scholarly journals Mechanisms of MTOC clustering and spindle positioning in the budding yeast Cryptococcus neoformans: a microtubule grow-and-catch model

2019 ◽  
Author(s):  
Saptarshi Chatterjee ◽  
Subhendu Som ◽  
Neha Varshney ◽  
Kaustuv Sanyal ◽  
Raja Paul
Keyword(s):  
Nuclear Envelope ◽  
Cryptococcus Neoformans ◽  
Mitotic Spindle ◽  
Computational Models ◽  
Budding Yeast ◽  
Cell Cortex ◽  
Critical Determinant ◽  
Spindle Formation ◽  
Spindle Positioning ◽  
Septin Ring

AbstractMitotic spindle formation in the pathogenic budding yeast, Cryptococcus neoformans, depends on multitudes of inter-dependent interactions involving kinetochores (KTs), microtubules (MTs), spindle pole bodies (SPBs), and molecular motors. Before the formation of the mitotic spindle, multiple visible microtubule organizing centers (MTOCs), coalesce into a single focus to serve as an SPB. We propose a ‘grow-and-catch’ model, in which cytoplasmic MTs (cMTs) nucleated by MTOCs grow and catch each other to promote MTOC clustering. Our quantitative modeling identifies multiple redundant mechanisms mediated by a combination of cMT-cell cortex interactions and inter-cMT coupling to facilitate MTOC clustering within the physiological time limit as determined by time-lapse live-cell microscopy. Besides, we screened various possible mechanisms by computational modeling and propose optimal conditions that favor proper spindle positioning - a critical determinant for timely chromosome segregation. These analyses also reveal that a combined effect of MT buckling, dynein pull, and cortical push maintain spatiotemporal spindle localization.Author summaryCells actively self-assemble a bipolar spindle to facilitate chromosomal segregation. Multiple MTOCs, on the outer nuclear envelope, cluster into a single SPB before spindle formation during semi-open mitosis of the budding yeast Cryptococcus neoformans. Eventually, the SPB duplicates and organizes the spindle to position it within the daughter bud near the septin ring during anaphase. In this work, we tested various computational models to match physiological phenomena in an attempt to find plausible mechanisms of MTOC clustering and spindle positioning in C. neoformans. Notably, we propose an MT ‘grow-and-catch’ model that relies on possible redundant mechanisms for timely MTOC clustering mediated by (a) minus end-directed motors that crosslink and slide anti-parallel cMTs from different MTOCs on the nuclear envelope and (b) a Bim1 mediated biased sliding of cMTs along the cell cortex toward the septin ring that pulls MTOCs in the presence of suppressed dynein activity. By combining an analytical model and stochastic MT dynamics simulations, we screened various MT-based forces to detect steady spindle positioning. By screening the outputs of various models, it is revealed that proper spindle positioning near the septin ring requires MT buckling from the cell cortex.

scholarly journals Mitochondria-driven assembly of a cortical anchor for mitochondria and dynein

2017 ◽  
Vol 216 (10) ◽  
pp. 3061-3071 ◽  
Author(s):  
Lauren M. Kraft ◽  
Laura L. Lackner
Keyword(s):  
Plasma Membrane ◽  
Mitotic Spindle ◽  
Cell Cortex ◽  
Dual Function ◽  
Mitochondrial Inheritance ◽  
Cellular Functions ◽  
Core Component ◽  
Spindle Positioning ◽  
The Core ◽  
Contact Sites

Interorganelle contacts facilitate communication between organelles and impact fundamental cellular functions. In this study, we examine the assembly of the MECA (mitochondria–endoplasmic reticulum [ER]–cortex anchor), which tethers mitochondria to the ER and plasma membrane. We find that the assembly of Num1, the core component of MECA, requires mitochondria. Once assembled, Num1 clusters persistently anchor mitochondria to the cell cortex. Num1 clusters also function to anchor dynein to the plasma membrane, where dynein captures and walks along astral microtubules to help orient the mitotic spindle. We find that dynein is anchored by Num1 clusters that have been assembled by mitochondria. When mitochondrial inheritance is inhibited, Num1 clusters are not assembled in the bud, and defects in dynein-mediated spindle positioning are observed. The mitochondria-dependent assembly of a dual-function cortical anchor provides a mechanism to integrate the positioning and inheritance of the two essential organelles and expands the function of organelle contact sites.

