A Guide to Fluorescent Protein FRET Pairs

Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies

Cell-type-Specific Patterned Stimulus-Independent Neuronal Activity in the Drosophila Visual System during Synapse Formation

Stereotyped synaptic connections define the neural circuits of the brain. In vertebrates, stimulus-independent activity contributes to neural circuit formation. It is unknown whether this type of activity is a general feature of nervous system development. Here, we report patterned, stimulus-independent neural activity in the Drosophila visual system during synaptogenesis. Using in vivo calcium, voltage, and glutamate imaging, we found that all neurons participate in this spontaneous activity, which is characterized by brain-wide periodic active and silent phases. Glia are active in a complementary pattern. Each of the 15 of over 100 specific neuron types in the fly visual system examined exhibited a unique activity signature. The activity of neurons that are synaptic partners in the adult was highly correlated during development. We propose that this cell-type-specific activity coordinates the development of the functional circuitry of the adult brain

Blocking paracrine signaling by TNF across all concentrations and preparations.

<p>The average time course for N number of active cells is plotted for cells stimulated in the absence (blue trace, top value of n) and presence (orange trace, bottom value) of sTNFRII, which competes to bind TNF. Concentrations are indicated at left, and the preparation at top.</p

Comparison of single-cell NF-κB activation dynamics for three different LPS preparations.

<p>Intensity represents the relative nuclear localization of p65-dsRed fusion protein, calculated as mean nuclear intensity divided by initial mean cytoplasmic intensity. The concentration for all three preparations was 0.5 µg/mL. The black line shows the average time course for all cells; the light blue traces are ten randomly selected individual cells. The number of active cells (N), as well as the maximum peak amplitude (Peak Amp), and time elapsed until the maximum amplitude is reached (Time to Peak) are also shown. The maximum intensity is indicated by a dot and the two dashed lines indicate how Peak Amp and Time to Peak are determined. The duration of the first peak (Peak Width) is also shown. This value is determined by drawing a horizontal line at the intensity that is halfway between the minimum and maximum peak value. The region above the line and shaded in green denotes the time during which the p65-dsRed nuclear intensity is more than half of the maximum p65-dsRed nuclear intensity. Below each plot, corresponding representative microscope images are shown for the first 200 minutes after stimulation, as labeled.</p

The potency window for each of the LPS preparations.

<p>The fraction of active cells is plotted as a function of concentration for values spanning nine orders of magnitude, as shown.</p

The effect of blocking TNF on the NF-κB activation time courses as a function of concentration and preparation.

<p>The cosine vector distances between the average time courses in the presence and absence of sTNFRII are shown. Almost no cells were activated by UP LPS at low concentrations; hence the dashed light blue line is included for completeness but is not statistically defensible.</p

Activation dynamics for each of the LPS preparations and several concentrations and summary statistical comparisons.

<p>(A) As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g001" target="_blank">Figure 1</a>, the average and ten randomly selected traces of active cells are shown, as well as Time to Peak, Peak Amp and N. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g001" target="_blank">Figure 1</a> legend for more details. Concentrations are indicated at left, and the preparation at top. The very lowest and highest concentrations are not shown here (but appear in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g004" target="_blank">Figure 4</a>), because virtually no cells were found to be active in the first case, and the traces are essentially identical to their nearest neighbor in the second. (B) Time-to-peak values for each LPS preparation are shown with standard deviations, across each concentration. LPS preparations are indicated with different colors as labeled in D. (C) Peak amplitude values for each LPS preparation are shown with standard deviations across each concentration. (D) The correspondence between time-to-peak and peak amplitude is shown for each LPS preparation, including all concentrations.</p

CLSTN3β enforces adipocyte multilocularity to facilitate lipid utilization.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3β) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3β is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3β have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3β is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3β associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3β-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes