Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion

Sadovsky, Rotem Gura; Brielle, Shlomi; Kaganovich, Daniel; England, Jeremy L.

doi:10.1016/j.celrep.2017.02.063

Cited by 38 publications

(35 citation statements)

References 51 publications

(52 reference statements)

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“…S4A,B). In mocktreated larval muscles the average GFP diffusion rate was 15.2 ± 4.7μm 2 /s, which is similar to GFP diffusion rates measured with various techniques in mammalian cells (Gura Sadovsky et al, 2017). The GFP diffusion rate in larval muscles was not significantly altered by KCl stimulation (Fig.…”

Section: Syp Expression and Protein Dynamics Are Modulated By Neural supporting

confidence: 85%

Syncrip/hnRNPQ is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation

Titlow

Robertson

Järvelin

et al. 2019

Preprint

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Titlow et al. find that Syncrip (hnRNPQ RNA binding protein) acts directly on msp300 to modulate activity-dependent synaptic plasticity. In vivo biophysical experiments reveal activity-dependent changes in RNP complex sizes compatible with an increase in translation at the synapse. AbstractMemory and learning involve activity-driven expression of proteins and cytoskeletal reorganisation at new synapses, often requiring post-transcriptional regulation a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organises actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is not yet known. Here, we show that the local translation of msp300 is promoted during activity-dependent plasticity by the conserved RNA binding protein Syncrip/hnRNP Q, which binds to msp300 transcripts and is essential for plasticity. Single molecule imaging shows that Syncrip is associated in vivo with msp300 mRNA in ribosome-rich particles. Elevated neural activity alters the dynamics of Syncrip RNP granules at the synapse, suggesting a change in particle composition or binding that facilitates translation. These results introduce Syncrip as an important early-acting activitydependent translational regulator of a plasticity gene that is strongly associated with human ataxias.

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Section: Syp Expression and Protein Dynamics Are Modulated By Neural supporting

confidence: 85%

Syncrip/hnRNPQ is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation

Titlow

Robertson

Järvelin

et al. 2019

Preprint

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“…Data fitting (Eqs 1 and 2), revealed two diffusion components, with the slower (49% ± 10%) GFP-tagged population (D = 0.54µm 2 /s ± 0.26) within the cell being common among all the subunits and cross-correlating. The faster component, which does not crosscorrelate, has a median diffusion rate of 14.5µm 2 /s, reminiscent of freely diffusing molecules in cells 42 . Importantly, Exo70-GFP, Sec8-GFP and Sec3-GFP did not show any crosscorrelation with the negative control, Akt-Halo ( Fig.…”

Section: Fluorescence Cross-correlation Spectroscopy Of Exocyst Subunmentioning

confidence: 99%

Exocyst Dynamics During Vesicle Tethering and Fusion

Ahmed

Nishida-Fukuda

et al. 2018

Preprint

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The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that, unexpectedly, can associate independently with vesicles and plasma membrane and are in dynamic equilibrium. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of Sec3 occurs prior to fusion, whereas other subunits depart just after fusion. Single molecule counting indicates ~9 exocyst complexes associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important new insights into assembly/disassembly mechanisms.Key words: vesicles, membrane fusion, dynamics, TIRFM, CRISPR, gene-editing, protein complex.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/354449 doi: bioRxiv preprint first posted online Jun. 22, 2018; 3 Traffic between membrane-bound compartments requires the docking of cargo vesicles at target membranes, and their subsequent fusion through the interactions of SNARE proteins. The capture and fusion of vesicles are both promoted by molecular tethers known as multisubunit tethering complexes 1 . One group of such tethers, sometimes called CATCHR (complexes associate with tethering containing helical rods) comprises multisubunit complexes required for fusion in the secretory pathway, and includes COG, Dsl1p, GARP and the exocyst 2 . The endolysosomal pathway contains two different tethering complexes, CORVET and HOPS, with similar overall structures to the CATCHR group 3 .COG consists of two subcomplexes, each containing four subunits, which function together within the Golgi 4-6 . The exocyst is also octameric, and is necessary for exocytic vesicle fusion to the plasma membrane (PM), but the organization of the complex has been controversial [7][8][9][10] . Several studies in yeast suggest that one (Sec3) or two (Sec3 and Exo70)subunits associate with the PM and recruit a vesicle-bound subcomplex of the other subunits, but other work argues that the exocyst consists of 2 subcomplexes of 4 subunits each that form a stable octamer or, in mammalian cells, that 5 subunits at the PM recruit 3 other subunits on the vesicle [11][12][13][14][15][16][17][18][19][20][21][22] . Rab GTPases promote exocyst binding to the vesicle, and SNARES, Rho family GTPases, the PAR3 polarity protein, and phospho-inositide binding domains are all involved ...

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“…Hence each protein is packed with average inter-particle distance ranging from 6 to 0.3 times its own radius. Macromolecular crowding means that proteins diffusion is considerably decreased (18,19) while metabolites and ions diffusing through the protein network exhibit enhanced percolation (18). The same water that bathes the proteins is confined in a thin interface and hence exhibits properties different from bulk behavior (20).…”

Section: Theorymentioning

confidence: 99%

“…The experimental data are fitted assuming the geometries shown inFig.2and using equation16. The two ligands are combined into forming multiplexed polymersomes and their binding is measured in brain endothelial cells (c), the data are thus fitted using equation19. Fitting parameters for angiopep: δP= 0.25, δG= 0.7, NPEO= 10, aPEO= 0.35nm,σ0= 3.14nm2, R = 50nm, d = 5nm, LRP1 tip volume VP =188.4nm3, angiopep KD= 313nM.…”

mentioning

confidence: 99%

On the design of precision nanomedicines

Tian¹,

Agioletti-Uberti²,

Battaglia

2019

Preprint

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TeaserWe proposed the design of nanomedicines that target cells phenotypically enabling binding only when a precise receptor(s) composition is present. AbstractTight control on the selectivity of nanoparticles' interaction with biological systems is paramount for the development of targeted therapies. However, the large number of synthetically tunable parameters makes it difficult to identify optimal design ``sweet spots'' without rational guiding principles. Here we address this problem combining super-selectivity theory (SST) with analytical models from soft matter and polymer physics into a unified theoretical framework. Starting from an archetypal polymersome functionalized with targeting ligands, we use our model to identify the most selective combination of parameters in terms of particle size, brush polymerization degree and grafting density, as well as tether length, binding affinity and ligands number. We further show how to combine multivalent interactions into multiplexed systems which act holistically as a function of the density of more than one receptor type, so as to achieve binding only when multiple receptors are expressed above a threshold density. We show that theory can be used to effectively fit experimental data and, hence confirming its suitability. We thus propose the design of "barcoding" targeting approach that can be tailor-made to unique cell populations enabling personalized therapies.

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Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion

Cited by 38 publications

References 51 publications

Syncrip/hnRNPQ is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation

Syncrip/hnRNPQ is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation

Exocyst Dynamics During Vesicle Tethering and Fusion

On the design of precision nanomedicines

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