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 Ordered Kinetochore Assembly in the Human-Pathogenic Basidiomycetous Yeast Cryptococcus neoformans

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Lukasz Kozubowski ◽  
Vikas Yadav ◽  
Gautam Chatterjee ◽  
Shreyas Sridhar ◽  
Masashi Yamaguchi ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Cryptococcus Neoformans ◽  
Budding Yeast ◽  
Genetic Material ◽  
Time Lapse ◽  
Content Type ◽  
Nuclear Envelope Breakdown ◽  
Kinetochore Assembly ◽  
Transmission Electron ◽  
Partial Opening

ABSTRACT Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes. IMPORTANCE Successful propagation of genetic material in progeny is essential for the survival of any organism. A proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process can lead to loss or gain of chromosomes, a common feature of most solid cancers. Several proteins assemble on centromere DNA to form a kinetochore. However, significant differences in the process of kinetochore assembly exist between unicellular yeasts and multicellular metaozoa. Here, we examined the key events that lead to formation of a proper kinetochore in a basidiomycetous budding yeast, Cryptococcus neoformans. We found that, during the progression of the cell cycle, nonclustered centromeres gradually clustered and kinetochores assembled in an ordered manner concomitant with partial opening of the nuclear envelope in this organism. These events have higher similarity to mitotic events of metazoans than to those previously described in other yeasts.

scholarly journals Cdk1-dependent destabilization of long astral microtubules is required for spindle orientation

2020 ◽  
Author(s):  
Divya Singh ◽  
Nadine Schmidt ◽  
Franziska Müller ◽  
Tanja Bange ◽  
Alexander W. Bird
Keyword(s):  
Mitotic Spindle ◽  
Cell Shape ◽  
Microtubule Dynamics ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Tissue Organization ◽  
Microtubule Stability ◽  
Astral Microtubule ◽  
Early Mitosis

AbstractThe precise execution of mitotic spindle orientation in response to cell shape cues is important for tissue organization and development. The presence of astral microtubules extending from the centrosome towards the cell cortex is essential for this process, but little is understood about the contribution of astral microtubule dynamics to spindle positioning, or how astral microtubule dynamics are regulated spatiotemporally. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells transition from interphase to mitosis, but how Cdk1 activity specifically modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules to ensure spindle reorientation in response to cell shape. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus-ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus-ends in mitosis. This decreases the catastrophe frequency of astral microtubules, and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules must thus not only be present, but also dynamic to allow the spindle to reorient in response to cell shape, a state achieved by selective destabilization of long astral microtubules via Cdk1.

scholarly journals Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast

2020 ◽  
Author(s):  
Safia Omer ◽  
Katia Brock ◽  
John Beckford ◽  
Wei-Lih Lee
Keyword(s):  
Budding Yeast ◽  
Current Model ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Direct Imaging ◽  
Spindle Positioning ◽  
Sliding Mechanism ◽  
Pulling Forces ◽  
Pulling Force

ABSTRACTCurrent model for spindle positioning requires attachment of the microtubule (MT) motor cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect spindle displacement. How dynein is anchored by cortical attachment machinery to generate large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the yeast dynein attachment molecule, is limited by Mdm36. Overexpression of Mdm36 results in an overall enhancement of Num1 clustering but reveals a population of dim Num1 clusters that mediate dynein-anchoring at the cell cortex. Direct imaging shows that bud-localized, dim Num1 clusters containing only ∼6 copies of Num1 molecules mediate dynein-dependent spindle pulling via lateral MT sliding mechanism. Mutations affecting Num1 clustering interfere with mitochondrial tethering but not dynein-based spindle-pulling function of Num1. We propose that formation of small ensembles of attachment molecules is sufficient for dynein anchorage and cortical generation of large spindle-pulling force.

Targeting of Ran: variation on a common theme?

2001 ◽  
Vol 114 (18) ◽  
pp. 3233-3241 ◽  
Author(s):  
Markus Künzler ◽  
Ed Hurt
Keyword(s):  
Nuclear Envelope ◽  
Mitotic Spindle ◽  
Nucleocytoplasmic Transport ◽  
Common Theme ◽  
Spindle Formation ◽  
Ran Gtpase ◽  
Cellular Processes ◽  
Important Target ◽  
Transport Receptors ◽  
Nuclear Envelope Formation

The Ran GTPase plays a key role in nucleocytoplasmic transport. In its GTP-bound form, it directly interacts with members of the importin β family of nuclear transport receptors and modulates their association with cargo. Work in cell-free higher-eukaryote systems has demonstrated additional roles for Ran in spindle and nuclear envelope formation during mitosis. However, until recently, no Ran-target proteins in these cellular processes were known. Several groups have now identified importin β as one important target of Ran during mitotic spindle formation. This finding suggests that Ran uses the same effectors to regulate different cellular processes.

