scholarly journals Hec1 and Nuf2 Are Core Components of the Kinetochore Outer Plate Essential for Organizing Microtubule Attachment Sites

2005 ◽  
Vol 16 (2) ◽  
pp. 519-531 ◽  
Author(s):  
Jennifer G. DeLuca ◽  
Yimin Dong ◽  
Polla Hergert ◽  
Joshua Strauss ◽  
Jennifer M. Hickey ◽  
...  
Keyword(s):  
Spindle Checkpoint ◽  
Attachment Site ◽  
Outer Plate ◽  
Microtubule Attachment ◽  
Attachment Sites ◽  
Outer Domain ◽  
Core Components ◽  
Fluorescence Light ◽  
Microtubule Formation ◽  
Stable Core

A major goal in the study of vertebrate mitosis is to identify proteins that create the kinetochore-microtubule attachment site. Attachment sites within the kinetochore outer plate generate microtubule dependent forces for chromosome movement and regulate spindle checkpoint protein assembly at the kinetochore. The Ndc80 complex, comprised of Ndc80 (Hec1), Nuf2, Spc24, and Spc25, is essential for metaphase chromosome alignment and anaphase chromosome segregation. It has also been suggested to have roles in kinetochore microtubule formation, production of kinetochore tension, and the spindle checkpoint. Here we show that Nuf2 and Hec1 localize throughout the outer plate, and not the corona, of the vertebrate kinetochore. They are part of a stable “core” region whose assembly dynamics are distinct from other outer domain spindle checkpoint and motor proteins. Furthermore, Nuf2 and Hec1 are required for formation and/or maintenance of the outer plate structure itself. Fluorescence light microscopy, live cell imaging, and electron microscopy provide quantitative data demonstrating that Nuf2 and Hec1 are essential for normal kinetochore microtubule attachment. Our results indicate that Nuf2 and Hec1 are required for organization of stable microtubule plus-end binding sites in the outer plate that are needed for the sustained poleward forces required for biorientation at kinetochores.

scholarly journals Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

2013 ◽  
Vol 202 (5) ◽  
pp. 735-746 ◽  
Author(s):  
Dileep Varma ◽  
Xiaohu Wan ◽  
Dhanya Cheerambathur ◽  
Reto Gassmann ◽  
Aussie Suzuki ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Attachment Site ◽  
Spindle Assembly ◽  
Nanometer Scale ◽  
Outer Plate ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Microtubule Attachment ◽  
C Terminus ◽  
Attachment Sites

Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1–Mis12–Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1–Zwint1 complex is required to recruit the Rod–Zwilch–Zw10 (RZZ) and Mad1–Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1–Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation.

scholarly journals Individual pericentromeres display coordinated motion and stretching in the yeast spindle

2013 ◽  
Vol 203 (3) ◽  
pp. 407-416 ◽  
Author(s):  
Andrew D. Stephens ◽  
Chloe E. Snider ◽  
Julian Haase ◽  
Rachel A. Haggerty ◽  
Paula A. Vasquez ◽  
...  
Keyword(s):  
Stochastic Dynamics ◽  
Attachment Site ◽  
Coordinated Motion ◽  
Daughter Cells ◽  
Microtubule Attachment ◽  
Sister Chromatids ◽  
Attachment Sites ◽  
Physical Linkage ◽  
Pulling Force ◽  
Structural Maintenance Of Chromosomes

The mitotic segregation apparatus composed of microtubules and chromatin functions to faithfully partition a duplicated genome into two daughter cells. Microtubules exert extensional pulling force on sister chromatids toward opposite poles, whereas pericentric chromatin resists with contractile springlike properties. Tension generated from these opposing forces silences the spindle checkpoint to ensure accurate chromosome segregation. It is unknown how the cell senses tension across multiple microtubule attachment sites, considering the stochastic dynamics of microtubule growth and shortening. In budding yeast, there is one microtubule attachment site per chromosome. By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase. The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes. Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8. Linking of pericentric chromatin through cohesin, condensin, and kinetochore microtubules functions to coordinate dynamics across multiple attachment sites.

scholarly journals hNuf2 inhibition blocks stable kinetochore–microtubule attachment and induces mitotic cell death in HeLa cells

2002 ◽  
Vol 159 (4) ◽  
pp. 549-555 ◽  
Author(s):  
Jennifer G. DeLuca ◽  
Ben Moree ◽  
Jennifer M. Hickey ◽  
John V. Kilmartin ◽  
E.D. Salmon
Keyword(s):  
Cell Death ◽  
Hela Cells ◽  
Spindle Checkpoint ◽  
Attachment Site ◽  
Mitotic Cell ◽  
Cytoplasmic Dynein ◽  
Microtubule Attachment ◽  
Microtubule Motors ◽  
Spindle Microtubules ◽  
Cell Cycle Block

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore–microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.

scholarly journals The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination.

