[Show abstract][Hide abstract] ABSTRACT: Chromosome bi-orientation occurs after conversion of initial lateral attachments between kinetochores and spindle microtubules into stable end-on attachments near the cell equator. After bi-orientation, chromosomes experience tension from spindle forces, which plays a key role in the stabilization of correct kinetochore-microtubule attachments. However, how end-on kinetochore-microtubule attachments are first stabilized in the absence of tension remains a key unanswered question. To address this, we generated Drosophila S2 cells undergoing mitosis with unreplicated genomes (SMUGs). SMUGs retained single condensed chromatids that attached laterally to spindle microtubules. Over time, laterally attached kinetochores converted into end-on attachments and experienced intra-kinetochore stretch/structural deformation, and SMUGs eventually exited a delayed mitosis with mono-oriented chromosomes after satisfying the spindle-assembly checkpoint (SAC). Polar ejection forces (PEFs) generated by Chromokinesins promoted the conversion from lateral to end-on kinetochore-microtubule attachments that satisfied the SAC in SMUGs. Thus, PEFs convert lateral to stable end-on kinetochore-microtubule attachments, independently of chromosome bi-orientation.
[Show abstract][Hide abstract] ABSTRACT: Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope
reassembly (NER). However, how these two processes are coordinated remains unknown. Here, we identified a conserved feedback
control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase.
A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature
chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases counteracted this gradient and promoted chromosome
decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation
and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging
chromosomes in the main nuclei before completion of NER.
[Show abstract][Hide abstract] ABSTRACT: During mitosis, human cells round up, decreasing their adhesion to extracellular substrates. This must be quickly reestablished by poorly understood cytoskeleton remodeling mechanisms that prevent detachment from epithelia, while ensuring the successful completion of cytokinesis. Here we show that the microtubule end-binding (EB) proteins EB1 and EB3 play temporally distinct roles throughout cell division. Whereas EB1 was involved in spindle orientation before anaphase, EB3 was required for stabilization of focal adhesions and coordinated daughter cell spreading during mitotic exit. Additionally, EB3 promoted midbody microtubule stability and, consequently, midbody stabilization necessary for efficient cytokinesis. Importantly, daughter cell adhesion and cytokinesis completion were spatially regulated by distinct states of EB3 phosphorylation on serine 176 by Aurora B. This EB3 phosphorylation was enriched at the midbody and shown to control cortical microtubule growth. These findings uncover differential roles of EB proteins and explain the importance of an Aurora B phosphorylation gradient for the spatiotemporal regulation of microtubule function during mitotic exit and cytokinesis.
Full-text · Article · May 2013 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: After slightly more than a decade since it was first established, fluorescent speckle microscopy (FSM) has been intensively used to investigate macromolecular dynamics, such as microtubule flux in mitosis and meiosis, microtubule translocation in neurons, microtubule-binding proteins, and focal adhesion proteins, as well as the assembly of actin filaments. This state-of-the-art technique is based on nonuniform distribution of fluorescently labeled subunits diluted in the endogenous, unlabeled ones, resulting in microscopy-detectable speckled patterns. In order to enable sufficient contrast between neighboring diffraction-limited image regions, a low ratio between labeled and endogenous molecules is required, which can be achieved either by microinjection or by expression of limited amounts of fluorescently labeled subunits in cells. Over the years, the initial settings for FSM have been significantly improved by introduction of more sensitive cameras and spinning-disk confocal units, as well as by the development of specialized algorithms for image analysis. In this chapter, we describe our current FSM setup and detail on the necessary experimental approaches for its use in cultured cells, while discussing the present and future challenges of this powerful technique.
