RESUMO
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.
Assuntos
Cálcio , Temperatura Alta , Cálcio/metabolismo , Regulação para Cima , Neurônios/metabolismo , Metabolismo Energético , Cálcio da DietaRESUMO
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.
Assuntos
Temperatura Alta , Água , Células HeLa , Humanos , TemperaturaRESUMO
Plant root absorbs water and nutrients from the soil, and the root apoplastic fluid (AF) is an important intermediate between cells and the surrounding environment. The acid growth theory suggests that an acidic AF is needed for cell wall expansion during root growth. However, technical limitations have precluded the quantification of root apoplastic fluid pH (AF-pH). Here, we used Green-enhanced Nano-lantern (GeNL), a chimeric protein of the luciferase NanoLuc (Nluc) and the green fluorescent protein mNeonGreen (mNG), as a ratiometric pH indicator based on the pH dependency of bioluminescence resonance energy transfer efficiency from Nluc to mNG. Luminescence spectrum of GeNL changed reciprocally from pH 4.5 to 7.5, with a pKa of 5.5. By fusing GeNL to a novel signal peptide from Arabidopsis thaliana Cellulase 1, we localised GeNL in A. thaliana AF. We visualised AF dynamics at subcellular resolution over 30 min and determined flow velocity in the maturation zone to be 0.97± 0.06 µm/s. We confirmed that the developing root AF is acidic in the pH range of 5.1-5.7, suggesting that the AF-pH is tightly regulated during root elongation. These results support the acid growth theory and provide evidence for AF-pH maintenance despite changes in ambient pH.
Assuntos
Arabidopsis , Arabidopsis/metabolismo , Concentração de Íons de Hidrogênio , Luciferases/metabolismo , Raízes de Plantas/metabolismoRESUMO
Hemostasis is an essential function that repairs tissues and maintains the survival of living organisms. To prevent diseases caused by the abnormality of the blood coagulation mechanism, it is important to carry out a blood test periodically by a method that is convenient and less burdensome for examiners. Thrombin is a protease that catalyzes the conversion of fibrinogen, and its cleavage activity can be an index of coagulation activity. Here, we developed a ratiometric bioluminescent indicator, Thrombastor (thrombin activity sensing indicator), which reflects the thrombin cleavage activity in blood by changing the emission color from green to blue. Compared to the current thrombin activity indicator, the rapid color change of the emission indicated a 2.5-fold decrease in the Km for thrombin, and the cleavage rate was more than two times faster. By improving the absolute bioluminescence intensity, detection from a small amount of plasma could be achieved with a smartphone camera. Using Thrombastor and a versatile device, an effective diagnosis for preventing coagulation disorders can be provided.
Assuntos
Smartphone , Trombina , Coagulação Sanguínea , Fibrinogênio , PlasmaRESUMO
We investigate the effects of urea and its derivatives on the ATPase activity and on the in vitro motility of chicken skeletal muscle actomyosin. Mg-ATPase rate of myosin subfragment-1 (S1) is increased by 4-fold by 0.3M 1,3-diethylurea (DEU), but it is unaffected by urea, thiourea, and 1,3-dimethylurea at ≤1M concentration. Thus, we further examine the effects of DEU in comparison to those of urea as reference. In in vitro motility assay, we find that in the presence of 0.3M DEU, the sliding speeds of actin filaments driven by myosin and heavy meromyosin (HMM) are significantly decreased to 1/16 and 1/6.6, respectively, compared with the controls. However, the measurement of the actin-activated ATPase activity of HMM shows that the maximal rate, Vmax, is almost unchanged with DEU. Thus, the myosin-driven sliding motility of actin filaments is significantly impeded in the presence of 0.3M DEU, whereas the cyclic interaction of myosin with F-actin occurs during the ATP turnover, the rate of which is close to that without DEU. In contrast to DEU, 0.3M urea exhibits only modest effects on both actin-activated ATPase and sliding motility of actomyosin. Thus, DEU has the effect of uncoupling the sliding motility of actomyosin from its ATP turnover.
