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Vacuole inheritance Strain expressing either GFP-Snc1 bud polarized plasma membrane protein and sfGFP-Hof1 bud neck specific protein, combined with pulse and chase staining with FM4-64 to image the vacuole. Micrographs show each channel for the vacuole in red (FM4-64) and local marker in cyan (GFP & sfGFP), as well as the merge. White arrows to indicate bud/daughter cell position. Scale bar: 1 μm.
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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 buddin...
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... forming an apparent protrusion migrating toward the bud neck region. Then, following by a crossing event, where the vacuole starts occupying the bud neck region and cross the bud neck cell with progressive transport and relaxation toward the daughter cell cytoplasm. Finally, the scission event occurs freeing almost instantly at the bud neck site (Fig. 9 and Supplemental Videos 4a and 4b). We also noticed some rare events of maternal vacuole retraction at the earlier stage of the crossing phase, and thus failure of efficient vacuolar inheritance during these experiments (Supplemental Video 5a and 5b). Stable and timely occupancy of the near bud neck region appears therefore critical to ...
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... aging harmful component such as protein aggregates, and other aging by product (Supplemental fig. S2). As opposed to metabolically transported complexes such as the vacuole inherited from the mother cell to the daughter with acute organization and control, mainly via polarised cytoskeleton dependant transport processes involving molecular motors (Fig. 9). Noticeably, essentials cellular compartment are reported inherited via metabolic active transport such as the nucleus (Spichal & Fabre, 2017), the endoplasmic reticulum ( Du et al., 2004) and even mitochondria ( Boldogh et al., 2001) shown to interact with the cytoskeleton. These observations supporting the presence of a diffusion ...
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... in the mother cell was Bud1-GTPase and sphingolipid dependent ( Clay et al., 2014). Meanwhile other studies have shown that a diffusion barrier does not exist for the vesicle membrane receptor Snc1 which is locally retained via diffusion at the daughter cell membrane independent from the bud neck structure ( (Valdez-Taubas & Pelham, 2003) - Fig. 9). Very little is known about these diffusion barrier dynamics in cells and how they are regulated. It however raises lots of questions and hypotheses regarding crowding modulation at the plasma membrane and cooperation between plasma membrane and cytoplasm dynamics. These powerful and complex processes tied to cellular organization and ...
Citations
... With these considerations in mind, we binned cells at the G1, S, and G2-M stages by their total Hoechst intensity in the nucleus and found no significant difference in protein or lipid density in any of the compartments between any 2 cell cycle stages in either HeLa or NIH3T3 cells (Figures 3B-E,G-J). This result is consistent with the previous density measurement by refractive index (Kim and Guck, 2020) but contrasts with measurements of the cell cycle-dependent molecular crowding (Lecinski et al., 2021;Yamamoto et al., 2021) and diffusion rate (Pradeep and Zangle, 2022). Although mass density, molecular crowding, and diffusion rate are related, they are evaluated by molecules of very different size, which may result in distinct behaviors. ...
Cell dry mass is principally determined by the sum of biosynthesis and degradation. Measurable change in dry mass occurs on a time scale of hours. By contrast, cell volume can change in minutes by altering the osmotic conditions. How changes in dry mass and volume are coupled is a fundamental question in cell size control. If cell volume were proportional to cell dry mass during growth, the cell would always maintain the same cellular mass density, defined as cell dry mass dividing by cell volume. The accuracy and stability against perturbation of this proportionality has never been stringently tested. Normalized Raman Imaging (NoRI), can measure both protein and lipid dry mass density directly. Using this new technique, we have been able to investigate the stability of mass density in response to pharmaceutical and physiological perturbations in three cultured mammalian cell lines. We find a remarkably narrow mass density distribution within cells, that is, significantly tighter than the variability of mass or volume distribution. The measured mass density is independent of the cell cycle. We find that mass density can be modulated directly by extracellular osmolytes or by disruptions of the cytoskeleton. Yet, mass density is surprisingly resistant to pharmacological perturbations of protein synthesis or protein degradation, suggesting there must be some form of feedback control to maintain the homeostasis of mass density when mass is altered. By contrast, physiological perturbations such as starvation or senescence induce significant shifts in mass density. We have begun to shed light on how and why cell mass density remains fixed against some perturbations and yet is sensitive during transitions in physiological state.
... The experimental state-of-the art is to use a Förster resonance energy transfer (FRET)-based probe to measure crowding. One such that we have used previously is called crGE2.3 [26][27][28], consisting of a donor fluorophore, mGFP and acceptor, mScarlet-I, linked by a flexible amino acid hairpin, forming an efficient FRET pair system as reported and characterized in the literature [29][30][31]. As molecular crowding increases, the donor and fluorophore are pushed closer together, and the FRET signal increases [26,32,33]. ...
In eukaryotes, intracellular physicochemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion, and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalised in cells is tracked and physicochemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Fӧrster Resonance Energy Transfer (FRET) biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder.
Here, we present correlative, multidimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterise in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae. It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalise fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells, and discuss limitations of this approach including impairment of cellular function.
It is now generally accepted that macromolecules do not act in isolation but “live” in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.
Cells employ myriad regulatory mechanisms to maintain protein homeostasis, termed proteostasis, to ensure correct cellular function. Dysregulation of proteostasis, which is often induced by physiological stress and ageing, often results in Protein Aggregation in cells. These aggregated structures can perturb normal physiological function, compromising cell integrity and viability, a prime example being early onset of several neurodegenerative diseases. Understanding aggregate dynamics in vivo is therefore of strong interest for biomedicine and pharmacology. However, factors involved in formation, distribution and clearance of intracellular aggregates are poorly understood. Here, we report an improved methodology for production of fluorescent aggregates in model budding yeast which can be detected, tracked and quantified using fluorescence microscopy in live cells. This new openly-available technology, iPAR (inducible Protein Aggregation Reporter), involves monomeric fluorescent protein reporters fused to a ΔssCPY* aggregation biomarker, with expression controlled under the copper-regulated CUP1 promoter. Monomeric tags overcome challenges associated with non-physiological aggregation, whilst CUP1 provides more precise control of protein production. We show that iPAR enables quantitative study of cytoplasmic aggregate kinetics and inheritance features in vivo . If suitably adapted, iPAR offers new potential for studying diseases in other model cellular systems.
