Miranda couples oskar mRNA/Staufen complexes to the bicoid mRNA localization pathway

The double-stranded RNA binding protein Staufen is required for the microtubule-dependent localization of bicoid and oskar mRNAs to opposite poles of the Drosophila oocyte and also mediates the actin-dependent localization of prospero mRNA during the asymmetric neuroblast divisions. The posterior localization of oskar mRNA requires Staufen RNA binding domain 2, whereas prospero mRNA localization mediated the binding of Miranda to RNA binding domain 5, suggesting that different Staufen domains couple mRNAs to distinct localization pathways. Here, we show that the expression of Miranda during mid-oogenesis targets Staufen/oskar mRNA complexes to the anterior of the oocyte, resulting in bicaudal embryos that develop an abdomen and pole cells instead of the head and thorax. Anterior Miranda localization requires microtubules, rather than actin, and depends on the function of Exuperantia and Swallow, indicating that Miranda links Staufen/oskar mRNA complexes to the bicoid mRNA localization pathway. Since Miranda is expressed in late oocytes and bicoid mRNA localization requires the Miranda-binding domain of Staufen, Miranda may play a redundant role in the final step of bicoid mRNA localization. Our results demonstrate that different Staufen-interacting proteins couple Staufen/mRNA complexes to distinct localization pathways and reveal that Miranda mediates both actin- and microtubule-dependent mRNA localization.     

The I/LWEQ Domain in RapGAP3 Required for Posterior Localization in Migrating Cells

Cell migration requires a defined cell polarity which is formed by diverse cytoskeletal components differentially localized to the poles of cells to extracellular signals. Rap-GAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation and localizes to the leading edge of migrating cells. Here, we examined localization of truncated RapGAP3 proteins and found that the I/LWEQ domain in the central region of RapGAP3 was sufficient for posterior localization in migrating cells, as opposed to leading-edge localization of full-length Rap-GAP3. All truncated proteins accumulated at the leading edge of migrating cells exhibited clear translocation to the cell cortex in response to stimulation, whereas proteins localized to the posterior in migrating cells displayed no translocation to the cortex. The I/LWEQ domain appears to passively accumulate at the posterior region in migrating cells due to exclusion from the extended front region in response to chemoattractant stimulation rather than actively being localized to the back of cells. Our results suggest that posterior localization of the I/LWEQ domain of RapGAP3 is likely related to F-actin, which has probably different properties compared to newly formed F-actin at the leading edge of migrating cells, at the lateral and posterior regions of the cell.     

受精蛋白β在人精子表面的免疫组织化学定位

Fertilin is a kind of sperm plasma membrane protein that mimics snake venom protein. It belongs to the ADAMs family of surface proteins that contain a disintegrin and a metalloprotease domain. Fertilin functions in the sperm-egg binding process by connecting the sperm to the egg plasma membrane via a binding site in the disintegrin domain of fertilin beta (HF93). Its localization on the sperm is in the change. In this study, the monoclonal antibody against human fertilin beta was prepared and used to analyze the localization of fertilin beta on capacitated and acrosome-reacted sperm by immunofluorescence and immunoelectron microscopy techniques. The results were as follows: (1) fertilin beta became restricted to the anterior head during the course of capacitation. (2) During the course of acrosome reaction, the expression and localization of fertilin beta changed immensely on the anterior head and restricted to the lateral of posterior head at last. The restrictions of fertilin beta to the anterior head of capacitated sperm of human beings indicated that fertilin beta may be involved in the binding the sperm to the epithelial cells of the oviduct; the restrictions of fertilin beta to the posterior head domain of acrosome-reacted sperm implied its function in sperm-egg binding and fusion.     

The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.     

Evidence that proteolysis of the surface is an initial step in the mechanism of formation of sperm cell surface domains

On terminally differentiated sperm cells, surface proteins are segregated into distinct surface domains that include the anterior and posterior head domains. We have analyzed the formation of the anterior and posterior head domains of guinea pig sperm in terms of both the timing of protein localization and the mechanism(s) responsible. On testicular sperm, the surface proteins PH-20, PH-30 and AH-50 were found to be present on the whole cell (PH-20) or whole head surface (PH- 30, AH-50). On sperm that have completed differentiation (cauda epididymal sperm), PH-20 and PH-30 proteins were restricted to the posterior head domain and AH-50 was restricted to the anterior head domain. Thus these proteins become restricted in their distribution late in sperm differentiation, after sperm leave the testis. We discovered that the differentiation process that localizes these proteins can be mimicked in vitro by treating testicular sperm with trypsin. After testicular sperm were treated with 20 micrograms/ml trypsin for 5 min at room temperature, PH-20, PH-30, and AH-50 were found localized to the same domains to which they are restricted during in vivo differentiation. The in vitro trypsin-induced localization of PH-20 to the posterior head mimicked the in vivo differentiation process quantitatively as well as qualitatively. The quantitative analysis showed the process of PH-20 localization involves the migration of surface PH-20 from other regions to the posterior head domain. Immunoprecipitation experiments confirmed that there is protease action in vivo on the sperm surface during the late stages of sperm differentiation. Both the PH-20 and PH-30 proteins were shown to be proteolytically cleaved late in sperm differentiation. These findings strongly implicate proteolysis of surface molecules as an initial step in the mechanism of formation of sperm head surface domains.     

The planar cell polarity protein Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells in Drosophila

Cell fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell division. In the Drosophila bristle lineage, the sensory organ precursor (pI) cell is polarized along the anteroposterior (AP) axis by Frizzled (Fz) receptor signaling. We show here that Fz localizes at the posterior apical cortex of the pI cell prior to mitosis, whereas Strabismus (Stbm) and Prickle (Pk), which are also required for AP polarization of the pI cell, co-localize at the anterior apical cortex. Thus, asymmetric localization of Fz, Stbm and Pk define two opposite cortical domains prior to mitosis of the pI cell. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Partner of Inscuteable (Pins) and Discs-large (Dlg), two components of the anterior Dlg-Pins-Galphai complex that regulates the localization of cell-fate determinants. At prophase, Stbm promotes the anterior localization of Pins. By contrast, Dishevelled (Dsh) acts antagonistically to Stbm by excluding Pins from the posterior cortex. We propose that the Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell is a novel read-out of planar cell polarity.     

Nanos is the localized posterior determinant in Drosophila

Segmental pattern in the Drosophila embryo is established by two maternal factors localized to the anterior and posterior poles of the egg cell. Here we provide molecular evidence that the localized posterior factor is the RNA of the nanos (nos) gene. nos RNA is localized to the posterior pole of early embryos, and nos protein acts at a distance to direct abdomen formation. Synthetic nos RNA has biological activity identical to that of the posterior pole plasm. Injection of nos RNA rescues the segmentation defect of embryos derived from females mutant for all nine known posterior group genes. Injection of nos RNA into the anterior is able to direct formation of ectopic posterior structures. Our results demonstrate that a localized source of nos RNA is sufficient to specify abdominal segmentation and imply that other posterior group genes are required for localization, stabilization, or distribution of the nos gene product.     

PAR-3 and PAR-1 inhibit LET-99 localization to generate a cortical band important for spindle positioning in Caenorhabditis elegans embryos

The conserved PAR proteins are localized in asymmetric cortical domains and are required for the polarized localization of cell fate determinants in many organisms. In Caenorhabditis elegans embryos, LET-99 and G protein signaling act downstream of the PARs to regulate spindle positioning and ensure asymmetric division. PAR-3 and PAR-2 localize LET-99 to a posterior cortical band through an unknown mechanism. Here we report that LET-99 asymmetry depends on cortically localized PAR-1 and PAR-4 but not on cytoplasmic polarity effectors. In par-1 and par-4 embryos, LET-99 accumulates at the entire posterior cortex, but remains at low levels at the anterior cortex occupied by PAR-3. Further, PAR-3 and PAR-1 have graded cortical distributions with the highest levels at the anterior and posterior poles, respectively, and the lowest levels of these proteins correlate with high LET-99 accumulation. These results suggest that PAR-3 and PAR-1 inhibit the localization of LET-99 to generate a band pattern. In addition, PAR-1 kinase activity is required for the inhibition of LET-99 localization, and PAR-1 associates with LET-99. Finally, examination of par-1 embryos suggests that the banded pattern of LET-99 is critical for normal posterior spindle displacement and to prevent spindle misorientation caused by cell shape constraints.     