Genetics ◽  
1989 ◽  
Vol 122 (4) ◽  
pp. 727-736
Author(s):  
C E Bauer ◽  
J F Gardner ◽  
R I Gumport ◽  
R A Weisberg
Keyword(s):  
Attachment Site ◽  
Strand Exchange ◽  
Segregation Pattern ◽  
Base Substitution ◽  
Base Pairs ◽  
Multiple Mutation ◽  
Attachment Sites ◽  
Dna Strands ◽  
Substitution Mutations ◽  
Overlap Region

Abstract Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.

AN ANATOMICAL STUDY OF THE LIGAMENTS OF THE ULNAR COMPARTMENT OF THE WRIST

Hand Surgery ◽  
2003 ◽  
Vol 08 (02) ◽  
pp. 219-226 ◽  
Author(s):  
Saburo Sasao ◽  
Moroe Beppu ◽  
Hitoshi Kihara ◽  
Kazuaki Hirata ◽  
Masayuki Takagi
Keyword(s):  
Three Dimensional ◽  
Attachment Site ◽  
Ulnar Collateral Ligament ◽  
Anatomical Study ◽  
Styloid Process ◽  
Dimensional Structure ◽  
Collateral Ligament ◽  
Ulnar Styloid ◽  
Attachment Sites ◽  
Triangular Fibrocartilage

The ligamentous structures of the triangular fibrocartilage complex (TFCC) and their attachments were examined anatomically and histologically using fresh and embalmed cadavers. The TFCC was observed to have a three-dimensional structure consisting of three palmar ligaments — the short radiolunate (SRL), ulnolunate (UL), and ulnotriquetral (UT) ligaments. In addition, the attachment site of the ulnocarpal ligament (UC), which had been previously unknown, was identified. The dorsal components of the TFCC have been previously reported to consist solely of the extensor carpi ulnaris (ECU) subsheath; however, the ligamentous components running from the ulnar styloid process to the triquetrum were found at a layer deeper than the floor of the ECU subsheath. The UC has been reported previously as a two-dimensional structure, but there has been some disagreement as to its attachment sites.2–6,14,15 It is suggested that the dorsal UT ligament should be considered as a separate ligament, based on its different direction and distal attachment site as compared with those of the ulnar collateral ligament (UCL) and ECU subsheath.

Comparison Between Wrist-Worn and Waist-Worn Accelerometry

2017 ◽  
Vol 14 (7) ◽  
pp. 539-545 ◽  
Author(s):  
Paul D. Loprinzi ◽  
Brandee Smith
Keyword(s):  
Pearson Correlation ◽  
Attachment Site ◽  
Free Living ◽  
Extreme Caution ◽  
Attachment Sites ◽  
Midaxillary Line

Objective:To use the most recent ActiGraph model (GT9X) to compare counts per minute (CPM) estimates between wrist-worn and waist-worn attachment sites.Methods:Participants completed 2 conditions (laboratory [N = 13] and free-living conditions [N = 9]), in which during both of these conditions they wore 2 ActiGraph GT9X accelerometers on their nondominant wrist (side-by-side) and 2 ActiGraph GT9X accelerometers on their right hip in line with the midaxillary line (side-by-side). During the laboratory visit, participants completed 5 treadmill-based trials all lasting 5 min: walk at 3 mph, 3.5 mph, 4 mph, and a jog at 6 mph and 6.5 mph. During the free-living setting, participants wore the monitors for 8 hours. Paired t test, Pearson correlation and Bland-Altman analyses were employed to evaluate agreement of CPM between the attachment sites.Results:Across all intensity levels and setting (laboratory and free-living), CPM were statistically significantly and substantively different between waist- and wrist-mounted accelerometry.Conclusion:Attachment site drastically influences CPM. As such, extreme caution should be exercised when comparing CPM estimates among studies employing different attachment site methodologies, particularly waist versus wrist.

scholarly journals MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling

2019 ◽  
Vol 218 (4) ◽  
pp. 1108-1117 ◽  
Author(s):  
Tatiana Alfonso-Pérez ◽  
Daniel Hayward ◽  
James Holder ◽  
Ulrike Gruneberg ◽  
Francis A. Barr
Keyword(s):  
Feedback Loop ◽  
Spindle Checkpoint ◽  
Mitotic Exit ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Mitotic Entry ◽  
Upstream Regulator ◽  
Microtubule Attachment ◽  
Integral Component ◽  
Checkpoint Pathway

Cyclin B–dependent kinase (CDK1-CCNB1) promotes entry into mitosis. Additionally, it inhibits mitotic exit by activating the spindle checkpoint. This latter role is mediated through phosphorylation of the checkpoint kinase MPS1 and other spindle checkpoint proteins. We find that CDK1-CCNB1 localizes to unattached kinetochores and like MPS1 is lost from these structures upon microtubule attachment. This suggests that CDK1-CCNB1 is an integral component and not only an upstream regulator of the spindle checkpoint pathway. Complementary proteomic and cell biological analysis demonstrate that the spindle checkpoint protein MAD1 is one of the major components of CCNB1 complexes, and that CCNB1 is recruited to unattached kinetochores in an MPS1-dependent fashion through interaction with the first 100 amino acids of MAD1. This MPS1 and MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop necessary for timely recruitment of MPS1 to kinetochores during mitotic entry and for sustained spindle checkpoint arrest. CDK1-CCNB1 is therefore an integral component of the spindle checkpoint, ensuring the fidelity of mitosis.

