Autophagy-Related Proteins GABARAP and LC3B Label Structures of Similar Size but Different Shape in Super-Resolution Imaging.

Subcellular structures containing autophagy-related proteins of the Atg8 protein family have been investigated with conventional wide-field fluorescence and single molecule localisation microscopy. Fusion proteins of GABARAP and LC3B, respectively, with EYFP were overexpressed in HEK293 cells. While size distributions of structures labelled by the two proteins were found to be similar, shape distributions appeared quite disparate, with EYFP-GABARAP favouring circular structures and elliptical structures being dominant for EYFP-LC3B. The latter also featured a nearly doubled fraction of U-shape structures. The experimental results point towards highly differential localisation of the two proteins, which appear to label structures representing distinct stages or even specific channels of vesicular trafficking pathways. Our data also demonstrate that the application of super-resolution techniques expands the possibilities of fluorescence-based methods in autophagy studies and in some cases can rectify conclusions obtained from conventional fluorescence microscopy with diffraction-limited resolution.

Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular Proteins.

Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation. To explore the key factors of proteofection processes, the complex formation of fusogenic liposomes and proteins of interest and the size and zeta potential of the formed fusogenic proteoliposoms were monitored. Intracellular protein delivery was analyzed using fluorescence microscopy and flow cytometry. Proteins such as EGFP, Dendra2, and R-phycoerythrin or peptides such as LifeAct-FITC and NTF2-AlexaFluor488 were successfully incorporated into mammalian cells with high efficiency. Moreover, correct functionality and faithful transport to binding sites were also proven for the imported proteins.

Direct interaction of CaVß with actin up-regulates L-type calcium currents in HL-1 cardiomyocytes.

Expression of the ß-subunit (CaVß) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. CaVß binds to the α1 pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although CaVß encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between CaVß and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by CaVß2 that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of CaVß2-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. CaVß mediated L-type up-regulation, and CaVß-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of CaVß that is directly involved in vesicular trafficking. We propose a model in which CaVß promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane.

Tryptophan fluorescence as a reporter for structural changes in photoactive yellow protein elicited by photo-activation.

Light-activation of photoactive yellow protein (PYP) is followed by a series of dynamical transitions in the structure of the protein. Tryptophan fluorescence is well-suited as a tool to study selected aspects of these. Using site-directed mutagenesis eight 'single-tryptophan' mutants of PYP were made by replacement of either a tyrosine, phenylalanine or histidine residue by tryptophan, while simultaneously eliminating the endogenous W119. Surprisingly, only three of these eight mutants turn out to emit measurable tryptophan fluorescence: F6W/W119F, F96W/W119F and H108W/W119F. Significantly, all three show altered tryptophan fluorescence upon formation of the pB state. As F96 is located very close to the chromophore, the F96W/W119F mutant protein is particularly suitable for further studies on the dynamical changes of the polarity in the chromophore-binding pocket of PYP. Furthermore, WT PYP can be photo-activated by a UV photon via the highly conserved W119 and subsequent Förster resonance energy transfer. Placing a unique tryptophan residue elsewhere in the protein shows that position 119 is favoured for UV-activation of PYP.

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli.

Bacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli. Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli, there is little change in ribosome abundance for µ <0.4 h-1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.

Spatial filter and its application in three-dimensional single molecule localization microscopy.

Single molecule localization microscopy (SMLM) allows the imaging of cellular structures with resolutions five to ten times below the diffraction limit of optical microscopy. It was originally introduced as a two-dimensional technique based on the localization of single emitters as projection onto the x-y imaging plane. The determination of the axial position of a fluorescent emitter is only possible by additional information. Here we report a method (spatial filter SMLM (SFSMLM)) that allows to determine the axial positions of fluorescent molecules and nanoparticles on the nanometer scale by the usage of two spatial filters, which are placed in two otherwise identical emission detection channels. SFSMLM allows axial localization in a range of ca. 1.5 µm with a localization precision of 15 - 30 nm in axial direction. The technique was utilized for localizing and imaging small cellular structures - e.g. actin filaments, vesicles and mitochondria - in three dimensions.

Dissociation kinetics of the GroEL-gp31 chaperonin complex studied with Förster resonance energy transfer.

Propagation of bacteriophage T4 in its host Escherichia coli involves the folding of the major capsid protein gp23, which is facilitated by a hybrid chaperone complex consisting of the bacterial chaperonin GroEL and the phage-encoded co-chaperonin, gp31. It has been well established that the GroEL-gp31 complex is capable of folding gp23 whereas the homologous GroEL-GroES complex cannot perform this function. To assess whether this is a consequence of differences in the interactions of the proteins within the chaperonin complex, we have investigated the dissociation kinetics of GroEL-gp31 and GroEL-GroES complexes using Forster resonance energy transfer. Here we report that the dissociation of gp31 from GroEL is slightly faster than that of GroES from GroEL and is further accelerated by the binding of gp23. In contrast to what had been observed previously, we found that gp23 is able to interact with the GroEL-GroES complex, which might explain how bacteriophage T4 redirects the folding machinery of Escherichia coli during morphogenesis.

