Biology subjects

Maiato, H.

Publications and source records attributed to Maiato, H..

18 recordsLinked to original sources

bioRxiv2026

Microtubule occupancy at kinetochores links checkpoint silencing with mitotic memory

The spindle assembly checkpoint (SAC) promotes faithful chromosome segregation by delaying mitosis until all kinetochores attach to spindle microtubules. Paradoxically, a p53-dependent memory mechanism--the mitotic stopwatch" --blocks daughter cell proliferation after unusually prolonged mitoses. How the SAC coordinates with the mitotic stopwatch remains unknown. Here, we found that microtubule occupancy at kinetochores is a cornerstone linking SAC silencing with mitotic memory. By combining live-cell with super-resolution microscopy, FRAP, laser microsurgery, biochemistry and molecular perturbations in Indian muntjac fibroblasts, we show that SAC silencing at kinetochores is gradual, non-uniform and confined to highly-localized microtubule attachments. Augmin promotes timely SAC silencing with high microtubule occupancy at kinetochores, whereas MPS1/CDK1 inhibition bypasses this requirement. Conversely, low microtubule occupancy delays SAC silencing, increases segregation errors and blocks daughter cell proliferation due to mitotic stopwatch surveillance. Thus, timely SAC silencing with high microtubule occupancy avoids "bad memories" of mitosis to allow daughter cell proliferation.

cell biology↗

bioRxiv2024

The Chromosome Periphery is an Essential Compartment of Oocyte Chromosomes

Chromosomes and their constituent compartments are core elements of the cell division machinery. In mammalian oocytes, defects in the structure or composition of chromosomes are a leading cause of aberrant or failed meiosis, and by extension, can cause miscarriage and infertility. The underlying mechanisms are poorly understood, but are critical for development of novel diagnostics and treatments. The chromosome periphery, the least understood chromosome compartment, has recently emerged as an essential component of mitotic chromosomes and somatic cell division. However, in female meiosis it remains completely unexplored. This study provides the first comprehensive survey of a meiotic chromosome periphery compartment in both human and mouse oocytes. Using a combination of time-lapse microscopy, super-resolution imaging and 3DCLEM we show that removing the chromosome periphery, via Ki67 depletion, has substantial negative impact on chromosome structure, spatial organization and positional awareness, with many oocytes stalling and arresting in meiosis I. Importantly, we also reveal key differences between the mitotic and oocyte meiotic chromosome periphery compartments, most remarkably in the retention of Ki67 in the oocyte through anaphase I, where unwanted chromosomes are stripped of Ki67 before being ejected from the oocyte. This work presents the discovery of an exciting new pathway operating during female meiosis and a provides a platform for future work exploring the meiotic chromosome periphery for therapeutic vulnerabilities.

cell biology↗

bioRxiv2022

The deubiquitylase USP31 controls the Chromosomal Passenger Complex and spindle dynamics

We have identified USP31 as a microtubule and centrosome associated deubiquitylase, depletion of which leads to an increase in individual cell mass and defective proliferation. We have examined its dynamics and impact during mitosis. GFP-USP31 associates with the mitotic and central spindles, its levels are increased 2-3-fold in prometaphase compared to asynchronous cells and it is dynamically phosphorylated in a CDK1 dependent manner. We find that USP31 depleted cells display altered spindle morphology and chromosomal segregation errors, whilst stable expression of a catalytically inactive form of USP31 leads to polyploidy. At prometaphase, levels of multiple CPC components are destabilised, most prominently INCENP. Under anaphase conditions, depletion of USP31 impairs the translocation of both endogenous and ectopically expressed INCENP to the spindle mid-zone, whilst expression of catalytically inactive USP31 results in multiple ectopic cleavage furrows. In summary, our data indicate a multifaceted regulatory role for USP31 during mitosis, with a profound impact on chromosomal passenger complex abundance, dynamics and function.

cell biology↗

bioRxiv2022

Long-term mitotic DNA damage promotes chromokinesin-mediated missegregation of polar chromosomes in cancer cells

