Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes

<p>Abstract</p> <p>Background</p> <p>Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered.</p> <p>Results</p> <p>Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching.</p> <p>Conclusions</p> <p>We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (α-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this technique promises to further increase the general usability of photoconvertible PAFPs to track the dynamic movement of cells and proteins over time.</p

Bridging fluorescence microscopy and electron microscopy

Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakthrough in fluorescence microscopy in biology is the ability to follow specific targets on or in living cells, revealing dynamic localization and/or function of target molecules. One of the inherent limitations of fluorescence microscopy is the resolution. Several efforts are undertaken to overcome this limit. The traditional and most well-known way to achieve higher resolution imaging is by electron microscopy. Moreover, electron microscopy reveals organelles, membranes, macromolecules, and thus aids in the understanding of cellular complexity and localization of molecules of interest in relation to other structures. With the new probe development, a solid bridge between fluorescence microscopy and electron microscopy is being built, even leading to correlative imaging. This connection provides several benefits, both scientifically as well as practically. Here, I summarize recent developments in bridging microscopy

The dynamics of university units as a multi-level process. Credibility cycles and resource dependencies

This paper presents an analysis of resource acquisition and profile development of institutional units within universities. We conceptualize resource acquisition as a two level nested process, where units compete for external resources based on their credibility, but at the same time are granted faculty positions from the larger units (department) to which they belong. Our model implies that the growth of university units is constrained by the decisions of their parent department on the allocation of professorial positions, which represent the critical resource for most units’ activities. In our field of study this allocation is largely based on educational activities, and therefore, units with high scientific credibility are not necessarily able to grow, despite an increasing reliance on external funds. Our paper therefore sheds light on the implications that the dual funding system of European universities has for the development of units, while taking into account the interaction between institutional funding and third-party funding

Connexin43 Modulates Cell Polarity and Directional Cell Migration by Regulating Microtubule Dynamics

Knockout mice deficient in the gap junction gene connexin43 exhibit developmental anomalies associated with abnormal neural crest, primordial germ cell, and proepicardial cell migration. These migration defects are due to a loss of directional cell movement, and are associated with abnormal actin stress fiber organization and a loss of polarized cell morphology. To elucidate the mechanism by which Cx43 regulates cell polarity, we used a wound closure assays with mouse embryonic fibroblasts (MEFs) to examine polarized cell morphology and directional cell movement. Studies using embryonic fibroblasts from Cx43 knockout (Cx43KO) mice showed Cx43 deficiency caused cell polarity defects as characterized by a failure of the Golgi apparatus and the microtubule organizing center to reorient with the direction of wound closure. Actin stress fibers at the wound edge also failed to appropriately align, and stabilized microtubule (Glu-tubulin) levels were markedly reduced. Forced expression of Cx43 with deletion of its tubulin-binding domain (Cx43dT) in both wildtype MEFs and neural crest cell explants recapitulated the cell migration defects seen in Cx43KO cells. However, forced expression of Cx43 with point mutation causing gap junction channel closure had no effect on cell motility. TIRF imaging revealed increased microtubule instability in Cx43KO cells, and microtubule targeting of membrane localized Cx43 was reduced with expression of Cx43dT construct in wildtype cells. Together, these findings suggest the essential role of Cx43 gap junctions in development is mediated by regulation of the tubulin cytoskeleton and cell polarity by Cx43 via a nonchannel function

Astrocytic Mechanisms Explaining Neural-Activity-Induced Shrinkage of Extraneuronal Space

Neuronal stimulation causes ∼30% shrinkage of the extracellular space (ECS) between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance and astrocyte function, the phenomenon remains unexplained. Here we present a dynamic model that accounts for current experimental data related to the shrinkage phenomenon in wild-type as well as in gene knockout individuals. We find that neuronal release of potassium and uptake of sodium during stimulation, astrocyte uptake of potassium, sodium, and chloride in passive channels, action of the Na/K/ATPase pump, and osmotically driven transport of water through the astrocyte membrane together seem sufficient for generating ECS shrinkage as such. However, when taking into account ECS and astrocyte ion concentrations observed in connection with neuronal stimulation, the actions of the Na+/K+/Cl− (NKCC1) and the Na+/HCO3− (NBC) cotransporters appear to be critical determinants for achieving observed quantitative levels of ECS shrinkage. Considering the current state of knowledge, the model framework appears sufficiently detailed and constrained to guide future key experiments and pave the way for more comprehensive astroglia–neuron interaction models for normal as well as pathophysiological situations