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[
Elife,
2015]
Nuclear pore complexes (NPCs) conduct massive transport mediated by shuttling nuclear transport receptors (NTRs), while keeping nuclear and cytoplasmic contents separated. The NPC barrier in Xenopus relies primarily on the intrinsically disordered FG domain of Nup98. We now observed that Nup98 FG domains of mammals, lancelets, insects, nematodes, fungi, plants, amoebas, ciliates, and excavates spontaneously and rapidly phase-separate from dilute (submicromolar) aqueous solutions into characteristic 'FG particles'. This required neither sophisticated experimental conditions nor auxiliary eukaryotic factors. Instead, it occurred already during FG domain expression in bacteria. All Nup98 FG phases rejected inert macromolecules and yet allowed far larger NTR cargo complexes to rapidly enter. They even recapitulated the observations that large cargo-domains counteract NPC passage of NTRcargo complexes, while cargo shielding and increased NTRcargo surface-ratios override this inhibition. Their exquisite NPC-typical sorting selectivity and strong intrinsic assembly propensity suggest that Nup98 FG phases can form in authentic NPCs and indeed account for the permeability properties of the pore.
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[
Neurochem Res,
2013]
To perform their diverse biological functions the adhesion activities of the cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) might be regulated by local clustering, proteolytical shedding of their ectodomains or rapid recycling to and from the plasma membrane. Another form of regulation of adhesion might be obtained through flexible ectodomains of IgCAMs which adopt distinct conformations and which in turn modulate their adhesion activity. Here, we discuss variations in the conformation of the extracellular domains of CEACAM1 and CAR that might influence their binding and signaling activities. Furthermore, we concentrate on alternative splicing of single domains and short segments in the extracellular regions of L1 subfamily members that might affect the organization of the N-terminal located Ig-like domains. In particular, we discuss variations of the linker sequence between Ig-like domains 2 and 3 (D2 and D3) that is required for the horseshoe conformation.
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[
EMBO J,
2023]
Nucleoporins (Nups) assemble nuclear pores that form the permeability barrier between nucleoplasm and cytoplasm. Nucleoporins also localize in cytoplasmic foci proposed to function as pore pre-assembly intermediates. Here, we characterize the composition and incidence of cytoplasmic Nup foci in an intact animal, C. elegans. We find that, in young non-stressed animals, Nup foci only appear in developing sperm, oocytes and embryos, tissues that express high levels of nucleoporins. The foci are condensates of highly cohesive FG repeat-containing nucleoporins (FG-Nups), which are maintained near their solubility limit in the cytoplasm by posttranslational modifications and chaperone activity. Only a minor fraction of FG-Nup molecules concentrate in Nup foci, which dissolve during M phase and are dispensable for nuclear pore assembly. Nucleoporin condensation is enhanced by stress and advancing age, and overexpression of a single FG-Nup in post-mitotic neurons is sufficient to induce ectopic condensation and organismal paralysis. We speculate that Nup foci are non-essential and potentially toxic condensates whose assembly is actively suppressed in healthy cells.
