Biology subjects

Su, K.-C.

Publications and source records attributed to Su, K.-C..

9 recordsLinked to original sources

bioRxiv2026

Functional genetics reveals a role for the proteasome in regulating centromere assembly

Centromeres link chromosomes to spindle microtubules to direct their segregation during mitosis. The vertebrate centromere is built on two critical DNA-binding proteins, the histone H3-variant CENP-A and histone fold protein CENP-T. CENP-T is a potent scaffold for kinetochore assembly, such that concentrating it at ectopic sites is sufficient to build kinetochore-like structures. How cells prevent excess CENP-T from acting at non-centromere sites remains unclear. Here, using optical pooled screening to systematically define the requirements for CENP-A and CENP-T localization, we recover the established CENP-A deposition machinery and reveal a specific role for the proteasome in controlling CENP-T levels. We find that non-centromere CENP-T is degraded in a ubiquitin- and proteasome-dependent manner. Whereas centromere-incorporated CENP-T is stable, CENP-T that is not bound to centromeres is rapidly turned over, with the CENP-T histone fold region sufficient to confer proteasome sensitivity. Quiescent cells, which disassemble their centromeres, continue to express CENP-T but eliminate it through continuous degradation. Thus, whereas CENP-A is controlled primarily through regulated deposition, cells additionally control CENP-T by selectively degrading the unincorporated protein.

cell biology↗

bioRxiv2026

Systematic functional genetic analysis of cell-substrate adhesion

Cellular adhesion is critical for tissue organization and integrity, but the full complement of proteins required for proper adhesion remains unresolved. Here, we define the requirements for cell-substrate adhesion in cultured human cells using orthogonal, large-scale functional genetic approaches. Using mechanical assays to test the maintenance ("shake-off") or formation of adhesion and parallel large-scale assays of cell morphology, we identify dozens of gene targets with roles in adhesion. Our analyses reveal dynamic requirements for adhesion across timepoints and cell lines. We additionally conduct targeted downstream mechanistic analyses to resolve the molecular basis for altered adhesion. Collectively, we identify established and uncharacterized regulators of adhesion, including genes involved in mitosis, focal adhesions, actin regulation, and membrane trafficking. Unexpectedly, we find that cells that fail cytokinesis display impaired adhesion, with strongly altered actin organization and nuclear dynamics. Together, this work provides a comprehensive view of the genetic requirements for cell-substrate adhesion.

cell biology↗

bioRxiv2025

The dynamics of centromere assembly and disassembly during quiescence

Quiescence is a state in which cells undergo a prolonged proliferative arrest while maintaining their capacity to reenter the cell cycle. Here, we analyze entry and exit from quiescence, focusing on how cells regulate the centromere, a structure involved in chromosome segregation. Despite the constitutive localization of centromere proteins throughout the cell cycle, we find that cells rapidly disassemble most centromere proteins during quiescence entry, while preserving those required to maintain centromere identity. During quiescence exit, the centromere is reassembled and rapidly regains normal homeostatic levels of centromere proteins. Although the histone variant CENP-A is typically deposited during G1, we find that CENP-A deposition does not occur during the G1 immediately following quiescence exit, and instead occurs after cells complete their first mitosis. In contrast, other centromere proteins relocalize during the first S phase independent of DNA replication. These findings reveal centromere dynamics during quiescence entry and exit and highlight paradigms for the timing and control of centromere protein deposition.

cell biology↗

bioRxiv2025

Functional genetics with a hypomorphic CENP-C mutant reveals a regulatory system for chromosome congression