Assuntos
Actinas/química , Actomiosina/química , Trifosfato de Adenosina/química , Movimento , Subfragmentos de Miosina/química , Miosinas de Músculo Esquelético/química , Actinas/metabolismo , Actomiosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Galinhas , Subfragmentos de Miosina/metabolismo , Miosinas de Músculo Esquelético/metabolismoRESUMO
A double hydrophilic block copolymer, poly(ethylene glycol)-poly(3-dimethyl (methacryloyloxyethyl) ammonium propane sulfonate) (PEG-SB), is synthesized by reversible addition-fragmentation transfer (RAFT) polymerization using PEG methyl ether (4-cyano-4-pentanoate dodecyl trithiocarbonate) as a chain transfer agent. PEG-SB forms multi-layered microspheres with dipole-dipole interactions of the SB side chains as the driving force. The PEG-SB polymers show an upper critical solution temperature (UCST) and the UCST is controllable by the polymerization degree. The PEG-SB microspheres are dissociated above the UCST and then monodispersed microspheres (â¼1 µm) are obtained when the solution temperature is decreased below the UCST again. The disassociation/association of the microspheres is also controllable using the concentration of NaCl. These multi-responsive microspheres could be a powerful tool in the field of nano-biotechnology.
Assuntos
Microesferas , Polímeros/química , Água/química , Biotecnologia , Microscopia Eletrônica de Transmissão , NanotecnologiaRESUMO
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.
RESUMO
Bacterial phytochromes are attractive molecular templates for engineering fluorescent proteins (FPs) because their near-infrared (NIR) emission significantly extends the spectral coverage of GFP-like FPs. Existing phytochrome-based FPs covalently bind heme-derived tetrapyrrole chromophores and exhibit constitutive fluorescence. Here we introduce Rep-miRFP, an NIR imaging probe derived from bacterial phytochrome, which interacts non-covalently and reversibly with biliverdin chromophore. In Rep-miRFP, the photobleached non-covalent adduct can be replenished with fresh biliverdin, restoring fluorescence. By exploiting this chromophore renewal capability, we demonstrate NIR PAINT nanoscopy in mammalian cells using Rep-miRFP.
Assuntos
Microscopia , Fitocromo , Animais , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Bactérias/metabolismo , Biliverdina/metabolismo , Bactérias/metabolismo , MamíferosRESUMO
We investigate the hydration state of horse-heart cytochrome c (hh cyt c) in the unfolding process induced by trifluoroacetic acid (TFA). The conformation of hh cyt c changes from the native (N) state (2.9 < pH < 6.0) to the acid-unfolded (U(A)) state (1.7 < pH < 2.0) to the acid-induced molten globule (A) state (pH â¼1.2). Hydration properties of hh cyt c during this process are measured at 20°C by high-resolution dielectric relaxation (DR) spectroscopy, UV-vis absorbance, and circular dichroism spectroscopy. Constrained water of hh cyt c is observed at every pH as an â¼5-GHz Debye component (DC) (DR time, τ(D) â¼30 ps) and its DR amplitude (DRA) is increased by 77% upon N-to-U(A) transition, when pH changes from 6.0 to 2.0. Even in the N state, the DRA of the constrained-water component is found to be increased by 22% with decreasing pH from 6.0 to 2.9, suggesting an increase in the accessible surface area of native hh cyt c. Moreover, hypermobile water around native hh cyt c is detected at pH 6.0 as a 19-GHz DC (τ(D) â¼ 8.4 ps <τ(DW) = 9.4 ps), but is not found at other pH values. The DRA signal of constrained water is found to return to the pH 2.9 (N-state) level upon U(A)-to-A transition. Fast-response water (slightly slower than bulk) around A-state hh cyt c is detected at pH 1.2, and this suggests some accumulation of TFA(-) ions around the peptide chain. Thus, this high-resolution DR spectroscopy study reveals that hh cyt c exhibits significant hydration-state change in the TFA-unfolding process.
