Biology subjects

Maiato, H.

Publications and source records attributed to Maiato, H..

3 recordsLinked to original sources

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↗