A sensor for quantification of macromolecular crowding in living cells

Boersma, Arnold J.; Zuhorn, Inge S.; Poolman, Bert

doi:10.1038/nmeth.3257

Cited by 194 publications

(221 citation statements)

References 21 publications

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“…The here determined lateral diffusion coefficients suggest a stronger dependence on protein radius than predicted by the Saffman-Delbrück model. We note that the cytoplasm and periplasm are highly crowded and more viscous than water3839. Since MscS has large cytoplasmic domains and MscK has large cytoplasmic and periplasmic domains, these proteins will experience more drag than MscL, which is not accounted for in the Saffman-Delbrück model.…”

Section: Discussionmentioning

confidence: 94%

On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli

Berg

Galbiati

Rasmussen

et al. 2016

Sci Rep

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Bacterial mechanosensitive channels protect cells from structural damage during hypoosmotic shock. MscS, MscL and MscK are the most abundant channels in E. coli and arguably the most important ones in osmoprotection. By combining physiological assays with quantitative photo-activated localization microscopy (qPALM), we find an almost linear relationship between channel abundance and cell survival. A minimum of 100 MscL (or MscS) channels is needed for protection when a single type of channel is expressed. Under native-like conditions MscL, MscS as well as MscK distribute homogeneously over the cytoplasmic membrane and the lateral diffusion of the channels is in accordance with their relative protein mass. However, we observe cluster formation and a reduced mobility of MscL when the majority of the subunits of the pentameric channel contain the fluorescent mEos3.2 protein. These data provide new insights into the quantitative biology of mechanosensitive channels and emphasizes the need for care in analysing protein complexes even when the fluorescent tag has been optimized for monomeric behaviour.

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Section: Discussionmentioning

confidence: 94%

On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli

Berg

Galbiati

Rasmussen

et al. 2016

Sci Rep

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“…However, the agreement between the ratiometric and the STORM data (Figure S3H) shows its applicability to samples of known topology. It is hoped that future studies of AZ organization will benefit from novel imaging tools, e.g., direct measurement of local macromolecular crowding (Boersma et al., 2015). …”

Section: Discussionmentioning

confidence: 99%

Nanoscale Structural Plasticity of the Active Zone Matrix Modulates Presynaptic Function

et al. 2017

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SummaryThe active zone (AZ) matrix of presynaptic terminals coordinates the recruitment of voltage-gated calcium channels (VGCCs) and synaptic vesicles to orchestrate neurotransmitter release. However, the spatial organization of the AZ and how it controls vesicle fusion remain poorly understood. Here, we employ super-resolution microscopy and ratiometric imaging to visualize the AZ structure on the nanoscale, revealing segregation between the AZ matrix, VGCCs, and putative release sites. Long-term blockade of neuronal activity leads to reversible AZ matrix unclustering and presynaptic actin depolymerization, allowing for enrichment of AZ machinery. Conversely, patterned optogenetic stimulation of postsynaptic neurons retrogradely enhanced AZ clustering. In individual synapses, AZ clustering was inversely correlated with local VGCC recruitment and vesicle cycling. Acute actin depolymerization led to rapid (5 min) nanoscale AZ matrix unclustering. We propose a model whereby neuronal activity modulates presynaptic function in a homeostatic manner by altering the clustering state of the AZ matrix.

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“…In addition, FRET-based “crowding sensors” consisting of donor-acceptor pairs linked by a flexible polymer 56 or a polypeptide consisting of two helical regions linked by a flexible “hinge” 54 have been designed. It is expected that the intensity of observed FRET, which is a function of the average distance between the donor and acceptor, will be a measure of the degree to which a particular crowded environment promotes or inhibits association of the donor/acceptor pair (contraction of the polymer chain or closing of the “hinge”).…”

Section: What Have We Learned From “In Vivo” Experiments?mentioning

confidence: 99%

Macromolecular Crowding In Vitro , In Vivo , and In Between

Rivas

Minton

2016

Trends in Biochemical Sciences

371

366

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Biochemical processes take place in heterogeneous and highly volume occupied or crowded environments that can considerably influence the reactivity and distribution of participating macromolecules. Here we summarize the thermodynamic consequences of excluded volume and longer-ranged nonspecific intermolecular interactions for macromolecular reactions in volume-occupied media. In addition, we summarize and compare the information content of studies of crowding in vitro and in vivo. We emphasize the importance of characterizing the behavior not only of labeled tracer macromolecules, but also the composition and behavior of unlabeled macromolecules in the immediate vicinity of the tracer. Finally, we propose strategies for extending quantitative analyses of crowding in simple model systems to increasingly complex media up to and including intact cells.

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A sensor for quantification of macromolecular crowding in living cells

Cited by 194 publications

References 21 publications

On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli

On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli

Nanoscale Structural Plasticity of the Active Zone Matrix Modulates Presynaptic Function

Macromolecular Crowding In Vitro , In Vivo , and In Between

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