Assuntos
Citocromos c/metabolismo , Cavalos/metabolismo , Miocárdio/metabolismo , Desdobramento de Proteína/efeitos dos fármacos , Ácido Trifluoracético/farmacologia , Água/química , Animais , Soluções Tampão , Dicroísmo Circular , Espectroscopia Dielétrica , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Modelos Moleculares , Peptídeos/química , Soluções , Espectrofotometria UltravioletaRESUMO
High-resolution microwave dielectric spectra of NaX, KX (X: F, Cl, Br, I) aqueous solutions of c = 0.05 and 0.1 M measured in the frequency range 0.2-26 GHz at 10 °C are analyzed. The dielectric relaxation (DR) spectrum of each solution, which deviates slightly from the bulk-water spectrum, is mathematically divided into the bulk-water spectrum and the spectrum of solute particles covered with a water layer using a mixture theory by assuming the existence of continuous bulk-water phase. The solute spectra above 3 GHz are fitted with a linear series of pure water component (γ dispersion with DR frequency fw), fast Debye component-1 with DR frequency f1 (>fw), and slow Debye component-2 with DR frequency f2 (Assuntos
Halogênios/química
, Micro-Ondas
, Potássio/química
, Sódio/química
, Ânions/química
, Soluções
RESUMO
Ion currents associated with channel proteins in the presence of membrane potential are ubiquitous in cellular and organelle membranes. When an ion current occurs through a channel protein, Joule heating should occur. However, this Joule heating seems to have been largely overlooked in biology. Here we show theoretical investigation of Joule heating involving channel proteins in biological processes. We used electrochemical potential to derive the Joule's law for an ion current through an ion transport protein in the presence of membrane potential, and we suggest that heat production and absorption can occur. Simulation of temperature distribution around a single channel protein with the Joule heating revealed that the temperature increase was as small as <10-3 K, although an ensemble of channel proteins was suggested to exhibit a noticeable temperature increase. Thereby, we theoretically investigated the Joule heating of systems containing ensembles of channel proteins. Nerve is known to undergo rapid heat production followed by heat absorption during the action potential, and our simulation of Joule heating for a squid giant axon combined with the Hodgkin-Huxley model successfully reproduced the feature of the heat. Furthermore, we extended the theory of Joule heating to uncoupling protein 1 (UCP1), a solute carrier family transporter, which is important to the non-shivering thermogenesis in brown adipose tissue mitochondria (BATM). Our calculations showed that the Joule heat involving UCP1 was comparable to the literature calorimetry data of BATM. Joule heating of ion transport proteins is likely to be one of important mechanisms of cellular thermogenesis.
RESUMO
This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo (1)H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by â¼5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.
Assuntos
Citoesqueleto de Actina/química , Água/química , Animais , Soluções Tampão , Carbocianinas/química , Cisteína/química , Corantes Fluorescentes/química , Ressonância Magnética Nuclear Biomolecular , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Elastina/química , Peptídeos/química , Temperatura , Termometria/métodos , Elastina/genética , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Peptídeos/genéticaRESUMO
Bilirubin in human blood is highly important as a general index of one's physical condition because its concentration changes under the influence of several diseases. In particular, in newborns, jaundice is one of the most common diseases involving unconjugated bilirubin (UCBR), causing serious symptoms such as nuclear jaundice and deafness. Therefore, a frequent measurement of the UCBR levels in the blood is important. Here, we report a ratiometric bioluminescent indicator, BABI (bilirubin assessment with a bioluminescent indicator), that changes the emission color from blue to green depending on the UCBR concentration in a sample. Owing to the use of a bioluminescence signal that has a higher signal-to-noise ratio than the absorption and fluorescence signal, BABI enables highly sensitive and quantitative detection of UCBR for small blood samples using a smartphone camera. The establishment of a UCBR measurement assay using BABI provides the possibility of a simple and rapid method for blood-based diagnosis using bioluminescent indicators and a versatile mobile device.
Assuntos
Bilirrubina , Smartphone , Humanos , Recém-NascidoRESUMO
Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.
Assuntos
Proteínas Luminescentes/química , Imagem com Lapso de Tempo/métodos , MicroscopiaRESUMO
Reversibly switchable fluorescent proteins (RSFPs) are crucial tags for super-resolution observation of protein localization and dynamics inside living cells. However, due to the high fluorescence pKa (â¼5-6) of most RSFPs, their usage in acidic conditions (pH 4.5-6.0) has been limited. Here, we investigated a new photochromic mechanism in Gamillus, a recently developed green fluorescent protein with acid tolerance. Gamillus exhibits negative switching with especially high contrast in acidic conditions, and its off switching is caused by trans-to-cis isomerization of the chromophore hydroxyphenyl ring that accompanies protonation. Through a combination of rational design and saturation mutagenesis, we developed two variants with enhanced switching contrasts and off-switching speeds, designated rsGamillus-S and rsGamillus-F, respectively. The fluorescence intensity, off-switching speed, and switching contrast of the rsGamillus variants are only slightly affected by changes in pH between 4.5 and 7.5. Exploiting these properties, we succeeded in high-contrast super-resolution imaging of cellular architectures in acidic conditions.
