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Fereidouni F.,University Utrecht | Esposito A.,The Medical Research Council Cancer Cell Unit | Blab G.A.,University Utrecht | Gerritsen H.C.,University Utrecht
Journal of Microscopy | Year: 2011

Fluorescence lifetime imaging is a versatile tool that permits mapping the biochemical environment in the cell. Among various fluorescence lifetime imaging techniques, time-correlated single photon counting and time-gating methods have been demonstrated to be very efficient and robust for the imaging of biological specimens. Recently, the phasor representation of lifetime images became popular because it provides an intuitive graphical view of the fluorescence lifetime content of the images and, when used for global analysis, significantly improves the overall S/N of lifetime analysis. Compared to time-correlated single photon counting, time gating methods can provide higher count rates (∼10 MHz) but at the cost of truncating and under sampling the decay curve due to the limited number of gates commonly used. These limitations also complicate the implementation of the phasor analysis for time-gated data. In this work, we propose and validate a theoretical framework that overcomes these problems. This modified approach is tested on both simulated lifetime images and on cells. We demonstrate that this method is able to retrieve two lifetimes from time gating data that cannot be resolved using standard (non-global) fitting techniques. The new approach increases the information that can be obtained from typical measurements and simplifies the analysis of fluorescence lifetime imaging data. © 2011 Utrecht University Journal of Microscopy © 2011 Royal Microscopical Society.

Esposito A.,The Medical Research Council Cancer Cell Unit
Remote Sensing | Year: 2012

Time-of-Flight (ToF) technologies are developed mainly for range estimations in industrial applications or consumer products. Recently, it was realized that ToF sensors could also be used for the detection of fluorescence and of the minute changes in the nanosecond-lived electronic states of fluorescent molecules. This capability can be exploited to report on the biochemical processes occurring within living organisms. ToF technologies, therefore, provide new opportunities in molecular and cell biology, diagnostics, and drug discovery. In this short communication, the convergence of the engineering and biomedical communities onto ToF technologies and its potential impact on basic, applied and translational sciences are discussed. © 2012 by the author.

Mahen R.,The Medical Research Council Cancer Cell Unit | Venkitaraman A.R.,The Medical Research Council Cancer Cell Unit
Current Opinion in Cell Biology | Year: 2012

A striking but poorly explained feature of cell division is the ability to assemble and maintain organelles not bounded by membranes, from freely diffusing components in the cytosol. This process is driven by information transfer across biological scales such that interactions at the molecular scale allow pattern formation at the scale of the organelle. One important example of such an organelle is the centrosome, which is the main microtubule organising centre in the cell. Centrosomes consist of two centrioles surrounded by a cloud of proteins termed the pericentriolar material (PCM). Profound structural and proteomic transitions occur in the centrosome during specific cell cycle stages, underlying events such as centrosome maturation during mitosis, in which the PCM increases in size and microtubule nucleating capacity. Here we use recent insights into the spatio-temporal behaviour of key regulators of centrosomal maturation, including Polo-like kinase 1, CDK5RAP2 and Aurora-A, to propose a model for the assembly and maintenance of the PCM through the mobility and local interactions of its constituent proteins. We argue that PCM structure emerges as a pattern from decentralised self-organisation through a reaction-diffusion mechanism, with or without an underlying template, rather than being assembled from a central structural template alone. Self-organisation of this kind may have broad implications for the maintenance of mitotic structures, which, like the centrosome, exist stably as supramolecular assemblies on the micron scale, based on molecular interactions at the nanometer scale. © 2011 Elsevier Ltd.

Mahen R.,The Medical Research Council Cancer Cell Unit | Hattori H.,The Medical Research Council Cancer Cell Unit | Lee M.,The Medical Research Council Cancer Cell Unit | Sharma P.,The Medical Research Council Cancer Cell Unit | And 2 more authors.
PLoS ONE | Year: 2013

A-type lamins encoded by LMNA form a structural fibrillar meshwork within the mammalian nucleus. How this nuclear organization may influence the execution of biological processes involving DNA transactions remains unclear. Here, we characterize changes in the dynamics and biochemical interactions of lamin A/C after DNA damage. We find that DNA breakage reduces the mobility of nucleoplasmic GFP-lamin A throughout the nucleus as measured by dynamic fluorescence imaging and spectroscopy in living cells, suggestive of incorporation into stable macromolecular complexes, but does not induce the focal accumulation of GFP-lamin A at damage sites. Using a proximity ligation assay and biochemical analyses, we show that lamin A engages chromatin via histone H2AX and its phosphorylated form (γH2AX) induced by DNA damage, and that these interactions are enhanced after DNA damage. Finally, we use three-dimensional time-lapse imaging to show that LMNA inactivation significantly reduces the positional stability of DNA repair foci in living cells. This defect is partially rescued by the stable expression of GFP-lamin A. Thus collectively, our findings suggest that the dynamic structural meshwork formed by A-type lamins anchors sites of DNA repair in mammalian nuclei, providing fresh insight into the control of DNA transactions by nuclear structural organization. © 2013 Mahen et al.

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