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Funatsu, T.

Publications and source records attributed to Funatsu, T..

5 recordsLinked to original sources

Membrane dynamics-mediated heat retention dictates economical intracellular energy flow

Intracellular thermometry has revealed temperature gradients within living cells, indicating the presence of localized energy flow. Moreover, increasing evidence suggests that cells can autonomously regulate heat production and use this heat to support cellular functions. Although heat is generally assumed to dissipate rapidly, recent studies suggest that slow, non-diffusive heat dissipation can generate high local temperatures, implying that heat may be retained within cells. However, such intracellular heat retention at the single-cell level has not been experimentally examined. Here, we measured the dissipation of metabolically generated heat using calorimetry and evaluated cellular heat balance. We further separated the dissipated heat into heat transfer and heat production components using an intracellular temperature imaging-based method. Through this approach, we found that perturbation of the cell membrane accelerated intracellular heat transfer rates and increased heat dissipation. These findings provide the first experimental evidence of intracellular heat retention, showing that heat generated by catabolic metabolism is not fully dissipated but is partly retained within cells by the cell membrane, which separates the intracellular space from the external environment. Furthermore, we identified the mechanism underlying this heat retention, demonstrating that membrane lipid dynamics are a key molecular determinant and that the motions of membrane lipids form an energetic barrier to heat dissipation. Together, our findings redefine metabolic heat, long regarded as mere waste, as a functional resource that supports efficient energy use, and they establish membrane-controlled heat retention as a fundamental principle of bioenergetic optimization in living systems.

biophysics↗

High-speed Intracellular Temperature Mapping Reveals the Existence of Non-Conductive Dissipation of Energy by Heating

Recently, intracellular thermometry has revealed temperature variations within cells. Although the biological significance of intracellular temperature change is recognized, the physical principles of intracellular temperature change remain a mystery. Here, we investigate intracellular heat transfer through intracellular temperature mapping using a fluorescent polymeric thermometer and high-speed fluorescence lifetime imaging microscopy. Through infrared laser irradiation-assisted heating, we track changes in temperature distribution to examine the mechanism of intracellular heat dissipation in comparison with heat conduction. Continuous heating induces the significantly slower relaxation of the average temperature of single cells compared with that of liposomes containing homogeneous aqueous solutions of comparable size; to the scale of seconds. We additionally elucidate that these phenomena are impacted by intracellular structures and molecules. Finally, we discover that this slow intracellular temperature relaxation originates from non-diffusive heat dissipation distinct from the conventional heat conduction model. Our results provide insights into the mechanisms of temperature variation in cells that are unresolved based on our current understanding, establishing a framework for understanding intracellular thermodynamics under non-equilibrium conditions.

biophysics↗

Tuning of ribosome levels mediated by RNase I and hibernating ribosomes

Protein biosynthesis is an energy-hungry intracellular process that requires the stringent regulation of ribosome abundance under environmental conditions. In response to stress, some active ribosomes are degraded while others, in bacteria, enter a hibernation state to protect against degradation. RNase I, a conserved T2 family ribonuclease in Escherichia coli, degrades ribosomal RNA to suppress biofilm formation, whereas it interacts with ribosomes. However, how and why RNase I binds to ribosomes remains elusive. Here, we show that hibernating ribosomes bind to RNase I and inhibit its activity, thereby promoting biofilm formation. We determined the cryo-electron microscopy structure of the hibernating ribosome complexed with RNase I. RNase I interacts with helix 41 of the 16S rRNA and the ribosomal protein uS14 in the head domain of the 30S subunit of the hibernating ribosome and positions its active centre away from helix 41, resulting in its catalytic inactivation. Hibernating ribosomes are protected from RNase I-mediated cleavage, and our in vivo and in vitro analyses revealed that RNase I targets dissociated large and small ribosomal subunits for rRNA degradation. These findings reveal a previously uncharacterized regulatory strategy that ribosomes modulate RNase I activity, ensuring both the preservation and timely degradation of ribosomes during environmental stress adaptation.

molecular biology↗

Quantitative live-cell imaging of secretion activity reveals dynamic immune responses

The measurement of cytokine secretions has contributed to the development of immunology; however, new methods that enable highly sensitive and efficient analysis are required for the precise characterisation of dynamic secretion activity when using rare cells or limited human specimens. Here, we report a new technology for quantitative live-cell imaging of secretion activity (qLCI-S), that enables high-throughput and dual-colour detection of prolonged secretion activity at the single-cell level, followed by transcriptome analysis for individual cells based on their phenotype. The power of the qLCI-S was demonstrated by visualising the individual and longitudinal cytokine secretion patterns of group 2 innate lymphoid cells, which comprised <0.01% human peripheral blood mononuclear cells, and identifying their minor subpopulations. This new technology will provide new insights into the spatiotemporal dynamic nature of various secretory functions and the development of fundamental tools for phenotypic drug discovery and regenerative and precision medicine.

biophysics↗

Quantification of the effect of site-specific histone acetylation on chromatin remodeling rate

Eukaryotic transcription is epigenetically regulated by chromatin structure and post-translational modifications (PTMs). For example, lysine acetylation in histone H4 is correlated with activation of RNA polymerase I-, II-, and III-driven transcription from chromatin templates, which requires prior chromatin remodeling. However, quantitative understanding of the contribution of particular PTM states to the sequential steps of eukaryotic transcription has been hampered partially because reconstitution of a chromatin template with designed PTMs is difficult. In this study, we reconstituted a di-nucleosome with site-specifically acetylated or unmodified histone H4, which contained two copies of the Xenopus somatic 5S rRNA gene with addition of a unique sequence detectable by hybridization-assisted fluorescence correlation spectroscopy. Using a Xenopus oocyte nuclear extract, we analyzed the time course of accumulation of nascent 5S rRNA-derived transcripts generated on chromatin templates in vitro. Our mathematically described kinetic model and fitting analysis revealed that tetra-acetylation of histone H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation ~3-fold in comparison with the absence of acetylation. We provide a kinetic model for quantitative evaluation of the contribution of epigenetic modifications to chromatin transcription.

biophysics↗