Chromosome congression is a key process that acts to align chromosomes at the spindle equator via kinetochore-microtubule interactions, with defects in chromosome alignment leading to chromosomal instability. However, defining the mechanisms that underlie chromosome congression is limited due to the multiple factors that act in parallel to regulate chromosome movement. Here, we conducted a genome-wide Cas9-based functional genetics screen using a hypomorphic CENP-C mutant that affects its kinetochore interactions. Our analysis identified KIF18A, whose knockout resulted in synthetic lethality with the CENP-C mutant. Further analysis revealed that the synthetic defect was due to a reduction in CENP-E function in the CENP-C mutant. Our work suggests that KIF18A promotes chromosome alignment in cooperation with CENP-E downstream of CENP-C during early prometaphase. Thus, our analysis enables us to dissect parallel molecular mechanisms for chromosome congression and identify sensitivities and biomarkers that might guide anti-KIF18A chemotherapeutics.

cell biology↗

bioRxiv2025

Differential roles for the spindle assembly checkpoint in error surveillance and mitotic timing

The Spindle Assembly Checkpoint (SAC) plays critical roles in regulating mitotic fidelity and progression. Here, we utilized a SAC-deficient cell line lacking the full-length Cdc20 translational protein isoform (Cdc20 {Delta}FL) to define its differential genetic interactions using CRISPR/Cas9-based gene targeting. Cdc20 {Delta}FL cells display synthetic lethal relationships with gene targets required for proper chromosome segregation, highlighting the critical role of the SAC in error surveillance. Surprisingly, we found that the checkpoint component Mad2 becomes dispensable for viability in Cdc20 {Delta}FL cells. Prior work suggested that Mad2 acts as an essential mitotic "timer" to control mitotic duration in unperturbed cells. Instead, our functional analysis indicates that the mitotic timer depends on the interdependent and overlapping actions of: (1) Mad2 inhibition of APC/C-Cdc20, (2) Cdk1-mediated phosphorylation of Cdc20, and (3) total Cdc20 protein levels. Simultaneously perturbing these pathways results in near immediate mitotic exit and catastrophic chromosome mis-segregation.

cell biology↗

bioRxiv2024

Nuclear release of eIF1 globally increases stringency of start-codon selection to preserve mitotic arrest physiology

Regulated start-codon selection has the potential to reshape the proteome through the differential production of uORFs, canonical proteins, and alternative translational isoforms. However, conditions under which start-codon selection is altered remain poorly defined. Here, using transcriptome-wide translation initiation site profiling, we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This increased stringency of start-codon selection during mitosis results from increased interactions between the key regulator of start-codon selection, eIF1, and the 40S ribosome. We find that increased eIF1-40S ribosome interactions during mitosis are mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the changes to translational stringency during mitosis, resulting in altered mitotic proteome composition. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage following treatment with anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency with critical roles during the mammalian cell cycle to preserve mitotic cell physiology.

molecular biology↗

bioRxiv2024

Functional genetics reveals modulators of anti-microtubule drug sensitivity

Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to anti-microtubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug taxol. We further conducted a focused secondary screen to test drug sensitivity for [~]1400 gene targets across two distinct human cell lines and to additionally test sensitivity to the Kif11-inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to anti-microtubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.

cell biology↗

bioRxiv2021

The phenotypic landscape of essential human genes

Understanding the basis for cellular growth, proliferation, and function requires determining the contributions of essential genes to diverse cellular processes. Here, we combined pooled CRISPR/Cas9-based functional screening of 5,072 fitness-conferring genes in human cells with microscopy-based visualization of DNA, DNA damage, actin, and microtubules. Analysis of >31 million individual cells revealed measurable phenotypes for >90% of genes. Using multi-dimensional clustering based on hundreds of quantitative phenotypic parameters, we identified co-functional genes across diverse cellular activities, revealing novel gene functions and associations. Pooled live-cell screening of [~]450,000 cell division events for 239 genes further identified functional contributions to chromosome segregation. Our work creates a resource for the phenotypic analysis of core cellular processes and defines the functional landscape of essential human genes.

cell biology↗

bioRxiv2018

Quiescent cells actively replenish CENP-A nucleosomes to maintain centromere identity and proliferative potential

Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant CENP-A. Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres, but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery, but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential.

cell biology↗