scholarly journals APC and EB1 Function Together in Mitosis to Regulate Spindle Dynamics and Chromosome Alignment

2005 ◽  
Vol 16 (10) ◽  
pp. 4609-4622 ◽  
Author(s):  
Rebecca A. Green ◽  
Roy Wollman ◽  
Kenneth B. Kaplan
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Functional Relationship ◽  
Microtubule Dynamics ◽  
Dominant Negative ◽  
Mitotic Spindles ◽  
Spindle Dynamics ◽  
Chromosome Alignment ◽  
Spindle Function ◽  
Insight Into

Recently, we have shown that a cancer causing truncation in adenomatous polyposis coli (APC) (APC1–1450) dominantly interferes with mitotic spindle function, suggesting APC regulates microtubule dynamics during mitosis. Here, we examine the possibility that APC mutants interfere with the function of EB1, a plus-end microtubule-binding protein that interacts with APC and is required for normal microtubule dynamics. We show that siRNA-mediated inhibition of APC, EB1, or APC and EB1 together give rise to similar defects in mitotic spindles and chromosome alignment without arresting cells in mitosis; in contrast inhibition of CLIP170 or LIS1 cause distinct spindle defects and mitotic arrest. We show that APC1–1450 acts as a dominant negative by forming a hetero-oligomer with the full-length APC and preventing it from interacting with EB1, which is consistent with a functional relationship between APC and EB1. Live-imaging of mitotic cells expressing EB1-GFP demonstrates that APC1–1450 compromises the dynamics of EB1-comets, increasing the frequency of EB1-GFP pausing. Together these data provide novel insight into how APC may regulate mitotic spindle function and how errors in chromosome segregation are tolerated in tumor cells.

scholarly journals Spatial regulation of astral microtubule dynamics by Kif18B in PtK cells

2016 ◽  
Vol 27 (20) ◽  
pp. 3021-3030 ◽  
Author(s):  
Claire E. Walczak ◽  
Hailing Zong ◽  
Sachin Jain ◽  
Jane R. Stout
Keyword(s):  
Chromosome Segregation ◽  
Microtubule Dynamics ◽  
Cell Cortex ◽  
Microtubule Organization ◽  
Spatial Regulation ◽  
Astral Microtubule ◽  
Chromosome Alignment ◽  
Spindle Microtubules ◽  
Kinesin Superfamily ◽  
Insight Into

The spatial and temporal control of microtubule dynamics is fundamentally important for proper spindle assembly and chromosome segregation. This is achieved, in part, by the multitude of proteins that bind to and regulate spindle microtubules, including kinesin superfamily members, which act as microtubule-destabilizing enzymes. These fall into two general classes: the kinesin-13 proteins, which directly depolymerize microtubules, and the kinesin-8 proteins, which are plus end–directed motors that either destabilize microtubules or cap the microtubule plus ends. Here we analyze the contribution of a PtK kinesin-8 protein, Kif18B, in the control of mitotic microtubule dynamics. Knockdown of Kif18B causes defects in spindle microtubule organization and a dramatic increase in astral microtubules. Kif18B-knockdown cells had defects in chromosome alignment, but there were no defects in chromosome segregation. The long astral microtubules that occur in the absence of Kif18B are limited in length by the cell cortex. Using EB1 tracking, we show that Kif18B activity is spatially controlled, as loss of Kif18B has the most dramatic effect on the lifetimes of astral microtubules that extend toward the cell cortex. Together our studies provide new insight into how diverse kinesins contribute to spatial microtubule organization in the spindle.

scholarly journals A Minus-End–directed Kinesin with Plus-End Tracking Protein Activity Is Involved in Spindle Morphogenesis

2005 ◽  
Vol 16 (4) ◽  
pp. 1584-1592 ◽  
Author(s):  
J. Christian Ambrose ◽  
Wuxing Li ◽  
Adam Marcus ◽  
Hong Ma ◽  
Richard Cyr
Keyword(s):  
Motor Activity ◽  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Null Mutant ◽  
Mitotic Spindles ◽  
Protein Activity ◽  
Interphase Cells ◽  
Motor Activities ◽  
Spindle Function

Diverse kinesin motor proteins are involved in spindle function; however, the mechanisms by which they are targeted to specific sites within spindles are not well understood. Here, we show that a fusion between yellow fluorescent protein (YFP) and a minus-end–directed Kinesin-14 (C-terminal family) from Arabidopsis, ATK5, localizes to mitotic spindle midzones and regions rich in growing plus-ends within phragmoplasts. Notably, in Arabidopsis interphase cells, YFP::ATK5 localizes to microtubules with a preferential enrichment at growing plus-ends; indicating ATK5 is a plus-end tracking protein (+TIP). This +TIP activity is conferred by regions outside of the C-terminal motor domain, which reveals the presence of independent plus-end tracking and minus-end motor activities within ATK5. Furthermore, mitotic spindles of atk5 null mutant plants are abnormally broadened. Based on these data, we propose a model in which ATK5 uses plus-end tracking to reach spindle midzones, where it then organizes microtubules via minus-end–directed motor activity.

