pebble: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - pebble

Synonyms -

Cytological map position - 66A18-20

Function - signaling

Keywords - cell division, cytoskeleton, required for the formation of the contractile ring and the initiation of cytokinesis

Symbol - pbl

FlyBase ID: FBgn0003041

Genetic map position -

Classification - guanyl-nucleotide exchange factor, Rho GEF

Cellular location - cytoplasmic and nuclear



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Rosa, A., Vlassaks, E., Pichaud, F. and Baum, B. (2015). Ect2/Pbl acts via Rho and polarity proteins to direct the assembly of an isotropic actomyosin cortex upon mitotic entry. Dev Cell 32: 604-616. PubMed ID: 25703349
Summary:
Entry into mitosis is accompanied by profound changes in cortical actomyosin organization. This study delineate a pathway downstream of the RhoGEF Pbl/Ect2 that directs this process in Drosophila notum epithelial cells. The data suggest that the release of Pbl/Ect2 from the nucleus at mitotic entry drives Rho-dependent activation of Myosin-II and, in parallel, induces a switch from Arp2/3 (see Actin-related protein 2/3 complex, subunit 1) to Diaphanous-mediated cortical actin nucleation that depends on Cdc42, aPKC, and Par6. At the same time, the mitotic relocalization of these apical protein complexes to more lateral cell surfaces enables Cdc42/aPKC/Par6 to take on a mitosis-specific function-aiding the assembly of a relatively isotropic metaphase cortex. Together, these data reveal how the repolarization and remodeling of the actomyosin cortex are coordinated upon entry into mitosis to provide cells with the isotropic and rigid form they need to undergo faithful chromosome segregation and division in a crowded tissue environment.
Jeong, S., Yang, D. S., Hong, Y. G., Mitchell, S. P., Brown, M. P. and Kolodkin, A. L. (2017). Varicose and cheerio collaborate with pebble to mediate semaphorin-1a reverse signaling in Drosophila. Proc Natl Acad Sci U S A 114(39): E8254-e8263. PubMed ID: 28894005
Summary:
The transmembrane semaphorin Sema-1a acts as both a ligand and a receptor to regulate axon-axon repulsion during neural development. Pebble (Pbl), a Rho guanine nucleotide exchange factor, mediates Sema-1a reverse signaling through association with the N-terminal region of the Sema-1a intracellular domain (ICD), resulting in cytoskeletal reorganization. This study uncover two additional Sema-1a interacting proteins, varicose (Vari) and cheerio (Cher), each with neuronal functions required for motor axon pathfinding. Vari is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins, members of which can serve as scaffolds to organize signaling complexes. Cher is related to actin filament cross-linking proteins that regulate actin cytoskeleton dynamics. The PDZ domain binding motif found in the most C-terminal region of the Sema-1a ICD is necessary for interaction with Vari, but not Cher, indicative of distinct binding modalities. Pbl/Sema-1a-mediated repulsive guidance is potentiated by both vari and cher Genetic analyses further suggest that scaffolding functions of Vari and Cher play an important role in Pbl-mediated Sema-1a reverse signaling. These results define intracellular components critical for signal transduction from the Sema-1a receptor to the cytoskeleton and provide insight into mechanisms underlying semaphorin-induced localized changes in cytoskeletal organization.
BIOLOGICAL OVERVIEW

Cytokinesis is the final step in cell division during which the daughter nuclei are invested with their own plasma membranes and cytoplasms. Cytokinesis depends on the formation of a cortical actin- and myosin-based ('actomyosin') contractile ring, which constricts and pinches off the membrane to form the two daughter cells. Pebble is a Rho GEF required for the formation of the contractile ring and the initiation of cytokinesis. Targeted vesicle fusion at the cleavage plane is now known to play an important role in animal cell cytokinesis, with microtubule arrays perhaps helping to direct vesicle transport. Membrane addition may be coordinated with and/or guided by contraction of the actomyosin ring, with these two processes together forming the foundation of cytokinesis in both fungal and animal cells. Several conserved protein families, including the septins, the Cdc15p/PSTPIP proteins, and GTPases of the Rho/Rac/Cdc42 family, act quite broadly in cytokinesis and are likely to direct or coordinate aspects of actomyosin-ring function, membrane addition, or both in various organisms (reviewed by Hales, 1999).

