Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level.

Intracellular temperature is a fundamental parameter in biochemical reactions. Genetically encoded fluorescent temperature indicators (GETIs) have been developed to visualize intracellular thermogenesis; however, the temperature sensitivity or localization capability in specific organelles should have been further improved to clearly capture when and where intracellular temperature changes at the subcellular level occur. Here, we developed a new GETI, gMELT, composed of donor and acceptor subunits, in which cyan and yellow fluorescent proteins, respectively, as a Förster resonance energy transfer (FRET) pair were fused with temperature-sensitive domains. The donor and acceptor subunits associated and dissociated in response to temperature changes, altering the FRET efficiency. Consequently, gMELT functioned as a fluorescence ratiometric indicator. Untagged gMELT was expressed in the cytoplasm, whereas versions fused with specific localization signals were targeted to the endoplasmic reticulum (ER) or mitochondria. All gMELT variations enabled more sensitive temperature measurements in cellular compartments than those in previous GETIs. The gMELTs, tagged with ER or mitochondrial targeting sequences, were used to detect thermogenesis in organelles stimulated chemically, a method previously known to induce thermogenesis. The observed temperature changes were comparable to previous reports, assuming that the fluorescence readout changes were exclusively due to temperature variations. Furthermore, we demonstrated how macromolecular crowding influences gMELT fluorescence given that this factor can subtly affect the fluorescence readout. Investigating thermogenesis with gMELT, accounting for factors such as macromolecular crowding, will enhance our understanding of intracellular thermogenesis phenomena.

Motility Assays of Chloroflexus.

Chloroflexus is a thermophilic, filamentous, gliding bacterium. Its multicellular filaments of several hundred micrometer length move straightforward at a speed of approximately 1-3 µm/s and occasionally reverse the moving direction. In liquid media, filaments glide on each other to form cell aggregates without tight adhesion. The molecular machinery on the cell surface that forces the gliding movement has not yet been identified. Here, we describe the cultivation methods to characterize the gliding motility of Chlroflexus and the microscopic assays to determine its gliding speed, reversal frequency, and cell-surface movements.

A highly-sensitive genetically encoded temperature indicator exploiting a temperature-responsive elastin-like polypeptide.

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.

Tree of motility - A proposed history of motility systems in the tree of life.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

Emended description of the genus Tabrizicola and the species Tabrizicola aquatica as aerobic anoxygenic phototrophic bacteria.

The genus Tabrizicola with its type species and strain Tabrizicola aquatica RCRI19T was previously described as a purely chemotrophic genus of Gram-negative, aerobic, non-motile and rod-shaped bacteria. With the present study, we expand the description of the metabolic capabilities of this genus and the T. aquatica type strain to include chlorophyll-dependent phototrophy. Our results confirmed that T. aquatica, does not grow under anaerobic photoautotrophic or photoheterotrophic conditions. However, the presence of the photosynthesis-related genes pufL and pufM could be demonstrated in the genomes of several Tabrizicola strains. Additionally, photosynthetic pigments (bacteriochlorophyll a) were formed under aerobic, heterotrophic and low light conditions in T. aquatica strain RCRI19T. Furthermore, all the genes necessary for a fully operational photosynthetic apparatus and bacteriochlorophyll a are present in the T. aquatica type strain genome. Therefore, we suggest categorising T. aquatica RCRI19T, isolated from freshwater environment of Qurugöl Lake, as an aerobic anoxygenic phototrophic (AAP) bacterium.

The Ser/Thr Kinase PknH Is Essential for Maintaining Heterocyst Pattern in the Cyanobacterium Anabaena sp. Strain PCC 7120.

In the filamentous cyanobacterium Anabaena sp. strain, PCC 7120, heterocysts (which are nitrogen-fixing cells) are formed in the absence of combined nitrogen in the medium. Heterocysts are separated from one another by 10 to 15 vegetative cells along the filaments, which consist of a few hundred of cells. hetR is necessary for heterocyst differentiation; and patS and hetN, expressed in heterocysts, play important roles in heterocyst pattern formation by laterally inhibiting the expression of hetR in adjacent cells. The results of this study indicated that pknH, which encodes a Ser/Thr kinase, was also involved in heterocyst pattern formation. In the pknH mutant, the heterocyst pattern was normal within 24 h after nitrogen deprivation, but multiple contiguous heterocysts were formed from 24 to 48 h. A time-lapse analysis of reporter strains harboring a fusion between gfp and the hetR promoter indicated that pknH was required to suppress hetR expression in cells adjacent to the preexisting heterocysts. These results indicated that pknH was necessary for the lateral inhibition of heterocyst differentiation to maintain the heterocyst pattern.

Gliding motility driven by individual cell-surface movements in a multicellular filamentous bacterium Chloroflexus aggregans.

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.