Biology subjects

Thorpe, P. H.

Publications and source records attributed to Thorpe, P. H..

6 recordsLinked to original sources

bioRxiv2023

Synthetic cell-cycle regulation identifies Mif2CENP-C as a CDK phospho-target at the kinetochore.

Protein phosphorylation regulates multiple cellular processes including cell-cycle progression, which is driven by highly conserved cyclin-dependent kinases (CDKs). CDKs are controlled by the oscillating levels of activating cyclins and the activity peaks during mitosis to promote chromosome segregation. However, with some exceptions, we do not understand how the multitude of CDK-phosphorylated residues within the proteome drive cell-cycle progression nor which CDK phosphorylation events are necessary. To identify yeast proteins whose phospho-regulation is most critical for cell-cycle progression, we created a synthetic CDK complex and systematically recruited this to proteins involved in chromosome segregation using the Synthetic Physical Interactions (SPI) method. We found that targeted recruitment of synthetic CDK to the centromeric protein Mif2CENP-C leads to enrichment of Mif2CENP-C at centromeres and arrested cells in late mitosis. We then identified putative CDK consensus sites on Mif2CENP-C which aid Mif2CENP-C localisation at centromeres and showed that CDK- dependent Mif2CENP-C phosphorylation is important for its stable kinetochore localisation. SummaryTo identify cellular sites of functional cell cycle phospho-regulation we generated a synthetic cyclin-dependent kinase which can be recruited to any given GFP-tagged protein. Using this system with a set of proteins involved in chromosome segregation, we identified Mif2CENP-C as a kinetochore target of CDK and show that CDK stabilises Mif2s kinetochore localisation.

cell biology↗

bioRxiv2021

A novel multifunctional role for Hsp70 in binding post-translational modifications on clients

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and inability of current technologies to distinguish direct vs bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the budding yeast Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multi-point interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors which can be used to probe chaperone function in cells, we have also identified a suite of PTM-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically-important PTMs on client proteins. O_LIIn vivo confirmation of Hsp70 dimerization C_LIO_LIComprehensive direct interactome of Hsp70 C_LIO_LIMulti-domain interactions between Hsp70 and client proteins C_LIO_LIIdentification of novel biologically-important client protein PTMs C_LI

molecular biology↗

bioRxiv2021

Oncogenic c-Myc induces replication stress by increasing cohesins chromatin occupancy

Oncogene-induced replication stress is a major driver of genomic instability in cancer cells, with a central role in both cancer initiation and progression (1). Despite its critical role in cancer development, the mechanisms that lay at the basis of oncogene-induced replication stress remains poorly understood. Here, we investigate the mechanism of c-Myc-induced replication stress. Our data shows that c-Myc induces replication stress by increasing the amount of cohesins bound to chromatin in the G1 phase of the cell cycle. This is independent of previously suggested mechanisms involving deregulation of replication initiation and transcriptional interference. Restoring the amount of chromatin-bound cohesins to control levels, or preventing the accumulation of cohesins at CTCF sites, in cells experiencing oncogenic c-Myc activity prevents replication stress. Increased cohesins chromatin occupancy correlates with a c-Myc-dependent increase in the levels of the cohesion loader Mau2. Preventing c-Myc-induced increase in Mau2 reduces oncogene-induced replication stress. Together our data support a novel mechanism for oncogene-induced replication stress. Since c-Myc activation is a crucial event in many human cancers (2), identifying the mechanisms through which this oncogene promotes replication stress provides critical insights into cancer biology.

cancer biology↗

bioRxiv2021

ScreenGarden: A shinyR application for fast and easy analysis of plate-based high-throughput screens

Colony growth on solid media is a simple and effective measure for high-throughput genomic experiments such as yeast-two hybrid, Synthetic Genetic Arrays and Synthetic Physical Interaction screens. The development of robotic pinning tools has facilitated the experimental design of these assays, and different imaging software can be used to automatically measure colony sizes on plates. However, comparison to control plates and statistical data analysis is often laborious and pinning issues or plate specific growth effects can lead to the detection of false positive growth defects. We have developed ScreenGarden, a shinyR application, to enable easy, quick and robust data analysis of plate-based high throughput assays.

genomics↗

bioRxiv2020

Unifying the mechanism of mitotic exit control in a spatio-temporal logical model

1The transition from mitosis into the first gap phase of the cell cycle in budding yeast is controlled by the Mitotic Exit Network (MEN). The network interprets spatio-temporal cues about the progression of mitosis and ensures that release of Cdc14 phosphatase occurs only after completion of key mitotic events. The MEN has been studied intensively however a unified understanding of how localization and protein activity function together as a system is lacking. In this paper we present a compartmental, logical model of the MEN that is capable of representing spatial aspects of regulation in parallel to control of enzymatic activity. Through optimization of the model, we reveal insights into role of Cdc5 in Cdc15 localization and the importance of Lte1 regulation in control of Bfa1. We show that our model is capable of correctly predicting the phenotype of[~] 80% of mutants we tested, including mutants representing mislocalizing proteins. We use a continuous time implementation of the model to demonstrate the role of Cdc14 Early Anaphase Release (FEAR) to ensure robust timing of anaphase and verify our findings in living cells. We show that our model can represent measured cell-cell variation in Spindle Position Checkpoint (SPoC) mutants. Finally, we use the model to predict the impact of forced localization of MEN proteins and validate these predictions experimentally. This model represents a unified view of the mechanism of mitotic exit control.

systems biology↗

bioRxiv2019

Comparative analysis of Synthetic Physical Interactions with the yeast centrosome

1The yeast centrosome or Spindle Pole Body (SPB) is situated in the nuclear membrane, where it nucleates spindle microtubules and acts as a signalling hub. Previously, we used Synthetic Physical Interactions to map the regions of the cell that are sensitive to forced relocalization of proteins across the proteome [Berry et al., 2016]. Here, we expand on this work to show that the SPB, in particular, is sensitive to the relocalization of many proteins. This work inspired a new data analysis approach that indicates that relocalization screens may produce more growth defects than previously reported. A set of associations with the SPB result in elevated SPB number and since hyper-proliferation of centrosomes is a hallmark of cancer cells, these associations point the way for the use of yeast models in the study of spindle formation and chromosome segregation in cancer.

cell biology↗