scholarly journals Microtubules assemble near most kinetochores during early prometaphase in human cells

2018 ◽  
Vol 217 (8) ◽  
pp. 2647-2659 ◽  
Author(s):  
Vitali Sikirzhytski ◽  
Fioranna Renda ◽  
Irina Tikhonenko ◽  
Valentin Magidson ◽  
Bruce F. McEwen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
Spindle Assembly ◽  
Human Cells ◽  
Spindle Poles

For proper segregation during cell division, each chromosome must connect to the poles of the spindle via microtubule bundles termed kinetochore fibers (K-fibers). K-fibers form by two distinct mechanisms: (1) capture of astral microtubules nucleated at the centrosome by the chromosomes’ kinetochores or (2) attachment of kinetochores to noncentrosomal microtubules with subsequent transport of the minus ends of these microtubules toward the spindle poles. The relative contributions of these alternative mechanisms to normal spindle assembly remain unknown. In this study, we report that most kinetochores in human cells develop K-fibers via the second mechanism. Correlative light electron microscopy demonstrates that from the onset of spindle assembly, short randomly oriented noncentrosomal microtubules appear in the immediate vicinity of the kinetochores. Initially, these microtubules interact with the kinetochores laterally, but end-on attachments form rapidly in the first 3 min of prometaphase. Conversion from lateral to end-on interactions is impeded upon inhibition of the plus end–directed kinetochore-associated kinesin CenpE.

scholarly journals The 3D architecture and molecular foundations of de novo centriole assembly via bicentrioles

2020 ◽  
Author(s):  
Sónia Gomes Pereira ◽  
Ana Laura Sousa ◽  
Catarina Nabais ◽  
Tiago Paixão ◽  
Alexander. J. Holmes ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
De Novo ◽  
Cellular Functions ◽  
Spindle Poles ◽  
3D Architecture ◽  
Centriole Assembly ◽  
Work Supports ◽  
Wall Components

Abstract/SummaryCentrioles are structurally conserved organelles, composing both centrosomes and cilia. In animal cycling cells, centrioles often form through a highly characterized process termed canonical duplication. However, a large diversity of eukaryotes form centrioles de novo through uncharacterized pathways. This unexplored diversity is key to understanding centriole assembly mechanisms and how they evolved to assist specific cellular functions. Here, combining electron microscopy and tomography, we show that during spermatogenesis of the moss Physcomitrium patens, centrioles are born as a co-axially oriented centriole pair united by a cartwheel. We observe that microtubules emanate from those bicentrioles, which localize to the spindle poles during cell division. Thereafter, each bicentriole breaks apart, and the two resulting sister centrioles mature asymmetrically, elongating specific microtubule triplets and a naked cartwheel. Subsequently, two cilia are assembled which are capable of beating asynchronously. We further show that conserved cartwheel and centriole wall components, SAS6, BLD10 and POC1 are expressed during spermatogenesis and are required for this de novo biogenesis pathway. Our work supports a scenario where centriole biogenesis is more diverse than previously thought and that conserved molecular modules underlie diversification of this essential pathway.

scholarly journals Poleward transport of Eg5 by dynein–dynactin in Xenopus laevis egg extract spindles

2008 ◽  
Vol 182 (4) ◽  
pp. 715-726 ◽  
Author(s):  
Marianne Uteng ◽  
Christian Hentrich ◽  
Kota Miura ◽  
Peter Bieling ◽  
Thomas Surrey
Keyword(s):  
Cell Division ◽  
Xenopus Laevis ◽  
Molecular Motors ◽  
Motor Proteins ◽  
Motor Protein ◽  
Spindle Assembly ◽  
Cross Linking ◽  
Motor Movement ◽  
Spindle Poles ◽  
Egg Extract

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein–dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization.

scholarly journals SART1 localizes to spindle poles forming a SART1 cap and promotes centrosome and spindle assembly

2021 ◽  
Author(s):  
Hideki Yokoyama ◽  
Kaoru Takizawa ◽  
Jian Ma ◽  
Daniel Moreno-Andrés ◽  
Wolfram Antonin ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Physiological Function ◽  
Spindle Assembly ◽  
Scaffold Protein ◽  
Human Cells ◽  
Dependent Manner ◽  
Normal Cells ◽  
Spindle Poles ◽  
Egg Extracts ◽  
Centrosomal Proteins

Abstract SART1 is overexpressed in various cancers. However, its physiological function and cancer relevance remains elusive. Here we identify SART1 as a mitotic-specific and Ran-regulated microtubule-associated protein. SART1 downregulation in human cells as well as its depletion from frog egg extracts disrupts spindle assembly. While SART1 is nuclear in interphase, it localizes during mitosis to spindle poles in a microtubule-dependent manner. SART1 accumulates close to centrosomes forming a half circle which we designate as SART1 cap. Immunoprecipitation of SART1 identifies the centrosome scaffold protein Cep192 as an interaction partner. Accordingly, Cep192 downregulation abolishes SART1 localization to spindle poles, and SART1 downregulation displaces centrosomal proteins like Ninein from centrosomes, but does not affect γ-tubulin localization. Furthermore, SART1 downregulation selectively kills cancer cells and prevents normal cells from oncogenic transformation. Our data unravel a novel function of SART1 for centrosome organization and spotlight SRAT1 as a potential target for anticancer therapies.

