Calcium-induced upregulation of energy metabolism heats neurons during neural activity.

Cellular temperature affects every biochemical reaction, underscoring its critical role in cellular functions. In neurons, temperature not only modulates neurotransmission but is also a key determinant of neurodegenerative diseases. Considering that the brain consumes a disproportionately high amount of energy relative to its weight, neural circuits likely generate a lot of heat, which can increase cytosolic temperature. However, the changes in temperature within neurons and the mechanisms of heat generation during neural excitation remain unclear. In this study, we achieved simultaneous imaging of Ca2+ and temperature using the genetically encoded indicators, B-GECO and B-gTEMP. We then compared the spatiotemporal distributions of Ca2+ responses and temperature. Following neural excitation induced by veratridine, an activator of the voltage-gated Na+ channel, we observed an approximately 2 °C increase in cytosolic temperature occurring 30 s after the Ca2+ response. The temperature elevation was observed in the non-nuclear region, while Ca2+ increased throughout the cell body. Moreover, this temperature increase was suppressed under Ca2+-free conditions and by inhibitors of ATP synthesis. These results indicate that Ca2+-induced upregulation of energy metabolism serves as the heat source during neural excitation.

Intracellular Heat Transfer and Thermal Property Revealed by Kilohertz Temperature Imaging with a Genetically Encoded Nanothermometer.

Despite improved sensitivity of nanothermometers, direct observation of heat transport inside single cells has remained challenging for the lack of high-speed temperature imaging techniques. Here, we identified insufficient temperature resolution under short signal integration time and slow sensor kinetics as two major bottlenecks. To overcome the limitations, we developed B-gTEMP, a nanothermometer based on the tandem fusion of mNeonGreen and tdTomato fluorescent proteins. We visualized the propagation of heat inside intracellular space by tracking the temporal variation of local temperature at a time resolution of 155 µs and a temperature resolution 0.042 °C. By comparing the fast in situ temperature dynamics with computer-simulated heat diffusion, we estimated the thermal diffusivity of live HeLa cells. The present thermal diffusivity in cells was about 1/5.3 of that of water and much smaller than the values reported for bulk tissues, which may account for observations of heterogeneous intracellular temperature distributions.

Fluorescent Protein-Based Indicators for Functional Super-Resolution Imaging of Biomolecular Activities in Living Cells.

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM.

Quantitative Imaging of Genetically Encoded Fluorescence Lifetime Biosensors.

Genetically encoded fluorescence lifetime biosensors have emerged as powerful tools for quantitative imaging, enabling precise measurement of cellular metabolites, molecular interactions, and dynamic cellular processes. This review provides an overview of the principles, applications, and advancements in quantitative imaging with genetically encoded fluorescence lifetime biosensors using fluorescence lifetime imaging microscopy (go-FLIM). We highlighted the distinct advantages of fluorescence lifetime-based measurements, including independence from expression levels, excitation power, and focus drift, resulting in robust and reliable measurements compared to intensity-based approaches. Specifically, we focus on two types of go-FLIM, namely Förster resonance energy transfer (FRET)-FLIM and single-fluorescent protein (FP)-based FLIM biosensors, and discuss their unique characteristics and benefits. This review serves as a valuable resource for researchers interested in leveraging fluorescence lifetime imaging to study molecular interactions and cellular metabolism with high precision and accuracy.

Modulation of Local Cellular Activities using a Photothermal Dye-Based Subcellular-Sized Heat Spot.

Thermal engineering at the microscale, such as the regulation and precise evaluation of the temperature within cellular environments, is a major challenge for basic biological research and biomaterials development. We engineered a polymeric nanoparticle having a fluorescent temperature sensory dye and a photothermal dye embedded in the polymer matrix, named nanoheater-thermometer (nanoHT). When nanoHT is illuminated with a near-infrared laser at 808 nm, a subcellular-sized heat spot is generated in a live cell. Fluorescence thermometry allows the temperature increment to be read out concurrently at individual heat spots. Within a few seconds of an increase in temperature by approximately 11.4 °C from the base temperature (37 °C), we observed the death of HeLa cells. The cell death was observed to be triggered from the exact local heat spot at the subcellular level under the fluorescence microscope. Furthermore, we demonstrate the application of nanoHT for the induction of muscle contraction in C2C12 myotubes by heat release. We successfully showed heat-induced contraction to occur in a limited area of a single myotube based on the alteration of protein-protein interactions related to the contraction event. These results demonstrate that even a single heat spot provided by a photothermal material can be extremely effective in altering cellular functions.

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.

Effect of PDGF-B aptamer on PDGFRß/PDGF-B interaction: Molecular dynamics study.

PDGFRß/PDGF-B interaction plays a role in angiogenesis, and is mandatory in wound healing and cancer treatment. It has been reported that the PDGF-B aptamer was able to bind to PDGF-B, thus regulating the angiogenesis. However, the binding interaction between the aptamer and the growth factor, including the binding sites, has not been well investigated. This study applied a molecular dynamics (MD) simulation to investigate the aptamer-growth factor interaction in the presence or absence of a receptor (PDGFRß). Characterization of the structure of an aptamer-growth factor complex revealed binding sites from each section in the complex. Upon the complex formation, PDGF-B and its aptamer exhibited less flexibility in their molecular movement, as indicated by the minimum values of RMSD, RMSF, loop-to-loop distance, and the summation of PCA eigenvalues. Our study of residue pairwise interaction demonstrated that the binding interaction was mainly contributed by electrostatic interaction between the positively-charged amino acid and the negatively-charged phosphate backbone. The role of the PDGF-B aptamer in PDGFRß/PDGF-B interaction was also investigated. We demonstrated that the stability of the Apt-PDGF-B complex could prevent the presence of a competitor, of PDGFRß, interrupting the binding process. Because the aptamer was capable of binding with PDGF-B, and blocking the growth factor from the PDGFRß, it could down regulate the consequent signaling pathway. We provide evidence that the PDGF-BB aptamer is a promising molecule for regulation of angiogenesis. The MD study provides a molecular understanding to modification of the aptamer binding interaction, which could be used in a number of medical applications.

Oligonucleotide Hybridization Combined with Competitive Antibody Binding for the Truncation of a High-Affinity Aptamer.

Truncation can enhance the affinity of aptamers for their targets by limiting nonessential segments and therefore limiting the molecular degrees of freedom that must be overcome in the binding process. This study demonstrated a truncation protocol relying on competitive antibody binding and the hybridization of complementary oligonucleotides, using platelet derived growth factor BB (PDGF-BB) as the model target. On the basis of the immunoassay results, an initial long aptamer was truncated to a number of sequences with lengths of 36-40 nucleotides (nt). These sequences showed apparent KD values in the picomolar range, with the best case being a 36-nt truncated aptamer with a 150-fold increase in affinity over the full-length aptamer. The observed binding energies correlated well with relative energies calculated by molecular dynamics simulations. The effect of the truncated aptamer on PDGF-BB-stimulated fibroblasts was found to be equivalent to that of the full-length aptamer.