scholarly journals Antenna Protein Clustering In Vitro Unveiled by Fluorescence Correlation Spectroscopy

2021 ◽  
Vol 22 (6) ◽  
pp. 2969
Author(s):  
Aurélie Crepin ◽  
Edel Cunill-Semanat ◽  
Eliška Kuthanová Trsková ◽  
Erica Belgio ◽  
Radek Kaňa
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Higher Plants ◽  
Fluorescence Emission ◽  
Correlation Spectroscopy ◽  
Photochemical Quenching ◽  
High Ph ◽  
Fluorescence Correlation ◽  
Non Photochemical Quenching

Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.

scholarly journals A Comprehensive Review of Fluorescence Correlation Spectroscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Lan Yu ◽  
Yunze Lei ◽  
Ying Ma ◽  
Min Liu ◽  
Juanjuan Zheng ◽  
...  
Keyword(s):  
Diffusion Coefficient ◽  
Fluorescence Correlation Spectroscopy ◽  
Pair Correlation ◽  
Time Correlation ◽  
Correlation Spectroscopy ◽  
Local Concentration ◽  
Fluorescence Correlation ◽  
Pros And Cons

Fluorescence correlation spectroscopy (FCS) is a powerful technique for quantification of molecular dynamics, and it has been widely applied in diverse fields, e.g., biomedicine, biophysics, and chemistry. By time-correlation of the fluorescence fluctuations induced by molecules diffusing through a focused light, FCS can quantitatively evaluate the concentration, diffusion coefficient, and interaction of the molecules in vitro or in vivo. In this review, the basic principle and implementation of FCS are introduced. Then, the advances of FCS variants are reviewed, covering dual-color FCCS, multi-focus FCS, pair correlation function (pCF), scanning FCS, focus-reduced FCS, SPIM-FCS, and inverse-FCS. Besides, the applications of FCS are demonstrated with the measurement of local concentration, hydrodynamic radius, diffusion coefficient, and the interaction of different molecules. Lastly, a discussion is given by summarizing the pros and cons of different FCS techniques, as well as the outlooks and perspectives of FCS.

scholarly journals Studying molecular interactions in the intact organism: fluorescence correlation spectroscopy in the living zebrafish embryo

2020 ◽  
Vol 154 (5) ◽  
pp. 507-519 ◽  
Author(s):  
Michael L. Dawes ◽  
Christian Soeller ◽  
Steffen Scholpp
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Full Range ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Biophysical Studies ◽  
Living Organisms ◽  
And Function ◽  
Compare And Contrast

AbstractCell behaviour and function is determined through the interactions of a multitude of molecules working in concert. To observe these molecular dynamics, biophysical studies have been developed that track single interactions. Fluorescence correlation spectroscopy (FCS) is an optical biophysical technique that non-invasively resolves single molecules through recording the signal intensity at the femtolitre scale. However, recording the behaviour of these biomolecules using in vitro-based assays often fails to recapitulate the full range of variables in vivo that directly confer dynamics. Therefore, there has been an increasing interest in observing the state of these biomolecules within living organisms such as the zebrafish Danio rerio. In this review, we explore the advancements of FCS within the zebrafish and compare and contrast these findings to those found in vitro.

scholarly journals An Alternative Framework for Fluorescence Correlation Spectroscopy

2018 ◽  
Author(s):  
Sina Jazani ◽  
Ioannis Sgouralis ◽  
Omer M. Shafraz ◽  
Marcia Levitus ◽  
Sanjeevi Sivasankar ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Light Exposure ◽  
Correlation Spectroscopy ◽  
High Signal ◽  
Fluorescence Correlation ◽  
Fluorescence Confocal Microscopy ◽  
Long Time

ABSTRACTFluorescence correlation spectroscopy (FCS), is a flexible and widely used tool routinely exploited forin vivoandin vitroapplications. While FCS provides estimates of dynamical quantities, such as diffusion coefficients, it demands high signal to noise ratios and long time traces, typically in the minute range. In principle, the same information can be extracted fromµ-s long time traces; however, an appropriate analysis method is missing. To overcome these limitations, we adapt novel tools inspired by Bayesian non-parametrics, which starts from the direct analysis of the observed photon counts. With this approach, we are able to analyze time traces, which are too short to be analyzed by existing methods, including FCS. Our new analysis extends the capability of single molecule fluorescence confocal microscopy based approaches, to probe processes several orders of magnitude faster in time and permits a reduction of phototoxic effects on living samples induced by long periods of light exposure.

Regulation of plant light harvesting by thermal dissipation of excess energy

2010 ◽  
Vol 38 (2) ◽  
pp. 651-660 ◽  
Author(s):  
Silvia de Bianchi ◽  
Matteo Ballottari ◽  
Luca Dall’Osto ◽  
Roberto Bassi
Keyword(s):  
Higher Plants ◽  
Current Knowledge ◽  
Protein Complexes ◽  
Thermal Dissipation ◽  
Photochemical Quenching ◽  
Knockout Mutants ◽  
Pigment Protein Complexes ◽  
Non Photochemical Quenching

Elucidating the molecular details of qE (energy quenching) induction in higher plants has proven to be a major challenge. Identification of qE mutants has provided initial information on functional elements involved in the qE mechanism; furthermore, investigations on isolated pigment–protein complexes and analysis in vivo and in vitro by sophisticated spectroscopic methods have been used for the elucidation of mechanisms involved. The aim of the present review is to summarize the current knowledge of the phenotype of npq (non-photochemical quenching)-knockout mutants, the role of gene products involved in the qE process and compare the molecular models proposed for this process.

Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy

2000 ◽  
Vol 113 (22) ◽  
pp. 3921-3930 ◽  
Author(s):  
R.H. Kohler ◽  
P. Schwille ◽  
W.W. Webb ◽  
M.R. Hanson
Keyword(s):  
Active Transport ◽  
Fluorescence Correlation Spectroscopy ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Long Distance ◽  
Fluorescence Correlation ◽  
Photon Excitation ◽  
Green Fluorescent

Dynamic tubular projections emanate from plastids in certain cells of vascular plants and are especially prevalent in non-photosynthetic cells. Tubules sometimes connect two or more different plastids and can extend over long distances within a cell, observations that suggest that the tubules may function in distribution of molecules within, to and from plastids. In a new application of two-photon excitation (2PE) fluorescence correlation spectroscopy (FCS), we separated diffusion of fluorescent molecules from active transport in vivo. We quantified the velocities of diffusion versus active transport of green fluorescent protein (GFP) within plastid tubules and in the cytosol in vivo. GFP moves by 3-dimensional (3-D) diffusion both in the cytosol and plastid tubules, but diffusion in tubules is about 50 times and 100 times slower than in the cytosol and an aqueous solution, respectively. Unexpectedly larger GFP units within plastid tubules exhibited active transport with a velocity of about 0.12 microm/second. Active transport might play an important role in the long-distance distribution of large numbers of molecules within the highly viscous stroma of plastid tubules.

scholarly journals Identification of parallel pH- and zeaxanthin-dependent quenching of excess energy in LHCSR3 in Chlamydomonas reinhardtii

2020 ◽  
Author(s):  
Julianne M. Troiano ◽  
Federico Perozeni ◽  
Raymundo Moya ◽  
Luca Zuliani ◽  
Kwangryul Baek ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Light Harvesting ◽  
Complex Stress ◽  
Excess Energy ◽  
Photochemical Quenching ◽  
Light Harvesting Complexes ◽  
Light Harvesting Complex ◽  
Non Photochemical Quenching

AbstractUnder high light conditions, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating excess absorbed energy, which is called non-photochemical quenching (NPQ). In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via pH and serves as a quenching site. However, the mechanisms by which LHCSR3 functions have not been determined. Using a combined in vivo and in vitro approach, we identify two parallel yet distinct quenching processes, individually controlled by pH and carotenoid composition, and their likely molecular origin within LHCSR3 from Chlamydomonas reinhardtii. The pH-controlled quenching is removed within a mutant LHCSR3 that lacks the protonable residues responsible for sensing pH. Constitutive quenching in zeaxanthin-enriched systems demonstrates zeaxanthin-controlled quenching, which may be shared with other light-harvesting complexes. We show that both quenching processes prevent the formation of damaging reactive oxygen species, and thus provide distinct timescales and mechanisms of protection in a changing environment.

scholarly journals From ecophysiology to phenomics: some implications of photoprotection and shade–sun acclimation in situ for dynamics of thylakoids in vitro

2012 ◽  
Vol 367 (1608) ◽  
pp. 3503-3514 ◽  
Author(s):  
Shizue Matsubara ◽  
Britta Förster ◽  
Melinda Waterman ◽  
Sharon A. Robinson ◽  
Barry J. Pogson ◽  
...  
Keyword(s):  
Membrane Dynamics ◽  
Persea Americana ◽  
Physiological Data ◽  
Photochemical Quenching ◽  
Shade Acclimation ◽  
Non Photochemical Quenching ◽  
Time Frames ◽  
Shade Leaves

Half a century of research into the physiology and biochemistry of sun–shade acclimation in diverse plants has provided reality checks for contemporary understanding of thylakoid membrane dynamics. This paper reviews recent insights into photosynthetic efficiency and photoprotection from studies of two xanthophyll cycles in old shade leaves from the inner canopy of the tropical trees Inga sapindoides and Persea americana (avocado). It then presents new physiological data from avocado on the time frames of the slow coordinated photosynthetic development of sink leaves in sunlight and on the slow renovation of photosynthetic properties in old leaves during sun to shade and shade to sun acclimation. In so doing, it grapples with issues in vivo that seem relevant to our increasingly sophisticated understanding of Δ pH-dependent, xanthophyll-pigment-stabilized non-photochemical quenching in the antenna of PSII in thylakoid membranes in vitro .

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