Search for chromosomal instability aiding variants reveal naturally occurring kinetochore gene variants that perturb chromosome segregation.

Chromosomal instability (CIN) is a hallmark of cancers, and CIN-promoting mutations are not fully understood. Here, we report 141 chromosomal instability aiding variant (CIVa) candidates by assessing the prevalence of loss-of-function (LoF) variants in 135 chromosome segregation genes from over 150,000 humans. Unexpectedly, we observe both heterozygous and homozygous CIVa in Astrin and SKA3, two evolutionarily conserved kinetochore and microtubule-associated proteins essential for chromosome segregation. To stratify harmful versus harmless variants, we combine live-cell microscopy and controlled protein expression. We find the naturally occurring Astrin p.Q1012∗ variant is harmful as it fails to localize normally and induces chromosome misalignment and missegregation, in a dominant negative manner. In contrast, the Astrin p.L7Qfs∗21 variant generates a shorter isoform that localizes and functions normally, and the SKA3 p.Q70Kfs∗7 variant allows wild-type SKA complex localisation and function, revealing distinct resilience mechanisms that render these variants harmless. Thus, we present a scalable framework to predict and stratify naturally occurring CIVa, and provide insight into resilience mechanisms that compensate for naturally occurring CIVa.

Opportunities and challenges for deep learning in cell dynamics research.

The growth of artificial intelligence (AI) has led to an increase in the adoption of computer vision and deep learning (DL) techniques for the evaluation of microscopy images and movies. This adoption has not only addressed hurdles in quantitative analysis of dynamic cell biological processes but has also started to support advances in drug development, precision medicine, and genome-phenome mapping. We survey existing AI-based techniques and tools, as well as open-source datasets, with a specific focus on the computational tasks of segmentation, classification, and tracking of cellular and subcellular structures and dynamics. We summarise long-standing challenges in microscopy video analysis from a computational perspective and review emerging research frontiers and innovative applications for DL-guided automation in cell dynamics research.

Deep learning techniques and mathematical modeling allow 3D analysis of mitotic spindle dynamics.

Time-lapse microscopy movies have transformed the study of subcellular dynamics. However, manual analysis of movies can introduce bias and variability, obscuring important insights. While automation can overcome such limitations, spatial and temporal discontinuities in time-lapse movies render methods such as 3D object segmentation and tracking difficult. Here, we present SpinX, a framework for reconstructing gaps between successive image frames by combining deep learning and mathematical object modeling. By incorporating expert feedback through selective annotations, SpinX identifies subcellular structures, despite confounding neighbor-cell information, non-uniform illumination, and variable fluorophore marker intensities. The automation and continuity introduced here allows the precise 3D tracking and analysis of spindle movements with respect to the cell cortex for the first time. We demonstrate the utility of SpinX using distinct spindle markers, cell lines, microscopes, and drug treatments. In summary, SpinX provides an exciting opportunity to study spindle dynamics in a sophisticated way, creating a framework for step changes in studies using time-lapse microscopy.

Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation.

Defects in chromosome-microtubule attachment can cause chromosomal instability (CIN), frequently associated with infertility and aggressive cancers. Chromosome-microtubule attachment is mediated by a large macromolecular structure, the kinetochore. Sister kinetochores of each chromosome are pulled by microtubules from opposing spindle-poles, a state called biorientation which prevents chromosome missegregation. Kinetochore-microtubule attachments that lack the opposing-pull are detached by Aurora-B/Ipl1. It is unclear how mono-oriented attachments that precede biorientation are spared despite the lack of opposing-pull. Using an RNAi-screen, we uncover a unique role for the Astrin-SKAP complex in protecting mono-oriented attachments. We provide evidence of domains in the microtubule-end associated protein that sense changes specific to end-on kinetochore-microtubule attachments and assemble an outer-kinetochore crescent to stabilise attachments. We find that Astrin-PP1 and Cyclin-B-CDK1 pathways counteract each other to preserve mono-oriented attachments. Thus, CIN prevention pathways are not only surveying attachment defects but also actively recognising and stabilising mature attachments independent of biorientation.

Electrically tunable lenses - eliminating mechanical axial movements during high-speed 3D live imaging.

