rac1 were extended in vivo where Rac1 targeting was able to specifically impair p53-deficient lymphoma cell growth in mouse xenografts and postpone lymphomagenesis onset in murine transplantation models. Because the Rac1 signaling axis is a critical determinant of apoptosis and tumorigenesis, it may represent an important basis for therapy in the treatment of p53-deficient lymphomas.

Determination of respiratory phase during acquisition of airway cine MR images.

Subjects were imaged on a 1.5-T Signa MRI system using the head-neck vascular coil. An axial fast gradient echo cine, at the base of the second cervical vertebra, was obtained. A total of 128 images were acquired with a rapid image acquisition (one per second) over several respiratory cycles. The analog signal from the MR scanner (RF unblank) was utilized to determine the duration of the cine MR sequence. The phase of respiration was determined by analyzing the nasal air flow connected via pressure tubing to a pressure transducer outside the MR scanner room. We were thus able to determine the phase of respiration during acquisition of individual airway cine MR images. There was a wide range of airway volume measurements over the respiratory cycle with the lowest volume at end expiration and the highest at peak inspiration.