Apoptosis and the subsequent clearance of these dying cells occur throughout development and adult life in many tissues. Failure to promptly clear apoptotic cells has been linked to many diseases1-3. ELMO1 is an evolutionarily conserved cytoplasmic engulfment protein that functions downstream of the phosphatidylserine receptor BAI1, and, along with Dock180 and Rac1, promotes internalization of the dying cells4-7. Here, we generated ELMO1-deficient mice, and unexpectedly found them to be viable and grossly normal. However, ELMO1-deficient mice had a striking testicular pathology, with disrupted seminiferous epithelium, multi-nucleated giant cells, uncleared apoptotic germ cells, and decreased sperm output. Subsequent in vitro and in vivo analyses revealed a crucial role for ELMO1 in the phagocytic clearance of apoptotic germ cells by Sertoli cells lining the seminiferous epithelium. The engulfment receptor BAI1 and the GTPase Rac (upstream and downstream of ELMO1, respectively) were also important for Sertoli cell-mediated engulfment. Collectively, these findings uncover a selective requirement for ELMO1 in Sertoli cell-mediated removal of apoptotic germ cells and make a compelling case for a relationship between engulfment and tissue homeostasis in vivo.

SummaryFunctional stay‐green is a valuable trait that extends the photosynthetic period, increases source capacity and biomass and ultimately translates to higher grain yield. Selection for higher yields has increased stay‐green in modern maize hybrids. Here, we report a novel QTL controlling functional stay‐green that was discovered in a mapping population derived from the Illinois High Protein 1 (IHP1) and Illinois Low Protein 1 (ILP1) lines, which show very different rates of leaf senescence. This QTL was mapped to a single gene containing a NAC‐domain transcription factor that we named nac7. Transgenic maize lines where nac7 was down‐regulated by RNAi showed delayed senescence and increased both biomass and nitrogen accumulation in vegetative tissues, demonstrating NAC7 functions as a negative regulator of the stay‐green trait. More importantly, crosses between nac7 RNAi parents and two different elite inbred testers produced hybrids with prolonged stay‐green and increased grain yield by an average 0.29 megagram/hectare (4.6 bushel/acre), in 2 years of multi‐environment field trials. Subsequent RNAseq experiments, one employing nac7 RNAi leaves and the other using leaf protoplasts overexpressing Nac7, revealed an important role for NAC7 in regulating genes in photosynthesis, chlorophyll degradation and protein turnover pathways that each contribute to the functional stay‐green phenotype. We further determined the putative target of NAC7 and provided a logical extension for the role of NAC7 in regulating resource allocation from vegetative source to reproductive sink tissues. Collectively, our findings make a compelling case for NAC7 as a target for improving functional stay‐green and yields in maize and other crops.

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Many agents that activate hematopoietic cells use phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P 3 ) to initiate signaling cascades. The SH2 domain-containing inositol 5 phosphatase, SHIP1, regulates hematopoietic cell function by opposing the action of phosphatidylinositol 3-kinase and reducing the levels of PtdIns 3,4,5-P 3 . Activation of the cyclic AMPdependent protein kinase (PKA) also opposes many of the proinflammatory responses of hematopoietic cells. We tested to see whether the activity of SHIP1 was regulated via phosphorylation with PKA. We prepared pure recombinant SHIP1 from HEK-293 cells and found it can be rapidly phosphorylated by PKA to a stoichiometry of 0.6 mol of PO 4 /mol of SHIP1. In Overall, activation of G protein-coupled receptors that raise cyclic AMP cause SHIP1 to be phosphorylated and stimulate its inositol phosphatase activity. These results outline a novel mechanism of SHIP1 regulation. Activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase)2 is central to regulation of multiple cell functions including cell shape changes, cell migration, cell activation, and proliferation (1). PtdIns 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate in the inner leaflet of the plasma membrane to generate phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P 3 ) (2). PtdIns 3,4,5-P 3 then activates downstream signaling pathways by interacting with pleckstrin homology domain-containing proteins, such as phosphoinositide-dependent kinase 1 and the serine-threonine kinase Akt (3). The finding of abnormal activation of the PtdIns 3-kinase pathway in cancer cells has led to interest in the development of inhibitors for PtdIns 3-kinase (4).The level of PtdIns 3,4,5-P 3 is stimulated by multiple members of the PtdIns 3-kinase family (2) and is opposed by two phosphatidylinositol phosphatases: the Src homology 2 (SH2) domain-containing inositol 5Ј phosphatase (SHIP) and the 3Ј inositol phosphatase, phosphatase and tensin homolog (PTEN) (5). PTEN removes phosphate from the 3Ј position in the inositol ring of PtdIns 3,4,5-P 3 and converts it to phosphatidylinositol 4,5-bisphosphate (6). PTEN has a C2 domain, a PDZbinding motif, and a N-terminal phosphatidylinositol 4,5-bisphosphate binding motif essential for translocation to the membrane and interaction with other regulatory proteins (7). There are serine and threonine residues in PTEN that have been found to be phosphorylated, but their role in regulating the activity of the enzyme is not clear (8). Mutations in the PTEN protein have been observed in many tumors, suggesting a role for this enzyme in cancer (9).In contrast, SHIP dephosphorylates the 5Ј position on the inositol ring and produces phosphatidylinositol 3,4-bisphosphate (10). There are three isoforms of SHIP: the 145-kDa hematopoietic cell restricted SHIP (also known as SHIP1); the 104-kDa stem cell-restricted SHIP, sSHIP; and the more widely expressed 150-kDa SHIP2 (11). SHIP1 is the major inositol phosphatase regulating PtdIns 3,4,5-P 3 in monocytes, macrophages,...

