Investigating molecular crowding during cell division and hyperosmotic stress in budding yeast with FRET

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Publication details

Title of host publicationNew Methods and Sensors for Membrane and Cell Volume Research
DateAccepted/In press - 23 Oct 2021
DatePublished (current) - 30 Nov 2021
Pages75-118
Number of pages44
PublisherAcademic Press
EditorsMichael A. Model, Irena Levitan
Original languageEnglish
ISBN (Print)9780323911146

Publication series

NameCurrent Topics in Membranes
Volume88
ISSN (Print)1063-5823

Abstract

Cell division, aging, and stress recovery triggers spatial reorganization of cellular components in the cytoplasm, including membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model system to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a Förster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to cellular growth of both mother and daughter cells, in addition to osmotic stress, and reveal hot spots of crowding across the bud neck in the burgeoning daughter cell. This crowding might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding while simultaneously imaging a third color, which can be used as a marker that labels organelle membranes. Our approaches can be combined with synchronized cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.

    Research areas

  • Cellular stress, Confocal, FRET, Molecular crowding sensor, Slimfield, Vacuolar inheritance, Yeast

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