Rac1 activation can generate untemplated, lamellar membrane ruffles.

BACKGROUND: Membrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 controls the membrane protrusions that form and encapsulate extracellular volumes to perform pinocytic or phagocytic functions. RESULTS: Here, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold local concentration for sustained actin polymerization leading to ruffling. We show that Rac1 activation leads to actin assembly at the dorsal surface of the cell membrane that result in sheet-like protrusion formation without any requirement of a template. Further, this approach can be used to study the complex morpho-dynamics of the protrusions or to investigate specific proteins that may be enriched in the ruffles. Deactivating PA-Rac1 leads to complex contractile processes resulting in formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically is enriched in the ruffles. CONCLUSIONS: Combining LLSM and optogenetics enables superior spatial and temporal control for studying such dynamic mechanisms. Demonstrated here, the techniques implemented provide insight into the complex nature of the molecular interplay involved in dynamic actin machinery, revealing that Rac1 activation can generate untemplated, lamellar protrusions.

DNA condensation at freestanding cationic lipid bilayers.

We describe a previously unreported coil-globule transition of DNA electrostatically bound to a freestanding fluid cationic lipid membrane. The collapse of a DNA coil into a compact globule takes place after the DNA molecule attaches in an extended conformation to the membrane. DNA condensation is favored at a higher cationic lipid content, while at lower membrane charge densities coexistence of DNA random coils, partially collapsed conformations, and globules is observed.

Toward understanding the high PDT efficacy of chlorin e6-polyvinylpyrrolidone formulations: photophysical and molecular aspects of photosensitizer-polymer interaction in vitro.

It is recognized that chlorin e6-polyvinylpyrrolidone (Ce6-PVP) formulations are characterized by a high efficacy in photodynamic therapy of malignant tumors. Currently, a commercially available formulation of this type is Photolon (Fotolon) with Ce6:PVP=1:1 (w/w) and the weight-average molecular weight of PVP is 1.2x10(4). To gain a better understanding of the role played by PVP in Ce6-PVP formulations, we carry out experiments on IR and UV-VIS absorption, steady-state and time-resolved fluorescence, time-resolved triplet-triplet absorption, octanol-water partitioning, and solubility of chlorin e6 in buffer solutions at pH 6.3, 7.4, and 8.5 in presence of PVP with Ce6:PVP ratios ranging from 1:0 to 1:1000 (w/w) for PVP samples with weight-average molecular weights of 8x10(3), 1.2x10(4), and 4.2x10(4). We show that Ce6 interacts with PVP by forming molecular complexes via hydrophobic interactions and determine the Ce6-PVP binding constant, as well as the mean number of PVP monomers per binding site. We find that complexation of Ce6 with PVP prevents Ce6 aggregation in aqueous media and leads to an enhancement of Ce6 fluorescence quantum yield, while keeping the quantum yield of the intersystem crossing essentially unchanged. Possible scenarios of how the presence of PVP can favorably affect the PDT efficacy of chlorin e6 in Ce6-PVP formulations are discussed.

Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede.

Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins.

Diffusion and segmental dynamics of double-stranded DNA.

Diffusion and segmental dynamics of the double-stranded lambda-phage DNA polymer are quantitatively studied over the transition range from stiff to semiflexible chains. Spectroscopy of fluorescence fluctuations of single-end fluorescently labeled monodisperse DNA fragments unambiguously shows that double-stranded DNA in the length range of 10(2) - 2 x 10(4) base pairs behaves as a semiflexible polymer with segmental dynamics controlled by hydrodynamic interactions.

Investigation of tetherable distilbazolium compounds as fluorescent probes in nanostructured silica sol-gel materials.