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[
International Worm Meeting,
2017]
Tissue development and homeostasis depend on the correct axis and plane of cell division, which are determined by the position of the mitotic spindle. Studies of the C. elegans one-cell embryo have provided much insight in the regulation of spindle positioning during asymmetric cell division. Pulling forces that act from the cell cortex on astral microtubules were found to position the spindle. These pulling forces depend on a force-generating complex (FG) that anchors the dynein motor to the cortex. The FG is a conserved complex consisting of a membrane-bound Ga protein that, in the GDP-bound form, binds the GPR-1/2 linker protein, which interacts with the LIN-5 coiled-coil protein. The discovery of a non-canonical role of Ga as a membrane-anchor for the FG was surprising at the time, and still is only partly understood. The Guanine-nucleotide Exchange Factor (GEF) RIC-8 and GTPase-Activating Protein (GAP) RGS-7 regulate the GDP/GTP cycle of Ga and are also required for proper force generation. It remains unclear how this corresponds to Ga functioning as a membrane anchor. Hampering analysis, complete inactivation of
ric-8 and
rgs-7 appears difficult to achieve by RNAi and null mutants are not viable. We therefore developed an inducible, tissue-specific gene knock-out system based on Cre-Lox recombination. We introduced Lox sites in the endogenous
ric-8 and
rgs-7 loci to enable KO of the genes. To assure spatiotemporal control of the KO, we use a two-component recombinase system. Thus, gene KO can be induced in the tissue of choice, for example the germline, while maintaining a healthy strain. A complementary approach focuses on the individual functions of FG components. Hereto, we use the light-inducible heterodimerization of LOV and ePDZ to reconstitute cortical FG complexes that lack one or several components. Using a PH::LOV as membrane anchor and an ePDZ::dynein knock-in, we were able to recruit endogenous dynein directly to the membrane and bypass the FG. This was insufficient for pulling force generation and suggests that the FG is more than a dynein anchor. To test the role of Ga signaling, we generated and are currently analyzing an ePDZ::GPR-1 knock-in to reconstitute a cortical FG that lacks the Ga component. We will present our latest results at the meeting.
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[
EMBO Rep,
2002]
The mechanism by which macromolecules are translocated through the nuclear pore complex (NPC) is little understood. However, recent measurements of nuclear transport in permeabilized cells showed that molecules binding to phenylalanine-glycine-rich repeats (FG repeats) in NPC proteins were translocated much faster through the NPC than molecules not interacting with FG repeats. We have studied that substrate preference of the NPC in isolated oocyte nuclei and purified nuclear envelopes by optical single transporter recording. NTF2, the transport receptor of RanGDP, was exported approximately 30 times faster than green fluorescent protein, an inert molecule of approximately the same size. The data confirm that restricted diffusion of inert molecules and facilitated transport of FG-repeat binding proteins are basic types of translocation through the NPC, demonstrating that the functional integrity of the NPC can be conserved in isolated nuclei and nuclear envelopes and thus opening new avenues to the analysis of nucleocytoplasmic transport.
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[
International Worm Meeting,
2011]
C. elegans germline development relies heavily upon post-transcriptional regulation in the germ plasm. Within the germ plasm, ribonucleoprotein aggregates called P granules overlie nuclear pore clusters and receive mRNAs as they are exported from the nucleus. A number of P-granule components, including the Vasa-related proteins GLH-1, GLH-2, and GLH-4, contain phenylalanine-glycine (FG) repeat domains, which are a common feature of nuclear pore complex (NPC) proteins. Within the NPC, FG-rich domains form a cohesive meshwork of filaments through hydrophobic interactions involving the phenylalanines in the FG motifs, creating a size-exclusion barrier that prevents diffusion of molecules larger than 45 kDa between the nucleus and the cytoplasm. We demonstrated that P granules, like NPCs, are held together by weak hydrophobic interactions and that they also establish a size-exclusion barrier similar to that of NPCs within the germ plasm. By expressing P-granule components in heterologous (intestinal) cells, we show that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient to target intestinal PGL granules to the nuclear periphery. Our results provide insights into the roles of the PGL and GLH families of proteins and suggest that P granules extend the NPC environment in the germ line to create a specialized hydrophobic microenvironment that may facilitate post-transcriptional processing events while selectively excluding large protein complexes from gaining access to mRNAs and endogenous siRNAs.
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[
J Cell Biol,
2011]
The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)-like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.
