Yeast AMP-activated protein kinase monitors glucose concentration changes and absolute glucose levels.

Analysis of the time-dependent behavior of a signaling system can provide insight into its dynamic properties. We employed the nucleocytoplasmic shuttling of the transcriptional repressor Mig1 as readout to characterize Snf1-Mig1 dynamics in single yeast cells. Mig1 binds to promoters of target genes and mediates glucose repression.

Osmostress-induced cell volume loss delays yeast Hog1 signaling by limiting diffusion processes and by Hog1-specific effects.

Signal transmission progresses via a series of transient protein-protein interactions and protein movements, which require diffusion within a cell packed with different molecules. Yeast Hog1, the effector protein kinase of the High Osmolarity Glycerol pathway, translocates transiently from the cytosol to the nucleus during adaptation to high external osmolarity. We followed the dynamics of osmostress-induced cell volume loss and Hog1 nuclear accumulation upon exposure of cells to different NaCl concentrations.

Initiation of the transcriptional response to hyperosmotic shock correlates with the potential for volume recovery.

The control of activity and localization of transcription factors is critical for appropriate transcriptional responses. In eukaryotes, signal transduction components such as mitogen-activated protein kinase (MAPK) shuttle into the nucleus to activate transcription. It is not known in detail how different amounts of nuclear MAPK over time affect the transcriptional response.

Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular diffusion within human skin.

There is a need for tools enabling quantitative imaging of biological tissue for pharmaceutical applications. In this study, two-photon fluorescence microscopy (TPM) has been combined with fluorescence correlation spectroscopy (FCS), demonstrating proof-of-principle providing quantitative data of fluorophore concentration and diffusion in human skin. Measurements were performed on excised skin exposed to either rhodamine B (RB) or rhodamine B isothiocyanate (RBITC), chosen based on their similarity in fluorescence yield and molecular weight, but difference in chemical reactivity.

Anti-Stokes fluorescence from endogenously formed protoporphyrin IX--implications for clinical multiphoton diagnostics.

Multiphoton imaging based on two-photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce.

Quantification of cell volume changes upon hyperosmotic stress in Saccharomyces cerevisiae.

Cell volume is a biophysical property, which is of great importance for quantitative characterisations of biological processes, such as osmotic adaptation. It also is a crucial parameter in the most common type of mathematical description of cellular behaviour-ordinary differential equation (ODE) models, e.g.

A mathematical analysis of nuclear intensity dynamics for Mig1-GFP under consideration of bleaching effects and background noise in Saccharomyces cerevisiae.

Fluorescence microscopy is an imaging technique that provides insights into signal transduction pathways through the generation of quantitative data, such as the spatiotemporal distribution of GFP-tagged proteins in signaling pathways. The data acquired are, however, usually a composition of both the GFP-tagged proteins of interest and of an autofluorescent background, which both undergo photobleaching during imaging. We here present a mathematical model based on ordinary differential equations that successfully describes the shuttling of intracellular Mig1-GFP under changing environmental conditions regarding glucose concentration.

ARFGAP2 and ARFGAP3 are essential for COPI coat assembly on the Golgi membrane of living cells.

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle.

Two-photon fluorescence correlation microscopy combined with measurements of point spread function; investigations made in human skin.

Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been applied in connection to measurements of the point spread function (PSF) for quantitative analysis of sulphorhodamine B (SRB) in excised human skin. The PSF was measured using subresolution fluorescent beads embedded in the skin specimen. The PSF, measured as full width at half maximum (FWHM) was found to be 0.

Multiphoton laser scanning microscopy--a novel diagnostic method for superficial skin cancers.

The increasing incidence of skin cancer and the importance of early diagnosis is a challenge, which requires the development of reliable, cost-effective, noninvasive, diagnostic techniques. Several such methods based on optical imaging techniques are available and currently being investigated. A novel method in this field is multiphoton laser scanning microscopy (MPLSM).

2,6,8-Trisubstituted 3-hydroxychromone derivatives as fluorophores for live-cell imaging.

We present the synthesis and photophysical characterisation of a series of structurally diverse, fluorescent 2,6,8-trisubstituted 3-hydroxychromone derivatives with high fluorescence quantum yields and molar extinction coefficients. Two of these derivatives (9 and 10 a) have been studied as fluorophores for cellular imaging in HeLa cells and show excellent permeability and promising fluorescence properties in a cellular environment. In addition, we have demonstrated by photophysical characterisation of 3-isobutyroxychromone derivatives that esterification of the 3-hydroxyl group results in acceptable and useful fluorescence properties.

Early stages of Golgi vesicle and tubule formation require diacylglycerol.

We have investigated the role for diacylglycerol (DAG) in membrane bud formation in the Golgi apparatus. Addition of propranolol to specifically inhibit phosphatidate phosphohydrolase (PAP), an enzyme responsible for converting phosphatidic acid into DAG, effectively prevents formation of membrane buds. The effect of PAP inhibition on Golgi membranes is rapid and occurs within 3 min.

Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy.

The distribution of sulphorhodamine B (SRB), a fluorescent hydrophilic model drug, was investigated in human skin after passive diffusion using four different topical delivery systems. The delivery vehicles applied were two bicontinuous lipid cubic systems, a commercial ointment and water. The lipid cubic systems consisted of either monoolein (MO) or phytantriol (PT) and water.

Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics.

This study describes the morphologic features of human non-melanoma skin cancer obtained using multiphoton laser scanning microscopy (MPLSM) on freshly excised specimens from 14 patients. Optical sectioning parallel to the tissue surface was performed, resulting in en face autofluorescence images of the epidermis and upper dermis, reaching tissue depths of 135 microm. The microscopy was carried out ex vivo using a femtosecond pulsed laser at 780 nm and a x 40/0.