Biology subjects

Kops, G. J.

Publications and source records attributed to Kops, G. J..

11 recordsLinked to original sources

bioRxiv2026

Linking live-cell imaging to single-cell sequencing using genetic barcoding

Linking cellular phenotype to genotype and gene expression is a central goal in biomedical research. Live-cell imaging captures dynamic cellular phenotypes, whereas single-cell sequencing measures genome-wide molecular states, but pairing these readouts for the same cell remains challenging. Here we present Waldo, a genetically encoded single-cell barcoding strategy that can be read out by both live-cell imaging and single-cell sequencing to connect single cells across both modalities. Waldo barcodes are generated by combinatorial expression of synthetic cell-surface receptors, each detected by a cognate peptide probe coupled either to a fluorescent dye or a DNA barcode. Iterative fluorescent probe staining achieves large barcode diversity, while preserving fluorescence channels for phenotypic reporters. As a proof-of-principle, we use Waldo to link cell division phenotypes after chromosome missegregation to the karyotype and transcriptome of the same cells. Waldo provides a general strategy for connecting imaging phenotypes to genome-wide sequencing readouts in single cells.

cell biology↗

bioRxiv2026

Mitotic errors drive rapid clearance of polyploidy during intestinal regeneration despite robust centrosome clustering

Polyploid cells are increasingly recognized not only as hallmarks of cancer but also as features of regenerating tissues. During intestinal regeneration, polyploid cells are transient, yet the mechanisms underlying their clearance remain unknown. Using mouse intestinal organoids as a regeneration model, we show that, unlike in many cancer cell-line models, this clearance occurs without immediate cell-cycle arrest and is not driven by failure to establish spindle bipolarity. Instead, polyploid intestinal cells efficiently cluster supernumerary centrosomes to form bipolar spindles in an HSET-dependent manner, facilitated by delayed centrosome separation at mitotic onset. Despite this, polyploid divisions frequently produce chromosome segregation errors, including catastrophic chronocrisis. Lineage tracing reveals that progeny of such divisions is rapidly lost over subsequent generations. Increasing polyploidy during early regeneration disrupts organoid maturation, indicating that timely polyploidy clearance is required for successful regeneration. Polyploid cells are also detected in regenerating human colonic organoids, suggesting that transient polyploidy is a conserved feature of intestinal regeneration.

cell biology↗

bioRxiv2025

Non-cell-autonomous mechanisms of tumor initiation and relapse by chromosomal instability

Chromosomal instability (CIN) is a hallmark of cancer, and a primary cause of genetic heterogeneity in tumors. Depending on the degree of CIN and the affected tissue, CIN can promote or suppress tumor formation, and high CIN induction has been proposed as a therapeutic strategy. How CIN achieves these effects is unclear. Here we use a conditional mouse model of graded CIN in combination with longitudinal monitoring of DMBA/TPA-initiated skin tumors to show that low CIN increases the frequency of skin tumor initiation, while higher CIN accelerates onset and growth rates. Strikingly, gene recombination analysis of the tumors reveals that upon high CIN induction the fast-growing tumors originate from rare low CIN cells, suggesting a strong non-cell-autonomous effect of high CIN. Gene expression analysis and immunohistochemistry show that high CIN causes epidermal hyperplasia, immune evasion and a regenerative response that stimulates low CIN tumor growth beyond what is achieved by induction of low CIN alone. Such cell-extrinsic effects may be a common mechanism of tumor formation by CIN, as we observe it also in CIN-induced tumors of the intestine, breast and mesentery. When high CIN is induced in established skin tumors, mimicking CIN-based therapy, tumors regress but relapse quickly. Relapsed tumors, too, arose from rare low CIN cells. Our findings have implications for our understanding of the contributions of CIN to cancer initiation and progression and give caution to the rationale for CIN therapies. Significance statementChromosomal instability (CIN) is a source of genetic diversity in cancer. Different degrees of CIN can be highly oncogenic or tumor suppressive, the latter being a rationale for CIN-based therapies. Using a mouse model for precise control of CIN levels, we show that low CIN promotes tumor initiation, while moderate CIN accelerates both initiation and tumor growth. Surprisingly, moderate CIN acts through its effects on the surrounding tissue rather than directly within tumor cells, by triggering a regenerative response accompanied by immune evasion. When very high CIN is induced in existing tumors, tumors initially regress but rapidly relapse. These findings inform on the contributions of CIN to cancer initiation and progression and give caution to the rationale for CIN therapies.

