Deletion of Dicer in late erythroid cells results in impaired stress erythropoiesis in mice.

MicroRNAs (miRNAs) have been shown to influence erythroid lineage commitment and differentiation; however, our knowledge of miRNA function in terminal erythropoiesis remains limited. To address this issue, we generated a novel animal model, where the miRNA-processing enzyme, Dicer, is selectively inactivated in erythropoietin receptor positive erythroid cells beginning with CFU-e/proerythroblast cells. This results in significant depletion of all miRNAs from the proerythroblast stage onwards, with one exception, miR-451, which is processed by Ago2 in a Dicer-independent manner. We observed that mature Dicer-dependent miRNAs, like miR-451, are dispensable under steady-state conditions, but these mutants have an impaired response to stress erythropoiesis, as demonstrated by a delay in recovery from anemia. This defect was specific to later maturing erythroid cells, as progenitor numbers were unaffected. In addition to generating a novel mouse model to study miRNA function in late erythroid cells, we conclude that miRNAs (both Dicer-dependent and independent) act primarily to regulate the optimal response to stress among late erythroid cells.

Stage-specific functional roles of integrins in murine erythropoiesis.

When the erythroid integrins α5ß1 and α4ß1 were each deleted previously at the stem cell level, they yielded distinct physiologic responses to stress by affecting erythoid expansion and terminal differentiation or only the latter, respectively. To test at what stage of differentiation the integrin effects were exerted, we created mice with α4- or α5-integrin deletions only in erythroid cells and characterized them at homeostasis and after phenylhydrazine-induced hemolytic stress. Unlike our prior data, the phenotype of mice with α5-erythroid deletions was similar to controls, especially after stress. These outcomes seem to reconcile divergent prior views on the role of α5-integrin in erythropoiesis. By contrast, α4 integrins whether deleted early or late have a dominant effect on bone marrow retention of erythroblasts and on terminal erythroid maturation at homeostasis and after stress.

Erythroid cells generated in the absence of specific ß1-integrin heterodimers accumulate reactive oxygen species at homeostasis and are unable to mount effective antioxidant defenses.

We have previously reported that ß1(Δ/Δ) mice have a markedly impaired response to hemolytic stress, but the mechanisms of this were unclear. In the present study we explored in detail quantitative, phenotypic and functional aspects of erythropoiesis at homeostasis in a large number of animals for each of 3 murine models with specific ß1 heterodimer integrin deficiencies. We found that, at homeostasis, ß1-deficient mice have a modest uncompensated anemia with ineffective erythropoiesis and decreased red blood cell survival. Mice lacking only α4 integrins (α4ß1/α4ß7) do not share this phenotype. There is an increased tendency for reactive oxygen species accumulation in ß1(Δ/Δ) erythroid cells with decreased anti-oxidant defenses at homeostasis which are exaggerated after stress. Furthermore, expansion of erythroid cells in spleen post-stress is dependent on α5ß1, likely through mechanisms activating focal adhesion kinase complexes that are distinct from α4ß1-mediated responses. In vivo inhibition of focal adhesion kinase activation partially recapitulates the ß1(Δ/Δ) stress response. Mice lacking all α4 and ß1 integrins (double knockouts) had, at homeostasis, the most severe phenotype with selective impairment of erythroid responses. The fact that integrins participate in mitigating stress in erythroid cells through redox activation of distinct signaling pathways by specific integrin heterodimers is a link that has not been appreciated until now.

The phosphoinositide phosphatase Sjl2 is recruited to cortical actin patches in the control of vesicle formation and fission during endocytosis.

The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositide (PI) phosphatases that regulate PI metabolism in the control of actin organization and membrane trafficking. However, the primary sites of action for each of the yeast synaptojanin-like proteins remain unclear. In this study, we show that Sjl2 is localized to cortical actin patches, sites of endocytosis. Cortical recruitment of Sjl2 requires the actin patch component Abp1. Consistent with this, the SH3 domain-containing protein Abp1 physically associates with Sjl2 through its proline-rich domain. Furthermore, abp1Delta mutations confer defects resembling loss of SJL2; sjl1Delta abp1Delta double-mutant cells exhibit invaginated plasma membranes and impaired endocytosis, findings similar to those for sjl1Delta sjl2Delta mutant cells. Thus, Abp1 acts as an adaptor protein in the localization or concentration of Sjl2 during late stages of endocytic vesicle formation. Overexpression of the Hip1-related protein Sla2 delayed the formation of extended plasma membrane invaginations in sjl2ts cells, indicating that Sla2 may become limiting or misregulated in cells with impaired PI phosphatase activity. Consistent with this, the cortical actin patch protein Sla2 is mislocalized in sjl1Delta sjl2Delta mutant cells. Together, our studies suggest that PI metabolism by the synaptojanin-like proteins coordinately directs actin dynamics and membrane invagination, in part by regulation of Sla2.

Retromer function in endosome-to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase.

A direct role for phosphoinositides in vesicular trafficking has been demonstrated by the identification of the yeast VPS34 gene encoding the phosphatidylinositol 3-kinase responsible for the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P). Vps34p binds the protein kinase Vps15p, and it has recently been shown that Vps15p and Vps34p associate with Vps30p and Vps38p to form a multimeric complex, termed complex II. We observed that mutations in the VPS30 and VPS38 genes led to a selective sorting and maturation phenotype of the soluble vacuolar protease CPY. Localization studies revealed that the CPY receptor Vps10p and the Golgi-endoprotease Kex2p were mislocalized to vacuolar membranes in strains deficient for either Vps30p or Vps38p, respectively. Interestingly, we measured decreased PtdIns3P levels in Deltavps30 and Deltavps38 cells and observed redistribution of Vps5p and Vps17p to the cytoplasm in these mutants. Vps5p and Vps17p are subunits of the retromer complex that is required for endosome-to-Golgi retrograde transport. Both proteins contain the Phox homology (PX) domain, a recently identified phosphoinositide-binding motif. We demonstrate that the PX domains of Vps5p and Vps17p specifically bind to PtdIns3P in vitro and in vivo. On the basis of these and other observations, we propose that the PtdIns 3-kinase complex II directs the synthesis of a specific endosomal pool of PtdIns3P, which is required for recruitment/activation of the retromer complex, thereby ensuring efficient endosome-to-Golgi retrograde transport.