The successful investigation of photosensitive and dynamic biological events, such as those in a proliferating tissue or a dividing cell, requires non-intervening high-speed imaging techniques. Electrically tunable lenses (ETLs) are liquid lenses possessing shape-changing capabilities that enable rapid axial shifts of the focal plane, in turn achieving acquisition speeds within the millisecond regime. These human-eye-inspired liquid lenses can enable fast focusing and have been applied in a variety of cell biology studies. Here, we review the history, opportunities and challenges underpinning the use of cost-effective high-speed ETLs. Although other, more expensive solutions for three-dimensional imaging in the millisecond regime are available, ETLs continue to be a powerful, yet inexpensive, contender for live-cell microscopy.

Multinucleation associated DNA damage blocks proliferation in p53-compromised cells.

Nuclear atypia is one of the hallmarks of cancers. Here, we perform single-cell tracking studies to determine the immediate and long-term impact of nuclear atypia. Tracking the fate of newborn cells exhibiting nuclear atypia shows that multinucleation, unlike other forms of nuclear atypia, blocks proliferation in p53-compromised cells. Because ~50% of cancers display compromised p53, we explored how multinucleation blocks proliferation. Multinucleation increases 53BP1-decorated nuclear bodies (DNA damage repair platforms), along with a heterogeneous reduction in transcription and protein accumulation across the multi-nucleated compartments. Multinucleation Associated DNA Damage associated with 53BP1-bodies remains unresolved for days, despite an intact NHEJ machinery that repairs laser-induced DNA damage within minutes. Persistent DNA damage, a DNA replication block, and reduced phospho-Rb, reveal a novel replication stress independent cell cycle arrest caused by mitotic lesions. These findings call for segregating protective and prohibitive nuclear atypia to inform therapeutic approaches aimed at limiting tumour heterogeneity.

Kinetochores attached to microtubule-ends are stabilised by Astrin bound PP1 to ensure proper chromosome segregation.

Microtubules segregate chromosomes by attaching to macromolecular kinetochores. Only microtubule-end attached kinetochores can be pulled apart; how these end-on attachments are selectively recognised and stabilised is not known. Using the kinetochore and microtubule-associated protein, Astrin, as a molecular probe, we show that end-on attachments are rapidly stabilised by spatially-restricted delivery of PP1 near the C-terminus of Ndc80, a core kinetochore-microtubule linker. PP1 is delivered by the evolutionarily conserved tail of Astrin and this promotes Astrin's own enrichment creating a highly-responsive positive feedback, independent of biorientation. Abrogating Astrin:PP1-delivery disrupts attachment stability, which is not rescued by inhibiting Aurora-B, an attachment destabiliser, but is reversed by artificially tethering PP1 near the C-terminus of Ndc80. Constitutive Astrin:PP1-delivery disrupts chromosome congression and segregation, revealing a dynamic mechanism for stabilising attachments. Thus, Astrin-PP1 mediates a dynamic 'lock' that selectively and rapidly stabilises end-on attachments, independent of biorientation, and ensures proper chromosome segregation.

MARK2/Par1b kinase present at centrosomes and retraction fibres corrects spindle off-centring induced by actin disassembly.

Tissue maintenance and development requires a directed plane of cell division. While it is clear that the division plane can be determined by retraction fibres that guide spindle movements, the precise molecular components of retraction fibres that control spindle movements remain unclear. We report MARK2/Par1b kinase as a novel component of actin-rich retraction fibres. A kinase-dead mutant of MARK2 reveals MARK2's ability to monitor subcellular actin status during interphase. During mitosis, MARK2's localization at actin-rich retraction fibres, but not the rest of the cortical membrane or centrosome, is dependent on its activity, highlighting a specialized spatial regulation of MARK2. By subtly perturbing the actin cytoskeleton, we reveal MARK2's role in correcting mitotic spindle off-centring induced by actin disassembly. We propose that MARK2 provides a molecular framework to integrate cortical signals and cytoskeletal changes in mitosis and interphase.

Spindle rotation in human cells is reliant on a MARK2-mediated equatorial spindle-centering mechanism.

The plane of cell division is defined by the final position of the mitotic spindle. The spindle is pulled and rotated to the correct position by cortical dynein. However, it is unclear how the spindle's rotational center is maintained and what the consequences of an equatorially off centered spindle are in human cells. We analyzed spindle movements in 100s of cells exposed to protein depletions or drug treatments and uncovered a novel role for MARK2 in maintaining the spindle at the cell's geometric center. Following MARK2 depletion, spindles glide along the cell cortex, leading to a failure in identifying the correct division plane. Surprisingly, spindle off centering in MARK2-depleted cells is not caused by excessive pull by dynein. We show that MARK2 modulates mitotic microtubule growth and length and that codepleting mitotic centromere-associated protein (MCAK), a microtubule destabilizer, rescues spindle off centering in MARK2-depleted cells. Thus, we provide the first insight into a spindle-centering mechanism needed for proper spindle rotation and, in turn, the correct division plane in human cells.

