Biology subjects

Holland, A. J.

Publications and source records attributed to Holland, A. J..

12 recordsLinked to original sources

bioRxiv2026

Centrosome depletion rewires mitosis to impose dependence on the AURK-TPX2 axis

Centrosomes are key microtubule-organizing centers required for accurate spindle assembly and chromosome segregation, and their dysfunction in cancer creates therapeutic vulnerabilities. Prior work identified a synthetic lethal interaction between TRIM37 overexpression and Polo-like kinase 4 inhibition (PLK4i) in 17q23-amplified tumors, motivating the clinical development of centrosome-depleting PLK4 inhibitors. However, the broader determinants of sensitivity and resistance to PLK4 inhibition remain poorly defined. Using genome-wide CRISPR-Cas9 screening, we identify multiple genetic suppressors of sensitivity to centrosome depletion, including loss of PPP6C as a general escape mechanism, mediated by enhanced activation of Aurora kinase A (AURKA) on the spindle. This process requires NuMA, which scaffolds robust acentrosomal spindle assembly, and operates independently of the TRIM37-regulated pathway that restores pericentriolar material (PCM) foci to reconstitute microtubule-organizing center activity. We further show that centrosome depletion creates a dependence on the AURKA-TPX2 axis for spindle assembly, such that modulation of this pathway shapes cellular responses to PLK4 inhibition. Loss of PPP6C elevates AURKA activity and confers resistance, whereas disruption of the AURKA-TPX2 axis sensitizes cells to centrosome depletion. Together, these findings reveal how centrosome depletion rewires mitotic organization, rendering cells dependent on distinct adaptive spindle assembly pathways.

cancer biology↗

bioRxiv2025

The DNA damage response pathway is required for multiciliated cell differentiation

DNA damage can result from external sources or occur during programmed genome rearrangements in processes like immunity or meiosis. To maintain genome integrity, cells activate DNA repair pathways that prevent harmful outcomes such as cancer or immune dysfunction. In this study, we uncover a novel role for DNA damage during the terminal differentiation of multiciliated cells (MCCs). MCCs, which line the airways, reproductive tracts, and brain ventricles, produce hundreds of motile cilia, each anchored by a centriole. Therefore, MCCs must generate hundreds of centrioles during differentiation. Normally, centriole duplication is tightly linked to the S and G2 phases of the cell cycle, raising questions about how MCCs override numerical and temporal restrictions on centriole duplication. We find that differentiating MCCs accumulate extensive double-stranded DNA breaks during centriole amplification, with damage levels correlating with centriole number. DNA damage response (DDR) kinases are essential for supporting centriole biogenesis and ciliogenesis. We also observe that transcriptional activity, required for the expression of centriole and cilia genes, produces RNA-DNA hybrids (R-loops) that co-localize with DNA damage. This suggests that transcription-coupled DNA damage helps initiate a pseudo-cell cycle program, permitting centriole amplification without triggering full S/G2 phase processes. Our findings indicate that MCCs harness DDR signaling as part of their developmental program, revealing a broader principle in which the canonical cell cycle is adaptively rewired during differentiation.

cell biology↗

bioRxiv2024

Mesoscale regulation of MTOCs by the E3 ligase TRIM37

Centrosomes ensure accurate chromosome segregation during cell division. Although the regulation of centrosome number is well-established, less is known about the suppression of non-centrosomal MTOCs (ncMTOCs). The E3 ligase TRIM37, implicated in Mulibrey nanism and 17q23-amplified cancers, has emerged as a key regulator of both centrosomes and ncMTOCs. Yet, the mechanism by which TRIM37 achieves enzymatic activation to target these mesoscale structures had remained unknown. Here, we elucidate TRIM37s activation process, beginning with TRAF domain-directed substrate recognition, progressing through B-box domain-mediated oligomerization, and culminating in RING domain dimerization. Using optogenetics, we demonstrate that TRIM37s E3 activity is directly coupled to the assembly state of its substrates, activating only when centrosomal proteins cluster into higher-order assemblies resembling MTOCs. This regulatory framework provides a mechanistic basis for understanding TRIM37-driven pathologies and, by echoing TRIM5s restriction of the HIV capsid, unveils a conserved activation blueprint among TRIM proteins for controlling mesoscale assembly turnover.

molecular biology↗

bioRxiv2024

The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

Studying essential genes required for dynamic processes in live mice is challenging as genetic perturbations are irreversible and limited by slow protein depletion kinetics. The first-generation auxin-inducible-degron (AID) system is a powerful tool for analyzing inducible protein loss in cultured cells. However, auxin administration is toxic to mice, preventing its long-term use in animals. Here, we use an optimized second-generation AID system to achieve the conditional and reversible loss of the essential centrosomal protein CEP192 in live mice. We show that the auxin derivative 5-Ph-IAA is well tolerated over two weeks and drives near-complete CEP192-mAID degradation in less than one hour in vivo. Prolonged CEP192 loss led to cell division failure and cell death in proliferative tissues. Thus, the second-generation AID system is well suited for rapid and/or sustained protein depletion in live mice, offering a valuable new tool for interrogating protein function in vivo.

