NegFluo, a Fast and Efficient Method to Determine Starch Granule Size and Morphology in Plant Chloroplasts.

Starch granules that accumulate in the plastids of plants vary in size, shape, phosphate, or protein content according to their botanical origin. Depending on their size, the applications in food and nonfood industries differ. Being able to master starch granule size for a specific plant, without alteration of other characteristics (phosphate content, protein content, etc.

The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation.

The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis.

Optimization of ERK activity biosensors for both ratiometric and lifetime FRET measurements.

Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes.

Dendritic spine remodeling induced by hindlimb unloading in adult rat sensorimotor cortex.

A sensorimotor restriction, for instance in patients confined to bed, induces an impairment in motor function, which could be due to structural and functional reorganization of the sensorimotor cortex. Hindlimb unloading (HU) is a rodent model used to reproduce the chronic weightless bearing and reduction in hindlimb movement. In this study, we determined whether a 14-day period of HU in adult rats leads to dendritic spine plasticity.

From FRET imaging to practical methodology for kinase activity sensing in living cells.

Biological processes are intrinsically dynamic. Although traditional methods provide valuable insights for the understanding of many biological phenomena, the possibility of measuring, quantifying, and localizing proteins within a cell, a tissue, and even an embryo has revolutionized our train of thoughts and has encouraged scientists to develop molecular tools for the assessment of protein or protein complex dynamics within their physiological context. These ongoing efforts rest on the emergence of biophotonic techniques and the continuous improvement of fluorescent probes, allowing precise and reliable measurements of dynamic cellular functions.

Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm.

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate the local environment of fluorophores in living cells. To correctly estimate all lifetime parameters, time domain FLIM imaging requires a high number of photons and consequently long laser exposure times. This is an issue because long exposure times are incompatible with the observation of dynamic molecular events and induce cellular stress.

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles.

Short exposure to the DNA intercalator DRAQ5 dislocates the transcription machinery and induces cell death.

The fluorescent probe DRAQ5 which rapidly permeates cells and binds to DNA is potentially useful for functional studies of molecular dynamics and interactions in living nuclei. Within minutes after the incubation of human osteosarcoma U2OS cells with 5μm DRAQ5, the distributions of RNA polymerase II and some of its associated regulatory proteins HEXIM and cyclin T1 in the nucleus are severely impaired, and transcription is inhibited. Furthermore, 30min exposure to DRAQ5 induces death of U2OS cells 24h later.

The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours.

Background: Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability.

Galectin-4-regulated delivery of glycoproteins to the brush border membrane of enterocyte-like cells.

We have previously reported that silencing of galectin-4 expression in polarized HT-29 cells perturbed apical biosynthetic trafficking and resulted in a phenotype similar to the inhibitor of glycosylation, 1-benzyl-2-acetamido-2-deoxy-beta-d-galactopyranoside (GalNAcalpha-O-bn). We now present evidence of a lipid raft-based galectin-4-dependent mechanism of apical delivery of glycoproteins in these cells. First, galectin-4 recruits the apical glycoproteins in detergent-resistant membranes (DRMs) because these glycoproteins were depleted in DRMs isolated from galectin-4-knockdown (KD) HT-29 5M12 cells.

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements.

Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition.

Enhanced FRET contrast in lifetime imaging.

In combination with two photon excitation, FLIM is currently one of the best techniques to quantitatively study the subcellular localization of protein-protein interactions in living cells. An appropriate analysis procedure is crucial to obtain reliable results. TCSPC is an accurate method to measure FLIM.

Correlated fluorescence lifetime and spectral measurements in living cells.

Studies of proteins' interaction in cells by FRET can take benefit from two important photo-physical properties describing fluorescent proteins: fluorescence emission spectrum and fluorescence lifetime. These properties provide specific and complementary information about the tagged proteins and their environment. However, none of them taken individually can completely quantify the involved fluorophore characteristics due to their multiparametric dependency with molecular environment, experimental conditions, and interpretation complexity.