scholarly journals The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation

2006 ◽  
Vol 173 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Susan L. Kline ◽  
Iain M. Cheeseman ◽  
Tetsuya Hori ◽  
Tatsuo Fukagawa ◽  
Arshad Desai
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Essential Role ◽  
Stable Complex ◽  
Wild Type ◽  
Outer Plate ◽  
Checkpoint Protein ◽  
Kinetochore Assembly ◽  
Attachment Sites ◽  
Spindle Microtubules

During cell division, kinetochores form the primary chromosomal attachment sites for spindle microtubules. We previously identified a network of 10 interacting kinetochore proteins conserved between Caenorhabditis elegans and humans. In this study, we investigate three proteins in the human network (hDsn1Q9H410, hNnf1PMF1, and hNsl1DC31). Using coexpression in bacteria and fractionation of mitotic extracts, we demonstrate that these proteins form a stable complex with the conserved kinetochore component hMis12. Human or chicken cells depleted of Mis12 complex subunits are delayed in mitosis with misaligned chromosomes and defects in chromosome biorientation. Aligned chromosomes exhibited reduced centromere stretch and diminished kinetochore microtubule bundles. Consistent with this, localization of the outer plate constituent Ndc80HEC1 was severely reduced. The checkpoint protein BubR1, the fibrous corona component centromere protein (CENP) E, and the inner kinetochore proteins CENP-A and CENP-H also failed to accumulate to wild-type levels in depleted cells. These results indicate that a four-subunit Mis12 complex plays an essential role in chromosome segregation in vertebrates and contributes to mitotic kinetochore assembly.

scholarly journals Conserved, N-Linked Carbohydrates of Human Immunodeficiency Virus Type 1 gp41 Are Largely Dispensable for Viral Replication

2001 ◽  
Vol 75 (23) ◽  
pp. 11426-11436 ◽  
Author(s):  
Welkin E. Johnson ◽  
Jennifer M. Sauvron ◽  
Ronald C. Desrosiers
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rhesus Monkey ◽  
Viral Replication ◽  
Envelope Protein ◽  
Attachment Site ◽  
Attachment Sites ◽  
Immunodeficiency Virus ◽  
Quadruple Mutant ◽  
Hiv 1

ABSTRACT The transmembrane subunit (TM) of human immunodeficiency virus type 1 (HIV-1) envelope protein contains four well-conserved sites for the attachment of N-linked carbohydrates. To study the contribution of these N-glycans to the function of TM, we systematically mutated the sites individually and in all combinations and measured the effects of each on viral replication in culture. The mutants were derived from SHIV-KB9, a simian immunodeficiency virus/HIV chimera with an envelope sequence that originated from a primary HIV-1 isolate. The attachment site mutants were generated by replacing the asparagine codon of each N-X-S/T motif with a glutamine codon. The mobilities of the variant transmembrane proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that all four sites are utilized for carbohydrate attachment. Transfection of various cell lines with the resulting panel of mutant viral constructs revealed that the N-glycan attachment sites are largely dispensable for viral replication. Fourteen of the 15 mutants were replication competent, although the kinetics of replication varied depending on the mutant and the cell type. The four single mutants (g1, g2, g3, and g4) and all six double mutants (g12, g13, g14, g23, g24, and g34) replicated in both human and rhesus monkey T-cell lines, as well as in primary rhesus peripheral blood mononuclear cells. Three of the four triple mutants (g124, g134, and g234) replicated in all cell types tested. The triple mutant g123 replicated poorly in immortalized rhesus monkey T cells (221 cells) and did not replicate detectably in CEMx174 cells. However, at 3 weeks posttransfection of 221 cells, a variant of g123 emerged with a new N-glycan attachment site which compensated for the loss of sites 1, 2, and 3 and resulted in replication kinetics similar to those of the parental virus. The quadruple mutant (g1234) did not replicate in any cell line tested, and the g1234 envelope protein was nonfunctional in a quantitative cell-cell fusion assay. The synthesis and processing of the quadruple mutant envelope protein appeared similar in transient assays to those of the parental SHIV-KB9 envelope. Given their high degree of conservation, the four N-linked carbohydrate attachment sites on the external domain of gp41 are surprisingly dispensable for viral replication. The viral variants described in this report should prove useful for investigation of the contribution of carbohydrate moieties on gp41 to recognition by antibodies, shielding from antibody-mediated neutralization, and structure-function relationships.

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