Asymmetric cell division in fucoid algae: a role for cortical adhesions in alignment of the mitotic apparatus

2001 ◽  
Vol 114 (23) ◽  
pp. 4319-4328
Author(s):  
Sherryl R. Bisgrove ◽  
Darryl L. Kropf
Keyword(s):  
Cell Division ◽  
Asymmetric Cell Division ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Pharmacological Agents ◽  
Division Plane ◽  
Adhesion Sites ◽  
Spindle Poles ◽  
Pelvetia Compressa ◽  
Two Phases

The first cell division in zygotes of the fucoid brown alga Pelvetia compressa is asymmetric and we are interested in the mechanism controlling the alignment of this division. Since the division plane bisects the mitotic apparatus, we investigated the timing and mechanism of spindle alignments. Centrosomes, which give rise to spindle poles, aligned with the growth axis in two phases – a premetaphase rotation of the nucleus and centrosomes followed by a postmetaphase alignment that coincided with the separation of the mitotic spindle poles during anaphase and telophase. The roles of the cytoskeleton and cell cortex in the two phases of alignment were analyzed by treatment with pharmacological agents. Treatments that disrupted cytoskeleton or perturbed cortical adhesions inhibited pre-metaphase alignment and we propose that this rotational alignment is effected by microtubules anchored at cortical adhesion sites. Postmetaphase alignment was not affected by any of the treatments tested, and may be dependent on asymmetric cell morphology.

scholarly journals Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation

2015 ◽  
Vol 35 (15) ◽  
pp. 2626-2640 ◽  
Author(s):  
Lingjun Meng ◽  
Jung-Eun Park ◽  
Tae-Sung Kim ◽  
Eun Hye Lee ◽  
Suk-Youl Park ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Spindle Assembly ◽  
Human Cells ◽  
Mitotic Progression ◽  
Spindle Formation ◽  
Content Type ◽  
Bipolar Spindle ◽  
Egg Extracts ◽  
Cultured Human Cells ◽  
Bipolar Spindles

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study withXenopus laevisegg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif ofXenopusCep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous toXenopusp-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.

scholarly journals Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly

2013 ◽  
Vol 24 (20) ◽  
pp. 3238-3250 ◽  
Author(s):  
Elena Nazarova ◽  
Eileen O'Toole ◽  
Susi Kaitna ◽  
Paul Francois ◽  
Mark Winey ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Dynamical Behavior ◽  
Three Dimensional ◽  
Spindle Assembly ◽  
Intermediate Step ◽  
High Angle ◽  
Normal Number ◽  
Spindle Poles ◽  
Evolutionarily Conserved ◽  
Microtubule Formation

During spindle assembly, microtubules may attach to kinetochores or pair to form antiparallel pairs or interpolar microtubules, which span the two spindle poles and contribute to mitotic pole separation and chromosome segregation. Events in the specification of the interpolar microtubules are poorly understood. Using three-dimensional electron tomography and analysis of spindle dynamical behavior in living cells, we investigated the process of spindle assembly. Unexpectedly, we found that the phosphorylation state of an evolutionarily conserved Cdk1 site (S360) in γ-tubulin is correlated with the number and organization of interpolar microtubules. Mimicking S360 phosphorylation (S360D) results in bipolar spindles with a normal number of microtubules but lacking interpolar microtubules. Inhibiting S360 phosphorylation (S360A) results in spindles with interpolar microtubules and high-angle, antiparallel microtubule pairs. The latter are also detected in wild-type spindles <1 μm in length, suggesting that high-angle microtubule pairing represents an intermediate step in interpolar microtubule formation. Correlation of spindle architecture with dynamical behavior suggests that microtubule pairing is sufficient to separate the spindle poles, whereas interpolar microtubules maintain the velocity of pole displacement during early spindle assembly. Our findings suggest that the number of interpolar microtubules formed during spindle assembly is controlled in part through activities at the spindle poles.

scholarly journals Kinesin molecular motor Eg5 functions during polypeptide synthesis

2011 ◽  
Vol 22 (18) ◽  
pp. 3420-3430 ◽  
Author(s):  
Kristen M. Bartoli ◽  
Jelena Jakovljevic ◽  
John L. Woolford ◽  
William S. Saunders
Keyword(s):  
Cell Division ◽  
Molecular Motor ◽  
Spindle Assembly ◽  
Intact Cells ◽  
Nascent Polypeptide ◽  
Polypeptide Synthesis

The kinesin-related molecular motor Eg5 plays roles in cell division, promoting spindle assembly. We show that during interphase Eg5 is associated with ribosomes and is required for optimal nascent polypeptide synthesis. When Eg5 was inhibited, ribosomes no longer bound to microtubules in vitro, ribosome transit rates slowed, and polysomes accumulated in intact cells, suggesting defects in elongation or termination during polypeptide synthesis. These results demonstrate that the molecular motor Eg5 associates with ribosomes and enhances the efficiency of translation.

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