Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Beyond analytic solution: Analysis of FRAP experiments by spatial simulation of the forward problem.

Fluorescence redistribution after photobleaching is a commonly used method to understand the dynamic behavior of molecules within cells. Analytic solutions have been developed for specific, well-defined models of dynamic behavior in idealized geometries, but these solutions are inaccurate in complex geometries or when complex binding and diffusion behaviors exist. We demonstrate the use of numerical reaction-diffusion simulations using the Virtual Cell software platform to model fluorescence redistribution after photobleaching experiments. Multiple simulations employing parameter scans and varying bleaching locations and sizes can help to bracket diffusion coefficients and kinetic rate constants in complex image-based geometries. This approach is applied to problems in membrane surface diffusion as well as diffusion and binding in cytosolic volumes in complex cell geometries. In addition, we model diffusion and binding within phase-separated biomolecular condensates (liquid droplets). These are modeled as spherical low-affinity binding domains that also define a high viscosity medium for exchange of the free fluorescently labeled ligand with the external cytosol.

What's past is prologue: FRAP keeps delivering 50 years later.

Fluorescence recovery after photobleaching (FRAP) has emerged as one of the most widely utilized techniques to quantify binding and diffusion kinetics of biomolecules in biophysics. Since its inception in the mid-1970s, FRAP has been used to address an enormous array of questions including the characteristic features of lipid rafts, how cells regulate the viscosity of their cytoplasm, and the dynamics of biomolecules inside condensates formed by liquid-liquid phase separation. In this perspective, I briefly summarize the history of the field and discuss why FRAP has proven to be so incredibly versatile and popular. Next, I provide an overview of the extensive body of knowledge that has emerged on best practices for quantitative FRAP data analysis, followed by some recent examples of biological lessons learned using this powerful approach. Finally, I touch on new directions and opportunities for biophysicists to contribute to the continued development of this still-relevant research tool.

The disordered regions of the methyltransferase SETD2 govern its function by regulating its proteolysis and phase separation.

SETD2 is an important methyltransferase that methylates crucial substrates such as histone H3, tubulin, and STAT1 and also physically interacts with transcription and splicing regulators such as Pol II and various hnRNPs. Of note, SETD2 has a functionally uncharacterized extended N-terminal region, the removal of which leads to its stabilization. How this region regulates SETD2 half-life is unclear. Here we show that SETD2 consists of multiple long disordered regions across its length that cumulatively destabilize the protein by facilitating its proteasomal degradation. SETD2 disordered regions can reduce the half-life of the yeast homolog Set2 in mammalian cells as well as in yeast, demonstrating the importance of intrinsic structural features in regulating protein half-life. In addition to the shortened half-life, by performing fluorescence recovery after photobleaching assay we found that SETD2 forms liquid droplets in vivo, another property associated with proteins that contain disordered regions. The phase-separation behavior of SETD2 is exacerbated upon the removal of its N-terminal segment and results in activator-independent histone H3K36 methylation. Our findings reveal that disordered region-facilitated proteolysis is an important mechanism governing SETD2 function.

The brown algal mode of tip growth: Keeping stress under control.

Tip growth has been studied in pollen tubes, root hairs, and fungal and oomycete hyphae and is the most widely distributed unidirectional growth process on the planet. It ensures spatial colonization, nutrient predation, fertilization, and symbiosis with growth speeds of up to 800 µm h-1. Although turgor-driven growth is intuitively conceivable, a closer examination of the physical processes at work in tip growth raises a paradox: growth occurs where biophysical forces are low, because of the increase in curvature in the tip. All tip-growing cells studied so far rely on the modulation of cell wall extensibility via the polarized excretion of cell wall-loosening compounds at the tip. Here, we used a series of quantitative measurements at the cellular level and a biophysical simulation approach to show that the brown alga Ectocarpus has an original tip-growth mechanism. In this alga, the establishment of a steep gradient in cell wall thickness can compensate for the variation in tip curvature, thereby modulating wall stress within the tip cell. Bootstrap analyses support the robustness of the process, and experiments with fluorescence recovery after photobleaching (FRAP) confirmed the active vesicle trafficking in the shanks of the apical cell, as inferred from the model. In response to auxin, biophysical measurements change in agreement with the model. Although we cannot strictly exclude the involvement of a gradient in mechanical properties in Ectocarpus morphogenesis, the viscoplastic model of cell wall mechanics strongly suggests that brown algae have evolved an alternative strategy of tip growth. This strategy is largely based on the control of cell wall thickness rather than fluctuations in cell wall mechanical properties.

Regression Analysis of Confocal FRAP and its Application to Diffusion in Membranes.

In most biological processes, diffusion plays a critical role in transferring various bio-molecules to transfer desirable locations in an effective and energy-efficient manner. How fast molecules are transferred is measured by diffusion coefficients. Since each bio-molecules, in particular, signaling molecules have their unique diffusion coefficients and quantifying the diffusion coefficients help us to understand various time scales of both physiological and pathological processes in biological systems. Moreover, since diffusion profiles of a diffusant vary in different micro-environments of cell membranes, accurate diffusion coefficient also can provide a good picture of membrane landscapes as well as interactions of different membrane constituents. Currently, only a few experimental methods are available to assess the diffusion coefficient of a biomolecule of interest in live cells including Fluorescence Recovery After Photobleaching (FRAP). FRAP was developed to study diffusion processes of biomolecules in the cell membranes in the 1970s. Albeit its long history, the main principle of FRAP analysis has remained unchanged since its inception: fitting FRAP data to a theoretical diffusion model for the best fitting diffusion coefficient or using the relation between the half time of recovery and ROI size. In this study, we developed a flexible yet versatile confocal FRAP data analysis framework based on linear regression analysis which allows FRAP users to determine the diffusion from either single or multiple FRAP data points without data fitting. We also validated this approach for a series of fluorescently labeled soluble and membrane-bound proteins and lipids.

Determining the domain-level reaction-diffusion properties of an actin-binding protein transgelin-2 within cells.

Proteins in cells undergo repeated binding to other molecules, thereby reducing the apparent extent of their intracellular diffusion. While much effort has been made to analytically decouple these combined effects of pure diffusion and chemical binding, it is difficult with conventional approaches to attribute the measured quantities to the nature of specific domains of the proteins. Motivated by the common goal in cell signaling research aimed at identifying the domains responsible for particular intermolecular interactions, here we describe a framework for determining the local physicochemical properties of cellular proteins associated with immobile scaffolds. To validate this new approach, we apply it to transgelin-2, an actin-binding protein whose intracellular dynamics remains elusive. We develop a fluorescence recovery after photobleaching (FRAP)-based framework, in which comprehensive combinations of domain-deletion mutants are created, and the difference among them in FRAP response is analyzed. We demonstrate that transgelin-2 in actin stress fibers (SFs) interacts with F-actin via two separate domains, and the chemical properties are determined for the individual domains. Its pure diffusion properties independent of the association to F-actin is also obtained. Our approach will thus be useful, as presented here for transgelin-2, in addressing the signaling mechanism of cellular proteins associated with SFs.

Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells.

The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells.

An amidase is required for proper intercellular communication in the filamentous cyanobacterium Anabaena sp. PCC 7120.

Channels that cross cell walls and connect the cytoplasm of neighboring cells in multicellular cyanobacteria are pivotal for intercellular communication. We find that the product of the gene all1140 of the filamentous cyanobacterium Anabaena sp. PCC 7120 is required for proper channel formation. All1140 encodes an amidase that hydrolyses purified peptidoglycans. An All1140-GFP fusion protein is located at the Z-ring in the periplasmic space during most of the cell cycle. An all1140-null mutant (M40) was unable to grow diazotrophically, and no mature heterocysts were observed in the absence of combined nitrogen. Expression of two key genes, hetR and patS, was studied in M40 using GFP as a reporter. Upon nitrogen step-down, the patterned distribution of green fluorescent cells in filaments seen in the wild type were not observed in mutant M40. Intercellular communication in M40 was studied by measuring fluorescence recovery after photobleaching (FRAP). Movement of calcein (622 Da) was aborted in M40, suggesting that the channels connecting the cytoplasm of neighboring cells are impaired in the mutant. The channels were examined with electron tomography; their diameters were nearly identical, 12.7 nm for the wild type and 12.4 nm for M40, suggesting that AmiC3 is not required for channel formation. However, when the cell wall sacculi isolated by boiling were examined by EM, the average sizes of the channels of the wild type and M40 were 20 nm and 12 nm, respectively, suggesting that the channel walls of the wild type are expandable and that this expandability requires AmiC3.

A Highly Accurate Pixel-Based FRAP Model Based on Spectral-Domain Numerical Methods.

We introduce a new, to our knowledge, numerical model based on spectral methods for analysis of fluorescence recovery after photobleaching data. The model covers pure diffusion and diffusion and binding (reaction-diffusion) with immobile binding sites, as well as arbitrary bleach region shapes. Fitting of the model is supported using both conventional recovery-curve-based estimation and pixel-based estimation, in which all individual pixels in the data are utilized. The model explicitly accounts for multiple bleach frames, diffusion (and binding) during bleaching, and bleaching during imaging. To our knowledge, no other fluorescence recovery after photobleaching framework incorporates all these model features and estimation methods. We thoroughly validate the model by comparison to stochastic simulations of particle dynamics and find it to be highly accurate. We perform simulation studies to compare recovery-curve-based estimation and pixel-based estimation in realistic settings and show that pixel-based estimation is the better method for parameter estimation as well as for distinguishing pure diffusion from diffusion and binding. We show that accounting for multiple bleach frames is important and that the effect of neglecting this is qualitatively different for the two estimation methods. We perform a simple experimental validation showing that pixel-based estimation provides better agreement with literature values than recovery-curve-based estimation and that accounting for multiple bleach frames improves the result. Further, the software developed in this work is freely available online.

Spatial Cycling of Rab GTPase, Driven by the GTPase Cycle, Controls Rab's Subcellular Distribution.

Rab GTPases (>60 members in humans) function as master regulators of intracellular membrane trafficking. Correct and specific localization of Rab proteins is required for their function. How the distinct spatial distribution of Rab GTPases in the cell is regulated remains elusive. To globally assess the subcellular localization of Rab1, we determined kinetic parameters of two pathways that control the spatial cycles of Rab1, i.e., vesicular transport and GDP dissociation inhibitor (GDI)-mediated recycling. We demonstrate that the switching between GTP and GDP binding states, which is governed by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), GDI, and GDI displacement factor (GDF), is a major determinant of Rab1's ability to effectively cycle between cellular compartments and eventually its subcellular distribution. In silico perturbations of vesicular transport, GEFs, GAPs, GDI, and GDF using a mathematical model with simplified cellular geometries showed that these regulators play an important role in the subcellular distribution and activity of Rab1.

Investigating the effect of proteoglycan 4 on hyaluronan solution properties using confocal fluorescence recovery after photobleaching.

BACKGROUND: The objective of this study was to use confocal fluorescence recovery after photobleaching (FRAP) to examine the specific and dose-dependent effect of proteoglycan 4 (PRG4) on hyaluronan (HA) solutions of different molecular weight; and assess the effect of reduction and alkylation (R/A) of PRG4 on its effects on HA solutions. METHODS: Confocal FRAP was used to determine the diffusion coefficient of fluorescein isothiocyanate (FITC)-dextran tracer (Dt) through 1500 kDa and 500 kDa HA solutions (0-3.3 mg/ml) ± PRG4 or a control protein, bovine serum albumin (BSA), at physiological (450 µg/ml) or pathophysiological (45 µg/ml) concentrations. The effect of PRG4 or R/A PRG4 on 1500 kDa HA solutions was also investigated. Empirical constants obtained from fitting data to the universal scaling equation were used to calculate the average distribution of apparent mesh sizes. RESULTS: PRG4 at both 45 and 450 µg/ml slowed the diffusion of the FITC-dextran tracer for all concentrations of HA and caused a decrease in the apparent mesh size within the HA solution. This effect was specific to PRG4, not observed with BSA, but not dependent on its tertiary/quaternary structure as the effect remained after R/A of PRG4. CONCLUSIONS: These results demonstrate that PRG4 can significantly alter the solution properties of HA; PRG4 essentially reduced the permeability of the HA network. This effect may be due to PRG4 entangling HA molecules through binding and/or HA crowding PRG4 molecules into a self-assembled network. Collectively these findings contribute to the understanding of PRG4 and HA interaction(s) in solution and therefore the function of SF in diarthroidal joints.

EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements.

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100-1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 µm2/s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates.

Selection of Biophysical Methods for Characterisation of Membrane Proteins.

Over the years, there have been many developments and advances in the field of integral membrane protein research. As important pharmaceutical targets, it is paramount to understand the mechanisms of action that govern their structure-function relationships. However, the study of integral membrane proteins is still incredibly challenging, mostly due to their low expression and instability once extracted from the native biological membrane. Nevertheless, milligrams of pure, stable, and functional protein are always required for biochemical and structural studies. Many modern biophysical tools are available today that provide critical information regarding to the characterisation and behaviour of integral membrane proteins in solution. These biophysical approaches play an important role in both basic research and in early-stage drug discovery processes. In this review, it is not our objective to present a comprehensive list of all existing biophysical methods, but a selection of the most useful and easily applied to basic integral membrane protein research.

Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP.

Enhanced green fluorescent protein (EGFP)-one of the most widely applied genetically encoded fluorescent probes-carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion ("redding") with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants. In this contribution we aim to clarify the role of the first chromophore-forming amino acid in photoinduced behavior of these fluorescent proteins. To that end, we compared photobleaching and redding kinetics of EGFP, EYFP, and their mutants with reciprocally substituted chromophore residues, EGFP-T65G and EYFP-G65T. Measurements showed that T65G mutation significantly increases EGFP photostability and inhibits its excited-state oxidation efficiency. Remarkably, while EYFP-G65T demonstrated highly increased spectral sensitivity to chloride, it is also able to undergo redding chloride-independently. Atomistic calculations reveal that the GYG chromophore has an increased flexibility, which facilitates radiationless relaxation leading to the reduced fluorescence quantum yield in the T65G mutant. The GYG chromophore also has larger oscillator strength as compared to TYG, which leads to a shorter radiative lifetime (i.e., a faster rate of fluorescence). The faster fluorescence rate partially compensates for the loss of quantum efficiency due to radiationless relaxation. The shorter excited-state lifetime of the GYG chromophore is responsible for its increased photostability and resistance to redding. In EYFP and EYFP-G65T, the chromophore is stabilized by π-stacking with Tyr203, which suppresses its twisting motions relative to EGFP.

Antibacterial and antibiofilm properties of traditional medicinal plant from Sheikh Buddin range.

During current project, antibacterial and antibiofilm properties of traditional medicinal plant Ziziphus nummularia leaf extract and various fractions was investigated. The plant leaves were dried and extracted using 90% methanol followed by sequential fractionation using liquid-liquid fractionation. The fractions of a diverse polarity including chloroform, n-hexane, methanol and ethyl acetate and aqueous extracts were obtained that was further analysed by using HPLC. The phytochemical screening indicated presence of saponins, triterpenes and flavonoids. During DPPH assay, the methanolic fraction presented highest activity (IC50 193.1µg/mL), followed by ethyl acetate (IC50 220µg/mL) and chloroform (IC50 263µg/mL) fractions respectively. During FRAP assay, FRAP value for Z. nummularia extract 20.43µM. Among fractions, ethyl acetate fraction presented highest FRAP value (370.2µM), followed by chloroform (204µM) and methanolic (249µM) fractions. The antimicrobial activity of chloroform fraction was significantly high against P. aureginosa (6mm), L. monocytogenes, S. aureus (5mm), K. pneumoniae, B. Subtillus and E. coli (4mm). The ethyl acetate part presented significant activity (MIC 4mg/mL) against S. aureus, B. Subtillus and L. monocytogenes. The total extract and fractions were further tested for MBC and the MBC for ethyl acetate fractions was 4mg/mL, whereas all other fractions exhibited MBC >10mg/mL. No activity was recorded against Aspergillus niger. During antibiofilm assay, n-hexane fraction presented highest inhibition (88%) followed by ethyl acetate (69%) chloroform (65%) fractions. It was concluded that Z. nummularia possess moderate antimicrobial and antibiofilm activities. Further a synergistic effect is suggested in formulation having Z. nummularia.

The Development and Enhancement of FRAP as a Key Tool for Investigating Protein Dynamics.

The saga of fluorescence recovery after photobleaching (FRAP) illustrates how disparate technical developments impact science. Starting with the classic 1976 Axelrod et al. work in Biophysical Journal, FRAP (originally fluorescence photobleaching recovery) opened the door to extraction of quantitative information from photobleaching experiments, laying the experimental and theoretical groundwork for quantifying both the mobility and the mobile fraction of a labeled population of proteins. Over the ensuing years, FRAP's reach dramatically expanded, with new developments in GFP technology and turn-key confocal microscopy, which enabled measurement of protein diffusion and binding/dissociation rates in virtually every compartment within the cell. The FRAP technique and data catalyzed an exchange of ideas between biophysicists studying membrane dynamics, cell biologists focused on intracellular dynamics, and systems biologists modeling the dynamics of cell activity. The outcome transformed the field of cellular biology, leading to a fundamental rethinking of long-held theories of cellular dynamism. Here, we review the pivotal FRAP studies that made these developments and conceptual changes possible, which gave rise to current models of complex cell dynamics.

