scholarly journals CDK5RAP2 functions in centrosome to spindle pole attachment and DNA damage response

2010 ◽  
Vol 189 (1) ◽  
pp. 23-39 ◽  
Author(s):  
Alexis R. Barr ◽  
John V. Kilmartin ◽  
Fanni Gergely
Keyword(s):  
Dna Damage ◽  
Cell Fate ◽  
Mitotic Spindle ◽  
Brain Size ◽  
Neurodevelopmental Disorder ◽  
Spindle Pole ◽  
Spindle Positioning ◽  
Dna Damage Signaling ◽  
Spindle Poles ◽  
G2 Cell Cycle Arrest

The centrosomal protein, CDK5RAP2, is mutated in primary microcephaly, a neurodevelopmental disorder characterized by reduced brain size. The Drosophila melanogaster homologue of CDK5RAP2, centrosomin (Cnn), maintains the pericentriolar matrix (PCM) around centrioles during mitosis. In this study, we demonstrate a similar role for CDK5RAP2 in vertebrate cells. By disrupting two evolutionarily conserved domains of CDK5RAP2, CNN1 and CNN2, in the avian B cell line DT40, we find that both domains are essential for linking centrosomes to mitotic spindle poles. Although structurally intact, centrosomes lacking the CNN1 domain fail to recruit specific PCM components that mediate attachment to spindle poles. Furthermore, we show that the CNN1 domain enforces cohesion between parental centrioles during interphase and promotes efficient DNA damage–induced G2 cell cycle arrest. Because mitotic spindle positioning, asymmetric centrosome inheritance, and DNA damage signaling have all been implicated in cell fate determination during neurogenesis, our findings provide novel insight into how impaired CDK5RAP2 function could cause premature depletion of neural stem cells and thereby microcephaly.

scholarly journals Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

2015 ◽  
Vol 26 (7) ◽  
pp. 1286-1295 ◽  
Author(s):  
Francisco Lázaro-Diéguez ◽  
Iaroslav Ispolatov ◽  
Anne Müsch
Keyword(s):  
Mitotic Spindle ◽  
Cell Shape ◽  
Spindle Assembly Checkpoint ◽  
Mdck Cells ◽  
Spindle Pole ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Spindle Poles ◽  
Spindle Position

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell's height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.

scholarly journals Human Microcephaly Protein RTTN Is Required for Proper Mitotic Progression and Correct Spindle Position

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1441
Author(s):  
En-Ju Chou ◽  
Tang K. Tang
Keyword(s):  
Brain Size ◽  
Neurodevelopmental Disorder ◽  
Mitotic Progression ◽  
Centriole Duplication ◽  
Spindle Poles ◽  
Multipolar Spindles ◽  
Mitotic Abnormalities ◽  
Cortical Localization ◽  
Spindle Position ◽  
Autosomal Recessive Primary Microcephaly

Autosomal recessive primary microcephaly (MCPH) is a complex neurodevelopmental disorder characterized by a small brain size with mild to moderate intellectual disability. We previously demonstrated that human microcephaly RTTN played an important role in regulating centriole duplication during interphase, but the role of RTTN in mitosis is not fully understood. Here, we show that RTTN is required for normal mitotic progression and correct spindle position. The depletion of RTTN induces the dispersion of the pericentriolar protein γ-tubulin and multiple mitotic abnormalities, including monopolar, abnormal bipolar, and multipolar spindles. Importantly, the loss of RTTN altered NuMA/p150Glued congression to the spindle poles, perturbed NuMA cortical localization, and reduced the number and the length of astral microtubules. Together, our results provide a new insight into how RTTN functions in mitosis.

scholarly journals NuMA-microtubule interactions are critical for spindle orientation and the morphogenesis of diverse epidermal structures

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lindsey Seldin ◽  
Andrew Muroyama ◽  
Terry Lechler
Keyword(s):  
Cell Fate ◽  
Mitotic Spindle ◽  
Cell Types ◽  
Epidermal Differentiation ◽  
Spindle Orientation ◽  
Direct Function ◽  
Spindle Positioning ◽  
Adult Mice ◽  
Neonatal Lethality ◽  
The Matrix

Mitotic spindle orientation is used to generate cell fate diversity and drive proper tissue morphogenesis. A complex of NuMA and dynein/dynactin is required for robust spindle orientation in a number of cell types. Previous research proposed that cortical dynein/dynactin was sufficient to generate forces on astral microtubules (MTs) to orient the spindle, with NuMA acting as a passive tether. In this study, we demonstrate that dynein/dynactin is insufficient for spindle orientation establishment in keratinocytes and that NuMA’s MT-binding domain, which targets MT tips, is also required. Loss of NuMA-MT interactions in skin caused defects in spindle orientation and epidermal differentiation, leading to neonatal lethality. In addition, we show that NuMA-MT interactions are also required in adult mice for hair follicle morphogenesis and spindle orientation within the transit-amplifying cells of the matrix. Loss of spindle orientation in matrix cells results in defective differentiation of matrix-derived lineages. Our results reveal an additional and direct function of NuMA during mitotic spindle positioning, as well as a reiterative use of spindle orientation in the skin to build diverse structures.

scholarly journals Stu1p Is Physically Associated with β-Tubulin and Is Required for Structural Integrity of the Mitotic Spindle

2002 ◽  
Vol 13 (6) ◽  
pp. 1881-1892 ◽  
Author(s):  
Hongwei Yin ◽  
Liru You ◽  
Danielle Pasqualone ◽  
Kristen M. Kopski ◽  
Tim C. Huffaker
Keyword(s):  
Mitotic Spindle ◽  
Structural Integrity ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Poles ◽  
Balance Of Forces ◽  
Related Proteins ◽  
Β Tubulin

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1–5mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1–5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin–related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with β-tubulin and identify the domains required for this interaction on both Stu1p and β-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.

scholarly journals Mechanisms for focusing mitotic spindle poles by minus end–directed motor proteins

2005 ◽  
Vol 171 (2) ◽  
pp. 229-240 ◽  
Author(s):  
Gohta Goshima ◽  
François Nédélec ◽  
Ronald D. Vale
Keyword(s):  
Rna Interference ◽  
High Resolution ◽  
Computer Modeling ◽  
Mitotic Spindle ◽  
Motor Proteins ◽  
Spindle Pole ◽  
S2 Cells ◽  
Spindle Poles ◽  
Drosophila S2 Cells ◽  
High Resolution Microscopy

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end–directed motor proteins. Here, we have characterized the roles of two minus end–directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end–tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end–directed motors cooperate to ensure spindle pole coalescence during mitosis.

scholarly journals Chromosome misalignment is associated with PLK1 activity at cenexin-positive mitotic centrosomes

2019 ◽  
Vol 30 (13) ◽  
pp. 1598-1609 ◽  
Author(s):  
Erica G. Colicino ◽  
Katrina Stevens ◽  
Erin Curtis ◽  
Lindsay Rathbun ◽  
Michael Bates ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Dependent Manner ◽  
Binding Partner ◽  
Mitotic Kinase ◽  
Daughter Cells ◽  
Chromosome Distribution ◽  
Spindle Poles ◽  
Mother Centriole

The mitotic kinase, polo-like kinase 1 (PLK1), facilitates the assembly of the two mitotic spindle poles, which are required for the formation of the microtubule-based spindle that ensures appropriate chromosome distribution into the two forming daughter cells. Spindle poles are asymmetric in composition. One spindle pole contains the oldest mitotic centriole, the mother centriole, where the majority of cenexin, the mother centriole appendage protein and PLK1 binding partner, resides. We hypothesized that PLK1 activity is greater at the cenexin-positive older spindle pole. Our studies found that PLK1 asymmetrically localizes between spindle poles under conditions of chromosome misalignment, and chromosomes tend to misalign toward the oldest spindle pole in a cenexin- and PLK1-dependent manner. During chromosome misalignment, PLK1 activity is increased specifically at the oldest spindle pole, and this increase in activity is lost in cenexin-depleted cells. We propose a model where PLK1 activity elevates in response to misaligned chromosomes at the oldest spindle pole during metaphase.

Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin

2000 ◽  
Vol 113 (9) ◽  
pp. 1623-1633 ◽  
Author(s):  
K.P. McNally ◽  
O.A. Bazirgan ◽  
F.J. McNally
Keyword(s):  
Mitotic Spindle ◽  
Binding Proteins ◽  
Spindle Pole ◽  
Negative Regulator ◽  
Microtubule Binding ◽  
Microtubule Severing ◽  
Spindle Poles ◽  
Wd40 Domain

