Asymmetric neuroblast divisions producing apoptotic cells require the cytohesin GRP-1 in Caenorhabditis elegans.
ABSTRACT: Cytohesins are Arf guanine nucleotide exchange factors (GEFs) that regulate membrane trafficking and actin cytoskeletal dynamics. We report here that GRP-1, the sole Caenorhabditis elegans cytohesin, controls the asymmetric divisions of certain neuroblasts that divide to produce a larger neuronal precursor or neuron and a smaller cell fated to die. In the Q neuroblast lineage, loss of GRP-1 led to the production of daughter cells that are more similar in size and to the transformation of the normally apoptotic daughter into its sister, resulting in the production of extra neurons. Genetic interactions suggest that GRP-1 functions with the previously described Arf GAP CNT-2 and two other Arf GEFs, EFA-6 and BRIS-1, to regulate the activity of Arf GTPases. In agreement with this model, we show that GRP-1's GEF activity, mediated by its SEC7 domain, is necessary for the posterior Q cell (Q.p) neuroblast division and that both GRP-1 and CNT-2 function in the Q.posterior Q daughter cell (Q.p) to promote its asymmetry. Although functional GFP-tagged GRP-1 proteins localized to the nucleus, the extra cell defects were rescued by targeting the Arf GEF activity of GRP-1 to the plasma membrane, suggesting that GRP-1 acts at the plasma membrane. The detection of endogenous GRP-1 protein at cytokinesis remnants, or midbodies, is consistent with GRP-1 functioning at the plasma membrane and perhaps at the cytokinetic furrow to promote the asymmetry of the divisions that require its function.
Project description:During development, all cells make the decision to live or die. Although the molecular mechanisms that execute the apoptotic program are well defined, less is known about how cells decide whether to live or die. In C. elegans, this decision is linked to how cells divide asymmetrically [1, 2]. Several classes of molecules are known to regulate asymmetric cell divisions in metazoans, yet these molecules do not appear to control C. elegans divisions that produce apoptotic cells . We identified CNT-2, an Arf GTPase-activating protein (GAP) of the AGAP family, as a novel regulator of this type of neuroblast division. Loss of CNT-2 alters daughter cell size and causes the apoptotic cell to adopt the fate of its sister cell, resulting in extra neurons. CNT-2's Arf GAP activity is essential for its function in these divisions. The N terminus of CNT-2, which contains a GTPase-like domain that defines the AGAP class of Arf GAPs, negatively regulates CNT-2's function. We provide evidence that CNT-2 regulates receptor-mediated endocytosis and consider the implications of its role in asymmetric cell divisions.
Project description:Neuroblast divisions in the nematode Caenorhabditis elegans often give rise to a larger neuron and a smaller cell that dies. We have previously identified genes that, when mutated, result in neuroblast divisions that generate daughter cells that are more equivalent in size. This effect correlates with the survival of daughter cells that would normally die. We now describe a role for the DEP domain-containing protein TOE-2 in promoting the apoptotic fate in the Q lineage. TOE-2 localized at the plasma membrane and accumulated in the cleavage furrow of the Q.a and Q.p neuroblasts, suggesting that TOE-2 might position the cleavage furrow asymmetrically to generate daughter cells of different sizes. This appears to be the case for Q.a divisions where loss of TOE-2 led to a more symmetric division and to survival of the smaller Q.a daughter. Localization of TOE-2 to the membrane is required for this asymmetry, but, surprisingly, the DEP domain is dispensable. By contrast, loss of TOE-2 led to loss of the apoptotic fate in the smaller Q.p daughter but did not affect the size asymmetry of the Q.p daughters. This function of TOE-2 required the DEP domain but not localization to the membrane. We propose that TOE-2 ensures an apoptotic fate for the small Q.a daughter by promoting asymmetry in the daughter cell sizes of the Q.a neuroblast division but by a mechanism that is independent of cell size in the Q.p division.
