Dave Trinel

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

BackgroundHistone 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. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks.ResultsTo gain insights into the mechanisms of SUV420H2 recruitment at heterochromatin, we applied a tandem affinity purification approach coupled to mass spectrometry. We identified heterochromatin proteins HP1 as main interacting partners. The regions responsible for the binding were mapped to the heterochromatic targeting module of SUV420H2 and HP1 chromoshadow domain. We studied the dynamic properties of SUV420H2 and the HP1 in living cells using fluorescence recovery after photobleaching. Our results showed that HP1 proteins are highly mobile with different dynamics during the cell cycle, whereas SUV420H2 remains strongly bound to pericentric heterochromatin. An 88 amino-acids region of SUV420H2, the heterochromatic targeting module, recapitulates both, HP1 binding and strong association to heterochromatin.ConclusionFRAP experiments reveal that in contrast to HP1, SUV420H2 is strongly associated to pericentric heterochromatin. Then, the fraction of SUV420H2 captured and characterized by TAP/MS is a soluble fraction which may be in a stable association with HP1. Consequently, SUV420H2 may be recruited to heterochromatin in association with HP1, and stably maintained at its heterochromatin sites in an HP1-independent fashion.

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. For this purpose, we visualized a large number of spines on pyramidal neurons located in superficial and deep layers of the cortex within the hindpaw representation area, by means of confocal microscopy. Spines were classified according to their shape, as stubby, thin, mushroom, or filopodium. Spine density was increased (+26%) after HU. The increase concerned mainly filopodium spines (+82%) and mushrooms (+33%), whereas no change was noticed for stubby and thin spines. Spine length was decreased, whatever their shape. Head diameter evolved differently depending on the layer: it was increased in superficial layers and decreased in deeper ones. These results indicate that morphological changes accompany functional reorganization of motor cortex in response to a decrease in sensorimotor function during adulthood.

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 5 μm 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, 30 min exposure to DRAQ5 induces death of U2OS cells 24 h later. Incubation with Hoechst 33342 under similar conditions does not induce these effects. These results emphasize the importance of carefully examining the functional consequences of labeling DNA with intercalating fluorescent dyes before use.

Setup of a fluorescence lifetime and spectral correlated acquisition system for two-photon microscopy

In this article we present a complete laser scanning microscope designed for simultaneous spectral and lifetime measurements from every point of the specimen located within the field of view. The pulsed laser source used for two-photon excitation provides good spatial resolution with minimal invasivity. In addition, the detection module was optimized for minimal photon loss, allowing laser power minimization and further reduction of cells photodamage. Analysis of biological samples illustrates the performances of this configuration, particularly when applied to fluorescent resonance energy transfer (FRET) measurements. Indeed, multiparametric acquisition is particularly useful to discriminate between FRET and artifactual response due to acquisition invasivity or cell heterogeneity. Combined with adapted homemade driving software, this system is stable, portable, and optimized for living cell studies.

Shadow Technique Algorithm (STA) Sheds a New Light on DifferentialInterference Contrast (DIC) Microscopy

Diversity of biological samples is still partially considered by conventional Differential Interference Contrast (DIC) microscopy approaches. Here we propose a new algorithm (developed as an ImageJ macro), the STA (Shadow Technique Algorithm), whose originality relies in the 3D/4D visualization of a large range of biological objects, from bacteria, vegetal tissues to living cells in culture. This new approach does not need extensive calculations, systems modifications or in-depth knowledge of acquisition optics. STA, providing 3D DIC reconstruction every hundredth of ms, can be applied to dissect various cellular phenomena. In addition, we propose different methods of graphic representations, which unable to enlighten the specificities of each category of questioning. Specifically we here addressed: i) tissue imaging, ii) cell cycle and cell death imaging iii) vesicle tracking.