DNA damage response (DDR) during interphase involves active signalling and repair to ensure genomic stability. However, how mitotic cells respond to DNA damage remains poorly understood. Supported by correlative live-/fixed-cell microscopy analysis we found that mitotic cells exposed to several cancer chemotherapy compounds acquire and signal DNA damage, regardless of how they interact with DNA. In-depth analysis upon long-term DNA damage during mitosis revealed a spindle assembly checkpoint (SAC)-dependent, but DDR-independent, mitotic delay. This delay was due to the presence of misaligned chromosomes that ultimately satisfy the SAC and missegregate, leading to micronuclei formation. Mechanistically, we show that long-term mitotic DNA damage specifically stabilizes kinetochore-microtubule attachments in cancer cells, causing the missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesins. Overall, these findings unveil that long-term therapeutic DNA damage regimens contribute to genomic instability through a surprising link between the stabilization of kinetochore-microtubule attachments and chromokinesin-mediated missegregation of polar chromosomes in cancer cells.

cell biology↗

bioRxiv2022

Misaligned chromosomes that satisfy the spindle assembly checkpoint are a strong predictor of micronuclei formation in dividing cancer cells

Chromosome alignment to the spindle equator is a hallmark of mitosis that is thought to promote chromosome segregation fidelity in metazoans. Yet, chromosome alignment is only indirectly supervised by the spindle assembly checkpoint (SAC) as a byproduct of chromosome bi-orientation, and the consequences of defective chromosome alignment remain unclear. Here we investigated how human cells respond to chromosome alignment defects of distinct molecular nature by following the fate of live HeLa cells after RNAi-mediated depletion of 120 proteins previously implicated in chromosome alignment. Surprisingly, in all cases, cells frequently entered anaphase after a delay with chronically misaligned chromosomes. Using depletion of key proteins as prototypes for defective chromosome alignment, we show that chronically misaligned chromosomes often satisfy the SAC and directly missegregate. In-depth analysis of specific molecular perturbations that prevent proper kinetochore-microtubule attachments revealed that chronically misaligned chromosomes that missegregate frequently result in micronuclei. Higher-resolution live-cell imaging indicated that, contrary to most anaphase lagging chromosomes that correct and reintegrate the main nuclei, chronically misaligned chromosomes are a strong predictor of micronuclei formation in a cancer cell model of chromosomal instability, but not in normal near-diploid cells. We provide evidence supporting that intrinsic differences in kinetochore-microtubule attachment stability on misaligned chromosomes account for this distinct outcome. Thus, chronically misaligned chromosomes that satisfy the SAC may represent a previously overlooked mechanism driving chromosomal/genomic instability during cancer cell division, and we unveil genetic conditions predisposing for these events.

cell biology↗

bioRxiv2022

CLASP1 is essential for neonatal lung function and survival in mice

The first breath of air at birth marks the beginning of extrauterine life, and breathing problems due to incomplete lung development or acute respiratory distress are common in premature babies and respiratory diseases. However, the underlying molecular mechanisms remain poorly understood. Here we show that the microtubule plus-end-tracking protein CLASP1 is required for neonatal lung function and survival. CLASP1 is expressed in the lungs and associated respiratory structures throughout embryonic development. Clasp1 disruption in mice caused intrauterine growth restriction and neonatal lethality due to acute respiratory failure. Knockout animals showed impaired lung inflation associated with smaller rib cage formation and abnormal diaphragm innervation. Live-cell analysis of microtubule dynamics in cultured hippocampal neurons revealed an increased catastrophe rate, consistent with a role of CLASP1 in neurite outgrowth. Histological and gene expression studies indicated that CLASP1 is required for normal pneumocyte differentiation and fetal lung maturation. Thus, CLASP1-mediated regulation of microtubule dynamics assists multiple systems essential for neonatal lung function and survival.

developmental biology↗

bioRxiv2022

CLASP2 stabilizes GDP-associated terminal tubulins to prevent microtubule catastrophe

