Biology subjects

Kapadia, N.

Publications and source records attributed to Kapadia, N..

7 recordsLinked to original sources

bioRxiv2026

AlphaFold-Multimer reveals diverse cyclin-CDK substrate docking interactions

Cyclin-dependent kinases (CDKs) control eukaryotic cell-cycle progression by phosphorylating specific substrates with substrate recognition often involving cyclin-specific docking interactions. However, in minimal cell cycle control systems driven by a single cyclin-CDK complex, how docking interactions contribute to the differential timing of substrate phosphorylation remains unclear. Here, we used AlphaFold-Multimer to systematically predict interactions between the fission yeast mitotic cyclin-CDK fusion Cdc13-L-Cdc2 and its known in vivo CDK substrates. We found that many substrates are predicted to interact with the cyclin hydrophobic patch, and have identified a previously uncharacterised docking motif, [FVIPWGLAM](x)xER[LMV] (ERL motif), with features consistent with an atypical RxL motif. We show that ERL motifs can functionally substitute for canonical RxL motifs to promote phosphorylation of a model CDK substrate by Cdc13-Cdc2, while the S-phase cyclin-CDK Cig2-Cdc2 was found to preferentially phosphorylate substrates containing canonical RxL motifs. Finally, we investigated whether Cdc13-L-Cdc2 is predicted to preferentially bind DNA replication substrates over mitotic substrates but found no evidence of differential binding. These results reveal diversity in cyclin-CDK substrate recognition beyond established docking motifs.

Cell Biology↗

bioRxiv2024

Effectiveness of FLASH vs conventional dose rate radiotherapy in a model of orthotopic, murine breast cancer

PurposeRadiotherapy is an effective breast cancer treatment that enhances local tumor control and prolongs overall survival yet is associated with undesirable side effects which can impair quality of life. Ultra-high dose rate radiotherapy (FLASH) has been shown to induce less normal tissue toxicity while producing comparable tumor growth delay in a variety of preclinical tumor models when compared with conventional dose rate radiotherapy (CONV). However, growth delay is not a surrogate for tumor eradication, which is a critical endpoint of cancer therapy, and studies using FLASH in breast cancer are limited. We sought to evaluate whether FLASH produced comparable tumor control to CONV in a breast cancer model with tumor eradication as the primary endpoint. Methods and Materials106 cells from the radiation sensitive mammary tumor cell line Py117 were used to create non-metastatic, syngeneic, orthotopic tumors in the left 4th mammary fat pad of C57BL/6J mice (n=67). Tumors were established for two distinct sequential irradiation studies (Rounds 1 and 2), utilizing either large (7.5 mm into the body) or small (5 mm) treatment tumor margins, respectively. For Round 1, mice were divided into groups with either small (20-40 mm3) or large (250-800 mm3) tumors, whereas only small tumors were included in Round 2. Tumors were irradiated with FLASH (93, 192 and 200 Gy/s) or CONV (0.08 Gy/s) using 16.6 MeV FLASH and 15.7 MeV CONV electron beams. Mice in the small tumor cohort were treated with single fractions of 20, 25, or 30 Gy. The larger tumors were treated with a single fraction of 30 Gy. Tumor eradication was determined by palpation and with histology as needed to clarify physical findings. ResultsSingle fractions of FLASH and CONV demonstrated comparable treatment responses within matched cohorts of small and large tumors. A portion of small tumors treated with single fractions of 20 or 25 Gy were eradicated though most regrew within 2 to 3 weeks. Eradication of small tumors was best seen treated with 30 Gy and a large treatment tumor margin. These mice had no tumor regrowth at 30 days with either FLASH or CONV: however, euthanasia criteria were met at the 30-day time point due to concerns over skin toxicity for both FLASH and CONV groups. Small tumors treated with 30 Gy and a smaller treatment tumor margin had less skin toxicity with 75% of mice remaining tumor free at 48 days. 30 Gy FLASH and CONV applied to larger tumors demonstrated growth delay equally with a partial reduction in size but without tumor eradication. ConclusionsFLASH and CONV produced comparable tumor control in this model of orthotopic, murine breast tumors. Single fractions of 30 Gy with both FLASH and CONV applied to small tumors achieved the highest rates of tumor eradication in particular when delivered with a wider treatment margin. Skin toxicity seen at this dose and in this location could be ameliorated with the use of multiple fractions or different tangents in future studies. Efforts at eradicating larger tumors would require testing higher single fraction doses, multiple fractions, and/or hypofractionated treatment regimens. The equivalent effectiveness between FLASH and CONV in this study of murine breast tumors supports ongoing evaluation of FLASH for use in treating human breast cancer. To this end future efforts at tumor eradication with single fraction FLASH doses with comprehensive evaluation of the toxicity of organs at risk as compared to CONV will be necessary. Additionally, studies of dose-response in a range of tumor volumes with additional breast cancer cell lines and tumors, including human xenografts, along with refined target margins, will guide future studies into the use of FLASH in the adjuvant therapy of primary human breast cancer.

cancer biology↗

bioRxiv2024

Spatiotemporal Orchestration of Mitosis by Cyclin-Dependent Kinase

Mitotic onset is a critical transition for eukaryotic cell proliferation. The prevailing view for its control is that cyclin-dependent kinase (CDK) is activated first in the cytoplasm, at the centrosome, which then initiates mitosis1-3. Bistability in CDK activation ensures the transition is irreversible but how this unfolds in a spatially compartmentalized cell is unknown4-8. Here using fission yeast, we show that CDK is actually activated in the nucleus first, not the cytoplasm, and that the bistable responses dramatically differ within the nucleus and cytoplasm. There is a stronger response in the nucleus permitting mitotic signal propagation from there to the cytoplasm. Abolishing cyclin-CDK localization to the yeast centrosome led to activation occurring only in the nucleus, spatially uncoupling the nucleus and cytoplasm mitotically, suggesting centrosomal cyclin-CDK acts as a "signal relayer". We propose that the key mitotic regulatory system operates in the nucleus in proximity to DNA, allowing incomplete DNA replication and DNA damage to be effectively monitored to preserve genome integrity and for integration of ploidy within the CDK control network. This spatiotemporal regulatory framework establishes core principles for control of the onset of mitosis in vivo, which will help inform how CDK controls mitotic onset in other eukaryotes.

cell biology↗

bioRxiv2023

CDK activity at the centrosome regulates the cell cycle

Cyclin-dependent kinases (CDKs) complexed with cyclins drive progression through the eukaryotic cell cycle. From yeast to human cells, cyclin-CDK localises to the centrosome, but the importance of this localisation is unclear. The conserved hydrophobic patch substrate docking site on human Cyclin B1 and on the equivalent fission yeast Cdc13 mediates their localisation to the centrosome and the spindle-pole body (SPB, yeast centrosome equivalent). A hydrophobic patch mutant (HPM) of Cdc13 cannot enter mitosis, but whether this mitotic defect is due to defective SPB localisation or defective cyclin-substrate docking is unknown. Here we show that artificially restoring Cdc13HPM SPB localisation in fission yeast partially rescues both mitosis and defective CDK substrate phosphorylation at both the SPB and within the cytoplasm. In addition, we found that an HPM of the S-phase cyclin Cig2 has defective SPB localisation but is still able to perform bulk DNA synthesis. Our results demonstrate that the hydrophobic patch mediates the SPB localisation of both S- and M-phase cyclins, and that Cdc13 SPB localisation is essential for mitotic entry and for full phosphorylation of CDK substrates, supporting the view that the centrosome plays a role as a signalling hub regulating CDK cell cycle control.

cell biology↗

bioRxiv2020

Bound2Learn: A Machine Learning Approach for Classification of DNA-Bound Proteins from Single-Molecule Tracking Experiments

DNA-bound proteins are essential elements for the maintenance, regulation, and use of the genome. The time they spend bound to DNA provides useful information on their stability within protein complexes and insight into the understanding of biological processes. Single-particle tracking allows for direct visualization of protein-DNA kinetics, however, identifying whether a molecule is bound to DNA can be non-trivial. Further complications arise when tracking molecules for extended durations in processes with slow kinetics. We developed a machine learning approach, termed Bound2Learn, using output from a widely used tracking software, to robustly classify tracks in order to accurately estimate residence times. We validated our approach in silico, and in live-cell data from Escherichia coli and Saccharomyces cerevisiae. Our method has the potential for broad utility and is applicable to other organisms.

biophysics↗

bioRxiv2019

Processive activity of the replicative DNA polymerases in the replisome of live eukaryotic cells

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol {delta}, at every cycle of Okazaki fragment synthesis. Here we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol {delta}, hence redefining the architecture of the active eukaryotic replisome.

molecular biology↗

bioRxiv2018

AAK1 inhibits WNT signaling by promoting clathrin-mediated endocytosis of LRP6

{beta}-catenin-dependent WNT signal transduction governs normal development and adult tissue homeostasis. Inappropriate pathway activity mediates a vast array of human diseases, including bone density disorders, neurodegeneration and cancer. Although several WNT-directed therapeutics are in clinical trials, new targets, compounds and strategies are needed. We performed a gain-of-function screen of the human kinome to identify new druggable regulators of {beta}-catenin-dependent transcription. We found that over-expression of the AP2 Associated Kinase 1 (AAK1) strongly inhibited WNT signaling. Reciprocally, silencing of AAK1 expression or pharmacological inhibition of AAK1 kinase activity using a new, selective and potent small molecule inhibitor activated WNT signaling. This small molecule is a cell active dual AAK1/BMP2K inhibitor that represents the best available tool to study AAK1-dependent signaling pathways. We report that AAK1 and the WNT co-receptor LRP6 physically co-complex and that AAK1 promotes clathrin-mediated endocytosis of LRP6. Collectively, our data support a WNT-induced negative feedback loop mediated by AAK1-driven, clathrin-mediated endocytosis of LRP6.\n\nSummary StatementA gain-of-function screen of the human kinome revealed AAK1 as a negative regulator of WNT signaling. We show that AAK1 promotes clathrin-mediated endocytosis of LRP6, resulting in downregulation of WNT signaling. We use a new selective and potent AAK1/BMP2K small molecule probe to validate our findings.

cell biology↗