On the signaling mechanism and the absence of photoreversibility in the AppA BLUF domain.

The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal, FAD-binding BLUF photoreceptor domain. Upon illumination, the AppA BLUF domain forms a signaling state that is characterized by red-shifted absorbance by 10 nm, a state known as AppA(RED). We have applied ultrafast spectroscopy on the photoaccumulated AppA(RED) state to investigate the photoreversible properties of the AppA BLUF domain. On light absorption by AppA(RED), the FAD singlet excited state FAD(RED)* decays monoexponentially in 7 ps to form the neutral semiquinone radical FADH(*), which subsequently decays to the original AppA(RED) molecular ground state in 60 ps. Thus, FAD(RED)* is deactivated rapidly via electron and proton transfer, probably from the conserved tyrosine Tyr-21 to FAD, followed by radical-pair recombination. We conclude that, in contrast to many other photoreceptors, the AppA BLUF domain is not photoreversible and does not enter alternative reaction pathways upon absorption of a second photon. To explain these properties, we propose that a molecular configuration is formed upon excitation of AppA(RED) that corresponds to a forward reaction intermediate previously identified for the dark-state BLUF photoreaction. Upon excitation of AppA(RED), the BLUF domain therefore enters its forward reaction coordinate, readily re-forming the AppA(RED) ground state and suppressing reverse or side reactions. The monoexponential decay of FAD* indicates that the FAD-binding pocket in AppA(RED) is significantly more rigid than in dark-state AppA. Steady-state fluorescence experiments on wild-type, W104F, and W64F mutant BLUF domains show tryptophan fluorescence maxima that correspond with a buried conformation of Trp-104 in dark and light states. We conclude that Trp-104 does not become exposed to solvent during the BLUF photocycle.

Rapid Turnover of the Cardiac L-Type CaV1.2 Channel by Endocytic Recycling Regulates Its Cell Surface Availability.

Calcium entry through CaV1.2 L-type calcium channels regulates cardiac contractility. Here, we study the impact of exocytic and post-endocytic trafficking on cell surface channel abundance in cardiomyocytes. Single-molecule localization and confocal microscopy reveal an intracellular CaV1.2 pool tightly associated with microtubules from the perinuclear region to the cell periphery, and with actin filaments at the cell cortex. Channels newly inserted into the plasma membrane become internalized with an average time constant of 7.5 min and are sorted out to the Rab11a-recycling compartment. CaV1.2 recycling suffices for maintaining stable L-type current amplitudes over 20 hr independent of de novo channel transport along microtubules. Disruption of the actin cytoskeleton re-routes CaV1.2 from recycling toward lysosomal degradation. We identify endocytic recycling as essential for the homeostatic regulation of voltage-dependent calcium influx into cardiomyocytes. This mechanism provides the basis for a dynamic adjustment of the channel's surface availability and thus, of heart's contraction.

Hexamerization of the bacteriophage T4 capsid protein gp23 and its W13V mutant studied by time-resolved tryptophan fluorescence.

The bacteriophage T4 capsid protein gp23 was studied using time-resolved and steady-state fluorescence of the intrinsic protein fluorophore tryptophan. In-vitro gp23 consists mostly of monomers at low temperature but forms hexamers at room temperature. To extend our knowledge of the structure and hexamerization characteristics of gp23, the temperature-dependent fluorescence properties of a tryptophan mutant (W13V) were compared to those of wild-type gp23. The W13V mutation is located in the N-terminal part of the protein, which is cleaved off after prohead formation in the live bacteriophage. Results show that W13 plays a role in the hexamerization process but is not needed to stabilize the hexamer once it is formed. Furthermore, besides the monomer-to-hexamer temperature transition (15-23 degrees C and 12-43 degrees C for wild-type and W13V gp23, respectively), we were able to observe denaturation of the N-terminus in hexameric wild-type gp23 around 40 degrees C. In addition, with the aid of a recently published homology model of gp23, the lifetimes obtained from time-resolved fluorescence measurements could tentatively be assigned to specific tryptophan residues.

Automatic Bayesian single molecule identification for localization microscopy.

