Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells

Abstract

In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

“…Another perspective is the development of OCP-based biosensors, since the obtained fluorescent OCP-TMR complexes could be used as novel molecular sensors of light intensity, temperature, and viscosity, which would obviate the need for complex instrumentation, such as fluorescence correlation spectroscopy, to measure these parameters. The first intracellular temperature mapping methods based on fluorescent thermometers and fluorescence-lifetime imaging microscopy were developed recently (43)(44)(45). The observed dependence of the OCP conversion rate on temperature and viscosity is consistent with the general behavior expected for protein folding: whereas temperature dependence followed the Arrhenius law, the dependence on viscosity was in line with the Kramers theory.…”

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

“…For example, mechanical pressures from flanking cells accelerate the cell-cycle progression in epithelial cells (58), and mechanotransduction derived from substrate rigidity can affect stem cell differentiation (59). Another report has shown that local metabolic heat production affects the electrochemical gradient in mitochondria (60). And although it is quite challenging to visualize physical stresses in vivo, we believe that FRET biosensors hold great potential for this purpose.…”

Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

“…42,[50][51][52][53] The correlation between the signal intensity and temperature was exploited in several studies. It was demonstrated that°uorescence intensity of certain FPs exhibit thermal sensitivity, thus enabling intracellular temperature sensing.…”

“…Genetically encoded GFP-based thermosensor, named tsGFPs, was applied for monitoring the temperature of the plasma membrane, mitochondria and endoplasmic reticulum in living cells. 51 Temperature-sensitive conformational transformations in tsGFP in turn caused changes in the intensity ratio between two characteristics emission peaks of tsGFP at 400 nm and 480 nm. As a result, the thermal heterogeneity in mitochondria was detected.…”

“…Furthermore, the dependence between temperature and membrane potential was demonstrated. 51 Another group developed ratiometric thermometer (gTEMP) by using two FPs with di®erent temperature sensitivity. 52 gTEMP enabled fast tracking of the temperature changes in the range of 5-50 C with temporal resolution of 50 ms.…”