Aurora-B kinase pathway controls the lateral to end-on conversion of kinetochore-microtubule attachments in human cells.

Human chromosomes are captured along microtubule walls (lateral attachment) and then tethered to microtubule-ends (end-on attachment) through a multi-step end-on conversion process. Upstream regulators that orchestrate this remarkable change in the plane of kinetochore-microtubule attachment in human cells are not known. By tracking kinetochore movements and using kinetochore markers specific to attachment status, we reveal a spatially defined role for Aurora-B kinase in retarding the end-on conversion process. To understand how Aurora-B activity is counteracted, we compare the roles of two outer-kinetochore bound phosphatases and find that BubR1-associated PP2A, unlike KNL1-associated PP1, plays a significant role in end-on conversion. Finally, we uncover a novel role for Aurora-B regulated Astrin-SKAP complex in ensuring the correct plane of kinetochore-microtubule attachment. Thus, we identify Aurora-B as a key upstream regulator of end-on conversion in human cells and establish a late role for Astrin-SKAP complex in the end-on conversion process.Human chromosomes are captured along microtubule walls and then tethered to microtubule-ends through a multi-step end-on conversion process. Here the authors show that Aurora-B regulates end-on conversion in human cells and establish a late role for Astrin-SKAP complex in the end-on conversion process.

A Semi-Supervised Approach for Refining Transcriptional Signatures of Drug Response and Repositioning Predictions.

We present a novel strategy to identify drug-repositioning opportunities. The starting point of our method is the generation of a signature summarising the consensual transcriptional response of multiple human cell lines to a compound of interest (namely the seed compound). This signature can be derived from data in existing databases, such as the connectivity-map, and it is used at first instance to query a network interlinking all the connectivity-map compounds, based on the similarity of their transcriptional responses. This provides a drug neighbourhood, composed of compounds predicted to share some effects with the seed one. The original signature is then refined by systematically reducing its overlap with the transcriptional responses induced by drugs in this neighbourhood that are known to share a secondary effect with the seed compound. Finally, the drug network is queried again with the resulting refined signatures and the whole process is carried on for a number of iterations. Drugs in the final refined neighbourhood are then predicted to exert the principal mode of action of the seed compound. We illustrate our approach using paclitaxel (a microtubule stabilising agent) as seed compound. Our method predicts that glipizide and splitomicin perturb microtubule function in human cells: a result that could not be obtained through standard signature matching methods. In agreement, we find that glipizide and splitomicin reduce interphase microtubule growth rates and transiently increase the percentage of mitotic cells-consistent with our prediction. Finally, we validated the refined signatures of paclitaxel response by mining a large drug screening dataset, showing that human cancer cell lines whose basal transcriptional profile is anti-correlated to them are significantly more sensitive to paclitaxel and docetaxel.

Modeling of Noisy Spindle Dynamics Reveals Separable Contributions to Achieving Correct Orientation.

The mechanisms by which the mammalian mitotic spindle is guided to a predefined orientation through microtubule-cortex interactions have recently received considerable interest, but there has been no dynamic model that describes spindle movements toward the preferred axis in human cells. Here, we develop a dynamic model based on stochastic activity of cues anisotropically positioned around the cortex of the mitotic cell and we show that the mitotic spindle does not reach equilibrium before chromosome segregation. Our model successfully captures the characteristic experimental behavior of noisy spindle rotation dynamics in human epithelial cells, including a weak underlying bias in the direction of rotation, suppression of motion close to the alignment axis, and the effect of the aspect ratio of the interphase cell shape in defining the final alignment axis. We predict that the force exerted per cue has a value that minimizes the deviation of the spindle from the predefined axis. The model has allowed us to systematically explore the parameter space around experimentally relevant configurations, and predict the mechanistic function of a number of established regulators of spindle orientation, highlighting how physical modeling of a noisy system can lead to functional biological understanding. We provide key insights into measurable parameters in live cells that can help distinguish between mechanisms of microtubule and cortical-cue interactions that jointly control the final orientation of the spindle.

High-speed microscopy with an electrically tunable lens to image the dynamics of in vivo molecular complexes.