Project description:ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ?200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.
Project description:ADP-ribosylation factors (ARF) GTPases are activated by guanine nucleotide exchange factors (GEFs) to support cellular homeostasis. Key to understanding spatio-temporal regulation of ARF signaling is the mechanism of GEF recruitment to membranes. Small GEFs are recruited through phosphoinositide (PIP) binding by a pleckstrin homology (PH) domain downstream from the catalytic Sec7 domain (Sec7d). The large GEFs lack PH domains, and their recruitment mechanisms are poorly understood. We probed Golgi recruitment of GBF1, a GEF catalyzing ARF activation required for Golgi homeostasis. We show that the homology downstream of Sec7d-1 (HDS1) regulates Golgi recruitment of GBF1. We document that GBF1 binds phosphoinositides, preferentially PI3P, PI4P and PI(4,5)P2, and that lipid binding requires the HDS1 domain. Mutations within HDS1 that reduce GBF1 binding to specific PIPs in vitro inhibit GBF1 targeting to Golgi membranes in cells. Our data imply that HDS1 and PH domains are functionally analogous in that each uses lipid-based membrane information to regulate GEF recruitment. Lipid-based recruitment of GBF1 extends the paradigm of lipid regulation to small and large GEFs and suggests that lipid-based mechanisms evolved early during GEF diversification. This article has an associated First Person interview with the first author of the paper.
Project description:The action of guanine nucleotide exchange factors (GEFs) on the ADP-ribosylation factor (ARF) family of small GTPases initiates intracellular transport pathways. This role requires ARF GEFs to be recruited from the cytosol to intracellular membrane compartments. An ARF GEF known as General receptor for 3-phosphoinositides 1 (Grp1) is recruited to the plasma membrane through its pleckstrin homology (PH) domain that recognizes phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here, we find that the phosphorylation of Grp1 induces its PH domain to recognize instead phosphatidylinositol 4-phosphate (PI4P). This phosphorylation also releases an autoinhibitory mechanism that results in the coil-coil (CC) domain of Grp1 engaging two peripheral membrane proteins of the recycling endosome. Because the combination of these actions results in Grp1 being recruited preferentially to the recycling endosome rather than to the plasma membrane, our findings reveal the complexity of recruitment mechanisms that need to be coordinated in localizing an ARF GEF to an intracellular compartment to initiate a transport pathway. Our elucidation is also remarkable for having revealed that phosphoinositide recognition by a PH domain can be switched through its phosphorylation.
Project description:At the Golgi complex, the biosynthetic sorting center of the cell, the Arf GTPases are responsible for coordinating vesicle formation. The Arf-GEFs activate Arf GTPases and are therefore the key molecular decision-makers for trafficking from the Golgi. In <i>Saccharomyces cerevisiae</i>, three conserved Arf-GEFs function at the Golgi: Sec7, Gea1, and Gea2. Our group has described the regulation of Sec7, the <i>trans</i>-Golgi Arf-GEF, through autoinhibition, positive feedback, dimerization, and interactions with a suite of small GTPases. However, we lack a clear understanding of the regulation of the early Golgi Arf-GEFs Gea1 and Gea2. Here we demonstrate that Gea1 and Gea2 prefer neutral over anionic membrane surfaces in vitro, consistent with their localization to the early Golgi. We illustrate a requirement for a critical mass of either Gea1 or Gea2 for cell growth under stress conditions. We show that the C-terminal domains of Gea1 and Gea2 toggle roles in the cytosol and at the membrane surface, preventing membrane binding in the absence of a recruiting interaction but promoting maximum catalytic activity once recruited. We also identify the small GTPase Ypt1 as a recruiter for Gea1 and Gea2. Our findings illuminate core regulatory mechanisms unique to the early Golgi Arf-GEFs.
