Germ granules extend the nuclear pore complex environment in the C. elegans germ line

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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Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans

eLife ◽

10.7554/elife.72276 ◽

2021 ◽

Vol 10 ◽

Author(s):

Ian F Price ◽

Hannah L Hertz ◽

Benjamin Pastore ◽

Jillian Wagner ◽

Wen Tang

Keyword(s):

Germ Line ◽

Nuclear Periphery ◽

P Granules ◽

C Elegans ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Germ Granules ◽

Protein Components ◽

Labeling Method ◽

Disordered Regions

The germ line produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules-well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germ line. Using C. elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions. An RNAi-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two putative LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from the nuclear envelope, germline atrophy and reduced fertility. We show that intrinsically disordered regions of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote perinuclear localization of P granules. Together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.

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PLP-1 is essential for germ cell development and germline gene silencing in Caenorhabditiselegans

Development ◽

10.1242/dev.195578 ◽

2020 ◽

Vol 147 (22) ◽

pp. dev195578

Author(s):

Rajaram Vishnupriya ◽

Linitha Thomas ◽

Lamia Wahba ◽

Andrew Fire ◽

Kuppuswamy Subramaniam

Keyword(s):

Gene Silencing ◽

Small Rna ◽

Germ Line ◽

Nucleic Acid Binding ◽

Germline Gene ◽

C Elegans ◽

Rna Granules ◽

Germ Granules ◽

Rna Biogenesis ◽

Silencing Pathways

ABSTRACTThe germline genome is guarded against invading foreign genetic elements by small RNA-dependent gene-silencing pathways. Components of these pathways localize to, or form distinct aggregates in the vicinity of, germ granules. These components and their dynamics in and out of granules are currently being intensively studied. Here, we report the identification of PLP-1, a Caenorhabditiselegans protein related to the human single-stranded nucleic acid-binding protein Pur-alpha, as a component of germ granules in C. elegans. We show that PLP-1 is essential for silencing different types of transgenes in the germ line and for suppressing the expression of several endogenous genes controlled by the germline gene-silencing pathways. Our results reveal that PLP-1 functions downstream of small RNA biogenesis during initiation of gene silencing. Based on these results and the earlier findings that Pur-alpha proteins interact with both RNA and protein, we propose that PLP-1 couples certain RNAs with their protein partners in the silencing complex. PLP-1 orthologs localized on RNA granules may similarly contribute to germline gene silencing in other organisms.

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Characterization and Targeted Disruption of Murine Nup50, a p27Kip1-Interacting Component of the Nuclear Pore Complex

Molecular and Cellular Biology ◽

10.1128/mcb.20.15.5631-5642.2000 ◽

2000 ◽

Vol 20 (15) ◽

pp. 5631-5642 ◽

Cited By ~ 73

Author(s):

Matthew Smitherman ◽

Keesook Lee ◽

Jherek Swanger ◽

Raj Kapur ◽

Bruce E. Clurman

Keyword(s):

Neural Tube ◽

Nuclear Pore Complex ◽

Mammalian Cells ◽

Germ Line ◽

Nuclear Pore ◽

Null Mouse ◽

Mouse Development ◽

Genomic Locus ◽

Two Hybrid

ABSTRACT p27Kip1 is a member of the Cip-Kip family of cyclin-dependent kinase (Cdk) inhibitors that binds to cyclin-Cdk complexes and inhibits their catalytic activity in response to antiproliferative stimuli. p27Kip1 is regulated by several posttranscriptional mechanisms, including subcellular localization. We have identified a component of the nuclear pore complex (NPC), termed Nup50, through its two-hybrid interactions with p27Kip1. Nup50 is a nucleoplasmically oriented component of the nuclear pore complex with a role in protein export (T. Guan, R. H. Kehlenbach, E. C. Schirmer, A. Kehlenbach, F. Fan, B. E. Clurman, N. Arnheim, and L. Gerace, Mol. Cell. Biol. 20:5619–5630, 2000). We found that murine Nup50 is a widely expressed nucleoporin and that Nup50 expression is highest in the developing neural tube and adult testes. We have also examined interactions between Nup50 and the NPC and found specific two-hybrid interactions between Nup50 and several well-defined components of the NPC, as well as coimmunoprecipitation of Nup50 with the nucleoporin Nup153 from transfected mammalian cells. In order to study Nup50 function in vivo, we cloned the mouse Nup50 genomic locus and created a targeted Nup50 deletion in the mouse germ line. Nup50 disruption resulted in a complex phenotype characterized by late embryonic lethality, neural tube defects, and intrauterine growth retardation. Although Nup50-null mouse embryo fibroblasts exhibited no defects in either cell cycle control or p27Kip1 regulation, Nup50 deletion was associated with abnormalities in p27Kip1expression and cell proliferation in the developing neuroepithelium. We conclude that Nup50 is a nucleoporin with essential functions during mouse development.