Regulation of Microtubule Assembly: The View From The End

2000 ◽  
Vol 6 (S2) ◽  
pp. 80-81
Author(s):  
L. Cassimeris ◽  
C. Spittle ◽  
M. Kratzer
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Dynamic Instability ◽  
Intrinsic Property ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
Chromosome Movement ◽  
Spindle Formation ◽  
Associated Proteins

The mitotic spindle is responsible for chromosome movement during mitosis. It is composed of a dynamic array of microtubules and associated proteins whose assembly and constant turnover are required for both spindle formation and chromosome movement. Because microtubule assembly and turnover are necessary for chromosome segregation, we are studying how cells regulate microtubule dynamics. Microtubules are polarized polymers composed of tubulin subunits; they assemble by a process of dynamic instability where individual microtubules exist in persistent phases of elongation or rapid shortening with abrupt transitions between these two states. The switch from elongation to shortening is termed catastrophe, and the switch from shortening to elongation, rescue. Although dynamic instability is an intrinsic property of the tubulin subunits, cells use associated proteins to both speed elongation (∼ 10 fold) and regulate transitions.The only protein isolated to date capable of promoting fast polymerization consistent with rates in vivo is XMAP215, a 215 kD protein from Xenopus eggs.

scholarly journals A quantitative analysis of cohesin decay in mitotic fidelity

2018 ◽  
Vol 217 (10) ◽  
pp. 3343-3353 ◽  
Author(s):  
Sara Carvalhal ◽  
Alexandra Tavares ◽  
Mariana B. Santos ◽  
Mihailo Mirkovic ◽  
Raquel A. Oliveira
Keyword(s):  
Quantitative Analysis ◽  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Mitotic Spindle ◽  
Sister Chromatid Cohesion ◽  
Force Balance ◽  
Chromosome Organization ◽  
Centromeric Chromatin ◽  
Chromatid Cohesion ◽  
Chromosome Alignment

Sister chromatid cohesion mediated by cohesin is essential for mitotic fidelity. It counteracts spindle forces to prevent premature chromatid individualization and random genome segregation. However, it is unclear what effects a partial decline of cohesin may have on chromosome organization. In this study, we provide a quantitative analysis of cohesin decay by inducing acute removal of defined amounts of cohesin from metaphase-arrested chromosomes. We demonstrate that sister chromatid cohesion is very resistant to cohesin loss as chromatid disjunction is only observed when chromosomes lose >80% of bound cohesin. Removal close to this threshold leads to chromosomes that are still cohered but display compromised chromosome alignment and unstable spindle attachments. Partial cohesin decay leads to increased duration of mitosis and susceptibility to errors in chromosome segregation. We propose that high cohesin density ensures centromeric chromatin rigidity necessary to maintain a force balance with the mitotic spindle. Partial cohesin loss may lead to chromosome segregation errors even when sister chromatid cohesion is fulfilled.

scholarly journals Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells

1999 ◽  
Vol 10 (4) ◽  
pp. 947-959 ◽  
Author(s):  
Anne-Marie C. Yvon ◽  
Patricia Wadsworth ◽  
Mary Ann Jordan
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Dynamic Instability ◽  
Human Tumor ◽  
Mitotic Checkpoint ◽  
Microtubule Dynamics ◽  
Mitotic Spindles ◽  
Ovarian Adenocarcinoma ◽  
Adenocarcinoma Cells

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, inliving cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30–100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.

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.

A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4391-4402 ◽  
Author(s):  
J.M. Schumacher ◽  
N. Ashcroft ◽  
P.J. Donovan ◽  
A. Golden
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Centrosomal Protein ◽  
C Elegans ◽  
Developmental Factors ◽  
Centrosome Separation ◽  
Spindle Dynamics ◽  
Bipolar Spindles

S. cerevisiae Ipl1, Drosophila Aurora, and the mammalian centrosomal protein IAK-1 define a new subfamily of serine/threonine kinases that regulate chromosome segregation and mitotic spindle dynamics. Mutations in ipl1 and aurora result in the generation of severely aneuploid cells and, in the case of aurora, monopolar spindles arising from a failure in centrosome separation. Here we show that a related, essential protein from C. elegans, AIR-1 (Aurora/Ipl1 related), is localized to mitotic centrosomes. Disruption of AIR-1 protein expression in C. elegans embryos results in severe aneuploidy and embryonic lethality. Unlike aurora mutants, this aneuploidy does not arise from a failure in centrosome separation. Bipolar spindles are formed in the absence of AIR-1, but they appear to be disorganized and are nucleated by abnormal-looking centrosomes. In addition to its requirement during mitosis, AIR-1 may regulate microtubule-based developmental processes as well. Our data suggests AIR-1 plays a role in P-granule segregation and the association of the germline factor PIE-1 with centrosomes.

scholarly journals Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

1995 ◽  
Vol 129 (6) ◽  
pp. 1617-1628 ◽  
Author(s):  
R M Golsteyn ◽  
K E Mundt ◽  
A M Fry ◽  
E A Nigg
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Subcellular Localization ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Laser Scanning Microscopy ◽  
Human Protein ◽  
Vitro Assay ◽  
Human Protein Kinase ◽  
Spindle Function

Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

scholarly journals Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

2016 ◽  
Vol 27 (22) ◽  
pp. 3490-3514 ◽  
Author(s):  
Zachary R. Gergely ◽  
Ammon Crapo ◽  
Loren E. Hough ◽  
J. Richard McIntosh ◽  
Meredith D. Betterton
Keyword(s):  
Fission Yeast ◽  
Microtubule Dynamics ◽  
Chromosome Loss ◽  
Mitotic Spindles ◽  
Aberrant Chromosome ◽  
Large Fluctuations ◽  
Spindle Function ◽  
Chromosome Movements ◽  
Sliding Force

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen.

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