Biology subjects

Strong, M. A.

Publications and source records attributed to Strong, M. A..

5 recordsLinked to original sources

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

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↗

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↗

bioRxiv2018

TIN2 functions with TPP1/POT1 to stimulate telomerase processivity

Telomere length maintenance is crucial for cells that divide many times. TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short telomere syndromes. While the genetics underscore the importance of TIN2, the mechanism through which TIN2 regulates telomere length remains unclear. Here, we characterize the effects of TIN2 on telomerase activity. We identified a new isoform in human cells, TIN2M, that is expressed at similar levels to previously studied TIN2 isoforms. Additionally, we found that all three TIN2 isoforms stimulated telomerase processivity beyond the previously characterized stimulation by TPP1/POT1. Mutations in the TPP1 TEL-patch abrogated this stimulation, implicating TIN2 as a component of the TPP1/POT1 processivity complex. All three TIN2 isoforms localized to telomeres in vivo but had distinct effects on telomere length, suggesting they are functionally distinct. These data contrast previous descriptions of TIN2 a simple scaffolding protein, showing that TIN2 isoforms directly regulate telomerase.

molecular biology↗

bioRxiv2017

Diagnostic utility of telomere length measurement in a hospital setting

Very short telomere length (TL) provokes cellular senescence in vitro, but the clinical utility of TL measurement in a hospital-based setting has not been determined. We tested the diagnostic and prognostic value of TL measurement by flow cytometry and fluorescence in situ hybridization (flowFISH) in individuals with mutations in telomerase and telomere maintenance genes, and examined prospectively whether TL altered treatment decisions for patients with bone marrow failure. TL had a definable normal range across populations with discrete lower and upper boundaries. TL above the 50th age-adjusted percentile had a 100% negative predictive value for clinically relevant mutations in telomere maintenance genes, but the lower threshold for diagnosis was age-dependent. The extent of deviation from the age-adjusted median correlated with the age at diagnosis of a telomere syndrome as well as the predominant complication. Mild short telomere defects manifested in adults as pulmonary fibrosis-emphysema, while severely short TL manifested in children as bone marrow failure and immunodeficiency. Among 38 newly diagnosed patients with bone marrow failure, TL shorter than the 1st age-adjusted percentile enriched for patients with germline mutations in inherited bone marrow failure genes, such as RUNX1, in addition to telomere maintenance genes. The TL result modified the hematopoietic stem cell donor choice and/or treatment regimen in one-fourth of the cases (9 of 38,24%). TL testing by flowFISH has diagnostic and predictive value in definable clinical settings. In patients with bone marrow failure, it altered treatment decisions for a significant subset.

genetics↗