Macromolecular crowding acts as a physical regulator of intracellular transport

Nettesheim, Guilherme; Nabti, Ibtissem; Murade, C.U.; Jaffe, Gabriel R.; King, Stephen J.; Shubeita, George T.

doi:10.1038/s41567-020-0957-y

Cited by 39 publications

(42 citation statements)

References 52 publications

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“…These dynamic effects have raised new interest in the context of protein aggregation ( Jing et al, 2020 ), intracellular organization ( Löwe et al, 2020 ), and membrane-less compartments in the cytoplasm, for example the formation of liquid-liquid phase separation in the cytoplasm ( Franzmann et al, 2018 ; Jin et al, 2021 ; Park et al, 2020 ). More generally, macromolecular crowding has been linked to a number of biological process such as protein-protein interaction ( Bhattacharya et al, 2013a ), protein conformational changes ( Dong et al, 2010 ), stability and folding ( Despa et al, 2006 ; Stagg et al, 2007 ; Tokuriki, 2004 ), promoting signalling cascades ( Rohwer et al, 1998 ), regulating biomolecular diffusion ( Tabaka et al, 2014 ; Tan et al, 2013 ), and intracellular transport ( Nettesheim et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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

Lecinski

Shepherd

Frame

et al. 2021

New Methods and Sensors for Membrane and Cell Volume Research

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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 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 whilst simultaneously imaging a third color, which can be used as a marker that labels organelle membranes. Our approaches can be combined with synchronised cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.

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Section: Introductionmentioning

confidence: 99%

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

Lecinski

Shepherd

Frame

et al. 2021

New Methods and Sensors for Membrane and Cell Volume Research

View full text Add to dashboard Cite

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“…We note that, though we based our method on quadri-wave lateral shearing interferometry (36), there are a large number of alternative methods for QPI (31)(32)(33)(34)36), any of which should be amenable to QPV. Our viscometry results, in particular, show an increase in viscosity with cell cycle progression, which may be due to macromolecular crowding accompanying growth (65).…”

Section: Discussionmentioning

confidence: 59%

Quantitative phase velocimetry measures bulk intracellular transport of cell mass during the cell cycle

Pradeep

Zangle

2021

Preprint

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Transport of mass within cells helps maintain homeostasis and is disrupted by disease and stress. Here, we develop quantitative phase velocimetry (QPV) as a label-free approach to make the invisible flow of mass within cells visible and quantifiable. We benchmark our approach against alternative image registration methods, a theoretical error model, and synthetic data. Our method tracks not just individual labeled particles or molecules, but the entire flow of material through the cell. This enables us to measure diffusivity within distinct cell compartments using a single approach, which we use here for direct comparison of nuclear and cytoplasmic diffusivity. As a label-free method, QPV can be used for long-term tracking to capture dynamics through the cell cycle. Finally, based on the known effective particle size, we show that QPV is an accessible method to measure intracellular viscosity.

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“…At the anterior pole we have previously shown that aqp0a -/mutants show severe suture defects at older stages (21). ACDAN emission analysis in anterior lens planes revealed that aqp0a -/mutant lenses had lower DR (Figure 6A2) than other genotypes, which were all quite similar to one another (Figure 6A1, [3][4]. This lower DR in aqp0a -/compared to the other genotypes was confirmed by comparison of the mean DR of the fiber cells (Figure 6B3), including the suture (Figure 6B4).…”

Section: Loss Of Aqp0a Function Regionally Disrupts Macromolecular Crowding In the Lensmentioning

confidence: 60%

“…However, its physiological roles remain obscure and are challenging to study in living organisms. There has been a great effort to understand the roles of macromolecular crowding in enzymatic activity, cell physiology, and pathophysiology, primarily using in vitro and cell culture systems (1, [3][4][5]. However, efforts to study macromolecular crowding in vivo with non-invasive, highresolution spectroscopic tools remain challenging.…”

Section: Introductionmentioning

confidence: 99%

In vivo macromolecular crowding is differentially modulated by Aquaporin 0 in zebrafish lens: insights from a nano-environment sensor and spectral imaging

Vorontsova

Lees

Torrado

et al. 2021

Preprint

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Macromolecular crowding is crucial for cellular homeostasis. In vivo studies of macromolecular crowding and ultimately water-dynamics are needed to understand their role in cellular fates. The macromolecular crowding in the lens is essential for understanding normal optics of the lens, and moreover for understanding and prevention of cataract and presbyopia. Here we combine the use of the water nano-environmentally sensitive sensor (6-acetyl-2-dimethylaminonaphthalene, ACDAN) with in vivo studies of Aquaporin zero zebrafish mutants to understand the lens macromolecular crowding. Spectral phasor analysis of ACDAN fluorescence reveal the extent of water dipolar relaxation and demonstrate that the mutations in the duplicated zebrafish Aquaporin 0s, Aqp0a and Aqp0b, alter the water state and macromolecular crowding in the living zebrafish lens. Our results provide in vivo evidence that Aqp0a promotes fluid influx in the deeper lens cortex, whereas Aqp0b facilitates fluid efflux. This work opens new perspectives for in vivo studies on macromolecular crowding.

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Macromolecular crowding acts as a physical regulator of intracellular transport

Cited by 39 publications

References 52 publications

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

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

Quantitative phase velocimetry measures bulk intracellular transport of cell mass during the cell cycle

In vivo macromolecular crowding is differentially modulated by Aquaporin 0 in zebrafish lens: insights from a nano-environment sensor and spectral imaging

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