). Using a combination of approaches, we identified the serine residue regulating SHIP1 activity. After mass spectrometric identification of 17 serine and threonine residues on SHIP1 as being phosphorylated by PKA in vitro, studies with truncation mutants of SHIP1 narrowed the phosphorylation site to the catalytic region between residues 400 and 866. Of the two candidate phosphorylation sites located in this region (Ser 440 and Ser 2 is central to many intracellular signaling cascades (1). PtdIns-3,4,5-P 3 is produced in the inner leaflet of the plasma membrane by a family of phosphatidylinositol 3-kinases that are activated by receptor tyrosine kinases or G proteincoupled receptors (2). The PtdIns-3,4,5-P 3 localized at the plasma membrane forms a docking site to attract and regulate downstream signaling molecules containing pleckstrin homology domains, such as the serine kinase, Akt (PKB), that has an essential role in stimulating cell proliferation, growth, survival, and metabolism (2, 3). The level of PtdIns-3,4,5-P 3 in the membrane is opposed by two inositol lipid phosphatases, PTEN (phosphatase and tensin homologue) and SHIP (SH2 domaincontaining inositol 5Ј-phosphatase) (4). SHIP opposes the effects of phosphatidylinositol 3-kinases by dephosphorylating the 5Ј-position on the inositol ring of PtdIns-3,4,5-P 3 and producing phophatidylinositol 3,4-bisphosphate (5).There are two major members of the SHIP family, SHIP1 and SHIP2, that share a highly homologous catalytic region with less similarity in their C-terminal regions (6). SHIP2 is widely expressed, whereas SHIP1 is only expressed in hematopoietic cells (7). SHIP1 was originally identified based on its ability to bind to cytoplasmic adaptor proteins, such as Shc, Dab-1, and Dok-3 (8). SHIP1 contains an N-terminal SH2 domain that leads to interactions with immune receptors, such as Fc␥RIIB and Fc⑀RI (9), a central inositol 5Ј-phosphatase domain, and two tyrosines within NPXY motifs in the C-terminal prolinerich region (10). Genetic manipulations of SHIP1 in mice have shown that SHIP1 plays important roles in functions of myeloid cells and B lymphocytes; for example, in mature neutrophils and mast cells, PtdIns-3,4,5-P 3 levels and the phosphorylation of Akt activity are significantly elevated in the absence of SHIP1 (11). Similarly, B lymphocytes lacking SHIP1 are insensitive to inhibitory signaling via the Fc␥RIIB receptor (9). Moreover, SHIP1 Ϫ/Ϫ mice have a decreased viability due to an increased infiltration of myeloid cells into the lungs, perhaps due to activation of migration pathways because of the elevated levels of PtdIns-3,4,5-P 3 (12).Due to its importance in hematopoietic cells, the activity of SHIP1 is tightly regulated. One model for regulation of SHIP1 function envisions translocation of SHIP1 from the cytosol to the membrane (13). Upon stimulation by growth factors, cytokine receptors, or specific signaling receptors on lymphocytes, SHIP1 is recruited via its N-terminal SH2 domain to phosphorylated tyrosine residues in receptor kina...

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