scholarly journals A centriolar FGR1 oncogene partner-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes

Open Biology ◽  
2018 ◽  
Vol 8 (7) ◽  
pp. 170218 ◽  
Author(s):  
Jane Harmer ◽  
Katie Towers ◽  
Max Addison ◽  
Sue Vaughan ◽  
Michael L. Ginger ◽  
...  
Keyword(s):  
Basal Body ◽  
Mitotic Spindle ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Molecular Level ◽  
African Trypanosomes ◽  
Spindle Poles ◽  
Microtubule Organizing Centres ◽  
Polar Spindle ◽  
And Function

Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organizing centres (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the axoneme extends. We report conservation of FOP family proteins, Tb FOPL and Tb OFD1, in the evolutionarily divergent sleeping sickness parasite Trypanosoma brucei , showing (in contrast with mammalian cells, where FOP is essential for flagellum assembly) depletion of a trypanosome FOP homologue, Tb FOPL, affects neither axoneme nor flagellum elongation. Instead, Tb FOPL depletion causes catastrophic failure in assembly of a lineage-specific, extra-axonemal structure, the paraflagellar rod (PFR). That depletion of centriolar Tb FOPL causes failure in PFR assembly is surprising because PFR nucleation commences approximately 2 µm distal from the basal body. When over-expressed with a C-terminal myc-epitope, Tb FOPL was also observed at mitotic spindle poles. Little is known about bi-polar spindle assembly during closed trypanosome mitosis, but indication of a possible additional MTOC function for Tb FOPL parallels MTOC localization of FOP-like protein TONNEAU1 in acentriolar plants. More generally, our functional analysis of Tb FOPL emphasizes significant differences in evolutionary cell biology trajectories of FOP-family proteins. We discuss how at the molecular level FOP homologues may contribute to flagellum assembly and function in diverse flagellates.

scholarly journals TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle

2011 ◽  
Vol 195 (1) ◽  
pp. 87-98 ◽  
Author(s):  
Nan Ma ◽  
Janel Titus ◽  
Alyssa Gable ◽  
Jennifer L. Ross ◽  
Patricia Wadsworth
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Spindle Assembly ◽  
Artificial Chromosome ◽  
Fiber Formation ◽  
Interaction Domain ◽  
Spindle Poles ◽  
Spindle Elongation ◽  
Multiple Spindle ◽  
And Function

Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2–Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5.

scholarly journals Systems cell biology of the mitotic spindle

2010 ◽  
Vol 188 (1) ◽  
pp. 7-9
Author(s):  
Ramsey A. Saleem ◽  
John D. Aitchison
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Cell Biology ◽  
Systems Genetics ◽  
Mitotic Spindles ◽  
High Content Imaging ◽  
Controlled Assembly ◽  
Daughter Cells ◽  
And Function ◽  
Insight Into

Cell division depends critically on the temporally controlled assembly of mitotic spindles, which are responsible for the distribution of duplicated chromosomes to each of the two daughter cells. To gain insight into the process, Vizeacoumar et al., in this issue (Vizeacoumar et al. 2010. J. Cell Biol. doi:10.1083/jcb.200909013), have combined systems genetics with high-throughput and high-content imaging to comprehensively identify and classify novel components that contribute to the morphology and function of the mitotic spindle.

scholarly journals The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

2009 ◽  
Vol 29 (14) ◽  
pp. 3975-3990 ◽  
Author(s):  
Laura O'Regan ◽  
Andrew M. Fry
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Serine Threonine Kinase ◽  
Spindle Formation ◽  
Spindle Poles ◽  
And Function ◽  
Interfering Rna ◽  
Reduced Activity

ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

scholarly journals Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells

2002 ◽  
Vol 158 (5) ◽  
pp. 841-847 ◽  
Author(s):  
Marko J. Kallio ◽  
Victoria A. Beardmore ◽  
Jasminder Weinstein ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Anaphase Promoting Complex ◽  
Late Prophase ◽  
Checkpoint Signaling ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Late Telophase ◽  
Green Fluorescent

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20–GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.

scholarly journals K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

2021 ◽  
Author(s):  
Marcus A Begley ◽  
April L Solon ◽  
Elizabeth Mae Davis ◽  
Michael Grant Sherrill ◽  
Ryoma Ohi ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Binding Ability ◽  
Microtubule Binding ◽  
Mechanical Integrity ◽  
Spindle Poles ◽  
Rigor State ◽  
Physical Response

The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes during cell division. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) connect chromosomes to the spindle poles. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We detach k-fibers from poles by laser ablation-based cutting, thus revealing the contribution of pole-localized forces to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced by ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15-microtubule binding reduces the mechanical integrity of k-fibers. In contrast, inhibition of its motor activity but not its microtubule binding ability, i.e., locking Kif15 into a rigor state, does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors. [Media: see text] [Media: see text] [Media: see text]

Cytocentrin is a Ral-binding protein involved in the assembly and function of the mitotic apparatus

1999 ◽  
Vol 112 (5) ◽  
pp. 707-718
Author(s):  
A. Quaroni ◽  
E.C. Paul
Keyword(s):  
Mitotic Spindle ◽  
Binding Protein ◽  
Early Prophase ◽  
Mitotic Apparatus ◽  
Cytosolic Protein ◽  
Spindle Poles ◽  
Ral Gtpases ◽  
And Function ◽  
High Degree

