scholarly journals Yeast centrosome components form a noncanonical LINC complex at the nuclear envelope insertion site

2019 ◽  
Vol 218 (5) ◽  
pp. 1478-1490 ◽  
Author(s):  
Jingjing Chen ◽  
Jennifer M. Gardner ◽  
Zulin Yu ◽  
Sarah E. Smith ◽  
Sean McKinney ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Protein Interactions ◽  
Insertion Site ◽  
Spindle Pole ◽  
Bimolecular Fluorescence Complementation ◽  
Spin Component ◽  
Structured Illumination ◽  
Linc Complex ◽  
Protein Distribution ◽  
Structured Illumination Microscopy

Bipolar spindle formation in yeast requires insertion of centrosomes (known as spindle pole bodies [SPBs]) into fenestrated regions of the nuclear envelope (NE). Using structured illumination microscopy and bimolecular fluorescence complementation, we map protein distribution at SPB fenestrae and interrogate protein–protein interactions with high spatial resolution. We find that the Sad1-UNC-84 (SUN) protein Mps3 forms a ring-like structure around the SPB, similar to toroids seen for components of the SPB insertion network (SPIN). Mps3 and the SPIN component Mps2 (a Klarsicht-ANC-1-Syne-1 domain [KASH]–like protein) form a novel noncanonical linker of nucleoskeleton and cytoskeleton (LINC) complex that is connected in both luminal and extraluminal domains at the site of SPB insertion. The LINC complex also controls the distribution of a soluble SPIN component Bbp1. Taken together, our work shows that Mps3 is a fifth SPIN component and suggests both direct and indirect roles for the LINC complex in NE remodeling.

scholarly journals Membrane insertion function for SUN-KASH complex revealed by high resolution analysis of yeast centrosomes

2018 ◽  
Author(s):  
Jingjing Chen ◽  
Jennifer M. Gardner ◽  
Zulin Yu ◽  
Sarah E. Smith ◽  
Sean McKinney ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Spindle Pole ◽  
Bimolecular Fluorescence Complementation ◽  
Spin Component ◽  
Structured Illumination ◽  
Linc Complex ◽  
Protein Distribution ◽  
Protein Protein Interactions ◽  
Structured Illumination Microscopy ◽  
Complex Forms

AbstractBipolar spindle formation in yeast requires insertion of centrosomes (known as spindle pole bodies (SPBs)) into fenestrated regions of the nuclear envelope (NE). Using structured-illumination microscopy and bimolecular fluorescence complementation, we map protein distribution at SPB fenestra and interrogate protein-protein interactions with high spatial resolution. We find that the Sad1-UNC-84 (SUN) protein Mps3 forms a ring-like structure around the SPB, similar to toroids seen for components of the SPB insertion network (SPIN). Mps3 and the SPIN component Mps2 (a Klarsicht-ANC-1-Syne-1 domain (KASH)-like protein) form a novel non-canonical linker of nucleoskeleton and cytoskeleton (LINC) complex that is connected in both luminal and extraluminal domains. This hairpin-like LINC complex forms during SPB insertion, suggesting it functions in NE reorganization at the pore membrane. The LINC complex also controls the distribution of a soluble SPIN component Bbp1. Taken together our work shows that Mps3 is a fifth SPIN component and suggests both direct and indirect roles for the LINC complex in NE remodeling.

scholarly journals Redistribution of centrosomal proteins by centromeres and Polo kinase controls nuclear envelope breakdown

2020 ◽  
Author(s):  
Andrew J. Bestul ◽  
Zulin Yu ◽  
Jay R. Unruh ◽  
Sue L. Jaspersen
Keyword(s):  
Nuclear Envelope ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Ring Structure ◽  
Yeast Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spindle Formation ◽  
Nuclear Envelope Breakdown ◽  
Polo Kinase

AbstractProper mitotic progression in Schizosaccharomyces pombe requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB – yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy (SIM), we show that the conserved SUNprotein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for SPB ring protein redistribution and for complete NEBD to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate NEBD and spindle formation through building of an SPB ring structure.SummaryNuclear envelope breakdown is necessary for fission yeast cells to go through mitosis. Bestul et al. show that the SUN protein, Sad1, is vital in carrying out this breakdown and is regulated by the centromere and Polo kinase.

scholarly journals Redistribution of centrosomal proteins by centromeres and Polo kinase controls partial nuclear envelope breakdown in fission yeast

2021 ◽  
pp. mbc.E21-05-0239
Author(s):  
Andrew J. Bestul ◽  
Zulin Yu ◽  
Jay R. Unruh ◽  
Sue L. Jaspersen
Keyword(s):  
Nuclear Envelope ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Ring Structure ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spindle Formation ◽  
Nuclear Envelope Breakdown ◽  
Polo Kinase ◽  
Ring Complex

