Biology subjects

MacDonald, C.

Publications and source records attributed to MacDonald, C..

23 records · Page 2Linked to original sources

bioRxiv2021

Fur4 mediated uracil-scavenging to screen for surface protein regulators

Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the Fur4 uracil permease in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a haploid deletion library that identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multi-subunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterised genes. Bioinformatic analysis of expression profiles from many strains lacking identified transcription factors required for efficient uracil-scavenging revealed they control expression of other uracil-scavenging factors, in addition to membrane trafficking genes essential for viability, and therefore not represented in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorise factors implicated in uracil-scavenging, most of which are conserved throughout evolution.

cell biology↗

bioRxiv2021

Virtual partition digital PCR for high precision chromosomal counting applications

Digital PCR (dPCR) is the gold standard analytical platform for rapid high precision quantification of genomic fragments. However, current dPCR assays are generally limited to monitoring 1-2 analytes per sample, thereby limiting the platforms ability to address some clinical applications that require the simultaneous monitoring of 20 - 50 analytes per sample. Here we present Virtual Partition dPCR (VPdPCR), a novel analysis methodology enabling the detection of 10 or more target regions per color channel using conventional dPCR hardware and workflow. Furthermore, VPdPCR enables dPCR instruments to overcome upper quantitation limits caused by partitioning error. While traditional dPCR analysis establishes a single threshold to separate negative and positive partitions, VPdPCR establishes multiple thresholds to identify the number of unique targets present in each positive droplet based on fluorescent intensity. Each physical partition is then divided into a series of virtual partitions, and the resulting increase in partition count substantially decreases partitioning error. We present both a theoretical analysis of the advantages of VPdPCR and an experimental demonstration in the form of a 20-plex assay for non-invasive fetal aneuploidy testing. This demonstration assay - tested on 432 samples contrived from sheared cell-line DNA at multiple input concentrations and simulated fractions of euploid or trisomy-21 "fetal" DNA - is analyzed using both traditional dPCR thresholding and VPdPCR. VPdPCR analysis significantly lowers variance of chromosome ratio across replicates and increases the accuracy of trisomy identification when compared to traditional dPCR, yielding >98% single-well sensitivity and specificity. VPdPCR has substantial promise for increasing the utility of dPCR in applications requiring ultra-high-precision quantitation.

molecular biology↗

bioRxiv2021

Endosomal recycling to the surface mediated by Gpa1 and PI3-Kinase is inhibited by glucose starvation

Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalised protein and lipid cargoes recycle back to the surface efficiently in glucose replete conditions, but this trafficking is attenuated following glucose starvation. We find cells with either reduced or hyperactive phosphatidylinositol 3-kinase (PI3K) activity are defective for recycling. Furthermore, we find the yeast G subunit Gpa1, an endosomal PI3K effector, is required for surface recycling of cargoes. Following glucose starvation, mRNA and protein levels of a distinct G subunit Gpa2 are elevated following nuclear translocation of Mig1, which inhibits recycling of various cargoes. As Gpa1 and Gpa2 interact at the surface where Gpa2 concentrates during glucose starvation, we propose this disrupts PI3K activity required for recycling, potentially diverting Gpa1 to the surface and interfering with its endosomal role in recycling. In support of this model, glucose starvation and over-expression of Gpa2 alters PI3K endosomal phosphoinositide production. Glucose deprivation therefore triggers a survival mechanism to increase retention of surface cargoes in endosomes and promote their lysosomal degradation.

cell biology↗

bioRxiv2020

Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on ionic strength, glucose conditions, and copy number

The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress.

biophysics↗

bioRxiv2020

A glucose starvation response governs endocytic trafficking and eisosomal retention of surface cargoes

Eukaryotic cells adapt their metabolism to the extracellular environment. Downregulation of surface cargo proteins in response to nutrient stress reduces the burden of anabolic processes whilst elevating catabolic production in the lysosome. We use yeast to show that glucose starvation triggers a transcriptional response that simultaneously increases internalisation from the plasma membrane whilst supressing recycling from endosomes back to the surface. Nuclear export of the Mig1 transcriptional repressor in response to glucose starvation increases levels of the Yap1801/02 clathrin adaptors, which is sufficient to increase cargo internalisation. We also show Gpa1, which coordinates endosomal lipid homeostasis, is required for surface recycling of cargo. Nuclear translocation of Mig1 increases GPA2 levels and inhibits recycling, potentially by diverting Gpa1 to the surface and interfering with its endosomal role in recycling. Finally, we show glucose starvation results in transcriptional upregulation of many eisosomal factors, which serve to sequester a portion of nutrient transporters to persist the starvation period and maximise nutrient uptake upon return to replete conditions. This latter mechanism provides a physiological benefit for cells to rapidly recover from glucose starvation. Collectively, this remodelling of the surface protein landscape during glucose starvation calibrates metabolism to available nutrients.

cell biology↗