No abstract

Genetically encoded temperature indicators (GETIs) allow for real-time measurement of subcellular temperature dynamics in live cells. However, GETIs have suffered from poor temperature sensitivity, which may not be sufficient to resolve small heat production from a biological process. Here, we develop a highly-sensitive GETI, denoted as ELP-TEMP, comprised of a temperature-responsive elastin-like polypeptide (ELP) fused with a cyan fluorescent protein (FP), mTurquoise2 (mT), and a yellow FP, mVenus (mV), as the donor and acceptor, respectively, of Förster resonance energy transfer (FRET). At elevated temperatures, the ELP moiety in ELP-TEMP undergoes a phase transition leading to an increase in the FRET efficiency. In HeLa cells, ELP-TEMP responded to the temperature from 33 to 40 °C with a maximum temperature sensitivity of 45.1 ± 8.1%/°C, which was the highest ever temperature sensitivity among hitherto-developed fluorescent nanothermometers. Although ELP-TEMP showed sensitivity not only to temperature but also to macromolecular crowding and self-concentration, we were able to correct the output of ELP-TEMP to achieve accurate temperature measurements at a subcellular resolution. We successfully applied ELP-TEMP to accurately measure temperature changes in cells induced by a local heat spot, even if the temperature difference was as small as < 1 °C, and to visualize heat production from stimulated Ca2+ influx in live HeLa cells induced by a chemical stimulation. Furthermore, we investigated temperatures in the nucleus and cytoplasm of live HeLa cells and found that their temperatures were almost the same within the temperature resolution of our measurement. Our study would contribute to better understanding of cellular temperature dynamics, and ELP-TEMP would be a useful GETI for the investigation of cell thermobiology.

No abstract

Chloroflexus aggregans is an unbranched multicellular filamentous bacterium having the ability of gliding motility. The filament moves straightforward at a constant rate, ∼3 μm sec(-1) on solid surface and occasionally reverses the moving direction. In this study, we successfully detected movements of glass beads on the cell-surface along long axis of the filament indicating that the cell-surface movement was the direct force for gliding. Microscopic analyses found that the cell-surface movements were confined to a cell of the filament, and each cell independently moved and reversed the direction. To understand how the cellular movements determine the moving direction of the filament, we proposed a discrete-time stochastic model; sum of the directions of the cellular movements determines the moving direction of the filament only when the filament pauses, and after moving, the filament keeps the same directional movement until all the cells pause and/or move in the opposite direction. Monte Carlo simulation of this model showed that reversal frequency of longer filaments was relatively fixed to be low, but the frequency of shorter filaments varied widely. This simulation result appropriately explained the experimental observations. This study proposed the relevant mechanism adequately describing the motility of the multicellular filament in C. aggregans.