CLASPs are ubiquitous stabilizers of microtubule dynamics but their molecular targets at the microtubule plus-end are not understood. Using DNA origami-based reconstructions we show that clusters of human CLASP2 form a load-bearing bond with terminal GDP-tubulins at the stabilized microtubule tip. This activity relies on the unconventional TOG2 domain of CLASP2, which releases its high-affinity bond with the GDP-dimers upon their conversion into polymerization-competent GTP-tubulin. By tethering dynamic microtubule ends near immobilized CLASP2, we show that the targets for CLASP2 binding at the polymerizing tip arise stochastically, leading to nanoscale disruptions in microtubule tip integrity. The ability of CLASP2 to recognize nucleotide-specific tubulin conformation and stabilize the catastrophe-promoting GDP-tubulins intertwines with the previously underappreciated exchange between GDP and GTP at terminal tubulins, providing a distinct molecular mechanism to suppress microtubule catastrophe without affecting tubulin incorporation.

biophysics↗

bioRxiv2021

Mechanosensitive control of mitotic entry

As cells prepare to divide, they must ensure that enough space is available to assemble the mitotic machinery without perturbing tissue homeostasis1. To do so, cells undergo a series of biochemical reactions regulated by cyclin B1-CDK1 that trigger the reorganization of the actomyosin cytoskeleton2 and ensure the coordination of cytoplasmic and nuclear events3. Along with the biochemical events that control mitotic entry, mechanical forces have recently emerged as important players in the regulation of cell cycle events4-6. However, the exact link between mechanical forces and the biochemical pathways that control mitotic progression remains to be established. Here, we identify a mechanical signal on the nucleus that helps set the time for nuclear envelope permeabilization (NEP) and mitotic entry. This signal relies on actomyosin contractility, which leads to nuclear unfolding during the G2-M transition, activating the stretch-sensitive cPLA2 on the nuclear envelope. This contributes to the spatiotemporal translocation of cyclin B1 to the nucleus. Our data demonstrate how nuclear mechanics during the G2-M transition contribute to timely and efficient mitotic spindle assembly and prevents chromosomal instability.

cell biology↗

bioRxiv2021

A mitotic screen in Indian muntjac cells unveils how Augmin drives kinetochore fiber maturation

Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fibers. How k-fibers mature from initial kinetochore-microtubule attachments remains a fundamental question. By combining functional perturbations in Indian muntjac, a placental mammal with the lowest known chromosome number (n=3) and distinctively large kinetochores, with fixed- and subsecond live-cell super-resolution CH-STED nanoscopy, we uncovered the mechanism by which Augmin mediates k-fiber maturation. Augmin promoted kinetochore microtubule turnover by sustaining microtubule formation from kinetochores and poleward flux, regardless of pioneer centrosomal microtubules. Tracking of microtubule growth events within individual k-fibers revealed a wide angular dispersion, consistent with Augmin-mediated branched microtubule nucleation. Augmin depletion reduced the frequency of microtubule growth events within k-fibers and hampered normal repair after acute k-fiber injury by laser microsurgery. Our work underscores the contribution of microtubule formation from kinetochores and Augmin-mediated microtubule amplification for k-fiber maturation and spindle assembly in mammals.

cell biology↗

bioRxiv2021

An anaphase surveillance mechanism prevents micronuclei formation from mitotic errors

Micronuclei are a hallmark of cancer and other human disorders and have recently been implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reformation to protect against micronuclei formation during human cell division. Using high-resolution live-cell imaging of human cancer and non-cancer cells we found that anaphase lagging chromosomes are often transient and rarely formed micronuclei. This strong bias against micronuclei formation relied on a midzone-based Aurora B phosphorylation gradient that assisted the mechanical transduction of spindle forces at the kinetochore-microtubule interface required for anaphase error correction, while delaying nuclear envelope reformation on lagging chromosomes, independently of microtubules. Our results uncover a new layer of protection against genomic instability and provide a strategy for the rational design of micronuclei-targeting therapies.

cell biology↗

bioRxiv2020

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MT dynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. We show that both of these activities work in coordination with MT-crosslinking motors kinesin-5/EG5 and kinesin-12/KIF15. Our data further indicate that MT-flux driving force is transmitted from non-KT MTs to KT-MTs via MT-coupling by HSET and NuMA. Moreover, we found that MT-flux rate correlates with spindle size and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we revealed that flux is required to counteract the kinesin 13/MCAK-dependent MT-depolymerization to regulate spindle length. Thus, our study demonstrates that MT-flux in human cells is driven by the coordinated action of four kinesins, and is required to regulate mitotic spindle size in response to MCAK-mediated MT-depolymerizing activity at KTs.