No preview · Article · Dec 2012 · Methods in enzymology
[Show abstract][Hide abstract] ABSTRACT: Chromokinesins are microtubule plus end–directed motor proteins that bind to chromosome arms. In Xenopus egg cell-free extracts, Xkid and Xklp1 are essential for bipolar spindle formation but the functions of the human homologues,
hKID (KIF22) and KIF4A, are poorly understood. By using RNAi-mediated protein knockdown in human cells, we find that only
co-depletion delayed progression through mitosis in a Mad2-dependent manner. Depletion of hKID caused abnormal chromosome
arm orientation, delayed chromosome congression, and sensitized cells to nocodazole. Knockdown of KIF4A increased the number
and length of microtubules, altered kinetochore oscillations, and decreased kinetochore microtubule flux. These changes were
associated with failures in establishing a tight metaphase plate and an increase in anaphase lagging chromosomes. Co-depletion
of both chromokinesins aggravated chromosome attachment failures, which led to mitotic arrest. Thus, hKID and KIF4A contribute
independently to the rapid and correct attachment of chromosomes by controlling the positioning of chromosome arms and the
dynamics of microtubules, respectively.
Full-text · Article · Sep 2012 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Chromosome positioning at the equator of the mitotic spindle emerges out of a relatively entropic background. At this moment, termed metaphase, all kinetochores have typically captured microtubules leading to satisfaction of the spindle-assembly checkpoint, but the cell does not enter anaphase immediately. The waiting time in metaphase is related to the kinetics of securin and cyclin B1 degradation, which trigger sister-chromatid separation and promote anaphase processivity, respectively. Yet, as judged by metaphase duration, such kinetics vary widely between cell types and organisms, with no evident correlation to ploidy or cell size. During metaphase, many animal and plant spindles are also characterized by a conspicuous "flux" activity characterized by continuous poleward translocation of spindle microtubules, which maintain steady-state length and position. Whether spindle microtubule flux plays a specific role during metaphase remains arguable. Based on known experimental parameters, we have performed a comparative analysis amongst different cell types from different organisms and show that spindle length, metaphase duration and flux velocity combine within each system to obey a quasi-universal rule. As so, knowledge of two of these parameters is enough to estimate the third. This trend indicates that metaphase duration is tuned to allow approximately one kinetochore-to-pole round of microtubule flux. We propose that the time cells spend in metaphase evolved as a quality enhancement step that allows for the uniform stabilization/correction of kinetochore-microtubule attachments, thereby promoting mitotic fidelity.
Preview · Article · Jul 2012 · Chromosome Research
[Show abstract][Hide abstract] ABSTRACT: To study the mechanism of transfection mediated by imidazole-grafted chitosan (CHimi) nanoparticles, to propose new strategies to control and improve the expression of a delivered gene in the context of regenerative medicine.
Biochemical and microscopy methods were used to establish transfection efficiency and nanoparticle intracellular trafficking. The role of CHimi and degree of N-acetylation (DA) on transfection was explored.
CHimi was found to promote the expression of a delivered gene during a minimum 7-day period. Additionally, the production of a protein of interest could be upheld by consecutive transfections, without compromising cell viability. Transfection was found to be a time-dependent process, requiring CHimi-DNA complex disassembling. The DA was found to have an impact on transfection kinetics in line with the observation that the rate of lysozyme-mediated nanoparticle degradation increases with the polymer DA.
The adjustment of the CH degradation rate can be used as a tool for tuning the expression of a gene delivered by CH-based nanoparticle systems.
[Show abstract][Hide abstract] ABSTRACT: The centromere-specific histone H3 variant CENH3 (also known as CENP-A) is considered to be an epigenetic mark for establishment and propagation of centromere identity. Pulse induction of CENH3 (Drosophila CID) in Schneider S2 cells leads to its incorporation into non-centromeric regions and generates CID islands that resist clearing from chromosome arms for multiple cell generations. We demonstrate that CID islands represent functional ectopic kinetochores, which are non-randomly distributed on the chromosome and show a preferential localization near telomeres and pericentric heterochromatin in transcriptionally silent, intergenic chromatin domains. Although overexpression of heterochromatin protein 1 (HP1) or increasing histone acetylation interferes with CID island formation on a global scale, induction of a locally defined region of synthetic heterochromatin by targeting HP1-LacI fusions to stably integrated Lac operator arrays produces a proximal hotspot for CID deposition. These data indicate that the characteristics of regions bordering heterochromatin promote de novo kinetochore assembly and thereby contribute to centromere identity.