Cytocentrin is a cytosolic protein that transiently associates with the mitotic spindle poles in early prophase, and dissociates from them after completion of mitosis. Cloning of its cDNA demonstrated a high degree of homology with three proteins known to specifically interact with an activated form of Ral. Herein we demonstrate that overexpression of cytocentrin inhibits assembly of the mitotic spindle without affecting polymerization or distribution of interphase microtubules. Conversely, loss of cytocentrin expression leads to formation of monopolar spindles. These results indicate that association of cytocentrin with the centrosome may be essential for a timely separation of the diplosomes. They also implicate Ral GTPases and their related pathways in the assembly and function of the mitotic apparatus.

APPLICATIONS OF PIXE ANALYSIS TO STUDIES IN CELL BIOLOGY

1992 ◽  
Vol 02 (03) ◽  
pp. 417-420 ◽  
Author(s):  
SHINJI MATSUMOTO ◽  
MASAKO FURUSE ◽  
KENSUKE KITAO ◽  
TOYOYUKI ISHIHARA ◽  
KUNIHIRO SHIMA ◽  
...  
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
Environmental Changes ◽  
Molecular Level ◽  
Pixe Analysis ◽  
X Ray ◽  
Preliminary Results ◽  
Linear Relationships ◽  
Element Contents

PIXE application to the measurement of cellular elements is outlined on characteristic variations of the contents of yeast, CHO, V79 and MM46 cultured mammalian cells according to environmental changes. Cellular elements from P to Br were successfully analyzed at these cells after cautious preparation of samples in filtration steps with as possible as less deterioration. We confirmed linear relationships between the X-ray yields and these element contents. The experiment was extended to an analysis of cellular substances at molecular level by scanning of specimens. Preliminary results were included.

The Role of the Centrosome in Development and Progression of Breast Cancer

2001 ◽  
Vol 7 (S2) ◽  
pp. 582-583
Author(s):  
W. Lingle ◽  
J. Salisbury ◽  
S. Barrett ◽  
V. Negron ◽  
C. Whitehead
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Microtubule Organizing Center ◽  
Daughter Cells ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Close Proximity ◽  
Interphase Cells ◽  
Organizing Center

The centrosome is the major microtubule organizing center in most mammalian cells, and as such it determines the number, polarity, and spatial distribution of microtubules (MTs). Interphase MTs, together with actin and intermediate filaments, constitute the cell's cytoskeleton, which dynamically maintains cell polarity and tissue architecture. Interphase cells begin Gl of the cell cycle with one centrosome. During S phase, the centrosome duplicates concomitantly with DNA replication. Duplicated centrosomes usually remain in close proximity to one another until late G2, at which time they separate and then move during prophase to become the poles that organize the bipolar mitotic spindle. During the G2/M transition, interphase MTs depolymerize and a new population of highly dynamic mitotic MTs are nucleated at the spindle poles. The bipolar mitotic spindle apparatus constitutes the machinery that partitions and separates sister chromatids equally between two daughter cells.

scholarly journals A specific basal body linker protein provides the connection function for basal body inheritance in trypanosomes

2021 ◽  
Vol 118 (8) ◽  
pp. e2014040118
Author(s):  
De-Hua Lai ◽  
Flavia Moreira-Leite ◽  
Zhi-Shen Xu ◽  
Jiong Yang ◽  
Keith Gull
Keyword(s):  
Basal Body ◽  
Mammalian Cells ◽  
De Novo ◽  
Coiled Coil ◽  
Centriole Duplication ◽  
Cytoskeletal Component ◽  
Dividing Cells ◽  
Orthogonal Orientation ◽  
And Function ◽  
Additional Role

Centrioles and basal bodies (CBBs) are found in physically linked pairs, and in mammalian cells intercentriole connections (G1–G2 tether and S–M linker) regulate centriole duplication and function. In trypanosomes BBs are not associated with the spindle and function in flagellum/cilia nucleation with an additional role in mitochondrial genome (kinetoplast DNA [kDNA]) segregation. Here, we describe BBLP, a BB/pro-BB (pBB) linker protein in Trypanosoma brucei predicted to be a large coiled-coil protein conserved in the kinetoplastida. Colocalization with the centriole marker SAS6 showed that BBLP localizes between the BB/pBB pair, throughout the cell cycle, with a stronger signal in the old flagellum BB/pBB pair. Importantly, RNA interference (RNAi) depletion of BBLP leads to a conspicuous splitting of the BB/pBB pair associated only with the new flagellum. BBLP RNAi is lethal in the bloodstream form of the parasite and perturbs mitochondrial kDNA inheritance. Immunogold labeling confirmed that BBLP is localized to a cytoskeletal component of the BB/pBB linker, and tagged protein induction showed that BBLP is incorporated de novo in both new and old flagella BB pairs of dividing cells. We show that the two aspects of CBB disengagement—loss of orthogonal orientation and ability to separate and move apart—are consistent but separable events in evolutionarily diverse cells and we provide a unifying model explaining centriole/BB linkage differences between such cells.

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