The HhH2/NDD domain of the Drosophila Nod chromokinesin-like protein is required for binding to chromosomes in the oocyte nucleus

Nod is a chromokinesin-like protein that plays a critical role in segregating achiasmate chromosomes during female meiosis. The C-terminal half of the Nod protein contains two putative DNA-binding domains. The first of these domains, known as the HMGN domain, consists of three tandemly repeated high-mobility group N motifs. This domain was previously shown to be both necessary and sufficient for binding of the C-terminal half of Nod to mitotic chromosomes in embryos. The second putative DNA-binding domain, denoted HhH(2)/NDD, is a helix-hairpin-helix(2)/Nod-like DNA-binding domain. Although the HhH(2)/NDD domain is not required or sufficient for chromosome binding in embryos, several well-characterized nod mutations have been mapped in this domain. To characterize the role of the HhH(2)/NDD domain in mediating Nod function, we created a series of UAS-driven transgene constructs capable of expressing either a wild-type Nod-GFP fusion protein or proteins in which the HhH(2)/NDD domain had been altered by site-directed mutagenesis. Although wild-type Nod-GFP localizes to the oocyte chromosomes and rescues the segregation defect in nod mutant oocytes, two of three proteins carrying mutants in the HhH(2)/NDD domain fail to either rescue the nod mutant phenotype or bind to oocyte chromosomes. However, these mutant proteins do bind to the polytene chromosomes in nurse-cell nuclei and enter the oocyte nucleus. Thus, even though the HhH(2)/NDD domain is not essential for chromosome binding in other cell types, it is required for chromosome binding in the oocyte. These HhH(2)/NDD mutants also block the localization of Nod to the posterior pole of stage 9-10A oocytes, a process that is thought to facilitate the interaction of Nod with the plus ends of microtubules (Cui et al. 2005). This observation suggests that the Nod HhH2/NDD domain may play other roles in addition to binding Nod to meiotic chromosomes.     

The extracytoplasmic domain of the Mycobacterium tuberculosis Ser/Thr kinase PknB binds specific muropeptides and is required for PknB localization

The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA) domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division.     

Structure and RNA binding of the mouse Pumilio-2 Puf domain

Puf proteins control translation through the interaction of a C-terminal Puf domain with specific sequences present in the 3′ untranslated region of messenger RNAs. In Drosophila, binding of the protein Pumilio to mRNA leads to translational repression which is required for anterior/posterior patterning during embryogenesis. The vertebrate Pumilio homologue 2 (Pum2) has been implicated in controlling germ cell development through interactions with the RNA binding proteins deleted in azoospermia (DAZ), DAZ-like (DAZL) and BOULE. We present the 1.6 Å resolution X-ray crystal structure of the Puf domain from murine Pum2 and demonstrate that this domain is capable of binding with nanomolar affinity to RNA sequences from the hunchback Nanos response element (NRE) and a previously identified Pum2 binding element (PBE).     

The Fz-Dsh planar cell polarity pathway induces oriented cell division via Mud/NuMA in Drosophila and zebrafish

The Frizzled receptor and Dishevelled effector regulate mitotic spindle orientation in both vertebrates and invertebrates, but how Dishevelled orients the mitotic spindle is unknown. Using the Drosophila S2 cell "induced polarity" system, we find that Dishevelled cortical polarity is sufficient to orient the spindle and that Dishevelled's DEP domain mediates this function. This domain binds a C-terminal domain of Mud (the Drosophila NuMA ortholog), and Mud is required for Dishevelled-mediated spindle orientation. In Drosophila, Frizzled-Dishevelled planar cell polarity (PCP) orients the sensory organ precursor (pI) spindle along the anterior-posterior axis. We show that Dishevelled and Mud colocalize at the posterior cortex of pI, Mud localization at the posterior cortex requires Dsh, and Mud loss-of-function randomizes spindle orientation. During zebrafish gastrulation, the Wnt11-Frizzled-Dishevelled PCP pathway orients spindles along the animal-vegetal axis, and reducing NuMA levels disrupts spindle orientation. Overall, we describe a Frizzled-Dishevelled-NuMA pathway that orients division from Drosophila to vertebrates.     

Proteasomal activity modulates TGF-ss signaling in a gene-specific manner

Myosin heavy chain kinase A (MHCK A) modulates myosin II filament assembly in the amoeba Dictyostelium discoideum. MHCK A localization in vivo is dynamically regulated during chemotaxis, phagocytosis, and other polarized cell motility events, with preferential recruitment into anterior filamentous actin (F-actin)-rich structures. The current work reveals that an amino-terminal segment of MHCK A, previously identified as forming a coiled-coil, mediates anterior localization. MHCK A co-sediments with F-actin, and deletion of the amino-terminal domain eliminated actin binding. These results indicate that the anterior localization of MHCK A is mediated via direct binding to F-actin, and reveal the presence of an actin-binding function not previously detected by primary sequence evaluation of the coiled-coil domain.     

lin-17/Frizzled and lin-18 regulate POP-1/TCF-1 localization and cell type specification during C. elegans vulval development

The Caenorhabditis elegans vulva is comprised of highly similar anterior and posterior halves that are arranged in a mirror symmetric pattern. The cell lineages that form each half of the vulva are identical, except that they occur in opposite orientations with respect to the anterior/posterior axis. We show that most vulval cell divisions produce sister cells that have asymmetric levels of POP-1 and that the asymmetry has opposite orientations in the two halves of the vulva. We demonstrate that lin-17 (Frizzled type Wnt receptor) and lin-18 (Ryk) regulate the pattern of POP-1 localization and cell type specification in the posterior half of the vulva. In the absence of lin-17 and lin-18, posterior lineages are reversed and resemble anterior lineages. These experiments suggest that Wnt signaling pathways reorient cell lineages in the posterior half of the vulva from a default orientation displayed in the anterior half of the vulva.     

Bazooka and PAR-6 are required with PAR-1 for the maintenance of oocyte fate in Drosophila

The anterior-posterior axis of C. elegans is defined by the asymmetric division of the one-cell zygote, and this is controlled by the PAR proteins, including PAR-3 and PAR-6, which form a complex at the anterior of the cell, and PAR-1, which localizes at the posterior [1-4]. PAR-1 plays a similar role in axis formation in Drosophila: the protein localizes to the posterior of the oocyte and is necessary for the localization of the posterior and germline determinants [5, 6]. PAR-1 has recently been shown to have an earlier function in oogenesis, where it is required for the maintenance of oocyte fate and the posterior localization of oocyte-specific markers [7, 8]. Here, we show that the homologs of PAR-3 (Bazooka) and PAR-6 are also required to maintain oocyte fate. Germline clones of mutants in either gene give rise to egg chambers that develop 16 nurse cells and no oocyte. Furthermore, oocyte-specific factors, such as Orb protein and the centrosomes, still localize to one cell but fail to move from the anterior to the posterior cortex. Thus, PAR-1, Bazooka, and PAR-6 are required for the earliest polarity in the oocyte, providing the first example in Drosophila where the three homologs function in the same process. Although these PAR proteins therefore seem to play a conserved role in early anterior-posterior polarity in C. elegans and Drosophila, the relationships between them are different, as the localization of PAR-1 does not require Bazooka or PAR-6 in Drosophila, as it does in the worm.     

Identification of novel membrane-binding domains in multiple yeast Cdc42 effectors

The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP(2)-containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase-dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein-protein and protein-membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex.     

WRM-1 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans.

During C. elegans development, Wnt/WG signaling is required for differences in cell fate between sister cells born from anterior/posterior divisions. A beta-catenin-related gene, wrm-1, and the lit-1 gene are effectors of this signaling pathway and appear to downregulate the activity of POP-1, a TCF/LEF-related protein, in posterior daughter cells. We show here that lit-1 encodes a serine/threonine protein kinase homolog related to the Drosophila tissue polarity protein Nemo. We demonstrate that the WRM-1 protein binds to LIT-1 in vivo and that WRM-1 can activate the LIT-1 protein kinase when coexpressed in vertebrate tissue culture cells. This activation leads to phosphorylation of POP-1 and to apparent changes in its subcellular localization. Our findings provide evidence for novel regulatory avenues for an evolutionarily conserved Wnt/WG signaling pathway.     

PH Domain-Arf G Protein Interactions Localize the Arf-GEF Steppke for Cleavage Furrow Regulation in Drosophila

The recruitment of GDP/GTP exchange factors (GEFs) to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. Analyses of mammalian cytohesins have identified a number of recruitment mechanisms for these multi-domain proteins, but the conservation and developmental roles for these mechanisms are unclear. Here, we report how the pleckstrin homology (PH) domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. We found that the PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. Next, we examined the role of conserved PH domain amino acid residues that are required for mammalian cytohesins to bind PIP3 or GTP-bound Arf G proteins. We confirmed that the Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. By analyzing GFP-tagged Arf and Arf-like small G proteins, we found that Arf1-GFP, Arf6-GFP and Arl4-GFP, but not Arf4-GFP, localized to furrows. However, analyses of embryos depleted of Arf1, Arf6 or Arl4 revealed either earlier defects than occur in embryos depleted of Steppke, or no detectable furrow defects, possibly because of redundancies, and thus it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, our data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos.