scholarly journals NuMA is a major acceptor of poly(ADP-ribosyl)ation by tankyrase 1 in mitosis

2005 ◽  
Vol 391 (2) ◽  
pp. 177-184 ◽  
Author(s):  
William Chang ◽  
Jasmin N. Dynek ◽  
Susan Smith
Keyword(s):  
Small Interfering Rna ◽  
Complete Loss ◽  
Mitotic Progression ◽  
Mitotic Apparatus ◽  
Spindle Poles ◽  
Early Anaphase ◽  
Parp Activity ◽  
Sirna Knockdown ◽  
Tankyrase 1 ◽  
Interfering Rna

Tankyrase 1 is a PARP [poly(ADP-ribose) polymerase] that localizes to multiple subcellular sites, including telomeres and mitotic centrosomes. Previous studies demonstrated that cells deficient in tankyrase 1 suffered a block in resolution of sister telomeres and arrested in early anaphase [Dynek and Smith (2004) Science 304, 97–100]. This phenotype was dependent on the catalytic PARP activity of tankyrase 1. To identify critical acceptors of PARsylation [poly(ADP-ribosyl)ation] by tankyrase 1 in mitosis, tankyrase 1 immunoprecipitates were analysed for associated PARsylated proteins. We identified NuMA (nuclear mitotic apparatus protein) as a major acceptor of poly(ADP-ribose) from tankyrase 1 in mitosis. We showed by immunofluorescence and immunoprecipitation that association between tankyrase 1 and NuMA increases dramatically at the onset of mitosis, concomitant with PARsylation of NuMA. Knockdown of tankyrase 1 by siRNA (small interfering RNA) eliminates PARsylation of NuMA in mitosis, confirming tankyrase 1 as the PARP responsible for this modification. However, even in the absence of tankyrase 1 and PARsylation, NuMA localizes to spindle poles. By contrast, siRNA knockdown of NuMA results in complete loss of tankyrase 1 from spindle poles. We discuss our result in terms of a model where PARsylation of NuMA by tankyrase 1 in mitosis could play a role in sister telomere separation and/or mitotic progression.

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 ERK1c regulates Golgi fragmentation during mitosis

2006 ◽  
Vol 172 (6) ◽  
pp. 885-897 ◽  
Author(s):  
Yoav D. Shaul ◽  
Rony Seger
Keyword(s):  
Small Interfering Rna ◽  
Cell Cycle Regulation ◽  
Mitotic Progression ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Alternatively Spliced ◽  
Golgi Fragmentation ◽  
Cycle Regulation ◽  
Interfering Rna ◽  
Expression And Activity

Extracellular signal-regulated kinase 1c (ERK1c) is an alternatively spliced form of ERK1 that is regulated differently than other ERK isoforms. We studied the Golgi functions of ERK1c and found that it plays a role in MEK-induced mitotic Golgi fragmentation. Thus, in late G2 and mitosis of synchronized cells, the expression and activity of ERK1c was increased and it colocalized mainly with Golgi markers. Small interfering RNA of ERK1c significantly attenuated, whereas ERK1c overexpression facilitated, mitotic Golgi fragmentation. These effects were also reflected in mitotic progression, indicating that ERK1c is involved in cell cycle regulation via modulation of Golgi fragmentation. Although ERK1 was activated in mitosis as well, it could not replace ERK1c in regulating Golgi fragmentation. Therefore, MEKs regulate mitosis via all three ERK isoforms, where ERK1c acts specifically in the Golgi, whereas ERK1 and 2 regulate other mitosis-related processes. Thus, ERK1c extends the specificity of the Ras-MEK cascade by activating ERK1/2-independent processes.

scholarly journals Organization of spindle microtubules in Ochromonas danica.

1980 ◽  
Vol 87 (3) ◽  
pp. 531-545 ◽  
Author(s):  
D H Tippit ◽  
L Pillus ◽  
J Pickett-Heaps
Keyword(s):  
Average Length ◽  
Numerical Data ◽  
Tracking Errors ◽  
Mitotic Apparatus ◽  
Ochromonas Danica ◽  
Spindle Poles ◽  
Late Anaphase ◽  
Early Anaphase ◽  
Spindle Microtubules ◽  
Spindle Elongation

The entire framework of microtubules (MTs) in the mitotic apparatus of Ochromonas danica is reconstructed (except at the spindle poles) from transverse serial sections. Eleven spindles were sectioned and used for numerical data, but only four were reconstructed: a metaphase, an early anaphase, a late anaphase, and telophase. Four major classes of MTs are observed: (a) free MTs (MTs not attached to either pole); (b) interdigitated MTs (MTs attached to one pole which laterally associate with MTs from the opposite pole); (c) polar MTs (MTs attached to one pole); (d) kinetochore MTs (kMTs). Pole-to-pole MTs are rare and may be caused by tracking errors. During anaphase, the kMTs, free MTs, and polar MTs shorten until most disappear, while interdigitated MTs lengthen. In the four reconstructed spindles, the number of MTs decreases between early anaphase and telophase from 881 to 285, while their average length increases from 1.66 to 4.98 micron. The total length of all the MTs in the spindle (placed end to end) remains at 1.42 +/- 0.04 mm between these stages. At late anaphase and telophase the spindle is comprised mainly of groups of interdigitated MTs. Such MTs from opposite poles form a region of overlap in the middle of the spindle. During spindle elongation (separation of the poles), the length of the overlap region does not decrease. These results are compatible with theories that suggest that MTs directly provide the force that elongates the spindle, either by MT polymerization alone or by MT sliding with concomitant MT polymerization.

scholarly journals ZYG-9, A Caenorhabditis elegans Protein Required for Microtubule Organization and Function, Is a Component of Meiotic and Mitotic Spindle Poles

1998 ◽  
Vol 141 (5) ◽  
pp. 1159-1168 ◽  
Author(s):  
Lisa R. Matthews ◽  
Philip Carter ◽  
Danielle Thierry-Mieg ◽  
Ken Kemphues
Keyword(s):  
Caenorhabditis Elegans ◽  
Mitotic Spindle ◽  
Meiotic Spindle ◽  
Microtubule Organization ◽  
Mitotic Apparatus ◽  
Meiotic Chromosomes ◽  
Spindle Poles ◽  
Common Activity ◽  
Early Anaphase ◽  
And Function

We describe the molecular characterization of zyg-9, a maternally acting gene essential for microtubule organization and function in early Caenorhabditis elegans embryos. Defects in zyg-9 mutants suggest that the zyg-9 product functions in the organization of the meiotic spindle and the formation of long microtubules. One-cell zyg-9 embryos exhibit both meiotic and mitotic spindle defects. Meiotic spindles are disorganized, pronuclear migration fails, and the mitotic apparatus forms at the posterior, orients incorrectly, and contains unusually short microtubules. We find that zyg-9 encodes a component of the meiotic and mitotic spindle poles. In addition to the strong staining of spindle poles, we consistently detect staining in the region of the kinetochore microtubules at metaphase and early anaphase in mitotic spindles. The ZYG-9 signal at the mitotic centrosomes is not reduced by nocodazole treatment, indicating that ZYG-9 localization to the mitotic centrosomes is not dependent upon long astral microtubules. Interestingly, in embryos lacking an organized meiotic spindle, produced either by nocodazole treatment or mutations in the mei-1 gene, ZYG-9 forms a halo around the meiotic chromosomes. The protein sequence shows partial similarity to a small set of proteins that also localize to spindle poles, suggesting a common activity of the proteins.

scholarly journals Mutual Dependence of Mob1 and the Chromosomal Passenger Complex for Localization during Mitosis

2010 ◽  
Vol 21 (3) ◽  
pp. 380-392 ◽  
Author(s):  
Lori Jo Wilmeth ◽  
Sanjay Shrestha ◽  
Gilbert Montaño ◽  
Jennifer Rashe ◽  
Charles Bradley Shuster
Keyword(s):  
Mitotic Exit ◽  
Signaling Cascades ◽  
Mitotic Progression ◽  
Chromosomal Passenger Complex ◽  
Related Family ◽  
Cytoplasmic Material ◽  
Spindle Poles ◽  
Chromosomal Passenger ◽  
Early Anaphase ◽  
Spindle Midzone

The spatial and temporal coordination of chromosome segregation with cytokinesis is essential to ensure that each daughter cell receives the correct complement of chromosomal and cytoplasmic material. In yeast, mitotic exit and cytokinesis are coordinated by signaling cascades whose terminal components include a nuclear Dbf2-related family kinase and a noncatalytic subunit, Mps one binding (Mob) 1. There are five human Mob1 isoforms, all of which display redundant localization patterns at the spindle poles and kinetochores in early mitosis, and the spindle midzone during cytokinesis. Mob1 shares similar localization patterns to Polo-like kinase (Plk1) and the chromosomal passenger complex (CPC), and although depletion of Plk1 resulted in a loss of Mob1 from the spindle poles, Mob1 recruitment to kinetochores was unaffected. Conversely, disruption of CPC signaling resulted in a loss of Mob1 from kinetochores without disrupting recruitment to the spindle poles. In Mob1-depleted cells, the relocalization of the CPC and mitotic kinesin-like protein (MKLP) 2 to the spindle midzone was delayed during early anaphase, and as a consequence, the midzone recruitment of MKLP1 also was affected. Together, these results suggest that Mob1 and the other mammalian orthologues of the mitotic exit network regulate mitotic progression by facilitating the timely mobilization of the CPC to the spindle midzone.

scholarly journals Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission

2013 ◽  
Vol 24 (18) ◽  
pp. 2785-2794 ◽  
Author(s):  
Matthew K. Martz ◽  
Elda Grabocka ◽  
Neil Beeharry ◽  
Timothy J. Yen ◽  
Philip B. Wedegaertner
Keyword(s):  
Small Interfering Rna ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Aurora B ◽  
Intercellular Bridge ◽  
Concerted Effort ◽  
Central Spindle ◽  
Daughter Cells ◽  
Spindle Poles ◽  
Interfering Rna

Proper completion of mitosis requires the concerted effort of multiple RhoGEFs. Here we show that leukemia-associated RhoGEF (LARG), a RhoA-specific RGS-RhoGEF, is required for abscission, the final stage of cytokinesis, in which the intercellular membrane is cleaved between daughter cells. LARG colocalizes with α-tubulin at the spindle poles before localizing to the central spindle. During cytokinesis, LARG is condensed in the midbody, where it colocalizes with RhoA. HeLa cells depleted of LARG display apoptosis during cytokinesis with unresolved intercellular bridges, and rescue experiments show that expression of small interfering RNA–resistant LARG prevents this apoptosis. Moreover, live cell imaging of LARG-depleted cells reveals greatly delayed fission kinetics in abscission in which a population of cells with persistent bridges undergoes apoptosis; however, the delayed fission kinetics is rescued by Aurora-B inhibition. The formation of a Flemming body and thinning of microtubules in the intercellular bridge of cells depleted of LARG is consistent with a defect in late cytokinesis, just before the abscission event. In contrast to studies of other RhoGEFs, particularly Ect2 and GEF-H1, LARG depletion does not result in cytokinetic furrow regression nor does it affect internal mitotic timing. These results show that LARG is a novel and temporally distinct RhoGEF required for completion of abscission.

scholarly journals Regulation of mitosis by poly(ADP-ribosyl)ation

2005 ◽  
Vol 391 (2) ◽  
Author(s):  
Duane A. Compton
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Protein A ◽  
Mitotic Apparatus ◽  
Spindle Poles ◽  
Biochemical Journal ◽  
Dividing Cells ◽  
Cross Links ◽  
Tankyrase 1 ◽  
The Impact

The spindle is a dynamic, microtubule-based structure responsible for chromosome segregation during cell division. Spindles in mammalian cells contain several thousand microtubules that are arranged into highly symmetric bipolar arrays by the actions of numerous microtubule-associated motor and non-motor proteins. In addition to these protein constituents, recent work has demonstrated that poly(ADP-ribose) is a key spindle component. Of the multitude of poly(ADP-ribose) polymerase proteins encoded in the genome, tankyrase 1 appears to be the primary enzyme responsible for building poly(ADP-ribose) in spindles during mitosis. In this issue of the Biochemical Journal, Susan Smith and co-workers show that the primary target of tankyrase 1 in dividing cells is NuMA (nuclear mitotic apparatus protein), a protein that cross-links microtubule ends at spindle poles. The impact of poly(ADP-ribosyl)ation on the biochemical function of NuMA remains murky at this time, but these new results represent the first step to clearing the view as to how poly(ADP-ribosyl)ation regulates cell division.

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