Mitochondria-targeted ratiometric fluorescent probes for micropolarity and microviscosity and their applications

Two fluorescent“off-on”probes YYH1 and YYH2 were used for the detection of mitochondrial polarity and viscosity.Both probes have two emission bands:The peak of shorter wavelength was sensitive to the polarity,while that in longer wavelength region was sensitive to the viscosity,which enables us to detect medium polarity and viscosity simultaneously.The lg(Ishort/Ilong)of YYH1 exhibited linear relationship to the solvent polarity,while that of YYH2 was closely related to the solvent viscosity.Furthermore,both probes showed potential in bioimaging mitochondrial polarity or viscosity in living cells.

Fluorescence lifetime imaging of molecular rotors to map microviscosity in cells

Fluorescence liftime imaging （FLIM） of modified hydrophobic bodipy dyes that act as fluorescent molecular rotors shows that the fluorescence lifetime of these probes is a function of the microviscosity of their environment. Incubating cells with these dyes, we find a punctate and continuous distribution of the dye in cells. The viscosity value obtained in what appears to be endocytotic vesicles in living cells is around 100 times higher than that of water and of cellular cytoplasm.Time-resolved fluorescence anisotropy measurements also yield rotational correlation times consistent with large microviscosity values. In this way, we successfully develop a practical and versatile approach to map the microviscosity in cells based on imaging fluorescent molecular rotors.

Microviscosity in modified Nafion membranes measured by fluorescence probes

Sodium or quaternary ammonium modified Nafion membranes in forms of Nafion-Na+, Nafion-NMe+4:, Nafion-NEt+4 and Nafion-NBu+4: have bean prepared by neutralizing Nafion-H+ membrane with NaOH or tetraalkylammonium hydroxide aqueous solution. Intramolecular excimer formation and fluorescence polarisation methods using 1,3-di(1-pyrenyl)propane and acridine orange-as probes respectively have been employed for measurements of the microviscosity within those modified Nafion membranes. Results show that the probes are located in the fluorocarbon/water interface in the cluster of the membranes and the microviscosities around the probe molecules an in the range of ca. 120–1200 cp and increase in the order of Nafion-Na+ <Nafion-NMe+4<Nafion-NEt+4<Nafion-NBu+4. Furthermore, the microviscosity values of these membranes in dry form an higher than those in wet form.

An Investigation on the Polymerization of Styrene by Fluorescence Probe Technique

The polymerization of styrene is monitored by pyrene excimer formation. The ratio of monomer to excimer intensities ( I m/ I e) of pyrene increases as polymerization proceeds. The increase of I m/ I e is ascribed to the increase of microviscosity surrounding the probes forming excimer during polymerization. The linear relationship between the changing rate of I m/ I e and the polymerization rate of styrene is obtained. Therefore, I m/ I e may be used to monitor the progress of the polymerization of styrene.

A likely role for a novel PH-domain containing protein,PEPP2,in connecting membrane and cytoskeleton

PH domains(pleckstrin homology)are well known to bind membrane phosphoinositides with different specificities and direct PH domain-containing proteins to discrete subcellular apartments with assistances of alternative binding partners.PH domain-containing proteins are found to be involved in a wide range of cellular events,including signalling,cytoskeleton rearrangement and vesicular trafficking.Here we showed that a novel PH domain-containing protein,PEPP2,displayed moderate phosphoinositide binding specificity.Full length PEPP2 associated with both plasma membrane and microtubules.The membrane-associated PEPP2 nucleated at cell-cell contacts and the leading edge of migrating cells.Overexpression of PEPP2 increased membrane microviscosity,indicating a potential role of PEPP2 in regulating function of membrane and microtubules.

A likely role for the PH-domain containing protein, PEPP2/ PLEKHA5, at the membrane-microtubule cytoskeleton interface

PH(pleckstrin homology)domains are well known to bind membrane phosphoinositides with different specificities and direct PH domain-containing proteins to discrete subcellular compartments with assistances of alternative binding partners.PH domain-containing proteins have been found to be involved in a wide range of cellular events,including signalling,cytoskeleton rearrangement and vesicular trafficking.Here we showed that a novel PH domain-containing protein,PEPP2(also known as PLEKHA5),displays moderate phosphoinositide binding specificity.Full length PEPP2 was observed to variably associate with both the plasma membrane and microtubules.The membrane-associated PEPP2 nucleated at cell-cell contacts and the leading edge of migrating cells.Overexpression of PEPP2 increased membrane microviscosity,indicating a potential role for PEPP2 in regulating function of microtubule-dependent membrane functions.

Lipid-Protein Microinclusions in the Morphological Structures of Organelle Membranes Studied by Fluorescent Confocal Microscopy

Peculiar properties of morphological structures of organelle membranes were studied by fluorescent confocal microscopy. The list of objects in our experiments was represented by mitochondria, chloroplasts and vacuoles. During this study, identification of lipid microinclusions having the form of such lipid-protein structural microformations as lipid-protein microdomains, vesicles and membrane tubular structures (cytoplasmic transvacuolar strands and nanotubes) located in organelle membranes or bound up with them was conducted. Such membrane probes as laurdan, DPH, ANS and bis-ANS were used. Comparison of fluorescence intensity of these membrane probes was conducted. This investigation of the morphological properties of lipid-protein structural microformations was accompanied with analysis of 1) the phase state and 2) dynamics of microviscosity variations in the membrane elements of isolated plant cell organelles. Distributions of laurdan fluorescence generalized polarization (GP) values for the membrane on the whole and for the intensively fluorescing membrane segments were obtained. It was discovered that the microviscosity of intensively fluorescing membrane segments essentially differed from the microviscosity of the rest part of the membrane. In conclusion, some results of the study of peculiar properties of lipid-protein structural microformations related to the structure of organelle membranes and the discoveries made in this investigation are discussed.

Features of Cellular,Humoral and Activation Markers of Immune System and Biophysical Parameters of Superficial Membranes of Lymphocytes of Periphery Blood at Patients with a Breast Cancer

Studied features of cellular and humoral factors of immune system and activation markers of lymphocytes at breast cancer patients.Analyzed biophysical parameters of lymphocyte superficial membranes of periphery blood of breast cancer patients.Features of immune system reaction were:expressed Т - cellular immunodeficiency,deficiency of CD4+Т-helpers/inducers against raised expression of СD8+Т-lymphocyte,suppression of immunoregulatory index,imbalance of immunity humoral link,activity of expression CD16 + and CD56 + on natural killers and rising of expression CD38 + and CD95 + on lymphocytes.Studying of biophysical parameters of lymphocytes superficial membranes has allowed to reveal statistically significant changes characterized by intensifying of electric field and rising ofmicroviscosity,obviously leading to disappearance or weakening of intercellular interactions.