Proper mitotic progression in Schizosaccharomyces pombe requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB – yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy (SIM), we show that the conserved SUN-domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for redistribution of other SPB ring proteins and for complete NEBD at the SPB to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate partial NEBD and spindle formation through building of a SPB ring structure.

scholarly journals Structured illumination with particle averaging reveals novel roles for yeast centrosome components during duplication

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Shannon Burns ◽  
Jennifer S Avena ◽  
Jay R Unruh ◽  
Zulin Yu ◽  
Sarah E Smith ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Membrane Insertion ◽  
Structured Illumination ◽  
Wild Type ◽  
Structured Illumination Microscopy ◽  
Pole Body ◽  
Intensity Information

Duplication of the yeast centrosome (called the spindle pole body, SPB) is thought to occur through a series of discrete steps that culminate in insertion of the new SPB into the nuclear envelope (NE). To better understand this process, we developed a novel two-color structured illumination microscopy with single-particle averaging (SPA-SIM) approach to study the localization of all 18 SPB components during duplication using endogenously expressed fluorescent protein derivatives. The increased resolution and quantitative intensity information obtained using this method allowed us to demonstrate that SPB duplication begins by formation of an asymmetric Sfi1 filament at mitotic exit followed by Mps1-dependent assembly of a Spc29- and Spc42-dependent complex at its tip. Our observation that proteins involved in membrane insertion, such as Mps2, Bbp1, and Ndc1, also accumulate at the new SPB early in duplication suggests that SPB assembly and NE insertion are coupled events during SPB formation in wild-type cells.

scholarly journals Molecular model of fission yeast centrosome assembly determined by superresolution imaging

2017 ◽  
Vol 216 (8) ◽  
pp. 2409-2424 ◽  
Author(s):  
Andrew J. Bestul ◽  
Zulin Yu ◽  
Jay R. Unruh ◽  
Sue L. Jaspersen
Keyword(s):  
Molecular Model ◽  
Spindle Pole ◽  
Structured Illumination ◽  
Cellular Functions ◽  
Structured Illumination Microscopy ◽  
Superresolution Imaging ◽  
Daughter Cells ◽  
Spindle Pole Bodies ◽  
Relationship Of ◽  
The Relationship

Microtubule-organizing centers (MTOCs), known as centrosomes in animals and spindle pole bodies (SPBs) in fungi, are important for the faithful distribution of chromosomes between daughter cells during mitosis as well as for other cellular functions. The cytoplasmic duplication cycle and regulation of the Schizosaccharomyces pombe SPB is analogous to centrosomes, making it an ideal model to study MTOC assembly. Here, we use superresolution structured illumination microscopy with single-particle averaging to localize 14 S. pombe SPB components and regulators, determining both the relationship of proteins to each other within the SPB and how each protein is assembled into a new structure during SPB duplication. These data enabled us to build the first comprehensive molecular model of the S. pombe SPB, resulting in structural and functional insights not ascertained through investigations of individual subunits, including functional similarities between Ppc89 and the budding yeast SPB scaffold Spc42, distribution of Sad1 to a ring-like structure and multiple modes of Mto1 recruitment.

scholarly journals Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe

2021 ◽  
Author(s):  
Joseph M Varberg ◽  
Jay Unruh ◽  
Andrew J Bestul ◽  
Azqa A Khan ◽  
Sue Jaspersen
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Spindle Pole Body ◽  
Cell Types ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Wide Range ◽  
Pore Complexes

The number, distribution and composition of nuclear pore complexes (NPCs) in the nuclear envelope (NE) varies between cell types and changes during cellular differentiation and in disease. To understand how NPC density and organization is controlled, we analyzed NPC number and distribution in the fission yeast Schizosaccharomyces pombe using structured illumination microscopy. The small size of yeast nuclei, genetic features of fungi and our robust image analysis pipeline allowed us to study NPCs in intact nuclei under multiple conditions. Our data revealed that NPC density is maintained across a wide range of nuclear sizes. Regions of reduced NPC density are observed over the nucleolus and surrounding the spindle pole body (SPB). Lem2-mediated tethering of the centromeres to the SPB is required to maintain NPC exclusion, which is important for timely mitotic progression. These findings provide a quantitative understanding of NPC number and distribution in S. pombe and show that interactions between the centromere and the NE influences local NPC distribution.