Distibazolium dyes are investigated by steady-state and time-resolved fluorescence techniques in a series of low- and high-viscosity polar solvents and in a silica sol-gel matrix. In all solvents and the sol-gel matrix, an interplay of photoinduced switching between different cis-trans isomers and solvation dynamics is observed. Even in a viscous solution (glycerol) and in silica gel, cis-trans isomerization is solvent-controlled. Whereas in glycerol the solvation results in a time-dependent fluorescence Stokes shift, the solvation-induced spectral heterogeneity in silica gel is mostly static, possibly due to a close proximity of dye molecules to the silica surfaces of the nanopores. Compared to low-viscosity solvents, where the cis-trans isomerization process takes place with a solvent-dependent rate on the timescale of about 120-150 ps, it slows down to about 1100-1400 ps in glycerol and about 1500 ps in a sol-gel matrix. Additionally, fluorescence kinetics of the dyes in the sol-gel reveals the presence of a range of different "frozen-in" conformers exhibiting a broad spectrum of lifetimes from 20 to 300 ps.

Nanoassemblies designed from semiconductor quantum dots and molecular arrays.

The formation of nanoassemblies of CdSe/ZnS quantum dots (QD) and pyridyl-substituted free-base porphyrin (H(2)P) molecules has been spectroscopically identified by static and time-resolved techniques. The formation of nanoassemblies has been engineered by controlling the type and geometry of the H(2)P molecules. Pyridyl functionalization gives rise to a strong complex formation accompanied by QD photoluminescence (PL) quenching. For some of the systems, this quenching is partly related to fluorescence resonance energy transfer (FRET) from the QD to H(2)P and can be explained according to the Förster model. The quantitative interpretation of PL quenching due to complexation reveals that (i) on average only about (1)/(5) of the H(2)P molecules at a given H(2)P/QD molar ratio are assembled on the QD and (ii) only a limited number of "vacancies" accessible for H(2)P attachment exist on the QD surface.

Fluorescence decay time distribution analysis reveals two types of binding sites for 1,8-anilinonaphthalene sulfonate in native human oxyhemoglobin.

Binding of 1,8-anilinonaphthalene sulfonate (1,8-ANS) with native human oxyhemoglobin (Hb) in 50 mM potassium phosphate buffer (pH 7.4) was studied by steady-state fluorescence spectroscopy and by laser spectrofluorimetry with subnanosecond time resolution. The distribution of fluorescence decay times and parameters of two- and three-exponential deconvolution of the fluorescence kinetics of 1,8-ANS in Hb solution demonstrate that the emission at wavelengths lambdaem of 455-600 nm is not single-exponential and has components with mean decay times < 0.5, 3.1-5.5, and 12.4-15.1 nsec with the amplitudes depending on the emission wavelength. Analysis of time-resolved fluorescence spectra shows that the shortest-lived component should be assigned to 1,8-ANS molecules in the aqueous medium, whereas the two longer-lived components are assigned to two types of binding sites for 1,8-ANS in the Hb molecule characterized by different polarity and accessibility to water molecules.

Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin.

Time-resolved fluorescence of 1,8-anilinonaphthalene sulfonate (1,8-ANS) fluorescent probe bound to intact human oxyhemoglobin (HbO2) is investigated. Fluorescence emission spectra of 1,8-ANS in a potassium buffer solution (pH 7.4) of HbO2 undergo a substantial blue shift during first 6 ns after pulsed optical excitation at 337.1 nm. Nonexponential fluorescence kinetics of 1,8-ANS in the HbO2 solution are studied by the decay time distribution and conventional multiexponential analyses for a set of emission wavelength range of lambdaem = 455-600 nm. These fluorescence decays contain components with mean decay times of <0.5 ns, 3.1-5.5 ns, and 12.4-15.1 ns with spectrally-dependent relative contributions. The shortest decay component is assigned to free 1,8-ANS molecules in the bulk buffer environment, whereas the two longer decay components are assigned to two types of binding sites of 1,8-ANS in the HbO2 molecule presumably differing by polarity and accessibility to water molecules. The results represent the first experimental evidence of heterogeneous binding of 1,8-ANS to intact human oxyhemoglobin.