cancer biology↗

bioRxiv2025

Cancer type-specific association of p53 deficiency with aneuploidy and chromosome losses

Aneuploidy and TP53 mutations are among the most frequent genetic alterations in cancer. p53 inactivation is widely considered a key enabling event for aneuploidy emergence. However, whether p53 protects against specific types of aneuploidy, and whether this is universal across cancer types, remains unclear. By analyzing aneuploidy features across cancer types in the TCGA database, we show that p53 inactivation is neither sufficient nor necessary for cancer aneuploidy. While p53-deficient tumors show higher pan-cancer aneuploidy, within individual cancer types p53-proficient and -deficient tumors often have comparable aneuploidy levels, and p53-proficient cancers can be highly aneuploid. Instead, p53 deficiency is associated with a higher frequency of chromosome losses. Single-cell sequencing of non-transformed cells after induced chromosomal instability reveals preferential p53 pathway activation upon chromosome loss but not gain. Prolonged culture selectively enriches for monosomies when p53 is inactivated. p53 thus acts as a context-dependent safeguard against aneuploidy, particularly against chromosome losses.

cancer biology↗

bioRxiv2024

A unique interplay of access and selection shapes peritoneal metastasis evolution in colorectal cancer

Whether metastasis in humans can be accomplished by most primary tumor cells or requires the evolution of a specialized trait remains an open question. To evaluate whether metastases are founded by non-random subsets of primary tumor lineages requires extensive, difficult-to-implement sampling. We have realized an unusually dense multi-region sampling scheme in a cohort of 26 colorectal cancer patients with peritoneal metastases, reconstructing the evolutionary history of on average 28.8 tissue samples per patient with a microsatellite-based fingerprinting assay. To assess metastatic randomness, we evaluate inter- and intra-metastatic heterogeneity relative to the primary tumor and find that peritoneal metastases are more heterogeneous than liver metastases but less diverse than locoregional metastases. Metachronous peritoneal metastases exposed to systemic chemotherapy show significantly higher inter-lesion diversity than synchronous, untreated metastases. Projection of peritoneal metastasis origins onto a spatial map of the primary tumor reveals that they often originate at the deep-invading edge, in contrast to liver and lymph node metastases which exhibit no such preference. Furthermore, peritoneal metastases typically do not share a common subclonal origin with distant metastases in more remote organs. Synthesizing these insights into an evolutionary portrait of peritoneal metastases, we conclude that the peritoneal-metastatic process imposes milder selective pressures onto disseminating cancer cells than the liver-metastatic process. Peritoneal metastases unique evolutionary features have potential implications for staging and treatment.

genetics↗

bioRxiv2023

A farnesyl-dependent structural role for CENP-E in expansion of the fibrous corona

Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation and spindle checkpoint signaling. The molecular requirements for formation of the fibrous corona are not fully understood. Here we show that the fibrous corona depends on the mitotic kinesin CENP-E, and that poorly expanded fibrous coronas after CENP-E depletion are functionally compromised. This previously unrecognized role for CENP-E does not require its motor activity but instead is driven by farnesyl modification of its C-terminal kinetochore-and microtubule-binding domain. We show that in cells CENP-E interacts with RZZ-S complexes in a farnesyl-dependent manner. CENP-E is recruited to kinetochores following RZZ-S, and - while not required for RZZ-S oligomerization per se - promotes subsequent fibrous corona expansion. Our comparative genomics analyses suggest that the farnesylation motif in CENP-E orthologs emerged alongside the full RZZ-S module in an ancestral lineage close to the fungi-animal split (Obazoa), revealing potential conservation of the mechanisms for fibrous corona formation. Our results show that proper spindle assembly has a potentially conserved non-motor contribution from the kinesin CENP-E through stabilization of the fibrous corona meshwork during its formation.

cell biology↗

bioRxiv2023

Predicting CIN rates from single-cell whole genome sequencing data using an in silico model