scholarly journals Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase

2020 ◽  
Author(s):  
Marco Geymonat ◽  
Qiuran Peng ◽  
Zhiang Guo ◽  
Zulin Yu ◽  
Jay R. Unruh ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cyclin Dependent Kinase ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Microtubule Organization ◽  
Nucleation Sites ◽  
Stem Cell Divisions ◽  
Gamma Tubulin ◽  
Duplication Cycle

ABSTRACTAsymmetric astral microtubule organization drives the polarized orientation of the S. cerevisiae mitotic spindle and primes the invariant inheritance of the old spindle pole body (SPB, the yeast centrosome) by the bud. This model has anticipated analogous centrosome asymmetries featuring in self-renewing stem cell divisions. We previously implicated Spc72, the cytoplasmic receptor for the gamma-tubulin nucleation complex, as the most upstream determinant linking SPB age, functional asymmetry and fate. Here we used structured illumination microscopy and biochemical analysis to explore the asymmetric landscape of nucleation sites inherently built into the spindle pathway and under the control of cyclin-dependent kinase (CDK). We show that CDK enforces Spc72 asymmetric docking by phosphorylating Nud1/centriolin. Furthermore, CDK-imposed order in the construction of the new SPB promotes the correct balance of nucleation sites between the nuclear and cytoplasmic faces of the SPB. Together these contributions by CDK inherently link correct SPB morphogenesis, age and fate.

scholarly journals Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Marco Geymonat ◽  
Qiuran Peng ◽  
Zhiang Guo ◽  
Zulin Yu ◽  
Jay R Unruh ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cyclin Dependent Kinase ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Microtubule Organization ◽  
Nucleation Sites ◽  
Stem Cell Divisions ◽  
Gamma Tubulin ◽  
Duplication Cycle

Asymmetric astral microtubule organization drives the polarized orientation of the S. cerevisiae mitotic spindle and primes the invariant inheritance of the old spindle pole body (SPB, the yeast centrosome) by the bud. This model has anticipated analogous centrosome asymmetries featured in self-renewing stem cell divisions. We previously implicated Spc72, the cytoplasmic receptor for the gamma-tubulin nucleation complex, as the most upstream determinant linking SPB age, functional asymmetry and fate. Here we used structured illumination microscopy and biochemical analysis to explore the asymmetric landscape of nucleation sites inherently built into the spindle pathway and under the control of cyclin-dependent kinase (CDK). We show that CDK enforces Spc72 asymmetric docking by phosphorylating Nud1/centriolin. Furthermore, CDK-imposed order in the construction of the new SPB promotes the correct balance of nucleation sites between the nuclear and cytoplasmic faces of the SPB. Together these contributions by CDK inherently link correct SPB morphogenesis, age and fate.

scholarly journals Identification of RUVBL1 and RUVBL2 as Novel Cellular Interactors of the Ebola Virus Nucleoprotein

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 372 ◽  
Author(s):  
M. Jane Morwitzer ◽  
Sarah R. Tritsch ◽  
Lisa H. Cazares ◽  
Michael D. Ward ◽  
Jonathan E. Nuss ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Ebola Virus ◽  
Affinity Purification ◽  
Super Resolution ◽  
Hemorrhagic Fever ◽  
Host Protein ◽  
Structured Illumination ◽  
Global Public Health ◽  
Structured Illumination Microscopy ◽  
Capsid Formation

Ebola virus (EBOV) is a filovirus that has become a global public health threat in recent years. EBOV is the causative agent of a severe, often fatal hemorrhagic fever. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. To date, several investigations have discovered specific host-pathogen interactions for various EBOV proteins. However, relatively little is known about the EBOV nucleoprotein (NP) with regard to host interactions. In the present study, we aimed to elucidate NP-host protein-protein interactions (PPIs). Affinity purification-mass spectrometry (AP-MS) was used to identify candidate NP cellular interactors. Candidate interactors RUVBL1 and RUVBL2, partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) superfamily, were confirmed to interact with NP in co-immunoprecipitation (co-IP) and immunofluorescence (IF) experiments. Functional studies using a minigenome system revealed that the siRNA-mediated knockdown of RUVBL1 but not RUVBL2 moderately decreased EBOV minigenome activity. Super resolution structured illumination microscopy (SIM) was used to identify an association between NP and components of the R2TP complex, which includes RUVBL1, RUVBL2, RPAP3, and PIH1D1, suggesting a potential role for the R2TP complex in capsid formation. Moreover, the siRNA-mediated knockdown of RPAP3 and subsequent downregulation of PIH1D1 was shown to have no effect on minigenome activity, further suggesting a role in capsid formation. Overall, we identify RUVBL1 and RUVBL2 as novel interactors of EBOV NP and for the first time report EBOV NP recruitment of the R2TP complex, which may provide novel targets for broad-acting anti-EBOV therapeutics.

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