Time-resolved, in vivo studies of mitotic spindle formation and nuclear lamina breakdown in Drosophila early embryos

1996 ◽  
Vol 109 (3) ◽  
pp. 591-607 ◽  
Author(s):  
M.R. Paddy ◽  
H. Saumweber ◽  
D.A. Agard ◽  
J.W. Sedat
Keyword(s):  
Nuclear Envelope ◽  
Mitotic Spindle ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
In Vivo Studies ◽  
Time Interval ◽  
Microscopy Imaging ◽  
Spindle Formation ◽  
Time Resolved ◽  
Early Embryos

Time-resolved, two-component, three-dimensional fluorescence light microscopy imaging in living Drosophila early embryos is used to demonstrate that a large fraction of the nuclear envelope lamins remain localized to a rim in the nuclear periphery until well into metaphase. The process of lamin delocalization and dispersal, typical of ‘open’ forms of mitosis, does not begin until about the time the final, metaphase geometry of the mitotic spindle is attained. Lamin dispersal is completed about the time that the chromosomal movements of anaphase begin. This pattern of nuclear lamina breakdown appears to be intermediate between traditional designations of ‘open’ and ‘closed’ mitoses. These results thus clarify earlier observations of lamins in mitosis in fixed Drosophila early embryos, clearly showing that the observed lamin localization does not result from a structurally defined ‘spindle envelope’ that persists throughout mitosis. During this extended time interval of lamin localization in the nuclear periphery, the lamina undergoes an extensive series of structural rearrangements that are closely coupled to, and likely driven by, the movements of the centrosomes and microtubules that produce the mitotic spindle. Furthermore, throughout this time the nuclear envelope structure is permeable to large macromolecules, which are excluded in interphase. While the functional significance of these structural dynamics is not yet clear, it is consistent with a functional role for the lamina in mitotic spindle formation.

scholarly journals Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast

2020 ◽  
Vol 133 (20) ◽  
pp. jcs246363 ◽  
Author(s):  
Safia Omer ◽  
Katia Brock ◽  
John Beckford ◽  
Wei-Lih Lee
Keyword(s):  
Budding Yeast ◽  
Current Model ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Direct Imaging ◽  
Spindle Positioning ◽  
Sliding Mechanism ◽  
Pulling Forces ◽  
Assembly Factor ◽  
Pulling Force

ABSTRACTThe current model for spindle positioning requires attachment of the microtubule (MT) motor cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect spindle displacement. How dynein is anchored by cortical attachment machinery to generate large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the yeast dynein attachment molecule, is limited by its assembly factor Mdm36. Overexpression of Mdm36 results in an overall enhancement of Num1 clustering but reveals a population of dim Num1 clusters that mediate dynein anchoring at the cell cortex. Direct imaging shows that bud-localized, dim Num1 clusters containing around only six Num1 molecules mediate dynein-dependent spindle pulling via a lateral MT sliding mechanism. Mutations affecting Num1 clustering interfere with mitochondrial tethering but do not interfere with the dynein-based spindle-pulling function of Num1. We propose that formation of small ensembles of attachment molecules is sufficient for dynein anchorage and cortical generation of large spindle-pulling forces.This article has an associated First Person interview with the first author of the paper.

iASPP contributes to cell cortex rigidity, mitotic cell rounding, and spindle positioning

2021 ◽  
Vol 220 (12) ◽  
Author(s):  
Aurélie Mangon ◽  
Danièle Salaün ◽  
Mohamed Lala Bouali ◽  
Mira Kuzmić ◽  
Sabine Quitard ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mitotic Spindle ◽  
Spherical Geometry ◽  
Mitotic Cell ◽  
Microtubule Dynamics ◽  
Regulatory Subunit ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Chromosome Partitioning ◽  
Apoptotic Activity

iASPP is a protein mostly known as an inhibitor of p53 pro-apoptotic activity and a predicted regulatory subunit of the PP1 phosphatase, which is often overexpressed in tumors. We report that iASPP associates with the microtubule plus-end binding protein EB1, a central regulator of microtubule dynamics, via an SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the cortex of mitotic cells, leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of the membrane-to-cortex linker Myosin-Ic (Myo1c), which we identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up upon mitosis because of defective cortical stiffness. We propose that by increasing cortical rigidity, iASPP helps cancer cells maintain a spherical geometry suitable for proper mitotic spindle positioning and chromosome partitioning.

Mechanics of microtubule organizing center clustering and spindle positioning in budding yeast Cryptococcus neoformans

2021 ◽  
Vol 104 (3) ◽  
Author(s):  
Saptarshi Chatterjee ◽  
Subhendu Som ◽  
Neha Varshney ◽  
PVS Satyadev ◽  
Kaustuv Sanyal ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Budding Yeast ◽  
Microtubule Organizing Center ◽  
Spindle Positioning ◽  
Organizing Center
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