Chromosomal instability (CIN) drives the formation of karyotype aberrations in cancer cells and is a major contributor to intra-tumour heterogeneity, metastasis, and therapy resistance. Understanding how CIN contributes to tumour karyotype evolution requires quantification of CIN rates in primary tumours. Single-cell sequencing-based technologies enable the detection of karyotype heterogeneity, however deducing the actual CIN rates that underlie intra-tumour heterogeneity is still complicated. We have developed an in-silico model, called CINsim, to simulate the karyotype dynamics and validated our model in a murine mouse model for T-cell lymphoma (T-ALL) in which CIN is introduced by mutation of the Mps1 spindle assembly checkpoint protein. CINsim can simulate karyotype evolution within physiologically relevant timescales, across a range of CIN rates, and across a range of karyotype-imposed survival and proliferation effects. We find that CINsim can accurately predict the CIN rates in chromosomal instable mouse T-ALLs as well as in human colon cancer organoids as observed by live-cell time-lapse imaging. We conclude that CINsim is a powerful tool to estimate CIN rates from static single-cell DNA sequencing data by finding the most likely path from euploid founder cell to a heterogeneous tumour cell population.

cancer biology↗

bioRxiv2019

Cyclin B1 scaffolds MAD1 at the corona to activate the spindle assembly checkpoint

The Cyclin B:CDK1 kinase complex is the master regulator of mitosis that phosphorylates hundreds of proteins to coordinate mitotic progression. We show here that, in addition to these kinase functions, Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminus of MAD1, and point mutations in this region remove corona MAD1 and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness, in this context, arises because Cyclin B1-MAD1 localisation becomes MPS1-independent after the corona has been established. We demonstrate that this allows corona-MAD1 to persist at kinetochores when MPS1 activity falls, ensuring that it can still be phosphorylated on a key C-terminal catalytic site by MPS1. Therefore, this study explains how corona MAD1 generates a robust SAC signal and why stripping of this pool by dynein is essential for SAC silencing. It also reveals that the key mitotic kinase, Cyclin B1-Cdk1, scaffolds the pathway that inhibits its own degradation.

cell biology↗

bioRxiv2018

Interactions between N-terminal modules in MPS1 enable spindle checkpoint silencing

AO_SCPCAPBSTRACTC_SCPCAPFaithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until all chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to unattached kinetochores to initiate SAC signaling, and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here we show that a helical fragment within the kinetochore-targeting NTE module of MPS1 is required for interactions with kinetochores, and also forms intramolecular interactions with its adjacent TPR domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, ineffecient MPS1 delocalization from kinetochores upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MSP1-NDC80-C interactions.

cell biology↗

bioRxiv2018

Dynamic Kinetochore Size Regulation Promotes Microtubule Capture And Chromosome Biorientation In Mitosis

Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. An outer layer of the kinetochore known as the fibrous corona transiently expands in early mitosis and disassembles upon microtubule capture. Neither the functional importance nor the mechanistic basis for this are known. Here we show that the dynein adaptor Spindly and the RZZ kinetochore complex drive fibrous corona formation in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ:Spindly complexes into a corona-like meshwork in cells and in vitro. Concurrent with corona expansion, Spindly potentiates corona shedding by recruiting dynein via three conserved short linear motifs. Expanded, non-sheddable fibrous coronas engage in extensive, long-lived lateral microtubule interactions that persist to metaphase and result in fused sister kinetochores, formation of merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments.

cell biology↗

bioRxiv2016

Phylogenomics-guided discovery of a novel conserved cassette of short linear motifs in BubR1 essential for the spindle checkpoint

The spindle assembly checkpoint (SAC) maintains genomic integrity by preventing progression of mitotic cell division until all chromosomes are stably attached to spindle microtubules. The SAC critically relies on the paralogs Bub1 and BubR1/Mad3, which integrate kinetochore-spindle attachment status with generation of the anaphase inhibitory complex MCC. We previously reported on the widespread occurrences of independent gene duplications of an ancestral MadBub gene in eukaryotic evolution and the striking parallel subfunctionalization that lead to loss of kinase function in BubR1/Mad3-like paralogs. We now present an elaborate subfunctionalization analysis that includes all known motifs in Bub1 and BubR1, and show that ancestral features are consistently retained in the same functional paralog: GLEBS/CDI/CDII/kinase in the Bub1-like and KEN1/KEN2/D-Box in the BubR1/Mad3-like. The recently described ABBA motif can be found in either or both paralogs. We however discovered two additional ABBA motifs that flank KEN2. This cassette of ABBA1-KEN2-ABBA2 forms a strictly conserved module in all ancestral and BubR1/Mad3-like proteins, suggestive of a specific and crucial SAC function. Indeed, deletion of the ABBA motifs in human BUBR1 abrogates the SAC and affects APC/C-Cdc20 interactions. Our detailed comparative genomics analyses thus enabled discovery of a conserved cassette of motifs essential for the SAC and shows how this approach can be used to uncover hitherto unrecognized functional protein features.

cell biology↗