Assuntos
Proteínas de Fluorescência Verde/química , Cristalografia por Raios X , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Processos Fotoquímicos , Células Tumorais CultivadasRESUMO
Electrophysiological field potential dynamics have been widely used to investigate brain functions and related psychiatric disorders. Considering recent demand for its applicability to freely moving subjects, especially for animals in a group and socially interacting with each other, here we propose a new method based on a bioluminescent voltage indicator LOTUS-V. Using our fiber-free recording method based on the LOTUS-V, we succeeded in capturing dynamic change of brain activity in freely moving mice. Because LOTUS-V is the ratiometric indicator, motion and head-angle artifacts were not significantly detected. Taking advantage of our method as a fiber-free system, we further succeeded in simultaneously recording from multiple independently-locomotive mice that were freely interacting with one another. Importantly, this enabled us to find that the primary visual cortex, a center of visual processing, was activated during the interaction of mice. This methodology may further facilitate a wide range of studies in neurobiology and psychiatry.
Assuntos
Movimento , Optogenética/métodos , Córtex Visual/fisiologia , Animais , Células Cultivadas , Meio Ambiente , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp/métodos , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Córtex Visual/diagnóstico por imagem , Córtex Visual/metabolismoRESUMO
Electrolytes and their dissociated ions are thought to form positive or negative hydration layers around them. In this study, we have developed a method to determine the volume and the dielectric relaxation property (relaxation frequency f c, dispersion intensity delta) of the water hydrating ions in salt solutions. The method consists of four steps: (1) By use of a high-resolution microwave dielectric spectroscopy technique, the dielectric spectra of sample salt solution and bulk water are measured in pair. (2) The dielectric spectrum of solutes (ions) with water layers for a given volume fraction varphi is then calculated from each pair of dielectric spectra of a sample salt solution and reference water according to the Hanai mixture theory. (3) Each spectrum of solutes with water layers at a given varphi is decomposed into a few Debye relaxation functions and the bulk water component. (4) The volume fraction varphi is operationally decreased from 0.5, and steps (2) and (3) are repeated at each varphi until the bulk water component vanished. Then the volume fraction of the hydrated solutes (ions) in solution is determined. The method was applied to NaF and NaCl solutions. As a result the different spectral intensity was nearly proportional to the salt concentration below 0.2 M in the frequency range of 3-26 GHz. The hydration number N h and the dielectric relaxation property of the hydration layer for each salt solution was successfully determined as ( f c1, delta 1, N h)= (18.7, 44.9, 27.9) for NaCl and ( f c1, delta 1, f c2, delta 2, N h) = (26.0, 6.70, 5.64, 19.2) for NaF.
RESUMO
Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102-106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70-80 nm with illumination power densities as low as ~1 W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of super-resolution fluorescence microscopy for live-cell imaging.
Assuntos
Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Proteínas Recombinantes de Fusão/metabolismo , Linhagem Celular Tumoral , Clatrina/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Paxilina/metabolismo , Vimentina/metabolismoRESUMO
Fluorescence emission properties of intact and alkaline-treated chlorosomes containing bacteriochlorophyll(BChl)-c, d, and e, which were isolated from four species of green sulfur photosynthetic bacteria, were successfully studied at the single-unit level using a total internal reflection fluorescence microscope. Single intact chlorosomes containing BChl-c from Chlorobium (Chl.) tepidum exhibited heterogeneous emission bands of BChl-c self-aggregates. In contrast, fluorescence spectra of chlorosomal BChl self-aggregates in single intact chlorosomes from the other three Chlorobium species were less heterogeneous than those from Chi. tepidum. Removal of energy-accepting BChl-a/protein complexes called baseplates from the intact chlorosomes by treatments with alkaline media hardly changed spectral shapes of BChl aggregates and their peak distributions at the single-chlorosome level. The similarity of spectral properties at the single-unit level between intact and alkaline-treated chlorosomes of four Chlorobium species clearly indicated that the removal of base-plates from intact chlorosomes by the alkaline-treatment did not affect BChl self-aggregates inside single chlorosomes.