Single molecule localization microscopy (SMLM) is on its way to become a mainstream imaging technique in the life sciences. However, analysis of SMLM data is biased by user provided subjective parameters required by the analysis software. To remove this human bias we introduce here the Auto-Bayes method that executes the analysis of SMLM data automatically. We demonstrate the success of the method using the photoelectron count of an emitter as selection characteristic. Moreover, the principle can be used for any characteristic that is bimodally distributed with respect to false and true emitters. The method also allows generation of an emitter reliability map for estimating quality of SMLM-based structures. The potential of the Auto-Bayes method is shown by the fact that our first basic implementation was able to outperform all software packages that were compared in the ISBI online challenge in 2015, with respect to molecule detection (Jaccard index).

SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy.

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe's resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets.

Modeling the functioning of YtvA in the general stress response in Bacillus subtilis.

The blue-light photoreceptor YtvA activates the general stress response (GSR) of Bacillus subtilis by activating a large protein complex (the stressosome). We have constructed a model for the YtvA's photocycle, and derived an equation for the fraction of YtvA in the light-induced signaling state at a given light intensity. The model was verified experimentally in vitro on wild type YtvA and on an R63K mutant with faster recovery kinetics. Application of the model to the activation of the GSR at various light intensities in vivo revealed that the GSR is more sensitive to light than would be expected based on YtvA's in vitro kinetics. These results were confirmed with the R63K mutant and a slower-recovering V28I mutant. Additionally, we have demonstrated the presence of a near-UV-light-induced branching reaction that converts the signaling state of YtvA to the dark state. Extension of the model with this reaction shows that it does not contribute significantly to the in vivo blue-light response. The model represents an important step towards a complete systems biology model of the GSR.

Binding of hydrogen-citrate to photoactive yellow protein is affected by the structural changes related to signaling state formation.

The tricarboxylic acid citric acid is a key intermediary metabolite in organisms from all domains of the tree of life. Surprisingly, this metabolite specifically interacts with the light-induced signaling state of the photoactive yellow protein (PYP), such that, at 30 mM, it retards recovery of this state to the stable ground state of the protein with up to 30%, in the range from pH 4.5 to pH 7. We have performed a detailed UV/vis spectroscopic study of the recovery of the signaling state of wild type (WT) PYP and two mutants, H108F and Δ25-PYP, derived from this protein, as a function of pH and the concentration of citric acid. This revealed that it is the dianionic form of citric acid that binds to the pB state of PYP. Its binding site is located in between the N-terminal cap and central ß-sheet of PYP, which is accessible only in the signaling state of the protein. The obtained results show how changes in the distribution of subspecies of the signaling state of PYP influence the rate of ground state recovery.

On the midpoint potential of the FAD chromophore in a BLUF-domain containing photoreceptor protein.

The redox-midpoint potential of the FAD chromophore in the BLUF domain of anti-transcriptional regulator AppA from Rhodobacter sphaeroides equals ∼-260mV relative to the calomel electrode. Altering the structure of its chromophore-binding pocket through site-directed mutagenesis brings this midpoint potential closer to that of free flavin in aqueous solution. The redox-midpoint potential of this BLUF domain is intermediate between those of LOV domains and Cryptochromes, which may rationalize the primary photochemistry observed in these three flavin-containing photoreceptor families. These results also imply that LOV domains, among the flavin-containing photosensory receptors, are least sensitive to intracellular chemical reduction in the dark.

In vivo effects on photosynthesis gene expression of base pair exchanges in the gene encoding the light-responsive BLUF domain of AppA in Rhodobacter sphaeroides.

The Rhodobacter sphaeroides protein AppA has the unique quality of sensing and transmitting light and redox signals. By acting as antirepressor to the PpsR protein, it acts as a major regulator in photosynthesis gene expression. In this study, we show that by introducing amino acid exchanges into the AppA protein, the in vivo activity as an antirepressor can be greatly altered. The tryptophan 104 to phenylalanine (W104F) base exchange greatly diminished blue-light sensitivity of the BLUF domain. From the obtained in vivo data, the difference in thermal recovery rate of the signaling state of the BLUF domain between the wild type and mutated protein was calculated, predicting an about 10-fold faster recovery in the mutant, which is consistent with in vitro data. Introduction of a tyrosine 21 to phenylalanine (Y21F) or to cysteine (Y21C) mutation led to a complete loss of AppA antirepressor activity, while additionally leading to an increase of photosynthesis gene expression after illumination with high blue-light quantities. Interestingly, this effect is not visible in a W104F/Y21F double mutant that again shows a wild-type-like behavior of the BLUF domain after blue-light illumination, thus restoring the activity of AppA.

pH Dependence of the photoactive yellow protein photocycle recovery reaction reveals a new late photocycle intermediate with a deprotonated chromophore.