cell biology↗

bioRxiv2019

Microtubule tyrosination/detyrosination specifies a mitotic error code

Incorrect kinetochore-microtubule attachments during mitosis can lead to chromosomal instability, a hallmark of human cancers. Mitotic error correction relies on the kinesin-13 MCAK, a microtubule depolymerase whose activity in vitro is suppressed by -tubulin detyrosination - a post-translational modification enriched on long-lived microtubules. However, whether and how MCAK activity required for mitotic error correction is regulated by microtubule tyrosination/detyrosination remains unknown. Here we found that microtubule detyrosination accumulates on correct, more stable, kinetochore-microtubule attachments, whereas constitutively high microtubule detyrosination near kinetochores compromised efficient error correction. Rescue experiments suggest that mitotic errors due to excessive microtubule detyrosination result from suppression of MCAK activity, without globally affecting kinetochore microtubule half-life. Importantly, MCAK centromeric activity was required and sufficient to rescue mitotic errors due to excessive microtubule detyrosination. Thus, microtubules are not just passive elements during mitotic error correction, and their tyrosination/detyrosination works as a mitotic error code that allows centromeric MCAK to discriminate correct and incorrect kinetochore-microtubule attachments, thereby promoting mitotic fidelity.

cell biology↗

bioRxiv2019

CLASP2 lattice-binding near microtubule plus ends stabilizes kinetochore attachments

The fine regulation of kinetochore microtubule dynamics during mitosis ensures proper chromosome segregation by promoting error correction and spindle assembly checkpoint (SAC) satisfaction. CLASPs are widely conserved microtubule plus-end-tracking proteins that regulate microtubule dynamics throughout the cell cycle and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore microtubule dynamics, but the underlying molecular mechanism remains unknown. Here we found that human CLASP2 can dimerize through its C-terminal kinetochore-targeting domain, but kinetochore localization was independent of dimerization. CLASP2 kinetochore localization, microtubule plus-end-tracking and microtubule lattice binding through TOG2 and TOG3 (but not TOG1) domains, independently sustained normal spindle length, timely SAC satisfaction, chromosome congression and faithful segregation. Measurements of kinetochore microtubule half-life in living cells expressing RNAi-resistant mutants revealed that CLASP2 kinetochore localization, microtubule plus-end-tracking and lattice binding cooperatively modulate kinetochore microtubule stability during mitosis. Thus, CLASP2 regulates kinetochore microtubule dynamics by integrating distinctive microtubule-binding properties at the kinetochore-microtubule interface to ensure chromosome segregation fidelity.

cell biology↗

bioRxiv2019

Mitotic exit is controlled during anaphase by an Aurora B-Cyclin B1/Cdk1 crosstalk

According to the prevailing \"clock\" model, chromosome decondensation and nuclear envelope reassembly during mitotic exit are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the \"ruler\" model. Here we reconciled both models by showing that Cyclin B1 degradation continues during anaphase in Drosophila, mouse and human cells, including primary tissues. This required APC/CCdh1 activity, and failure to degrade Cyclin B1 during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1 localization and half-life during anaphase depended on kinesin-6, which targets Aurora B to the spindle midzone. Mechanistically, we show that anaphase duration is regulated by Aurora B-mediated phosphorylation of Cyclin B1. We propose that a crosstalk between molecular \"rulers\" and \"clocks\" licenses mitotic exit only after proper chromosome separation.

cell biology↗

bioRxiv2019

Spatial Asymmetries of the Prophase Nucleus Regulate Dynein Recruitment to Ensure Mitotic Fidelity

During prophase, centrosomes need to separate and position to correctly assemble the mitotic spindle. This process occurs through the action of molecular motors, cytoskeletal networks and the nucleus. How the combined activity of these different components is spatiotemporally regulated to ensure efficient spindle assembly remains unclear. Here we show that during prophase the centrosomes-nucleus axis reorients, so that centrosomes are positioned on the shortest nuclear axis at nuclear envelope (NE) breakdown. This centrosomes-nucleus configuration depends on mechanical cues generated by mitotic chromosome condensation on the prophase nucleus. We further show these mechanosensitive cues act through SUN1/2 and NudE+NudEL to enable the polarized loading of Dynein on the NE. Finally, we observe this centrosome configuration favors the establishment of an initial bipolar spindle scaffold, facilitating chromosome capture and accurate segregation, without compromising division plane orientation. We propose that chromosome segregation fidelity depends on the mechanical properties of the prophase nucleus that facilitate spindle assembly by regulating NE-Dynein localization.

