scholarly journals Cytoplasmic organization promotes protein diffusion

2021 ◽  
Author(s):  
William Y. C. Huang ◽  
Xianrui Cheng ◽  
James E. Ferrell
Keyword(s):  
Molecular Size ◽  
Correlation Spectroscopy ◽  
Self Organization ◽  
Protein Diffusion ◽  
Cytoplasmic Proteins ◽  
Diffusion Dynamics ◽  
Egg Extracts ◽  
Cytoplasmic Organization ◽  
Will Self ◽  
Cytoskeletal Elements

The cytoplasm is highly organized. However, the extent to which this organization influences the dynamics of cytoplasmic proteins is not well understood. Here, we used Xenopus laevis egg extracts as a model system to study diffusion dynamics in organized versus disorganized cytoplasm. Such extracts are initially homogenized and disorganized, and will self-organize into cell-like units over the course of 20-60 min. Using fluorescence correlation spectroscopy, we observed that self-organization is accompanied by changes in protein diffusivity; as the extract organizes, proteins diffuse about twice as quickly over a length scale of a few hundred nanometers. Even though the ordered cytoplasm contained organelles and cytoskeletal elements that might be expected to interfere with diffusion, after self-organization took place, the speed of protein diffusion approached that of organelle-depleted cytosolic extracts. This finding suggests that subcellular organization optimizes protein diffusivity. The effect of organization on diffusion varies with molecular size, with the effects being largest for protein-sized molecules. These results show that cytoplasmic organization promotes the efficient diffusion of protein molecules in a densely packed environment.

scholarly journals Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data

2017 ◽  
Author(s):  
Dominic Waithe ◽  
Falk Schneider ◽  
Jakub Chojnacki ◽  
Mathias Clausen ◽  
Dilip Shrestha ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Spatial Heterogeneity ◽  
Fluorescence Correlation Spectroscopy ◽  
Large Scale ◽  
Molecular Diffusion ◽  
Correlation Spectroscopy ◽  
The Other ◽  
Statistical Accuracy ◽  
Fluorescence Correlation ◽  
Diffusion Dynamics

AbstractScanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

scholarly journals Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

2020 ◽  
Author(s):  
Valentin Dunsing ◽  
Annett Petrich ◽  
Salvatore Chiantia
Keyword(s):  
Correlation Analysis ◽  
Influenza A ◽  
Cross Correlation ◽  
Matrix Protein ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Fluorescence Fluctuation Spectroscopy ◽  
Diffusion Dynamics ◽  
Cross Correlation Analysis

AbstractFluorescence fluctuation spectroscopy provides a powerful toolbox to quantify transport dynamics and interactions between biomolecules in living cells. For example, cross-correlation analysis of spectrally separated fluctuations allows the investigation of inter-molecular interactions. This analysis is conventionally limited to two fluorophore species that are excited with a single or two different laser lines and detected in two non-overlapping spectral channels. However, signaling pathways in biological systems often involve interactions between multiple biomolecules, e.g. formation of ternary or quaternary protein complexes. Here, we present a methodology to investigate such interactions at the plasma membrane (PM) of cells, as encountered for example in viral assembly or receptor-ligand interactions. To this aim, we introduce scanning fluorescence spectral correlation spectroscopy (SFSCS), a combination of scanning fluorescence correlation spectroscopy with spectrally resolved detection and decomposition. We first demonstrate that SFSCS allows cross-talk-free cross-correlation analysis of PM-associated proteins labeled with strongly overlapping fluorescent proteins (FPs), such as mEGFP and mEYFP, excited with a single excitation line. We then verify the applicability of SFSCS for quantifying diffusion dynamics and protein oligomerization (based on molecular brightness analysis) of two protein species tagged with spectrally overlapping FPs. Adding a second laser line, we demonstrate the possibility of three- and four-species (cross-) correlation analysis using mApple and mCherry2, as examples of strongly overlapping FP tags in the red spectral region. Next, we apply this scheme to investigate the interactions of influenza A virus (IAV) matrix protein 2 (M2) with two cellular host factors simultaneously. Using the same set of fluorophores, we furthermore extend the recently presented raster spectral image correlation spectroscopy (RSICS) approach to four species analysis, successfully demonstrating multiplexed RSICS measurements of protein interactions in the cell cytoplasm. Finally, we apply RSICS to investigate the assembly of the ternary IAV polymerase complex and report a 2:2:2 stoichiometry of these protein assemblies in the nucleus of living cells.

Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts

Nature ◽  
1996 ◽  
Vol 382 (6590) ◽  
pp. 420-425 ◽  
Author(s):  
Rebecca Heald ◽  
Régis Tournebize ◽  
Thiemo Blank ◽  
Raphael Sandaltzopoulos ◽  
Peter Becker ◽  
...  
Keyword(s):  
Self Organization ◽  
Artificial Chromosomes ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Bipolar Spindles

scholarly journals Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

2021 ◽  
Author(s):  
Silvia Galiani ◽  
Katharina Reglinski ◽  
Pablo Carravilla ◽  
Aurelien Barbotin ◽  
Iztok Urbančič ◽  
...  
Keyword(s):  
Molecular Interactions ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Interaction Dynamics ◽  
Diffusion Dynamics ◽  
Interaction Partners ◽  
Target Binding ◽  
Import Receptor

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics.

scholarly journals The Cytoskeleton and Its Roles in Self-Organization Phenomena: Insights from Xenopus Egg Extracts

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2197
Author(s):  
Zachary M. Geisterfer ◽  
Gabriel Guilloux ◽  
Jesse C. Gatlin ◽  
Romain Gibeaux
Keyword(s):  
Experimental System ◽  
Structure And Function ◽  
Self Organization ◽  
African Clawed Frog ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
The Many ◽  
And Function ◽  
Xenopus Egg Extracts ◽  
Clawed Frog

Self-organization of and by the cytoskeleton is central to the biology of the cell. Since their introduction in the early 1980s, cytoplasmic extracts derived from the eggs of the African clawed-frog, Xenopus laevis, have flourished as a major experimental system to study the various facets of cytoskeleton-dependent self-organization. Over the years, the many investigations that have used these extracts uniquely benefited from their simplified cell cycle, large experimental volumes, biochemical tractability and cell-free nature. Here, we review the contributions of egg extracts to our understanding of the cytoplasmic aspects of self-organization by the microtubule and the actomyosin cytoskeletons as well as the importance of cytoskeletal filaments in organizing nuclear structure and function.

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