The recovery reaction of the signaling state of photoactive yellow protein includes the following: (i) deprotonation of the p-coumaryl chromophore, (ii) refolding of the protein, and (iii) chromophore re-isomerization from the cis to the trans configuration. Through analysis of the pH dependence of this recovery reaction, we were able to provide proof for the existence of an additional photocycle intermediate. The spectral similarity between this new intermediate and the dark state indicates that the new intermediate has a deprotonated chromophore, which may facilitate chromophore re-isomerization. This spectral similarity also explains why this new intermediate has not been noticed in earlier studies. For our data analysis we introduce a photocycle model that takes into account the effect of the specific light regime selected, a model that was also used for simulations.

Tryptophan fluorescence monitors structural changes accompanying signalling state formation in the photocycle of photoactive yellow protein.

Photoactive yellow protein, a small, water-soluble blue-light absorbing photoreceptor protein from Ectothiorhodospira(Halorhodospira)[space]halophila has a structure with two hydrophobic cores, of which the main one houses its light-sensitive chromophore (p-coumaric acid), separated by a central [small beta]-sheet. This photoreceptor protein contains a single tryptophan residue (W119) that is situated at the interface between the central beta-sheet and its N-terminal cap. The fluorescence properties of W119 in the dark state pG (lambda(max)= 328 nm; Phi(fl)= 0.01; nearly pH-independent) are typical for a buried tryptophan in a hydrophobic environment with significant quenching by nearby amino acid residues. Signalling state formation leads to pH-dependent fluorescence changes: At pH values <6.5 the fluorescence emission increases, with a minor blue shift of the emission maximum. Above this pH, the emission maximum of the tryptophan shifts considerably to the red, whereas its total intensity decreases. These results further support the contention that signalling state formation in PYP leads to significant changes in the structure of this protein, even at sites that are at a considerable distance from the chromophore. The nature of these changes in pB, however, depend upon the pH imposed upon the protein: At slightly alkaline pH, which presumably is closest to the pH to which this protein is exposed in vivo, these changes lead to an exposure of the part of the central beta-sheet harbouring W119. At slightly acidic pH the polarity of the environment of W119 is hardly affected by the formation of the signalling state but the quenching of its fluorescence emission, possibly by nearby amino acids, is reduced. On the other hand, its accessibility for quenching by small molecules in the solution is enhanced at acidic and alkaline pH in the signalling state (pB) compared to the dark state (pG). This latter observation points towards a more flexible structure of the N-terminal cap, having a looser interaction with the central beta-sheet in pB.

Deuterium isotope effects in the photocycle transitions of the photoactive yellow protein.

The Photoactive Yellow Protein (PYP) from Halorhodospira halophila (formerly Ectothiorhodospira halophila) is increasingly used as a model system. As such, a thorough understanding of the photocycle of PYP is essential. In this study we have combined information from pOH- (or pH-) dependence and (kinetic) deuterium isotope effects to elaborate on existing photocycle models. For several characteristics of PYP we were able to make a distinction between pH- and pOH-dependence, a nontrivial distinction when comparing data from samples dissolved in H(2)O and D(2)O. It turns out that most characteristics of PYP are pOH-dependent. We confirmed the existence of a pB' intermediate in the pR to pB transition of the photocycle. In addition, we were able to show that the pR to pB' transition is reversible, which explains the previously observed biexponential character of the pR-to-pB photocycle step. Also, the absorption spectrum of pB' is slightly red-shifted with respect to pB. The recovery of the pG state is accompanied by an inverse kinetic deuterium isotope effect. Our interpretation of this is that before the chromophore can be isomerized, it is deprotonated by a hydroxide ion from solution. From this we propose a new photocycle intermediate, pB(deprot), from which pG is recovered and which is in equilibrium with pB. This is supported in our data through the combination of the observed pOH and pH dependence, together with the kinetic deuterium isotope effect.

Dynamics of protein and chromophore structural changes in the photocycle of photoactive yellow protein monitored by time-resolved optical rotatory dispersion.

The dynamics of the PYP photocycle have been studied using time-resolved optical rotatory dispersion (TRORD) spectroscopy in the visible and far-UV spectral regions to probe the changes in the chromophore configuration and the protein secondary structure, respectively. The changes in the secondary structure in PYP upon photoisomerization of the chromophore can be described by two exponential lifetimes of 2 +/- 0.8 and 650 +/- 100 ms that correspond to unfolding and refolding processes, respectively. The TRORD experiments that follow the dynamics of the chromophore report three exponential components, with lifetimes of 10 +/- 3 micros, 1.5 +/- 0.5 ms, and 515 +/- 110 ms. A comparison of the kinetic behaviors of the chromophore and protein shows that during the decay of pR(465) an initial relaxation that is localized to the chromophore hydrophobic pocket precedes the formation of the chromophore and protein structures found in pB(355). In contrast, the protein and chromophore processes occur with similar time constants during inactivation of the signaling state.