We provide an evaluation for an electrically tunable lens (ETL), combined with a microscope system, from the viewpoint of tracking intracellular protein complexes. We measured the correlation between the quantitative axial focus shift and the control current for ETL, and determined the stabilization time for refocusing to evaluate the electrical focusing behaviour of our system. We also confirmed that the change of relative magnification by the lens and associated resolution does not influence the ability to find intracellular targets. By applying the ETL system to observe intracellular structures and protein complexes, we confirmed that this system can obtain 10 nm order z-stacks, within video rate, while maintaining the quality of images and that this system has sufficient optical performance to detect the molecules.

A proteomic study of mitotic phase-specific interactors of EB1 reveals a role for SXIP-mediated protein interactions in anaphase onset.

Microtubules execute diverse mitotic events that are spatially and temporally separated; the underlying regulation is poorly understood. By combining drug treatments, large-scale immunoprecipitation and mass spectrometry, we report the first comprehensive map of mitotic phase-specific protein interactions of the microtubule-end binding protein, EB1. EB1 interacts with some, but not all, of its partners throughout mitosis. We show that the interaction of EB1 with Astrin-SKAP complex, a key regulator of chromosome segregation, is enhanced during prometaphase, compared to anaphase. We find that EB1 and EB3, another EB family member, can interact directly with SKAP, in an SXIP-motif dependent manner. Using an SXIP defective mutant that cannot interact with EB, we uncover two distinct pools of SKAP at spindle microtubules and kinetochores. We demonstrate the importance of SKAP's SXIP-motif in controlling microtubule growth rates and anaphase onset, without grossly disrupting spindle function. Thus, we provide the first comprehensive map of temporal changes in EB1 interactors during mitosis and highlight the importance of EB protein interactions in ensuring normal mitosis.

DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair.

XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.

TAO1 kinase maintains chromosomal stability by facilitating proper congression of chromosomes.

Chromosomal instability can arise from defects in chromosome-microtubule attachment. Using a variety of drug treatments, we show that TAO1 kinase is required for ensuring the normal congression of chromosomes. Depletion of TAO1 reduces the density of growing interphase and mitotic microtubules in human cells, showing TAO1's role in controlling microtubule dynamics. We demonstrate the aneugenic nature of chromosome-microtubule attachment defects in TAO1-depleted cells using an error-correction assay. Our model further strengthens the emerging paradigm that microtubule regulatory pathways are important for resolving erroneous kinetochore-microtubule attachments and maintaining the integrity of the genome, regardless of the spindle checkpoint status.

Automated tracking of mitotic spindle pole positions shows that LGN is required for spindle rotation but not orientation maintenance.

Spindle orientation defines the plane of cell division and, thereby, the spatial position of all daughter cells. Here, we develop a live cell microscopy-based methodology to extract spindle movements in human epithelial cell lines and study how spindles are brought to a pre-defined orientation. We show that spindles undergo two distinct regimes of movements. Spindles are first actively rotated toward the cells' long-axis and then maintained along this pre-defined axis. By quantifying spindle movements in cells depleted of LGN, we show that the first regime of rotational movements requires LGN that recruits cortical dynein. In contrast, the second regime of movements that maintains spindle orientation does not require LGN, but is sensitive to 2ME2 that suppresses microtubule dynamics. Our study sheds first insight into spatially defined spindle movement regimes in human cells, and supports the presence of LGN and dynein independent cortical anchors for astral microtubules.

Lateral to end-on conversion of chromosome-microtubule attachment requires kinesins CENP-E and MCAK.

BACKGROUND: Proper attachment of chromosomes to microtubules is crucial for the accurate segregation of chromosomes. Human chromosomes attach initially to lateral walls of microtubules. Subsequently, attachments to lateral walls disappear and attachments to microtubule ends (end-on attachments) predominate. While it is known in yeasts that lateral to end-on conversion of attachments occurs through a multistep process, equivalent conversion steps in humans remain unknown. RESULTS: By developing a high-resolution imaging assay to visualize intermediary steps of the lateral to end-on conversion process, we show that the mechanisms that bring a laterally bound chromosome and its microtubule end closer to each other are indispensable for proper end-on attachment because laterally attached chromosomes seldom detach. We show that end-on conversion requires (1) the plus-end-directed motor CENP-E to tether the lateral kinetochore onto microtubule walls and (2) the microtubule depolymerizer MCAK to release laterally attached microtubules after a partial end-on attachment is formed. CONCLUSIONS: By uncovering a CENP-E mediated wall-tethering event and a MCAK-mediated wall-removing event, we establish that human chromosome-microtubule attachment is achieved through a set of deterministic sequential events rather than stochastic direct capture of microtubule ends.