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[
International Worm Meeting,
2009]
Germ granules are large, non-membrane-bound, ribonucleoprotein (RNP) organelles found in the germline cytoplasm of most, if not all, animals[1]. Like germ granules across species, P granules in C. elegans are found at the nuclear periphery[2], and are closely associated with nuclear pores in the germ line[3]. The C. elegans VASA homologs, GLH-1, GLH-2, and GLH-4, which are constitutively associated with P granules, resemble nuclear pore (NUP) proteins in being rich in FG (PheGly) repeats[4]. We hypothesized that the association between P granules and nuclear pores is facilitated by hydrophobic interactions between the FG repeats of the GLHs and NUPs. Consistent with this, P granules are dispersed when hydrophobic interactions are disrupted by aliphatic alcohols. In nuclear pores, hydrophobic interactions between FG repeat domains create a size exclusion barrier. We have shown that P granules impose a similar size exclusion barrier. The integral connection between P granules and nuclear pores is supported by results from a genome-wide RNAi screen for components required for proper P granule assembly and localization, in which we identified several nuclear pore associated factors. We are currently using a sensitive assay to measure interactions between FG repeat domains from GLH-1, GLH-2, GLH-4 and nuclear pore components identified in our screen. We propose that P granules extend the nuclear pore environment and provide a unique cytoplamic domain for post-transcriptional regulation in the germ line. 1.Eddy, E.M., Germ plasm and the differentiation of the germ cell line. Int Rev Cytol, 1975. 43: p. 229-80. 2.Strome, S. and W.B. Wood, Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans. Proc Natl Acad Sci U S A, 1982. 79(5): p. 1558-62. 3.Pitt, J.N., J.A. Schisa, and J.R. Priess, P granules in the germ cells of Caenorhabditis elegans adults are associated with clusters of nuclear pores and contain RNA. Dev Biol, 2000. 219(2): p. 315-33. 4.Gruidl, M.E., et al., Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans. Proc Natl Acad Sci U S A, 1996. 93(24): p. 13837-42.
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[
International Worm Meeting,
2009]
Germline-specific P granules contain numerous RNA-binding proteins, but little detectable mRNA except under quiescent states. P granules are perinuclear during most of development, but are cytoplasmic in oocytes and early embryos. Although functions of cytoplasmic P granules have not been determined, loss of perinuclear P granules is associated with inappropriate expression of at least some "masked" maternal mRNAs and transformation of germ cells into somatic cells [1]. The perinuclear P granules are associated specifically with clusters of nuclear pore complexes (NPCs), representing about 75% of the total NPCs. Here, we wanted to determine whether the NPCs associated with P granules (Pg-NPCs) are active sites of mRNA export, and found that mRNA export factors such as NXF-1 are enriched at the base of the Pg-NPCs under normal growth conditions. We discovered that heat shock could be used to induce expression of transgenic mRNA in late pachytene nuclei, and used this assay to examine mRNAs containing several types of 3''UTRs. These include 3''UTRs from mRNAs that are (a) normally translated in the gonad, (b) not translated in the gonad, or (c) targeted for degradation by RNAi. None of these nascent mRNAs were retained in P granules, and instead each showed a similar, transient accumulation in P granules before entering the general cytoplasm. This transient accumulation depended on P granule proteins such as GLH-1, and was not seen in somatic cells. Thus, Pg-NPCs appear to contain a dynamic population of mRNAs. Although in time-lapse movies Pg-NPCs appear to be stable (>20 mins), we found in photobleaching experiments that P granule components such as PGL-1 are highly dynamic (<20 sec half-recovery times). We conclude that, under normal growth conditions, the Pg-NPCs do not appear to act as storage sites for masked mRNAs although it remains possible that cytoplasmic P granules have a role in storage. Instead, our results support the view that Pg-NPCs are sites of dynamic interactions between proteins and nascent mRNAs. We noted previously that the GLH family of P granule proteins contains numerous FG repeats [2], analogous to FG repeats in core nucleoporins that function in the export of mRNA. Interestingly, we found that a gonad-specific isoform of DDX-19 is highly enriched in FG repeats, and that DDX-19 and a novel C. elegans protein with FG repeats are enriched in P granules. Thus, we hypothesize that these repeats function in the extended export of mRNA/protein past the NPC and through P granules. [1] Ciosk et al., Science 311, 851(2006) [2] Schisa et al., Development 128, 1287 (2001).
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[
Elife,
2014]
In epithelial collective migration, leader and follower cells migrate while maintaining cell-cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG-GAP domains in the extracellular domain, which potentially folds into a -propeller structure resembling the -integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and -tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for
lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain.