Fluorescence Fluctuation Microscopy Research Articles

Response and adaptation of the transcriptional heat shock response to pressure.

The molecular mechanisms underlying pressure adaptation remain largely unexplored, despite their significance for understanding biological adaptation and improving sterilization methods in the food and beverage industry. The heat shock response leads to a global stabilization of the proteome. Prior research suggested that the heat shock regulon may exhibit a transcriptional response to high-pressure stress. In this study, we investigated the pressure-dependent heat shock response in E. coli strains using plasmid-borne green fluorescent protein (GFP) promoter fusions and fluorescence fluctuation microscopy. We quantitatively confirm that key heat shock genes-rpoH, rpoE, dnaK, and groEL - are transcriptionally upregulated following pressure shock in both piezosensitive Escherichia coli and a more piezotolerant laboratory-evolved strain, AN62. Our quantitative imaging results provide the first single cell resolution measurements for both the heat shock and pressure shock transcriptional responses, revealing not only the magnitude of the responses, but also the biological variance involved. Moreover, our results demonstrate distinct responses in the pressure-adapted strain. Specifically, PgroEL is upregulated more than PdnaK in AN62, while the reverse is true in the parental strain. Furthermore, unlike in the parental strain, the pressure-induced upregulation of PrpoE is highly stochastic in strain AN62, consistent with a strong feedback mechanism and suggesting that RpoE could act as a pressure sensor. Despite its capacity to grow at pressures up to 62 MPa, the AN62 genome shows minimal mutations, with notable single nucleotide substitutions in genes of the transcriptionally important b subunit of RNA polymerase and the Rho terminator. In particular, the mutation in RNAP is one of a cluster of mutations known to confer rifampicin resistance to E. coli via modification of RNAP pausing and termination efficiency. The observed differences in the pressure and heat shock responses between the parental MG1655 strain and the pressure-adapted strain AN62 could arise in part from functional differences in their RNAP molecules.

Influenza A M2 recruits M1 to the plasma membrane: A fluorescence fluctuation microscopy study

Influenza A virus (IAV) is a respiratory pathogen that causes seasonal epidemics with significant mortality. One of the most abundant proteins in IAV particles is the matrix protein 1 (M1), which is essential for the virus structural stability. M1 organizes virion assembly and budding at the plasma membrane (PM), where it interacts with other viral components. The recruitment of M1 to the PM as well as its interaction with the other viral envelope proteins (hemagglutinin [HA], neuraminidase, matrix protein 2 [M2]) is controversially discussed in previous studies. Therefore, we used fluorescence fluctuation microscopy techniques (i.e., scanning fluorescence cross-correlation spectroscopy and number and brightness) to quantify the oligomeric state of M1 and its interactions with other viral proteins in co-transfected as well as infected cells. Our results indicate that M1 is recruited to the PM by M2, as a consequence of the strong interaction between the two proteins. In contrast, only a weak interaction between M1 and HA was observed. M1-HA interaction occurred only in the event that M1 was already bound to the PM. We therefore conclude that M2 initiates the assembly of IAV by recruiting M1 to the PM, possibly allowing its further interaction with other viral proteins.

Measuring protein surface density on glass substrate using fluorescence fluctuation spectroscopy

Surface functionalization implies to know the surface density of proteins, which, for cellular biology, impacts on cells behavior (motility, spreading, and fate). Contradictory, the quantification of this parameter is lacking. We studied the surface density of deposited proteins on glass using the Image Correlation Spectroscopy (ICS) technique as part of the Fluorescence Fluctuation Microscopy (FFM) methods. As the confocal microscope scans the surface, the proteins deposited on it contribute to the image through the confocal point spread function (PSF). The width of the image autocorrelation is equal to the width of the PSF; its amplitude provides the average number of proteins within the PSF, proportional to the surface density. Nevertheless, the assessment of the surface density can be underestimated if the surface coverage is not uniform or overestimated if a background contributes to the fluorescence signal. Hence, we combine the ICS and photobleaching (pICS) to detect these artifacts and estimate the actual surface density. The pICS method makes it possible to have a better control of the estimation of the surface density. We noticed that the surface density of proteins increased with their initial concentration in solutions. Overall, FFM is a quantitative technique to infer the surface density of protein.

Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells.

High-end microscopy studies of G protein-coupled receptors (GPCRs) require installing onto the receptors bright and photostable dyes. Labeling must occur in quantitative yields, to allow stoichiometric data analysis, and in a minimally invasive fashion, to avoid perturbing GPCR function. We demonstrate here that the genetic incorporation of trans-cyclooct-2-ene lysine (TCO*) allows achieving quantitative single-residue labeling of the extracellular loops of the β2-adrenergic and the muscarinic M2 class A GPCRs, as well as of the corticotropin releasing factor class B GPCR. Labeling occurs within a few minutes by reaction with dye-tetrazine conjugates on the surface of live cells and preserves the functionality of the receptors. To precisely quantify the labeling yields, we devise a method based on fluorescence fluctuation microscopy that extracts the number of labeling sites at the single-cell level. Further, we show that single-residue labeling is better suited for studies of GPCR diffusion than fluorescent-protein tags, since the latter can affect the mobility of the receptor. Finally, by performing dual-color competitive labeling on a single TCO* site, we devise a method to estimate the oligomerization state of a GPCR without the need for a biological monomeric reference, which facilitates the application of fluorescence methods to oligomerization studies. As TCO* and the dye-tetrazines used in this study are commercially available and the described microscopy techniques can be performed on a commercial microscope, we expect our approach to be widely applicable to fluorescence microscopy studies of membrane proteins in general.

Quantification of G1-Cyclin Dynamics in Yeast by Scanning Number and Brightness

Cell size homeostasis reflects the coordination between cell growth and division and is vital to all living organisms. This coordination is achieved primarily at the commitment to cell division at the G1/S phase transition, termed Start in budding yeast. Cells pass Start upon activation of kinase Cdc28 by the G1 cyclin, Cln3, which phosphorylates the repressor Whi5, thereby activating expression of the downstream G1 cyclins CLN1/2 and ∼200 other genes in the G1/S regulon by the SBF/MBF transcription factor complexes. Recently, we demonstrated that SBF/MBF abundance, and consequently Cln1/2 levels, are differentially regulated by nutrients. However, it remains unclear how dynamics of Cln1/2 levels in G1 phase affect Start. Moreover, the mechanism by which Cln3 activates Start remains elusive due to its low expression level and extreme instability. We performed time-course Scanning Number and Brightness (sN&B) fluorescence fluctuation microscopy experiments on live yeast expressing Cln1/2/3 GFP fusions at their endogenous loci. We quantified single-cell protein concentrations and localization patterns of each G1 cyclin throughout G1 phase in rich medium and in response to nutrient shifts. In early G1 phase, Cln1/2 were expressed at low levels primarily in the cytoplasm. Cln1 cytoplasmic levels were generally higher (∼5 nM) in early G1 compared to Cln2 levels (∼1 nM). Unexpectedly, we observed low levels of Cln3 throughout G1 phase, with distribution between the nucleus (∼20 nM) and cytoplasm (∼5 nM) varying randomly with respect to Start onset. This result suggests that some aspect of Cln3 dynamics beyond its mere accumulation governs the timing of Start. These data have been used to constrain a dynamic mathematical model of Start.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies.

The promyelocytic leukemia (pml) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology.

Super-resolution photoacoustic imaging via flow-induced absorption fluctuations

In deep-tissue photoacoustic imaging, optical-contrast images of deep-lying structures are formed by recording acoustic waves that are generated by optical absorption. Although photoacoustics is perhaps the leading technique for high-resolution deep-tissue optical imaging, its spatial resolution is fundamentally limited by the acoustic wavelength, which is orders of magnitude longer than the optical diffraction limit. Here, we present an approach for surpassing the acoustic diffraction limit in photoacoustics by exploiting inherent temporal fluctuations in the photoacoustic signals due to sample dynamics, such as those induced by the flow of absorbing red blood cells. This was achieved using a conventional photoacoustic imaging system by adapting concepts from super-resolution fluorescence fluctuation microscopy to the statistical analysis of acoustic signals from flowing acoustic emitters. Specifically, we experimentally demonstrate that flow of absorbing particles and whole human blood yields super-resolved photoacoustic images, and provides static background reduction. By generalizing the statistical analysis to complex-valued signals, we demonstrate super-resolved photoacoustic images that are free from common photoacoustic imaging artifacts caused by band-limited acoustic detection. The presented technique holds potential for contrast-agent-free microvessel imaging, as red blood cells provide a strong endogenous source of naturally fluctuating absorption.

High pressure activation of the Mrr restriction endonuclease in Escherichia coli involves tetramer dissociation.

A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer.

Identifying Protein Complexes in the Nuclear Envelope by Fluorescence Fluctuation Microscopy

Identifying Protein Complexes in the Nuclear Envelope by Fluorescence Fluctuation Microscopy

Association of Endophilin B1 with Cytoplasmic Vesicles

Studying protein association with cytoplasmic vesicles within the live cell remains a general challenge for optical microscopy techniques. Here, we show that fluorescence fluctuation microscopy offers an effective approach to tackle this problem by using endophilins as a model system. Endophilins are SH3- and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2 promote the budding of endocytic vesicles from the plasma membrane, whereas endophilin B1 has been implicated in vesicle budding from intracellular organelles, including the trans-Golgi network and late endosomes. We previously reported that endophilins A1 and A2 exist almost exclusively as soluble dimers in the cytosol. Here we present results of fluorescence fluctuation spectroscopy analyses indicating that, in contrast, the majority of endophilin B1 is present in multiple copies on small, highly mobile cytoplasmic vesicles. Formation of these vesicles was enhanced by overexpression of wild-type dynamin 2, but suppressed by expression of a catalytically inactive dynamin 2 mutant. Using dual-color heterospecies partition analysis, we identified the epidermal growth factor receptor (EGFR) on EndoB1 vesicles. Moreover, a proportion of EndoB1 vesicles also contained caveolin, whereas clathrin was almost undetectable on those vesicles. These results raise the possibility that endophilin B1 participates in dynamin 2-dependent formation of a population of transport vesicles distinct from those generated by A-type endophilins. This work has been supported by a grant from the National Institutes of Health (R01 GM64589).

Multimodal combinational holographic and fluorescence fluctuation microscopy to obtain spatial super-resolution

Ways of combination of holographic and super-resolution fluorescent techniques in the same optical scheme are described. The key parameters influencing achievement of maximum possible resolution are considered. The possibility to choose different fluorescence technic for different types of fluorophores without any optical scheme changes is presented. As a result in case of visualization of the samples, which transparent in optical band, three-dimensional super resolution is received that significantly expands possibilities of the noninvasive analysis of biological samples.

High Pressure Induced DNA Damage in Escherichia Coli Involves Pressure-Mediated Dissociation of the Tetrameric Mrr Restriction Endonuclease

Pressure shock of ∼100 MPa elicits a bona fide DNA damage response in the mesophilic E. coli bacterium, despite the fact that pressure, itself, cannot compromise DNA integrity. Screens for resistance to pressure shock by Aertsen's group revealed an endogenous restriction endonuclease, Mrr (Methylated adenine Recognition and Restriction), that cleaves methylated DNA. Since expression of Mrr is not toxic for the cell in the absence of HP, pressure stress must somehow be able to elicit Mrr activity. We used fluorescence fluctuation microscopy of strains chromosomally expressing a GFPmut2-Mrr fusion to investigate HP activation of Mrr. In scanning mode, FFM provides a spatial map of the absolute concentration (=n/PSF) and stoichiometry (=brightness, e) of the fluorescent particles. Before HP shock GFPmut2-Mrr exhibits a stoichiometry consistent with a tetramer. It appears to be slightly preferentially localized around the chromosome, diffusing more slowly than free GFP, yet not being immobile. After 20 min at 100 MPa followed by pressure release, GFPmut2-Mrr is found accumulated in foci that co-localize strongly with the chromosome, as previously observed. Interestingly, the mobility of the Mrr in these foci is significantly decreased and their stoichiometry is consistent with a dimer. The Mrr molecules which are excluded from the foci remain tetrameric. We suggest that pressure-mediated triggering of Mrr activity and the concomitant generation of DNA damage involves the conversion of inactive Mrr tetramers to active Mrr dimers. This could occur either via a direct effect of pressure on the Mrr tetramer-dimer equilibrium, a common effect of pressure on oligomers, or via a pressure effect on chromosome structural that leads to more efficient recruitment of active dimeric Mrr.

Correlative Traction Force Microscopy and Fluorescence Fluctuation Analysis of Molecular Aggregation and Complex Formation in Cell Adhesions in Distinct Microenvironments

The mechanical properties of the cellular microenvironment regulate processes that include migration, proliferation, and differentiation. These interactions occur at adhesions, which serve as both traction points and signaling hubs and mediate bi-directional sensing and responses to specific features of the surrounding extracellular matrix (ECM). Adhesions execute these activities through an intricate network of putative molecular interactions that largely remain to be demonstrated and characterized functionally in living cells. The challenge is to capture the highly localized and transient associations that characterize these activities in adhesions and determine how they respond to different microenvironments. In this study, we use high-resolution fluorescence fluctuation microscopy to map the formation and stoichiometry of integrin-associated complexes in the adhesions that populate the leading edge of migrating cells. We focus on putative integrin activating (kindlin and talin) and actin-linking (talin, vinculin and a-actinin) molecules and show that all molecules are present in adhesions as soon as they are visible; however, they form integrin containing complexes hierarchically, at different times, with variable stoichiometry within the adhesion itself and change as the adhesion matures into larger structures. To parse out the effects of the mechanical properties of the ECM on the numbers, aggregation states, and associations of these molecules, we extend these measurements to substrates with variable stiffness and correlate them with high-resolution traction force microscopy (TFM) measurements. We show that individual and newly formed adhesions at the leading edge of protruding cells transmit forces on soft as well as stiff substrates, with force magnitudes that correlate with the integrated intensity of the adhesions and the total number of individual adhesion molecules. These measurements provide novel information on complex formation as adhesions evolve and respond to substrate rigidity.

Fluorescence Fluctuation Microscopy Techniques to Study mRNA Synthesis and Dynamics

1627-Pos Board B578 Probing Short-Range Protein Brownian Motion in the Cytoplasm of Living Cells Carmine Di Rienzo 1,2 , Enrico Gratton 3 , Fabio Beltram 1,2 , Francesco Cardarelli 2 . NEST, Scuola Normale Superiore, Pisa, Italy, 2 Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy, 3 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering., University of California, Irvine, CA, USA. The translational motion of small molecules in cells appears to be suppressed compared to what is observed in dilute solutions. Although, the rotation of small proteins is almost unhindered, pointing out a local aqueous environment. Different theoretical models provide explanations for this apparent discrepancy but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive experimental test of the nature of the translational motion in cells is still missing owing to the lack of techniques capable of probing protein motion with the required temporal and spatial resolution. We show that fluorescence-fluctuation analysis of raster scans at variable time scales can provide this information. By using GFP, we measure protein translational motion at the unprecedented time-scale of 1 micro- second, unveiling unobstructed Brownian motion from 25 to 100 nanometers, and partially-suppressed diffusion above 100 nm. Experiments on in vitro model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. In this regard, internal membranes (e.g. the ER sheets, vesicles, Golgi apparatus, etc.) appear to be the more likely candidates as selective disruption of the microtubules network by treatment with Nocodazole did not significantly alter GFP behavior in the cytoplasm. Also, the same measurement in a structurally-different (e.g devoid of membranes) intra- cellular environment, such as the nucleoplasm, yields a different behavior, in which GFP motion is never coincident with that in a dilute solution. Finally, we believe the present findings coupled with use of genetically-encoded fluores- cent markers pave the way to novel studies of biomolecular processes in live cells at the physiologically-relevant spatio-temporal scale. Supported by grants NIH P41-GM103540 and NIH P50-GM076516 (grants to EG), MIUR under FIRB- RBAP11X42L and Fondazione Monte dei Paschi di Siena (grants to FB).

Measuring protein dynamics in live cells: protocols and practical considerations for fluorescence fluctuation microscopy.

Quantitative analysis of protein complex stoichiometries and mobilities are critical for elucidating the mechanisms that regulate cellular pathways. Fluorescence fluctuation spectroscopy (FFS) techniques can measure protein dynamics, such as diffusion coefficients and formation of complexes, with extraordinary precision and sensitivity. Complete calibration and characterization of the microscope instrument is necessary in order to avoid artifacts during data acquisition and to capitalize on the full capabilities of FFS techniques. We provide an overview of the theory behind FFS techniques, discuss calibration procedures, provide protocols, and give practical considerations for performing FFS experiments. One important parameter recovered from FFS measurements is the relative molecular brightness that can correlate with oligomerization. Three methods for measuring molecular brightness (fluorescence correlation spectroscopy, photon-counting histogram, and number and brightness analysis) recover similar values when measuring samples under ideal conditions in vitro. However, examples are given illustrating that these different methods used for calculating molecular brightness of fluorescent molecules in cells are not always equivalent. Methods relying on spot measurements are more prone to bleaching and movement artifacts that can lead to underestimation of brightness values. We advocate for the use of multiple FFS techniques to study molecular brightnesses to overcome and compliment limitations of individual techniques.