cell biology↗

bioRxiv2024

Haspin kinase binds to a nucleosomal DNA supergroove

Phosphorylation of histone H3 threonine 3 (H3T3) by Haspin recruits the chromosomal passenger complex to the inner centromere and ensures proper cell cycle progression through mitosis. The mechanism by which Haspin binds to nucleosomes to phosphorylate H3T3 is not known. We report here cryo-EM structures of the Haspin kinase domain bound to a nucleosome. In contrast with previous structures of histone-modifying enzymes, Haspin solely contacts the nucleosomal DNA, inserting into a supergroove formed by apposing major grooves of two DNA gyres. This unique binding mode provides a plausible mechanism by which Haspin can bind to nucleosomes in a condensed chromatin environment to phosphorylate H3T3. We identify key basic residues in the Haspin kinase domain that are essential for phosphorylation of nucleosomal histone H3 and binding to mitotic chromatin. Our structure is the first of a kinase domain bound to a nucleosome and is the first example of a histone-modifying enzyme that binds to nucleosomes solely through DNA contacts.

biophysics↗

bioRxiv2024

Centriole structural integrity defects are a crucial feature of Hydrolethalus Syndrome

Hydrolethalus Syndrome (HLS) is a lethal, autosomal recessive ciliopathy caused by the mutation of the conserved centriole protein HYLS1. However, how HYLS1 facilitates the centriole-based templating of cilia is poorly understood. Here, we show that mice harboring the HYLS1 disease mutation die shortly after birth and exhibit developmental defects that recapitulate several manifestations of the human disease. These phenotypes arise from tissue-specific defects in cilia assembly and function caused by a loss of centriole integrity. We show that HYLS1 is recruited to the centriole by CEP120 and functions to recruit centriole inner scaffold proteins that stabilize the centriolar microtubule wall. The HLS mutation disrupts the interaction of HYLS1 with CEP120 leading to HYLS1 displacement and degeneration of the centriole distal end. We propose that tissue-specific defects in centriole integrity caused by the HYLS1 mutation prevent ciliogenesis and drive HLS phenotypes.

cell biology↗

bioRxiv2024

Exact centriole counts are critical for B cell development but not function

Centrioles define centrosome structure and function. Deregulation of centriole numbers can cause developmental defects and foster malignant disease. The p53 tumor suppressor limits the growth of cells lacking or harboring additional centrioles and can be engaged by the "mitotic surveillance" or the "PIDDosome pathway", respectively. Here, we show that early B cell progenitors frequently present extra centrioles that are rapidly lost during maturation. Increasing centriole counts beyond physiological levels by Polo-like kinase 4 (PLK4) overexpression induces apoptosis, suggesting clearance of such cells during development. Remarkably, this apoptotic response is independent of PIDD1 or p53, but can be blocked by excess BCL2. In contrast, loss of centrosomes upon Plk4 deletion arrests B cell development at the pro B cell stage. This defect can be rescued by co-deletion of Usp28, a critical component of the mitotic surveillance pathway that restores cell number and function in the absence of centrioles. In both scenarios, too many and too few centrosomes, mitochondrial apoptosis is engaged to kill B cells with abnormal centriole counts during their development with progenitor B cells being intolerant to centriole loss but permissive to centriole amplification. Unexpectedly, our findings show that centrioles are dispensable for mounting an effective humoral immune response.

cell biology↗

bioRxiv2024

Microtubule-dependent orchestration of centriole amplification in brain multiciliated cells

Multiciliated cell (MCC) differentiation is a calibrated version of the canonical cell cycle. The MCC cell cycle variant sustains amplification of centrioles for the nucleation of dozens of motile cilia, while avoiding cell division. In this study, we show that the MCC cell cycle variant is an accelerated version of the canonical cell cycle, which superposes two cycles of centriole biogenesis, in order to obtain multiple mature centrioles within a single -instead of double- cell cycle iteration. We further show that the precocious maturation of amplified procentrioles is even determinant for their spatial self-organization, disengagement and apical migration for cilia nucleation. Our findings collectively suggest that the decomposition of centriole biogenesis over two cycle iterations in dividing cells may have been adopted to ensure the growth of a solitary primary cilium, and exemplify how minimal deviations of the canonical cell cycle allow MCC progenitors to both amplify, and accelerate, centriole biogenesis for vital motile ciliogenesis.

cell biology↗

bioRxiv2023

The mitotic surveillance pathway requires PLK1-dependent 53BP1 displacement from kinetochores

53BP1 acts at the crossroads between DNA repair and p53-mediated stress response. With its interactor USP28, it is part of the mitotic surveillance pathway (MSP), a sensor that monitors the duration of cell division, promoting p53-dependent cell cycle arrest when a critical time threshold is surpassed. 53BP1 dynamically associates with kinetochores, being recruited during prophase, and then undergoing a time-dependent loss of affinity. However, the relevance of this behaviour remains unclear. Here, we identify CENP-F as an interaction partner and kinetochore receptor for 53BP1. By engineering human cells with a CENP-F point mutation, we demonstrate that preventing 53BP1 kinetochore localization does not reduce MSP proficiency. Strikingly, however, preventing the loss of 53BP1 from the kinetochore by inhibiting Polo-like kinase 1 (PLK1) restrains MSP activity, a phenomenon that is abrogated in the CENP-F mutant condition. Taken together, we demonstrate that kinetochore-loaded 53BP1 represents an MSP functionally inhibited state and that PLK1-dependent re-localization of 53BP1 represents an important layer of MSP regulation.

cell biology↗

bioRxiv2022

PLK4 drives centriole amplification and apical surface area expansion in multiciliated cells

Multiciliated cells (MCCs) are terminally differentiated epithelia that assemble multiple motile cilia used to promote fluid flow. To template these cilia, MCCs dramatically expand their centriole content during a process known as centriole amplification. In cycling cells, the master regulator of centriole assembly Polo-like kinase 4 (PLK4) is essential for centriole duplication; however recent work has questioned the role of PLK4 in centriole assembly in MCCs. To address this discrepancy, we created genetically engineered mouse models and demonstrated that both PLK4 protein and kinase activity are critical for centriole amplification in MCCs. Tracheal epithelial cells that fail centriole amplification accumulate large assemblies of centriole proteins and do not undergo apical surface area expansion. These results show that the initial stages of centriole assembly are conserved between cycling cells and MCCs and suggest that centriole amplification and surface area expansion are coordinated events.

cell biology↗

bioRxiv2021

Development and validation of inducible protein degradation and quantitative phosphoproteomics to identify kinase-substrate relationships.

Phosphorylation signaling is an essential post-translational regulatory mechanism that governs almost all eukaryotic biological processes and is controlled by an interplay between protein kinases and phosphatases. Knowledge of direct substrates of kinases provides evidence of mechanisms that relate activity to biological function. Linking kinases to their protein substrates can be achieved by inhibiting or reducing kinase activity and quantitative comparisons of phosphoproteomes in the presence and absence of kinase activity. Unfortunately, most of the human kinases lack chemical inhibitors with selectivity required to unambiguously assign protein substrates to their respective kinases. Here, we develop and validate a chemical proteomics strategy for linking kinase activities to protein substrates via targeted protein degradation and quantitative phosphoproteomics and apply it to the well-studied, essential mitotic regulator polo-like kinase 1 (Plk1). We leveraged the Tir1/auxin system to engineer HeLa cells with endogenously homozygous auxin-inducible degron (AID)-Plk1). We used HeLa cells and determined the impact of AID-tagging on Plk1 activity, localization, protein interactors, and substrate motifs. Using quantitative proteomics, we show that of over 8,000 proteins quantified, auxin addition is highly selective for degrading AID-Plk1 in mitotic cells. Comparison of phosphoproteome changes in response to chemical Plk1 inhibition to auxin-induced degradation revealed a striking degree of correlation. Finally, we explored basal protein turnover as a potential basis for clonal differences in auxin-induced degradation rates for AID-Plk1 cells. Taken together, our work provides a roadmap for the application of AID technology as a general strategy for the kinome-wide discovery of kinase-substrate relationships.

systems biology↗

bioRxiv2018

Autoamplification and competition drive symmetry breaking: Initiation of centriole duplication by the PLK4-STIL network

Symmetry breaking, a central principle of physics, has been hailed as the driver of self-organization in biological systems in general and biogenesis of cellular organelles in particular, but the molecular mechanisms of symmetry breaking only begin to become understood. Centrioles, the structural cores of centrosomes and cilia, must duplicate every cell cycle to ensure their faithful inheritance through cellular divisions. Work in model organisms identified conserved proteins required for centriole duplication and found that altering their abundance affects centriole number. However, the biophysical principles that ensure that, under physiological conditions, only a single procentriole is produced on each mother centriole remain enigmatic. Here we propose a mechanistic biophysical model for the initiation of procentriole formation in mammalian cells. We posit that interactions between the master regulatory kinase PLK4 and its activator-substrate STIL form the basis of the procentriole initiation network. The model faithfully recapitulates the experimentally observed transition from PLK4 uniformly distributed around the mother centriole, the \"ring\", to a unique PLK4 focus, the \"spot\", that triggers the assembly of a new procentriole. This symmetry breaking requires a dual positive feedback based on autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4-STIL complexes by phosphorylated STIL. We find that, contrary to previous proposals, in situ degradation of active PLK4 is insufficient to break symmetry. Instead, the model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes explains both the instability of the PLK4 ring and formation of the unique PLK4 spot. In the model, strong competition at physiologically normal parameters robustly produces a single procentriole, while increasing overexpression of PLK4 and STIL weakens the competition and causes progressive addition of procentrioles in agreement with experimental observations.

cell biology↗