The assembly and function of the mitotic spindle requires the activity of a number of microtubule-binding proteins. Some microtubule-binding proteins bind microtubules in vitro but do not co-localize with microtubules in interphase cells. Instead these proteins associate with specific subregions of the mitotic spindle. Katanin, a heterodimeric microtubule-severing ATPase, is found localized at mitotic spindle poles. In this paper we demonstrate that human p60 katanin and the C-terminal domain of human p80 katanin both bind microtubules in vitro. Association of these two proteins results in an increased microtubule affinity and increased microtubule-severing activity in vitro. Association of these subunits in transfected HeLa cells increases microtubule disassembly activity and targeting to spindle poles. The N-terminal WD40 domain of p80 katanin acts as a negative regulator of microtubule disassembly activity and is also required for spindle pole localization, possibly through interactions with another spindle-pole protein. These results support a model in which katanin is targeted to spindle poles through a combination of direct microtubule binding by the p60 subunit and through interactions between the WD40 domain and an unknown protein. We propose that both domains of p80 are essential in precisely regulating katanin's activity in vivo.

scholarly journals A Potential Role for Human Cohesin in Mitotic Spindle Aster Assembly

2001 ◽  
Vol 276 (50) ◽  
pp. 47575-47582 ◽  
Author(s):  
Heather C. Gregson ◽  
John A. Schmiesing ◽  
Jong-Soo Kim ◽  
Toshiki Kobayashi ◽  
Sharleen Zhou ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Mitotic Spindle ◽  
Potential Role ◽  
Metaphase Plate ◽  
Spindle Pole ◽  
Sister Chromatid Cohesion ◽  
Cycle Arrest ◽  
Spindle Poles ◽  
Chromatid Cohesion

The cohesin multiprotein complex containing SMC1, SMC3, Scc3 (SA), and Scc1 (Rad21) is required for sister chromatid cohesion in eukaryotes. Although metazoan cohesin associates with chromosomes and was shown to function in the establishment of sister chromatid cohesion during interphase, the majority of cohesin was found to be off chromosomes and reside in the cytoplasm in metaphase. Despite its dissociation from chromosomes, however, microinjection of an antibody against human SMC1 led to disorganization of the metaphase plate and cell cycle arrest, indicating that human cohesin still plays an important role in metaphase. To address the mitotic function of human cohesin, the subcellular localization of cohesin components was reexamined in human cells. Interestingly, we found that cohesin localizes to the spindle poles during mitosis and interacts with NuMA, a spindle pole-associated factor required for mitotic spindle organization. The interaction with NuMA persists during interphase. Similar to NuMA, a significant amount of cohesin was found to associate with the nuclear matrix. Furthermore, in the absence of cohesin, mitotic spindle asters failed to formin vitro. Our results raise the intriguing possibility that in addition to its well demonstrated function in sister chromatid cohesion, cohesin may be involved in spindle assembly during mitosis.

scholarly journals A novel mitotic spindle pole component that originates from the cytoplasm during prophase.

1986 ◽  
Vol 103 (5) ◽  
pp. 1863-1872 ◽  
Author(s):  
P R Sager ◽  
N L Rothfield ◽  
J M Oliver ◽  
R D Berlin
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Early Prophase ◽  
Microtubule Organizing Center ◽  
Spindle Poles ◽  
Interphase Cells ◽  
Organizing Center ◽  
Mitotic Spindle Pole ◽  
Pole Component

Several unique aspects of mitotic spindle formation have been revealed by investigation of an autoantibody present in the serum of a patient with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, schlerodacytyly, and telangiectasias) syndrome. This antibody was previously shown to label at the spindle poles of metaphase and anaphase cells and to be absent from interphase cells. We show here that the serum stained discrete cytoplasmic foci in early prophase cells and only later localized to the spindle poles. The cytoplasmic distribution of the antigen was also seen in nocodazole-arrested cells and prophase cells in populations treated with taxol. In normal and taxol-treated cells, the microtubules appeared to emanate from the cytoplasmic foci and polar stain, and in cells released from nocodazole block, microtubules regrew from antigen-containing centers. This characteristic distribution suggests that the antigen is part of a microtubule organizing center. Thus, we propose that a prophase originating polar antigen functions in spindle pole organization as a coalescing microtubule organizing center that is present only during mitosis. Characterization of the serum showed reactions with multiple proteins at 115, 110, 50, 36, 30, and 28 kD. However, affinity-eluted antibody from the 115/110-kD bands was shown to specifically label the spindle pole and cytosolic foci in prophase cells.

scholarly journals Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

2004 ◽  
Vol 167 (5) ◽  
pp. 831-840 ◽  
Author(s):  
Helder Maiato ◽  
Conly L. Rieder ◽  
Alexey Khodjakov
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
S2 Cells ◽  
Spindle Poles ◽  
Drosophila S2 Cells ◽  
Mitotic Spindle Assembly ◽  
Normal Mitosis ◽  
Proper Orientation ◽  
Dependent Mechanism

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

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