Characterization of Cell Boundary and Confocal Effects Improves Quantitative FRAP Analysis.

Fluorescence recovery after photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus, or cell membrane. Although many FRAP models have been developed over the past decades, the influence of the complex boundaries of 3D cellular geometries on the recovery curves, in conjunction with regions of interest and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied. Here, we developed a 3D computational model of the FRAP process that incorporates particle diffusion, cell boundary effects, and the optical properties of the scanning confocal microscope, and validated this model using the tip-growing cells of Physcomitrella patens. We then show how these cell boundary and optical effects confound the interpretation of FRAP recovery curves, including the number of dynamic states of a given fluorophore, in a wide range of cellular geometries-both in two and three dimensions-namely nuclei, filopodia, and lamellipodia of mammalian cells, and in cell types such as the budding yeast, Saccharomyces pombe, and tip-growing plant cells. We explored the performance of existing analytical and algorithmic FRAP models in these various cellular geometries, and determined that the VCell VirtualFRAP tool provides the best accuracy to measure diffusion coefficients. Our computational model is not limited only to these cells types, but can easily be extended to other cellular geometries via the graphical Java-based application we also provide. This particle-based simulation-called the Digital Confocal Microscopy Suite or DCMS-can also perform fluorescence dynamics assays, such as number and brightness, fluorescence correlation spectroscopy, and raster image correlation spectroscopy, and could help shape the way these techniques are interpreted.

Analysis of NTPDase2 in the cell membrane using fluorescence recovery after photobleaching (FRAP).

NTPDase2, a member of the CD39/NTPDase family, is an ecto-nucleotidase anchored to the plasma membrane by two transmembrane domains, with a catalytic site facing the extracellular space and preferentially hydrolyzing nucleoside triphosphates. While NTPDase2 is expressed in many cell types, its unique functionality, mobility and dynamics at the cell membrane remain unexplored. We therefore constructed a recombinant NTPDase2 linked to the yellow fluorescent protein (EYFP) to investigate its dynamics by confocal microscopy. The present study shows that the expression of EYFP-NTPDase2 in different cell lines does not affect its proliferation, migration and adhesion to extracellular matrices (ECM). Moreover, in human embryonic kidney cells 293 (HEK293) grown on collagen type I and fibronectin, EYFP-NTPDase2 fluorescence is greater in free plasma membrane regions than in cell-cell contacts, in comparison with cells grown on other substrates. Differences in the time required for fluorescence recovery after photobleaching (FRAP) in free membrane regions and cell-cell contacts indicate that the mobility of EYFP-NTPDase2 depends on the matrix to which the cells are attached. © 2018 International Society for Advancement of Cytometry.

Effective scheme of photolysis of GFP in live cell as revealed with confocal fluorescence microscopy.

We proposed an effective kinetics scheme of photolysis of green fluorescent protein (GFP) observed in live cells with a commercial confocal fluorescence microscope. We investigated the photolysis of GFP-tagged heterochromatin protein, HP1ß-GFP, in live nucleus with the pulse position modulation approach, which has several advantages over the classical pump-and-probe method. At the basis of the proposed scheme lies a process of photoswitching from the native fluorescence state to the intermediate fluorescence state, which has a lower fluorescence yield and recovers back to native state in the dark. This kinetics scheme includes four effective parameters (photoswitching, reverse switching, photodegradation rate constants, and relative brightness of the intermediate state) and covers the time scale from dozens of milliseconds to minutes of the experimental fluorescence kinetics. Additionally, the applicability of the scheme was demonstrated in the cases of continuous irradiation and the classical pump-and-probe approach using numerical calculations and analytical solutions. An interesting finding of experimental data analysis was that the overall photodegradation of GFP proceeds dominantly from the intermediate state, and demonstrated approximately the second-order reaction versus irradiation power. As a practical example, the proposed scheme elucidates the artifacts of fluorescence recovery after the photobleaching method, and allows us to propose some suggestions on how to diminish them.