Biology subjects

Flynn, M. J.

Publications and source records attributed to Flynn, M. J..

6 recordsLinked to original sources

bioRxiv2026

The role of cell growth rate on accumulation of the mitotic cyclin Cdc13 in fission yeast

Eukaryotic cells control their size by coordinating growth and division. Fission yeast divide at a reproducible cell size due to regulated activation of the cyclin-dependent kinase Cdk1. The nuclear concentration of mitotic cyclin Cdc13 increases in a time-dependent manner to promote Cdk1 activation as cells grow. Here, we show that interphase Cdc13 is stable against degradation and nuclear export, but is diluted by cell growth. Low glucose reduced cell growth rate but not time-dependent accumulation of Cdc13. Uncoupling the rates of cell growth and Cdc13 accumulation resulted in higher concentrations of nuclear Cdc13 despite reduced cell size. This change coincided with reduced activating phosphorylation of Cdk1-T167. Mathematical modeling and experiments showed that cells maintain size homeostasis under these conditions. In contrast to low glucose, poor nitrogen reduced both cell growth rate and Cdc13 accumulation rate. Therefore, Cdc13 accumulation is independent of cell growth rate but can be altered by nutrient-specific mechanisms.

cell biology↗

bioRxiv2025

Engineered protein circuits for cancer therapy

A central challenge in cancer therapy is selectively killing cancer cells while minimizing resistance. Here, we engineer modular protease-based protein circuits that sense mutant RAS, the most frequently mutated oncogene in cancer, and conditionally activate cell death. Delivered transiently as mRNA in lipid nanoparticles (LNPs), circuits selectively eliminated RAS-mutant cancer cells in culture and suppressed aggressive, multifocal RAS-driven liver tumors. Compared to RAS inhibitors, circuits killed cancer cells independently of oncogene addiction, achieved potent cytotoxicity rather than cytostasis, and functioned at low RAS occupancy through catalysis rather than stoichiometric inhibition. Critically, circuits acquired minimal to no resistance under prolonged selection, while remaining potent against prevalent drug-resistance mechanisms including RAS amplification and bypass signaling. Together, these results establish design principles for engineering therapeutic protein circuits and highlight their potential to overcome longstanding limitations of existing therapeutic modalities.

synthetic biology↗

bioRxiv2024

Calcineurin promotes adaptation to chronic stress through two distinct mechanisms

Adaptation to environmental stress requires coordination between stress-defense programs and cell cycle progression. The immediate response to many stressors has been well characterized, but how cells survive in challenging environments long-term is unknown. Here, we investigate the role of the stress-activated phosphatase calcineurin (CN) in adaptation to chronic CaCl2 stress in Saccharomyces cerevisiae. We find that prolonged exposure to CaCl2 impairs mitochondrial function and demonstrate that cells respond to this stressor using two CN-dependent mechanisms - one that requires the downstream transcription factor Crz1 and another that is Crz1-independent. Our data indicate that CN maintains cellular fitness by promoting cell cycle progression and preventing CaCl2-induced cell death. When Crz1 is present, transient CN activation suppresses cell death and promotes adaptation despite high levels of mitochondrial loss. However, in the absence of Crz1, prolonged activation of CN prevents mitochondrial loss and further cell death by upregulating glutathione (GSH) biosynthesis genes thereby mitigating damage from reactive oxygen species. These findings illustrate how cells maintain long-term fitness during chronic stress and suggest that CN promotes adaptation in challenging environments by multiple mechanisms.

cell biology↗

bioRxiv2024

Synthetic dosage-compensating miRNA circuits for quantitative gene therapy

A longstanding challenge in gene therapy is expressing a dosage-sensitive gene within a tight therapeutic window. For example, loss of MECP2 function causes Rett syndrome, while its duplication causes MECP2 duplication syndrome. Viral gene delivery methods generate variable numbers of gene copies in individual cells, creating a need for gene dosage-invariant expression systems. Here, we introduce a compact miRNA-based, incoherent feed-forward loop circuit that achieves precise control of Mecp2 expression in cells and brains, and improves outcomes in an AAV-based mouse model of Rett syndrome gene therapy. Single molecule analysis of endogenous and ectopic Mecp2 mRNA revealed precise, sustained expression across a broad range of gene dosages. Delivered systemically in a brain-targeting AAV capsid, the circuit strongly suppressed Rett behavioral symptoms for over 24 weeks, outperforming an unregulated gene therapy. These results demonstrate that synthetic miRNA-based regulatory circuits can enable precise in vivo expression to improve the safety and efficacy of gene therapy. One sentence description: A synthetic miRNA-based incoherent feed-forward loop circuit embedded in a gene delivery vector overcomes the challenge of overexpression toxicity in a mouse model of Rett syndrome gene therapy.

synthetic biology↗

bioRxiv2024

miRNA circuit modules for precise, tunable control of gene expression

Accurate control of transgene expression is important for research and therapy but challenging to achieve in most settings. miRNA-based regulatory circuits can be incorporated within transgenes for improved control. However, the design principles, performance limits, and applications of these circuits in research and biotechnology have not been systematically determined. Here, combining modeling and experiments, we introduce miRNA-based circuit modules, termed DIMMERs, that establish precise, tunable control of transgene expression across diverse cell types to facilitate imaging, editing, and gene therapy. The circuits use multivalent miRNA regulatory interactions to achieve nearly uniform, tunable, protein expression over two orders of magnitude variation in gene dosage. They function across diverse cell types, and can be multiplexed for independent regulation of multiple genes. DIMMERs reduce off-target CRISPR base editing, improve single-molecule imaging, and allow live tracking of AAV-delivered transgene expression in mouse cortical neurons. DIMMERs thus enable accurate regulation for research and biotechnology applications.

synthetic biology↗

bioRxiv2019

The coordinate actions of calcineurin and Hog1 mediate the response to cellular stress through multiple nodes of the cell cycle network

Upon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between diverse stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the stress-activated phosphatase calcineurin (CN) in this process and show that CN utilizes multiple pathways to control the cell cycle. Upon activation, CN inhibits transcription factors (TFs) that regulate the G1/S transition through activation of the stress-activated MAPK Hog1. In contrast, CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner at multiple nodes of the cell cycle-regulatory network to rewire gene expression and arrest cells in response to stress. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors.

cell biology↗