Project description:Budding of transport vesicles in the Golgi apparatus requires the recruitment of coat proteins and is regulated by ADP ribosylation factor (ARF) 1. ARF1 activation is promoted by guanine nucleotide exchange factors (GEFs), which catalyze the transition to GTP-bound ARF1. We recently have identified a human protein, ARNO (ARF nucleotide-binding-site opener), as an ARF1-GEF that shares a conserved domain with the yeast Sec7 protein. We now describe a human Sec7 domain-containing GEF referred to as ARNO3. ARNO and ARNO3, as well as a third GEF called cytohesin-1, form a family of highly related proteins with identical structural organization that consists of a central Sec7 domain and a carboxy-terminal pleckstrin homology domain. We show that all three proteins act as ARF1 GEF in vitro, whereas they have no effect on ARF6, an ARF protein implicated in the early endocytic pathway. Substrate specificity of ARNO-like GEFs for ARF1 depends solely on the Sec7 domain. Overexpression of ARNO3 in mammalian cells results in (i) fragmentation of the Golgi apparatus, (ii) redistribution of Golgi resident proteins as well as the coat component beta-COP, and (iii) inhibition of SEAP transport (secreted form of alkaline phosphatase). In contrast, the distribution of endocytic markers is not affected. This study indicates that Sec7 domain-containing GEFs control intracellular membrane compartment structure and function through the regulation of specific ARF proteins in mammalian cells.
Project description:Eukaryotic cells require selective sorting and transport of cargo between intracellular compartments. This is accomplished at least in part by vesicles that bud from a donor compartment, sequestering a subset of resident protein "cargos" destined for transport to an acceptor compartment. A key step in vesicle formation and targeting is the recruitment of specific proteins that form a coat on the outside of the vesicle in a process requiring the activation of regulatory GTPases of the ARF family. Like all such GTPases, ARFs cycle between inactive, GDP-bound, and membrane-associated active, GTP-bound, conformations. And like most regulatory GTPases the activating step is slow and thought to be rate limiting in cells, requiring the use of ARF guanine nucleotide exchange factor (GEFs). ARF GEFs are characterized by the presence of a conserved, catalytic Sec7 domain, though they also contain motifs or additional domains that confer specificity to localization and regulation of activity. These domains have been used to define and classify five different sub-families of ARF GEFs. One of these, the BIG/GBF1 family, includes three proteins that are each key regulators of the secretory pathway. GEF activity initiates the coating of nascent vesicles via the localized generation of activated ARFs and thus these GEFs are the upstream regulators that define the site and timing of vesicle production. Paradoxically, while we have detailed molecular knowledge of how GEFs activate ARFs, we know very little about how GEFs are recruited and/or activated at the right time and place to initiate transport. This review summarizes the current knowledge of GEF regulation and explores the still uncertain mechanisms that position GEFs at "budding ready" membrane sites to generate highly localized activated ARFs.
Project description:The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth.
Project description:ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates membrane trafficking and cytoskeleton remodelling, but how it assembles actin filaments is unknown. By reconstituting membrane-associated actin assembly mediated by the WASP family veroprolin homolog (WAVE) regulatory complex (WRC), we recapitulated an Arf6-driven actin polymerization pathway. We show that Arf6 is divergent from other Arf members, as it was incapable of directly recruiting WRC. We demonstrate that Arf6 triggers actin assembly at the membrane indirectly by recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO that activates Arf1 to enable WRC-dependent actin assembly. The pathogen Salmonella usurped Arf6 for host cell invasion by recruiting its canonical GEFs EFA6 and BRAG2. Arf6 and its GEFs facilitated membrane ruffling and pathogen invasion via ARNO, and triggered actin assembly by generating an Arf1-WRC signaling hub at the membrane in vitro and in cells. This study reconstitutes Arf6-dependent actin assembly to reveal a mechanism by which related Arf GTPases orchestrate distinct steps in the WRC cytoskeleton remodelling pathway.