cell biology↗

bioRxiv2018

Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast

Resolution in microscopy is not limited by diffraction as long as a nonlinear sample response is exploited. In a paradigmatic example, stimulated-emission depletion (STED) fluorescence microscopy fundamentally breaks the diffraction limit by using a structured optical pattern to saturate depletion on a previously excited sample area. Two-dimensional (2D) STED, the canonical low-noise STED mode, structures the STED beam by using a vortex phase mask, achieving a significant lateral resolution improvement over confocal fluorescence microscopy. However, axial resolution and optical sectioning remain bound to diffraction. Here we use a tunable coherent-hybrid (CH) beam to improve optical sectioning, markedly reducing background fluorescence. CH-STED, which inherits the 2D-STED immunity to spherical aberration, diversifies the depletion strategy, allowing an optimal balance between two key metrics (lateral resolution and background suppression) to be found. CH-STED is used to perform high-contrast imaging of complex biological structures, such as the mitotic spindle and the neuron cell body.

biophysics↗

bioRxiv2018

Mitotic progression, arrest, exit or death is determined by centromere integrity and independent of de novo transcription

Recent studies have challenged the prevailing dogma that transcription is repressed during mitosis. Transcription was also proposed to sustain the spindle assembly checkpoint (SAC) for several hours in response to unattached kinetochores. Here we used live-cell imaging of human cells in culture, combined with RNA-seq and qPCR, to investigate the requirement for de novo transcription during mitosis. Under conditions of persistently unattached kinetochores, transcription inhibition with actinomycin D, or treatment with other DNA-intercalating drugs, delocalized the chromosomal passenger complex (CPC) protein Aurora B from centromeres, compromising SAC robustness and cell fate. However, we were unable to detect significant changes in transcript levels. Moreover, inhibition of transcription independently of DNA intercalation had no effect on SAC response, mitotic progression, exit or death. Mechanistically, we show that DNA intercalating agents reduce the interaction of the CPC with nucleosomes. Thus, the capacity of human cells to progress, sustain, exit or die in mitosis relies on centromere integrity, rather than de novo transcription.

cell biology↗

bioRxiv2018

Chromosome (mis)segregation is biased by kinetochore size

Summary:\n\nAneuploidy, the gain or loss of chromosomes, arises through problems in chromosome segregation during mitosis or meiosis and has been implicated in cancer and developmental abnormalities in humans [1]. Possible routes to aneuploidy include a compromised spindle assembly checkpoint (SAC), cohesion defects, centrosome amplification, as well as improper kinetochore-microtubule attachments [2]. However, none of these established routes takes into account the intrinsic features of the kinetochore - the critical chromosomal interface with spindle microtubules. Kinetochore size and respective microtubule binding capacity varies between different animal and plant species [3-10], among different chromosomes from the same species (including humans) [3, 11-15], and in response to microtubule attachments throughout mitosis [16-18]. How kinetochore size impacts chromosome segregation remains unknown. Addressing this fundamental question in human cells is virtually impossible, because most of the 23 pairs of chromosomes cannot be morphologically distinguished, while detection of 2-3 fold differences in kinetochore size is limited by diffraction and cannot be resolved by conventional light microscopy in living cells. Here we used the unique cytological attributes of female Indian muntjac, the mammal with the lowest known chromosome number (n=3), to track individual chromosomes with distinct kinetochore sizes throughout mitosis. We found that chromosomes with larger kinetochores bi-orient more easily and are biased to congress to the equator in a motor-independent manner. However, they are also more prone to establish erroneous merotelic attachments and lag behind during anaphase. Thus, we uncovered an intrinsic kinetochore feature - size - as an important determinant of chromosome segregation fidelity.

cell biology↗