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Sm protein down-regulation leads to defects in nuclear pore complex disassembly and distribution in C. elegans embryos

Developmental Biology ◽

10.1016/j.ydbio.2012.02.036 ◽

2012 ◽

Vol 365 (2) ◽

pp. 445-457 ◽

Cited By ~ 6

Author(s):

Daphna Joseph-Strauss ◽

Mátyás Gorjánácz ◽

Rachel Santarella-Mellwig ◽

Ekaterina Voronina ◽

Anjon Audhya ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Pore ◽

Down Regulation ◽

C Elegans ◽

Sm Protein

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The Formation, Structure, Distribution and Frequency of Nuclear Pores

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066644 ◽

1971 ◽

Vol 29 ◽

pp. 518-519

Author(s):

G. G. Maul

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Membrane ◽

Dynamic Structure ◽

Nuclear Pore ◽

Nuclear Pores ◽

Filter Function ◽

Etching Technique ◽

Selective Filter ◽

Membranous Part

The chromatin of eukaryotic cells is separated from the cytoplasm by a double membrane. One obvious structural specialization of the nuclear membrane is the presence of pores which have been implicated to facilitate the selective nucleocytoplasmic exchange of a variety of large molecules. Thus, the function of nuclear pores has mainly been regarded to be a passive one. Non-membranous diaphragms, radiating fibers, central rings, and other pore-associated structures were thought to play a role in the selective filter function of the nuclear pore complex. Evidence will be presented that suggests that the nuclear pore is a dynamic structure which is non-randomly distributed and can be formed during interphase, and that a close relationship exists between chromatin and the membranous part of the nuclear pore complex.Octagonality of the nuclear pore complex has been confirmed by a variety of techniques. Using the freeze-etching technique, it was possible to show that the membranous part of the pore complex has an eight-sided outline in human melanoma cells in vitro. Fibers which traverse the pore proper at its corners are continuous and indistinguishable from chromatin at the nucleoplasmic side, as seen in conventionally fixed and sectioned material. Chromatin can be seen in octagonal outline if serial sections are analyzed which are parallel but do not include nuclear membranes (Fig. 1). It is concluded that the shape of the pore rim is due to fibrous material traversing the pore, and may not have any functional significance. In many pores one can recognize a central ring with eight fibers radiating to the corners of the pore rim. Such a structural arrangement is also found to connect eight ribosomes at the nuclear membrane.

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Structure, assembly and interactions of the nuclear lamina and the nuclear pore complex

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012285x ◽

1992 ◽

Vol 50 (1) ◽

pp. 490-491

Author(s):

N. Panté ◽

M. Jarnik ◽

E. Heitlinger ◽

U. Aebi

Keyword(s):

Nuclear Pore Complex ◽

Divalent Cations ◽

Nuclear Lamina ◽

Dynamic Structure ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Cytoplasmic Filaments ◽

Radial Spokes ◽

Nuclear Ring ◽

Central Plug

The nuclear pore complex (NPC) is a ∼120 MD supramolecular machine implicated in nucleocytoplasmic transport, that is embedded in the double-membraned nuclear envelope (NE). The basic framework of the ∼120 nm diameter NPC consists of a 32 MD cytoplasmic ring, a 66 MD ‘plug-spoke’ assembly, and a 21 MD nuclear ring. The ‘central plug’ seen in en face views of the NPC reveals a rather variable appearance indicating that it is a dynamic structure. Projecting from the cytoplasmic ring are 8 short, twisted filaments (Fig. 1a), whereas the nuclear ring is topped with a ‘fishtrap’ made of 8 thin filaments that join distally to form a fragile, 30-50 nm distal diameter ring centered above the NPC proper (Fig. 1b). While the cytoplasmic filaments are sensitive to proteases, they as well as the nuclear fishtraps are resistant to RNase treatment. Removal of divalent cations destabilizes the distal rings and thereby opens the fishtraps, addition causes them to reform. Protruding from the tips of the radial spokes into perinuclear space are ‘knobs’ that might represent the large lumenal domain of gp210, a membrane-spanning glycoprotein (Fig. 1c) which, in turn, may play a topogenic role in membrane folding and/or act as a membrane-anchoring site for the NPC. The lectin wheat germ agglutinin (WGA) which is known to recognize the ‘nucleoporins’, a family of glycoproteins having O-linked N-acetyl-glucosamine, is found in two locations on the NPC (Fig. 1. d-f): (i) whereas the cytoplasmic filaments appear unlabelled (Fig. 1d&e), WGA-gold labels sites between the central plug and the cytoplasmic ring (Fig. le; i.e., at a radius of 25-35 nm), and (ii) it decorates the distal ring of the nuclear fishtraps (Fig. 1, d&f; arrowheads).

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