Rho proteins are GTPases that act as molecular switches, cycling between inactive (GDP-bound) and active (GTP-bound) states. Rho is activated by guanine nucleotide exchange factors (GEFs), which enhance the exchange of bound GDP for GTP, and are inactivated by GTPase-activating proteins (GAPs), which increase the intrinsic GTPase activity of Rho proteins. Activation of Rho results in a conformational change of the protein revealing structural domains required for the interaction with downstream target proteins. Thus, the intracellular ratio of the GTP/GDP-bound forms of Rho proteins determines the activation of signal transduction pathways regulating the spatial and temporal reorganization of cytoskeletal architecture (Prokopenko, 1999).

The dynamics of Pbl expression and its distribution during mitosis, as well as structure-function analysis, indicate that it is a key regulatory component of the pathway leading to cytokinesis. In Drosophila, Rho1 has also been identified as a putative effector of DRhoGEF2 (Barrett, 1997; H├Ącker, 1998), indicating that Rho1 is regulated by Pebble and DrhoGEF2, and potentially by other RhoGEFs. pbl interacts genetically with Rho1, but not with Rac1 or Cdc42, and Pbl and Rho1 proteins interact in vivo in yeast. Similar to mutations in pbl, loss of Rho1 or expression of a dominant-negative Rho1 blocks cytokinesis. These results identify Pbl as a RhoGEF specifically required for cytokinesis and linked through Rho1 activity to the reorganization of the actin cytoskeleton at the cleavage furrow (Prokopenko, 1999).

Genetic analysis and biochemical studies carried out in Drosophila have led to the identification of a number of genes required for the assembly or function of the contractile ring (Hale, 1999 and Field, 1999). Structural components of the contractile ring include actin, the regulatory light chain of nonmuscle myosin [encoded by spaghetti-squash (sqh)], septins [Peanut (Pnut), Septin-1, and Septin-2], and actin-binding proteins cofilin [encoded by twinstar (tsr), profilin [encoded by chickadee (chic), and anillin. Mutations in sqh, pnut, tsr, and chic result in a failure of cytokinesis, most likely due to defects in contractile ring organization or function. Yet little is known about the roles of these proteins in contractile ring assembly, stabilization, constriction, and disassembly or about the molecular machinery regulating actin cytoskeleton reorganization during cytokinesis (Prokopenko, 1999).

Pbl redistribution during mitosis parallels the onset of cytokinesis. Double stainings of wild-type embryos undergoing mitotic cycle 14 with anti Pbl antibody and with anti-phospho-histone H3 antibody, which marks chromosomes during mitosis, reveals that Pbl is expressed dynamically during the cell cycle. No Pbl protein is observed during late prophase, metaphase, and early anaphase. Pbl protein is first detected in late anaphase, when it colocalizes with separating daughter chromosomes. The highest levels of Pbl staining were found in telophase nuclei, and this staining persists during interphase of the following mitotic cycle. Since Pbl is never observed at the onset of mitosis, it appears that it is rapidly degraded during each cycle of cell division, presumably after being released from the nucleus upon disassembly of the nuclear envelope in prophase. Further analyses with antibodies against lamin to mark nuclei and alpha-spectrin to stain plasma membranes demonstrates that in late anaphase, there are low levels of Pbl protein at the plasma membrane and in the cytoplasm. Initiation of cytokinesis, as judged by the appearance of the cleavage furrow and reassembly of nuclear laminae, coincides with the accumulation of Pbl at the cleavage furrow. As the cleavage furrow progresses, Pbl accumulates at the equator between dividing cells and in the nuclei of the daughter cells. Pbl translocation to the equatorial region beneath the cleavage furrow parallels the accumulation of anillin and Pnut at the equator where the contractile ring is assembled. In telophase and early interphase cells, the protein is strongly enriched in the nuclei and is found at low levels at the cortex. Hence, during mitosis, subcellular distribution of Pbl undergoes dynamic changes and Pbl is associated with the cleavage furrow during cytokinesis. In addition, the levels of protein are known to cycle during each cell cycle with the highest levels of Pbl in late telophase and interphase (Prokopenko, 1999).

Embryos homozygous for the pbl2 null allele as well as cytological deficiencies removing pbl [Df(3L)pbl-NR] and [Df(3L)25] have greatly reduced levels of staining in cycle 15-16 interphase cells, which is likely to correspond to the remaining maternal contribution. In contrast, pbl5 embryos that carry the V531D missense mutation affecting a conserved valine have protein levels comparable with wild type. It is likely that the reduction in the levels of cytoplasmic, but not nuclear Pbl, is responsible for failure of cytokinesis in mitotic cycles 14-16, because Pbl has never been observed at the cortex or at the cleavage furrow during anaphase and telophase in cycle 14 pbl mutant cells (pbl2 and pbl5 alleles) (Prokopenko, 1999).

The dramatic increase in Pbl levels at the onset of cytokinesis and localization of Pbl to the cleavage furrow suggests that Pbl may play a regulatory role in cytokinesis. As a protein, which presumably regulates the activity of Rho GTPases, Pbl may be required for the initiation of cytokinesis or may couple cytokinesis with the mitotic cycle. To investigate this hypothesis, the effects of overexpression of full-length Pbl and of Pbl protein in which the BRCT domains and the NLS sequence were deleted (DeltaPbl325-853 allele) were compared. The amino-terminal truncation in DeltaPbl325-853 resembles a similar mutation in a number of mammalian RhoGEFs (for review, see Whitehead, 1997), including Ect2 (Miki, 1993), that results in the constitutive activation of their transforming potential. However, the amino-terminal sequences that are lost in different RhoGEFs do not share common domains. Hence, their role in controlling the activity of the full-length protein remains elusive (Prokopenko, 1999).

Ectopic expression of full-length Pbl (with pblEP3415 or the UAS-Pbl3.2line) in stage 8-11 embryos with the paired-GAL4 (prd-GAL4) driver does not result in a detectable phenotype. Although all cells in segments overexpressing Pbl have high levels of exogenous protein in their nuclei and cytoplasm, this does not interfere with cytokinesis, completion of mitosis, or viability. In contrast, expression of DeltaPbl325-853 with the same driver leads to 100% embryonic lethality. Most cells within the embryonic segments expressing DeltaPbl325-853 become multinucleate. This polyploid phenotype is caused by a failure of cytokinesis, because the cleavage furrow is not initiated, and, likewise, the contractile ring is not assembled. The same approach was used to overexpress the amino-terminally truncated form of Ect2, DeltaEct2. As for DeltaPbl325-853, ectopic expression of DeltaEct2 results in the absence of the contractile ring and cleavage furrow, leading to formation of multinucleate cells, and lethality. In contrast, flies expressing full-length Ect2 exhibit no phenotype and are viable (Prokopenko, 1999).

Thus, it appears that full-length Pbl (as well as Ect2) does not behave in vivo as an active protein, because it does not cause a cytokinetic phenotype when expressed in the embryonic ectoderm. This suggests that activation of the exchange activity of Pbl during cytokinesis may require some initiating event. In contrast, amino-terminally truncated Pbl, when overexpressed in the wild-type embryo, has a phenotype similar to lack of pbl. It is proposed that DeltaPbl may behave as an activated protein competing with endogenous Pbl for a rate-limiting target protein (such as Rho GTPase), which results in an inappropriate activation of downstream effectors. Furthermore, the amino-terminal BRCT domains of Pbl, lacking in DeltaPbl, and known to be protein-protein interaction modules, may be required for its regulation by an upstream signal. Such regulation may be essential for the spatial and temporal control of cytokinesis and for the coupling of signaling pathways initiating cytokinesis with other mitotic events (Prokopenko, 1999).

To investigate the possibility that mutations in Rho1 cause defects during cell division, Rho1 embryos were stained with antibodies against lamin and alpha-spectrin. Embryos homozygous for null alleles of Rho1 (Rho172O and Rho172R) show many binucleate cells in the head region. Occasionally, a few polyploid cells are also observed in thoracic or abdominal segments. Although the latter phenotype is observed with low penetrance, binucleate ectodermal cells could never be found in wild-type embryos. The relatively weak phenotype caused by Rho1 mutations (unlike that of pbl) is most likely due to maternally provided Rho1 protein. Thus, it appears that Rho1 is required for cytokinesis, and that decreased levels of Rho1 may account for the observed phenotype in cytokinesis (Prokopenko, 1999).

To further demonstrate the role of Rho1 in cytokinesis, the effects of expression of a dominant-negative form of Rho1, Rho1N19 were analyzed. A dominant-negative form of H-Ras, H-RasN17, cannot interact with downstream target proteins. In addition, H-RasN17 competes with normal H-Ras for binding to rasGEF, which results in formation of inactive H-RasN17-rasGEF complexes and depletion of the pool of the endogenous rasGEF leading to a dominant-negative effect. Therefore, a dominant-negative Rho1 may produce a phenotype similar to or stronger than loss-of-function alleles of Rho1. Ectopic expression of Rho1N19 creates a phenotype that is much worse than loss of zygotic Rho1. Expression of Rho1N19 in ectodermal stripes leads to a complete block of cytokinesis leading to polyploidy of almost every cell within the affected segment. In contrast, expression of wild-type Rho1 or dominant-negative Rac1 (Rac1N17 or Rac1L89) does not affect cytokinesis or completion of mitosis. Interestingly, coexpression of Rho1N19 and Pbl (unlike Pbl alone) results in the mislocalization of Pbl to the cell cortex in interphase and late mitotic cells, further indicating that the two proteins interact during cytokinesis. These results, together with other data, suggest that activation of the Rho1 GTPase by Pbl, its putative exchange factor, is required for the initiation of cytokinesis, because mutations in either protein or overexpression of an inactive protein result in accumulation of multinucleate cells (Prokopenko, 1999).

It is proposed that Pbl, acting at the cleavage furrow, is required for the initiation of cytokinesis. Pbl would function by interacting with and regulating proteins involved in the assembly of the contractile ring. Although the highest levels of Pbl are found in late telophase and young postmitotic nuclei, nuclear Pbl does not seem to play a role in the initiation of cytokinesis. The accumulation of Pbl in telophase nuclei follows the initiation of the cleavage furrow and the beginning of the reassembly of the nuclear envelope and, hence, occurs too late in mitosis to be an instructive signal for cytokinesis. This conclusion is further supported by the observation that amino-terminally truncated Pbl, DeltaPbl, lacking the NLS and localized to the cell cortex and cytoplasm, is able to block cytokinesis. DeltaPbl may compete with the endogenous Pbl at the cleavage furrow for Rho1 or, possibly, other proteins that bind to Pbl (Prokopenko, 1999).

What is the function of nuclear Pbl? The translocation of Pbl to the nuclei may be a regulatory mechanism to turn off the downstream signaling cascade by removing a RhoGEF from the cellular compartment in which the cascade is initiated. Alternatively, there may be two pools of Pbl protein -- a cytoplasmic/cortical pool required for the initiation of cytokinesis and another pool targeted to nuclei in which Pbl plays some other role, unrelated to cytokinesis (Prokopenko, 1999).

Previously, Drosophila Rho1 has been shown to be required for cellularization, gastrulation, dorsal closure, and generation of tissue polarity, but not for cytokinesis. The data suggest that the molecular basis of the genetic interaction between pbl and Rho1 is a physical interaction between Pbl and Rho1 proteins, because the two proteins interact in a yeast two-hybrid assay. Both genetic data and two-hybrid assay results indicate that this interaction is specific for Rho1, but not Rac1 or Cdc42. This interaction presumably results in the activation of the Rho1 GTPase by Pbl and induction of the signaling cascade that initiates the assembly of the contractile ring (Prokopenko, 1999 and references therein).

These results illustrate the complexity of the intracellular signaling pathways involving Rho GTPases and their GEFs. In Drosophila, Rho1 has also been identified as a putative effector of DRhoGEF2, indicating that Rho1 is regulated by at least two RhoGEFs. Given the diversity of developmental roles of Rho1, one would expect that there is a complex hierarchy of molecules regulating Rho1 activity, both spatially and temporally, within a cell. Such differential regulation of Rho1 by upstream signals may be achieved through tissue-specific expression of regulatory molecules (such as GEFs and GAPs) restricted to a particular developmental or cell cycle stage. An additional level of regulation within a cell can be achieved through subcellular compartmentalization of the molecular machinery (such as Rho effectors) required for the activation of distinct pathways mediated by Rho GTPases (Prokopenko, 1999 and references therein).

These observations suggest that Pbl is a key regulatory component of the signaling pathway initiating cytokinesis and therefore, Pbl plays a unique role, different from other cytoskeletal and structural proteins known to be required for cytokinesis. (1) Unlike other proteins required for cytokinesis, the levels of Pbl protein cycle during each round of cell division. (2) Pbl accumulates at the cleavage furrow at the time when cytokinesis is initiated. (3) Pbl is a putative RhoGEF and knowledge about mammalian RhoGEFs clearly implicates them in the regulation of signaling pathways involving Rho GTPases. (4) Mutations within the catalytic DH domain inactivate Pbl function (the pbl5 allele) and abolish Pbl interaction with the Rho1 (PblDeltaDH497-549) allele. In addition, ectopic expression of PblDeltaDH497-549 in the embryo or in the eye imaginal disc blocks cytokinesis and results in the formation of polyploid cells (Prokopenko, 1999).

The data presented here together with earlier observations allow for a proposal of a signaling pathway required for cytokinesis. It is suggested that activation of Rho1 GTPase by Pbl is required for initiation of a molecular pathway leading to assembly of a contractile ring. Such activation is likely to be achieved through direct binding of Pbl and Rho1 at the cleavage furrow. This is consistent with data from other species, in which Rho proteins have been shown to localize to the plasma membrane or cytosol in resting cells, but to translocate to the cleavage furrow and midbody during cytokinesis. Pbl has a similar distribution during mitosis, being initially cortical but accumulating at the cleavage furrow at the onset of cytokinesis. Significantly, coexpression of Pbl and a dominant-negative form of Rho1 results in the accumulation of Pbl at the cell cortex in late mitotic cells, suggesting that the two proteins interact during cytokinesis (Prokopenko, 1999).

Interaction of Rho1 with its effectors is likely to occur at the mouse cleavage furrow as suggested previously for mouse RhoA and p140mDia, a formin-related protein required for cytokinesis (Watanabe. 1997). Both p140mDia and its Drosophila homolog Diaphanous (Castrillon, 1994) localize to the cleavage furrow during cytokinesis. Furthermore, mutations in dia result in the absence of a contractile ring and the failure of cytokinesis. Interestingly, null alleles of dia dominantly suppress the rough eye phenotype of the GMR-Rho1 and GMR-Pbl flies, but dominantly enhance the eye phenotype of GMR-PblDeltaDH flies, suggesting that the three genes are in the same pathway. In addition, ectopic expression of activated Rho1, Rho1V14, but not wild-type Rho1, abolishes cytokinesis. Because mouse p140mDia interacts preferentially with activated RhoA (RhoAV14) but not with wild-type RhoA, it has been suggested that Rho1V14 blocks cytokinesis by titrating out one of its limiting downstream effectors, such as Dia, required for the initiation of cytokinesis. Mouse and Drosophila Dia, as well as other formin-related proteins implicated in cytokinesis (Bni1p in Saccharomyces cerevisiae and cdc12p in Schizosaccharomyces pombe) bind profilin, an actin-binding protein that regulates F-actin polymerization, in vitro and in vivo. In Drosophila, mutations in profilin (encoded by the chic gene) block the assembly of a contractile ring during meiotic cytokinesis (Giansanti. 1998), and in fission yeast, mutations in both genes -- cdc12 (formin) and cdc3(profilin) -- show a synthetic lethal genetic interaction (Prokopenko, 1999 and references therein).

In conclusion, a linear signaling pathway is proposed linking a RhoGEFwith the actin cytoskeleton and, hence, the actomyosin contractile ring. All of the components of this pathway have been implicated in cytokinesis and have been shown to localize to the cleavage furrow during cytokinesis in Drosophila or other organisms. Furthermore, all of the proteins in the pathway interact in vivo or in vitro with their neighboring partners. It is concluded that the activation of Rho1 by Pbl is likely to be an initiating event for the assembly of the contractile ring at the onset of cytokinesis. Identification of upstream components of this pathway will shed light on how cytokinesis is coordinated with the mitotic cycle and on the nature of the signal required for cytokinesis (Prokopenko, 1999).


PROTEIN STRUCTURE

Amino Acids - 853

Structural Domains

pbl encodes a novel RhoGEF related to the mouse Ect2 oncoprotein. The lengths of Pbl and Ect2 are 853 and 738 amino acids, respectively. Overall, the two proteins are 40% identical and 61% similar. Common functional domains of Pbl and Ect2 include the Dbl homology (DH) and pleckstrin homology (PH) domains (in Pbl, amino acids 390-574 and 600-718, respectively) that are found in tandem in all RhoGEFs. Mammalian members of the RhoGEF family are considered to be potential oncogenes because their truncated forms often induce transformed foci when overexpressed in cultured fibroblasts. The DH domain catalyzes GDP/GTP exchange through direct binding to its effector GTPase (Hart, 1994; Zheng, 1995; Liu, 1998). The PH domain (Shaw, 1996), always located carboxy-terminal to the DH domain in RhoGEFs, is thought to be required for membrane or cytoskeletal targeting of the protein and in enhancement of the catalytic activity of the adjacent DH domain (Whitehead, 1995; Zheng, 1996; Liu, 1998; Sterpetti, 1999). At the amino terminus, Pbl and Ect2 share two BRCT (BRCA1 carboxyl terminus) domains (in Pbl, amino acids 113-198 and 208-291) initially identified in the familial breast and ovarian cancer susceptibility gene BRCA1 (Callebaut, 1997). BRCT domains are found in proteins involved in DNA metabolism and cell cycle checkpoint control and are thought to be protein-protein interaction modules. In addition, Pbl contains a putative nuclear localization signal (NLS) (amino acids 306-322) and a PEST sequence (amino acids 371-383), which often serves as a proteolytic signal to target proteins for rapid degradation (Prokopenko, 1999 and references therein).


pebble: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 11 May 2000

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