Spatial heterogeneity of the cytosol revealed by machine learning-based 3D particle tracking

McLaughlin, Grace A.; Langdon, Erin M.; Crutchley, John; Holt, Liam J.; Forest, M. Gregory; Newby, Jay M.; Gladfelter, Amy S.

doi:10.1091/mbc.e20-03-0210

Cited by 20 publications

(20 citation statements)

References 29 publications

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“…While cell-averaged concentrations provide a single easily interpretable number, its obvious limitation is that it is just cell-averaged. Mitochondria, lysosomes, and nucleoli have a higher protein content than the cytosol or the nucleus [6], and the space immediately next to the membrane may experience a different extent of crowding (A. Minton, personal communication); even the bulk cytosol is highly heterogenous [40]. Thus, not all processes can be equally well captured by space-averaged measurements.…”

Section: Quantification Of Macromolecular Crowding In Living Cellsmentioning

confidence: 99%

Macromolecular Crowding: a Hidden Link Between Cell Volume and Everything Else

Model

Hollembeak

Kurokawa

2021

Cell Physiol Biochem

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High density of intracellular macromolecules creates a special condition known as macromolecular crowding (MC). One well-established consequence of MC is that only a slight change in the concentration of macromolecules (e.g., proteins) results in a shift of chemical equilibria towards the formation of macromolecular complexes and oligomers. This suggests a physiological mechanism of converting cell density changes into cellular responses. In this review, we start by providing a general overview of MC; then we examine the available experimental evidence that MC may act as a direct signaling factor in several types of cellular activities: mechano- and osmosensing, cell volume recovery in anisosmotic solutions, and apoptotic shrinkage. The latter phenomenon is analyzed in particular detail, as persistent shrinkage is known both to cause apoptosis and to occur during apoptosis resulting from other stimuli. We point to specific apoptotic reactions that involve formation of macromolecular complexes and, therefore, may provide a link between shrinkage and downstream responses.

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Section: Quantification Of Macromolecular Crowding In Living Cellsmentioning

confidence: 99%

Macromolecular Crowding: a Hidden Link Between Cell Volume and Everything Else

Model

Hollembeak

Kurokawa

2021

Cell Physiol Biochem

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“…The complexity of the cytoplasmic milieu could influence molecules’ behavior locally. Indeed, spatiotemporal heterogeneity in the diffusion of particles has been observed in multiple contexts such as E. coli, fungi, mammalian cells and even Xenopus egg extract using methods ranging from fluorescence correlation spectroscopy (FCS) to particle tracking (Bakshi et al, 2011; Baum et al, 2014; Dross et al, 2009; Huang et al, 2021; Manley et al, 2008; McLaughlin et al, 2020; Scipioni et al, 2018; Xiang et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…Because each particle contains tens of fluorescent proteins, they can be tracked for relatively long periods of time without photobleaching. Additionally, the near-diffusive movements of GEMs suggest they do not interact strongly with eukaryotic cellular components, making them ideal reagents for rheological studies (Delarue et al, 2018; McLaughlin et al, 2020). Critically, their relatively large size and slow diffusion rates - comparable to large protein complexes such as ribosomes - allow GEMs to be tracked using modern high-speed cameras (which is still not attainable for individual fluorescent proteins).…”

Section: Introductionmentioning

confidence: 99%

Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelgänger simulations

Garner

Molines

Theriot

et al. 2022

Preprint

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The cytoplasm is a complex, crowded, actively-driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem-cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nano-rheology on genetically encoded multimeric 40-nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of the fission yeast Schizosaccharomyces pombe. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the average particle diffusivity for each individual cell spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger Simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume an equally wide intra- and inter-cellular variation in cytoplasmic viscosity. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, cytoskeletal effects, cell cycle stage and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not “well-mixed” but represents a highly heterogeneous environment in which subcellular components at the 40-nm size-scale experience dramatically different effective viscosities within an individual cell, as well as in different cells in the population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels.Graphical abstract

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“…The characterization of the rheological properties of an ensemble of biomolecules and their surrounding environment is important to understand molecule dynamics and macromolecular assembly (Delarue et al, 2018;Golkaram and Loos, 2019). Taking advantage of a recently developed technique that uses genetically encoded multimeric nanoparticles (GEMs), it was observed that the polarized growing zone of fungi is a crowded environment packed with macromolecules, resulting in a low local diffusivity for cytoplasmic biomolecules (Delarue et al, 2018;McLaughlin et al, 2020); this might favor the interaction of each macromolecular component with others and the formation of a functional complex that persists over a long period of time (Grimaldo et al, 2019). In S. cerevisiae, the polarisome components display local condensation behavior at the polarized tip (dense phase), which shows a dynamic exchange of polarisome proteins with the cytosolic pool (dilute phase), such as described above during cell cycle progression.…”

Section: Coordination Between Polarisome Condensation and Actin Polymerizationmentioning

confidence: 99%

Polarisome assembly mediates actin remodeling during polarized yeast and fungal growth

Xie

Miao

2021

Journal of Cell Science

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Dynamic assembly and remodeling of actin is critical for many cellular processes during development and stress adaptation. In filamentous fungi and budding yeast, actin cables align in a polarized manner along the mother-to-daughter cell axis, and are essential for the establishment and maintenance of polarity; moreover, they rapidly remodel in response to environmental cues to achieve an optimal system response. A formin at the tip region within a macromolecular complex, called the polarisome, is responsible for driving actin cable polymerization during polarity establishment. This polarisome undergoes dynamic assembly through spatial and temporally regulated interactions between its components. Understanding this process is important to comprehend the tuneable activities of the formin-centered nucleation core, which are regulated through divergent molecular interactions and assembly modes within the polarisome. In this Review, we focus on how intrinsically disordered regions (IDRs) orchestrate the condensation of the polarisome components and the dynamic assembly of the complex. In addition, we address how these components are dynamically distributed in and out of the assembly zone, thereby regulating polarized growth. We also discuss the potential mechanical feedback mechanisms by which the force-induced actin polymerization at the tip of the budding yeast regulates the assembly and function of the polarisome.

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Spatial heterogeneity of the cytosol revealed by machine learning-based 3D particle tracking

Cited by 20 publications

References 29 publications

Macromolecular Crowding: a Hidden Link Between Cell Volume and Everything Else

Macromolecular Crowding: a Hidden Link Between Cell Volume and Everything Else

Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelgänger simulations

Polarisome assembly mediates actin remodeling during polarized yeast and fungal growth

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