scholarly journals Control of the spindle checkpoint by lateral kinetochore attachment and limited Mad1 recruitment

2015 ◽  
Vol 26 (14) ◽  
pp. 2620-2639 ◽  
Author(s):  
Nathaniel I. Krefman ◽  
David G. Drubin ◽  
Georjana Barnes
Keyword(s):  
Binding Sites ◽  
De Novo ◽  
Spindle Checkpoint ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Fully Depleted ◽  
Checkpoint Proteins ◽  
Kinetochore Assembly ◽  
Sister Chromatids ◽  
Pore Complexes

We observed the dynamic recruitment of spindle checkpoint proteins Mad1 and Bub1 to detached kinetochores in budding yeast using real-time live-cell imaging and quantified recruitment in fixed cells. After induced de novo kinetochore assembly at one pair of sister centromeres, Mad1 appeared after the kinetochore protein Mtw1. Detached kinetochores were not associated with the nuclear envelope, so Mad1 does not anchor them to nuclear pore complexes (NPCs). Disrupting Mad1's NPC localization increased Mad1 recruitment to detached sister kinetochores. Conversely, increasing the number of detached kinetochores reduced the amount of Mad1 per detached kinetochore. Bub1 also relocalized completely from the spindle to detached sister centromeres after kinetochore assembly. After their capture by microtubules, Mad1 and Bub1 progressively disappeared from kinetochores. Sister chromatids that arrested with a lateral attachment to one microtubule exhibited half the Mad1 of fully detached sisters. We propose that detached kinetochores compete with alternate binding sites in the nucleus to recruit Mad1 and Bub1 from available pools that are small enough to be fully depleted by just one pair of detached kinetochores and that lateral attachment licenses Mad1 removal from kinetochores after a kinetic delay.

scholarly journals A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sarah A. Nordeen ◽  
Kasper R. Andersen ◽  
Kevin E. Knockenhauer ◽  
Jessica R. Ingram ◽  
Hidde L. Ploegh ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Complex Structure ◽  
Constitutive Expression ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Modular Assembly ◽  
High Affinity ◽  
Ring Complex ◽  
Pore Complexes

AbstractNuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

scholarly journals Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase

2001 ◽  
Vol 114 (5) ◽  
pp. 953-963 ◽  
Author(s):  
M.S. Campbell ◽  
G.K. Chan ◽  
T.J. Yen
Keyword(s):  
Mammalian Cells ◽  
Metaphase Plate ◽  
Mitotic Checkpoint ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Checkpoint Proteins ◽  
Anaphase Onset ◽  
Human Proteins ◽  
Tax Interaction ◽  
Pore Complexes

Mad1 was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset. Using antibodies to the human homologue of Mad1 (HsMAD1), we have begun to characterize this protein in mammalian cells. HsMad1 is found localized at kinetochores in mitosis. The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase. In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled. We find HsMad1 associated with HsMad2. Association with p55CDC, a protein previously shown to bind HsMad2, was not detected. Surprisingly, unlike any other known mitotic checkpoint proteins, HsMad1 and HsMAD2 were found localized at nuclear pores throughout interphase. This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions. HsMad1 was identified serendipitously by its binding to a viral protein, HTLV-1 Tax, which affects transcription of viral and human proteins. The localization of HsMad1 to nuclear pore complexes suggests an alternate, non-mitotic role for the Mad1/Tax interaction in the viral transformation of cells.

scholarly journals Nuclear Export of mRNA by TAP/NXF1 Requires Two Nucleoporin-Binding Sites but Not p15

2002 ◽  
Vol 22 (15) ◽  
pp. 5405-5418 ◽  
Author(s):  
Isabelle C. Braun ◽  
Andrea Herold ◽  
Michaela Rode ◽  
Elisa Izaurralde
Keyword(s):  
Binding Sites ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Wild Type ◽  
Structural Domains ◽  
Export Activity ◽  
In The Wild ◽  
Pore Complexes ◽  
Directional Transport

ABSTRACT Metazoan NXF1/p15 heterodimers promote export of bulk mRNA through nuclear pore complexes (NPC). NXF1 interacts with the NPC via two distinct structural domains, the UBA-like domain and the NTF2-like scaffold, which results from the heterodimerization of the NTF2-like domain of NXF1 with p15. Both domains feature a single nucleoporin-binding site, and they act synergistically to promote NPC translocation. Whether the NTF2-like scaffold (and thereby p15) contributes only to NXF1/NPC association or is also required for other functions, e.g., to impart directionality to the export process by regulating NXF1/NPC or NXF1/cargo interactions, remains unresolved. Here we show that a minimum of two nucleoporin-binding sites is required for NXF1-mediated export of cellular mRNA. These binding sites can be provided by an NTF2-like scaffold followed by a UBA-like domain (as in the wild-type protein) or by two NTF2-like scaffolds or two UBA-like domains in tandem. In the latter case, the export activity of NXF1 is independent of p15. Thus, as for the UBA-like domain, the function of the NTF2-like scaffold is confined to nucleoporin binding. More importantly, two copies of either of these domains are sufficient to promote directional transport of mRNA cargoes across the NPC.

scholarly journals A nanobody suite for yeast scaffold nucleoporins provides details of the Nuclear Pore Complex structure

2020 ◽  
Author(s):  
Sarah A Nordeen ◽  
Kasper Andersen ◽  
Kevin E Knockenhauer ◽  
Jessica R Ingram ◽  
Hidde Ploegh ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Complex Structure ◽  
Constitutive Expression ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Modular Assembly ◽  
High Affinity ◽  
Ring Complex ◽  
Pore Complexes

AbstractNuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we developed a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae revealed accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

scholarly journals Subcellular analyses of planarian meiosis implicates a novel, double-membraned vesiculation process in nuclear envelope breakdown

2019 ◽  
Author(s):  
Longhua Guo ◽  
Fengli Guo ◽  
Shasha Zhang ◽  
Kexi Yi ◽  
Melainia McClain ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
De Novo ◽  
Germinal Vesicle ◽  
Protein Composition ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cell Nuclei ◽  
Germinal Vesicle Breakdown ◽  
Evolutionary Origins ◽  
Pore Complexes

AbstractThe cell nuclei of Ophisthokonts, the eukaryotic supergroup defined by fungi and metazoans, is remarkable in the constancy of both their double-membraned structure and protein composition. Such remarkable structural conservation underscores common and ancient evolutionary origins. Yet, the dynamics of disassembly and reassembly displayed by Ophisthokont nuclei vary extensively. Besides closed mitosis in fungi and open mitosis in some animals, little is known about the evolution of nuclear envelope break down (NEBD) during cell division. Here, we uncovered a novel form of NEBD in primary oocytes of the flatworm Schmidtea mediterranea. From zygotene to metaphase II, both nuclear envelope (NE) and peripheral endoplasmic reticulum (ER) expand notably in size, likely involving de novo membrane synthesis. 3-D electron microscopy reconstructions demonstrated that the NE transforms itself into numerous double-membraned vesicles similar in membrane architecture to NE doublets in mammalian oocytes after germinal vesicle breakdown. The vesicles are devoid of nuclear pore complexes and DNA, yet are loaded with nuclear proteins, including a planarian homologue of PIWI, a protein essential for the maintenance of stem cells in this and other organisms. Our data contribute a new model to the canonical view of NE dynamics and support that NEBD is an evolutionarily adaptable trait in multicellular organisms.

scholarly journals The transmission of nuclear pore complexes to daughter cells requires a cytoplasmic pool of Nsp1

2013 ◽  
Vol 203 (2) ◽  
pp. 215-232 ◽  
Author(s):  
Paolo Colombi ◽  
Brant M. Webster ◽  
Florian Fröhlich ◽  
C. Patrick Lusk
Keyword(s):  
De Novo ◽  
Selective Inhibition ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Protein Assemblies ◽  
Class V ◽  
Specific Loss ◽  
Daughter Cells ◽  
Mother And Daughter ◽  
Pore Complexes

Nuclear pore complexes (NPCs) are essential protein assemblies that span the nuclear envelope and establish nuclear–cytoplasmic compartmentalization. We have investigated mechanisms that control NPC number in mother and daughter cells during the asymmetric division of budding yeast. By simultaneously tracking existing NPCs and newly synthesized NPC protomers (nups) through anaphase, we uncovered a pool of the central channel nup Nsp1 that is actively targeted to the bud in association with endoplasmic reticulum. Bud targeting required an intact actin cytoskeleton and the class V myosin, Myo2. Selective inhibition of cytoplasmic Nsp1 or inactivation of Myo2 reduced the inheritance of NPCs in daughter cells, leading to a daughter-specific loss of viability. Our data are consistent with a model in which Nsp1 releases a barrier that otherwise prevents NPC passage through the bud neck. It further supports the finding that NPC inheritance, not de novo NPC assembly, is primarily responsible for controlling NPC number in daughter cells.

scholarly journals In Vivo Dynamics of Drosophila Nuclear Envelope Components

2008 ◽  
Vol 19 (9) ◽  
pp. 3652-3666 ◽  
Author(s):  
Katerina R. Katsani ◽  
Roger E. Karess ◽  
Nathalie Dostatni ◽  
Valérie Doye
Keyword(s):  
Nuclear Envelope ◽  
Mammalian Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Checkpoint Proteins ◽  
Early Anaphase ◽  
Associated Proteins ◽  
Pore Complexes ◽  
Drosophila Cells

Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells.

Nuclear Envelope Dynamics During Mitosis

1988 ◽  
Vol 46 ◽  
pp. 224-225
Author(s):  
Brian Burke
Keyword(s):  
Nuclear Envelope ◽  
Membrane Vesicles ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Animal Cells ◽  
Interphase Chromosome ◽  
Lamin B ◽  
Chromosome Architecture ◽  
Complex Membrane ◽  
Pore Complexes

The nuclear envelope is a complex membrane structure that forms the boundary of the nuclear compartment in eukaryotes. It regulates the passage of macromolecules between the two compartments and may be important for organizing interphase chromosome architecture. In interphase animal cells it forms a remarkably stable structure consisting of a double membrane ouerlying a protein meshwork or lamina and penetrated by nuclear pore complexes. The latter form the channels for nucleocytoplasmic exchange of macromolecules, At the onset of mitosis, however, it rapidly disassembles, the membranes fragment to yield small vesicles and the lamina, which is composed of predominantly three polypeptides, lamins R, B and C (MW approx. 74, 68 and 65 kDa respectiuely), breaks down. Lamins B and C are dispersed as monomers throughout the mitotic cytoplasm, while lamin B remains associated with the nuclear membrane vesicles.

Pore relations: nuclear pore complexes and nucleocytoplasmic exchange

2000 ◽  
Vol 36 ◽  
pp. 75-88 ◽  
Author(s):  
Michael P. Rout ◽  
John D. Aitchison
Keyword(s):  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Pore Complexes
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