Fluorescence fluctuation microscopy: a diversified arsenal of methods to investigate molecular dynamics inside cells

Fluorescence microscopy provides insight into the subcellular organization of biological functions. However, images are snap shots averaging over a highly dynamic molecular system. Fluorescence fluctuation microscopy, employing similar detection technology, encompasses a powerful arsenal of analysis tools that investigate the molecular heterogeneity in space and time. Analyzing signal fluctuations from small ensembles (several hundred particles) reveals their concentration, the stoichiometry, the stochastic motion, as well as superimposed signatures of the environment such as spatial confinement and binding events. Thus, fluctuation analysis provides access to dynamic molecular properties that can be used to build physical models of cellular processes. In the last decade these methods experienced a remarkable diversification, which we revisit here with a particular focus on live cell applications.

The stoichiometry of scaffold complexes in living neurons - DLC2 functions as a dimerization engine for GKAP.

Quantitative spatio-temporal characterization of protein interactions in living cells remains a major challenge facing modern biology. We have investigated in living neurons the spatial dependence of the stoichiometry of interactions between two core proteins of the N-methyl-D-aspartate (NMDA)-receptor-associated scaffolding complex, GKAP (also known as DLGAP1) and DLC2 (also known as DYNLL2), using a novel variation of fluorescence fluctuation microscopy called two-photon scanning number and brightness (sN&B). We found that dimerization of DLC2 was required for its interaction with GKAP, which, in turn, potentiated GKAP self-association. In the dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of two DLC2 and two GKAP monomers, whereas, in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP monomers. Disruption of the GKAP-DLC2 interaction strongly destabilized the oligomers, decreasing the spine-preferential localization of GKAP and inhibiting NMDA receptor activity. Hence, DLC2 serves a hub function in the control of glutamatergic transmission by ordering GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2-GKAP interactions in glutamatergic signaling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes.

Recruitment, Assembly, and Molecular Architecture of the SpoIIIE DNA Pump Revealed by Superresolution Microscopy

Author SummaryMolecular motors are implicated in myriad cellular processes, notably in the transcription, replication, and segregation of DNA. Segregation or packaging of DNA is essential for production of viable viral particles, proper division of bacterial cells, and production of spores. A dramatic example of this process occurs during sporulation in Bacillus subtilis, in which a large proportion of the chromosome is actively transferred across a division septum by the SpoIIIE motor protein. Here, we use advanced microscopy methods to study the mechanism of recruitment and assembly of the SpoIIIE pump and the architecture of its complex with DNA. We found that SpoIIIE complexes are recruited before the beginning of cell division, and are subsequently escorted by the constriction machinery to the center of the septum. We show that the directionality of DNA transport by SpoIIIE results in the establishment of an asymmetric complex that exports DNA into the nascent spore. Our data are inconsistent with previous models that predicted the formation of a membrane-spanning DNA-conducting channel. Instead, the greater resolution afforded by our approach supports a model in which DNA movement occurs through an open pore structurally maintained by the division apparatus, with SpoIIIE working as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed model may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.

Defining the subcellular interface of nanoparticles by live-cell imaging.

Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes.

Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states

Assessing gene expression noise in order to obtain mechanistic insights requires accurate quantification of gene expression on many individual cells over a large dynamic range. We used a unique method based on 2-photon fluorescence fluctuation microscopy to measure directly, at the single cell level and with single-molecule sensitivity, the absolute concentration of fluorescent proteins produced from the two Bacillus subtilis promoters that control the switch between glycolysis and gluconeogenesis. We quantified cell-to-cell variations in GFP concentrations in reporter strains grown on glucose or malate, including very weakly transcribed genes under strong catabolite repression. Results revealed strong transcriptional bursting, particularly for the glycolytic promoter. Noise pattern parameters of the two antagonistic promoters controlling the nutrient switch were differentially affected on glycolytic and gluconeogenic carbon sources, discriminating between the different mechanisms that control their activity. Our stochastic model for the transcription events reproduced the observed noise patterns and identified the critical parameters responsible for the differences in expression profiles of the promoters. The model also resolved apparent contradictions between in vitro operator affinity and in vivo repressor activity at these promoters. Finally, our results demonstrate that negative feedback is not noise-reducing in the case of strong transcriptional bursting.