Modest increase of KIF11 expression exposes fragilities in the mitotic spindle, causing chromosomal instability.

Chromosomal instability (CIN), the process of increased chromosomal alterations, compromises genomic integrity and has profound consequences on human health. Yet, our understanding of the molecular and mechanistic basis of CIN initiation remains limited. We developed a high-throughput, single-cell, image-based pipeline employing deep-learning and spot-counting models to detect CIN by automatically counting chromosomes and micronuclei. To identify CIN-initiating conditions, we used CRISPR activation in human diploid cells to upregulate, at physiologically relevant levels, 14 genes that are functionally important in cancer. We found that upregulation of CCND1, FOXA1 and NEK2 resulted in pronounced changes in chromosome counts, and KIF11 upregulation resulted in micronuclei formation. We identified KIF11-dependent fragilities within the mitotic spindle; increased levels of KIF11 caused centrosome fragmentation, higher microtubule stability, lagging chromosomes or mitotic catastrophe. Our findings demonstrate that even modest changes in the average expression of single genes in a karyotypically stable background are sufficient for initiating CIN by exposing fragilities of the mitotic spindle, which can lead to a genomically diverse cell population.

Probing microtubule polymerisation state at single kinetochores during metaphase chromosome motion.

Kinetochores regulate the dynamics of attached microtubule bundles (kinetochore-fibres, K-fibres) to generate the forces necessary for chromosome movements in mitosis. Current models suggest that poleward-moving kinetochores are attached to depolymerising K-fibres and anti-poleward-moving kinetochores to polymerising K-fibres. How the dynamics of individual microtubules within the K-fibre relate to poleward and anti-poleward movements is poorly understood. To investigate this, we developed a live-cell imaging assay combined with computational image analysis that allows eGFP-tagged EB3 (also known as MAPRE3) to be quantified at thousands of individual metaphase kinetochores as they undergo poleward and anti-poleward motion. Surprisingly, we found that K-fibres are incoherent, containing both polymerising and depolymerising microtubules ­ with a small polymerisation bias for anti-poleward-moving kinetochores. K-fibres also display bursts of EB3 intensity, predominantly on anti-poleward-moving kinetochores, equivalent to more coherent polymerisation, and this was associated with more regular oscillations. The frequency of bursts and the polymerisation bias decreased upon loss of kinesin-13, whereas loss of kinesin-8 elevated polymerisation bias. Thus, kinetochores actively set the balance of microtubule polymerisation dynamics in the K-fibre while remaining largely robust to fluctuations in microtubule polymerisation.

KiT: a MATLAB package for kinetochore tracking.

UNLABELLED: During mitosis, chromosomes are attached to the mitotic spindle via large protein complexes called kinetochores. The motion of kinetochores throughout mitosis is intricate and automated quantitative tracking of their motion has already revealed many surprising facets of their behaviour. Here, we present 'KiT' (Kinetochore Tracking)-an easy-to-use, open-source software package for tracking kinetochores from live-cell fluorescent movies. KiT supports 2D, 3D and multi-colour movies, quantification of fluorescence, integrated deconvolution, parallel execution and multiple algorithms for particle localization. AVAILABILITY AND IMPLEMENTATION: KiT is free, open-source software implemented in MATLAB and runs on all MATLAB supported platforms. KiT can be downloaded as a package from http://www.mechanochemistry.org/mcainsh/software.php The source repository is available at https://bitbucket.org/jarmond/kit and under continuing development. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: jonathan.armond@warwick.ac.uk.

Springs, clutches and motors: driving forward kinetochore mechanism by modelling.

As a mechanical system, the kinetochore can be viewed as a set of interacting springs, clutches and motors; the problem of kinetochore mechanism is now one of understanding how these functional modules assemble, disassemble and interact with one another to give rise to the emergent properties of the system. The sheer complexity of the kinetochore system points to a future requirement for data-driven mathematical modelling and statistical analysis based on quantitative empirical measurement of sister kinetochore trajectories. Here, we review existing models of chromosome motion in the context of recent advances in our understanding of kinetochore molecular biology.

Perspective: Potential Impact and Therapeutic Implications of Oncogenic PI3K Activation on Chromosomal Instability.