Full-text · Article · Jun 2011 · Nature Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Although applicability of kymographs is limited to nearly one-dimensional (1D) processes, they have been instrumental in the analysis and interpretation of a wide range of dynamic biological processes. We focus here on some applications of kymography in the study of one among the range of 'nearly-1D' processes -mitosis. Using this biological context, we suggest generalized procedures in kymograph assembly that allow a partial retrieval of spatial information which is typically lost or distorted in conventional kymography. These kymograph variations, namely guided-kymography and chromo-kymography, are helpful in the determination of actual velocities and discrimination of structures when using thick regions of interest (ROIs). The method used to generate chromo-kymographs is generalized to other (non-kymograph) projection techniques, which include time-stack and z-stack projections.
[Show abstract][Hide abstract] ABSTRACT: The synchronous movement of chromosomes during anaphase ensures their correct inheritance in every cell division. This reflects the uniformity of spindle forces acting on chromosomes and their simultaneous entry into anaphase. Although anaphase onset is controlled by the spindle assembly checkpoint, it remains unknown how spindle forces are uniformly distributed among different chromosomes. In this paper, we show that tension uniformity at metaphase kinetochores and subsequent anaphase synchrony in Drosophila S2 cells are promoted by spindle microtubule flux. These results can be explained by a mechanical model of the spindle where microtubule poleward translocation events associated with flux reflect relaxation of the kinetochore-microtubule interface, which accounts for the redistribution and convergence of kinetochore tensions in a timescale comparable to typical metaphase duration. As predicted by the model, experimental acceleration of mitosis precludes tension equalization and anaphase synchrony. We propose that flux-dependent equalization of kinetochore tensions ensures a timely and uniform maturation of kinetochore-microtubule interfaces necessary for error-free and coordinated segregation of chromosomes in anaphase.
Full-text · Article · Aug 2009 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Establishment and maintenance of the mitotic spindle requires the balanced activity of microtubule-associated proteins and motors. In this study we have addressed how the microtubule plus-end tracking protein mast/orbit/CLASP and cytoplasmic dynein regulate this process in Drosophila melanogaster embryos and S2 cells. We show that mast accumulates at kinetochores early in mitosis, which is followed by a poleward streaming upon microtubule attachment. This leads to a reduction of mast levels at kinetochores during metaphase and anaphase that depends largely on the microtubule minus end-directed motor cytoplasmic dynein. Surprisingly, we also found that co-depletion of dynein rescues spindle bipolarity in mast-depleted cells, while restoring normal microtubule poleward flux. Our results suggest that mast and dynein have antagonistic roles in the local regulation of microtubule plus-end dynamics at kinetochores, which are important for the maintenance of spindle bipolarity and normal spindle length.
[Show abstract][Hide abstract] ABSTRACT: Progress from our present understanding of the mechanisms behind mitosis has been compromised by the fact that model systems that were ideal for molecular and genetic studies (such as yeasts, C. elegans, or Drosophila) were not suitable for intracellular micromanipulation. Unfortunately, those systems that were appropriate for micromanipulation (such as newt lung cells, PtK1 cells, or insect spermatocytes) are not amenable for molecular studies. We believe that we can significantly broaden this scenario by developing high-resolution live cell microscopy tools in a system where micromanipulation studies could be combined with modern gene-interference techniques. Here we describe a series of methodologies for the functional dissection of mitosis by the use of simultaneous live cell microscopy and state-of-the-art laser microsurgery, combined with RNA interference (RNAi) in Drosophila cell lines stably expressing fluorescent markers. This technological synergism allows the specific targeting and manipulation of several structural components of the mitotic apparatus in different genetic backgrounds, at the highest spatial and temporal resolution. Finally, we demonstrate the successful adaptation of agar overlay flattening techniques to human HeLa cells and discuss the advantages of its use for laser micromanipulation and molecular studies of mitosis in mammals.
Full-text · Article · Feb 2009 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.
Full-text · Article · Nov 2006 · Molecular Biology of the Cell