Genetic activation of the class I PI3K pathway is very common in cancer. This mostly results from oncogenic mutations in PIK3CA, the gene encoding the ubiquitously expressed PI3Kα catalytic subunit, or from inactivation of the PTEN tumour suppressor, a lipid phosphatase that opposes class I PI3K signalling. The clinical impact of PI3K inhibitors in solid tumours, aimed at dampening cancer-cell-intrinsic PI3K activity, has thus far been limited. Challenges include poor drug tolerance, incomplete pathway inhibition and pre-existing or inhibitor-induced resistance. The principle of pharmacologically targeting cancer-cell-intrinsic PI3K activity also assumes that all cancer-promoting effects of PI3K activation are reversible, which might not be the case. Emerging evidence suggests that genetic PI3K pathway activation can induce and/or allow cells to tolerate chromosomal instability, which-even if occurring in a low fraction of the cell population-might help to facilitate and/or drive tumour evolution. While it is clear that such genomic events cannot be reverted pharmacologically, a role for PI3K in the regulation of chromosomal instability could be exploited by using PI3K pathway inhibitors to prevent those genomic events from happening and/or reduce the pace at which they are occurring, thereby dampening cancer development or progression. Such an impact might be most effective in tumours with clonal PI3K activation and achievable at lower drug doses than the maximum-tolerated doses of PI3K inhibitors currently used in the clinic.

The secret lives of cancer cell lines.

The extent of genetic and epigenetic diversity between and within patient tumors is being mapped in ever more detail. It is clear that cancer is an evolutionary process in which tumor cell intrinsic and extrinsic forces shape clonal selection. The pre-clinical oncology pipeline uses model systems of human cancer - including mouse models, cell lines, patient-derived organoids and patient-derived xenografts - to study tumor biology and assess the efficacy of putative therapeutic agents. Model systems cannot completely replicate the environment of human tumors and, even within the same cancer model, data are often irreproducible between laboratories. One hypothesis is that ongoing evolutionary processes remain relevant in laboratory models, leading to divergence over time. In a recent edition of Nature, Ben-David and colleagues showed that different stocks of widely used cancer cell lines - a staple of cancer research over many decades - are highly heterogeneous in terms of their genetics, transcriptomics and responses to therapies. The authors find compelling evidence of positive selection based on ongoing mutational processes and chromosomal instability. Thus, the origin, culture conditions and cumulative number of population doublings of cell lines likely influence experimental outcomes. Here, we summarize the key findings of this important study and discuss the practical implications of this work for researchers using cell lines in the laboratory.

Non-random Mis-segregation of Human Chromosomes.

A common assumption is that human chromosomes carry equal chances of mis-segregation during compromised cell division. Human chromosomes vary in multiple parameters that might generate bias, but technological limitations have precluded a comprehensive analysis of chromosome-specific aneuploidy. Here, by imaging specific centromeres coupled with high-throughput single-cell analysis as well as single-cell sequencing, we show that aneuploidy occurs non-randomly following common treatments to elevate chromosome mis-segregation. Temporary spindle disruption leads to elevated mis-segregation and aneuploidy of a subset of chromosomes, particularly affecting chromosomes 1 and 2. Unexpectedly, we find that a period of mitotic delay weakens centromeric cohesion and promotes chromosome mis-segregation and that chromosomes 1 and 2 are particularly prone to suffer cohesion fatigue. Our findings demonstrate that inherent properties of individual chromosomes can bias chromosome mis-segregation and aneuploidy rates, with implications for studies on aneuploidy in human disease.

Nonautonomous movement of chromosomes in mitosis.

Kinetochores are the central force-generating machines that move chromosomes during cell division. It is generally assumed that kinetochores move in an autonomous manner. However, we reveal here that movements of neighboring sister-kinetochore pairs in metaphase are correlated in a distance-dependent manner. This correlation increases in the absence of kinetochore oscillations or stable end-on attachments. This suggests that periodic movements of bioriented chromosomes limit the correlated motion of nonsisters. Computer simulations show that these correlated movements can occur when elastic crosslinks are placed between the K-fibers of oscillating kinetochores. Strikingly, inhibition of the microtubule crosslinking motor kinesin-5 Eg5 leads to an increase in nonsister correlation and impairs periodic oscillations. These phenotypes are partially rescued by codepletion of the kinesin-12 Kif15, demonstrating a function for kinesin-5 and kinesin-12 motors in driving chromosome movements, possibly as part of a crosslinking structure that correlates the movements of nonsister kinetochores.

Diffusion of a membrane protein, Tat subunit Hcf106, is highly restricted within the chloroplast thylakoid network.

The thylakoid membrane forms stacked thylakoids interconnected by 'stromal' lamellae. Little is known about the mobility of proteins within this system. We studied a stromal lamellae protein, Hcf106, by targeting an Hcf106-GFP fusion protein to the thylakoids and photobleaching. We find that even small regions fail to recover Hcf106-GFP fluorescence over periods of up to 3 min after photobleaching. The protein is thus either immobile within the thylakoid membrane, or its diffusion is tightly restricted within distinct regions. Autofluorescence from the photosystem II light-harvesting complex in the granal stacks likewise fails to recover. Integral membrane proteins within both the stromal and granal membranes are therefore highly constrained, possibly forming 'microdomains' that are sharply separated.