RESUMEN
Carboxy terminal fragments (CTFs) of TDP-43 contain an intrinsically disordered region (IDR) and form cytoplasmic condensates containing amyloid fibrils. Such condensates are toxic and associated with pathogenicity in amyotrophic lateral sclerosis. However, the molecular details of how the domain of TDP-43 CTFs leads to condensation and cytotoxicity remain elusive. Here, we show that truncated RNA/DNA-recognition motif (RRM) at the N-terminus of TDP-43 CTFs leads to the structural transition of the IDR, whereas the IDR itself of TDP-43 CTFs is difficult to assemble even if they are proximate intermolecularly. Hetero-oligomers of TDP-43 CTFs that have recruited other proteins are more toxic than homo-oligomers, implicating loss-of-function of the endogenous proteins by such oligomers is associated with cytotoxicity. Furthermore, such toxicity of TDP-43 CTFs was cell-nonautonomously affected in the nematodes. Therefore, misfolding and oligomeric characteristics of the truncated RRM at the N-terminus of TDP-43 CTFs define their condensation properties and toxicity.
Asunto(s)
Proteínas de Unión al ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Animales , Multimerización de Proteína , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/genéticaRESUMEN
Alcadein α (Alcα) and amyloid-ß protein precursor (APP) are cargo receptors that associate vesicles with kinesin-1. These vesicles, which contain either Alcα or APP, transport various proteins/cargo molecules into axon nerve terminals. Here, we analyzed immune-isolated Alcα- and APP-containing vesicles of adult mouse brains with LC-MS/MS and identified proteins present in vesicles that contained either Alcα or APP. Among these proteins, Frizzled-5 (Fzd5), a Wnt receptor, was detected mainly in Alcα vesicles. Although colocalization ratios of Fzd5 with Alcα are low in the neurites of differentiating neurons by a low expression of Fzd5 in embryonic brains, the suppression of Alcα expression decreased the localization of Fzd5 in neurites of primary cultured neurons. Furthermore, Fzd5-EGFP expressed in primary cultured neurons was preferentially transported in axons with the transport velocities of Alcα vesicles. In synaptosomal fractions of adult-mice brains that express higher levels of Fzd5, the amount of Fzd5 and the phosphorylation level of calcium/calmodulin-dependent protein kinase-II were reduced in the Alcα-deficient mice. These results suggest that reduced transport of Fzd5 by Alcα-containing vesicles associated with kinesin-1 in axon terminals may impair the response to Wnt ligands in the noncanonical Ca2+-dependent signal transduction pathway at nerve terminals of mature neurons.
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Transporte Axonal , Cinesinas , Animales , Ratones , Precursor de Proteína beta-Amiloide/metabolismo , Transporte Axonal/fisiología , Cromatografía Liquida , Cinesinas/metabolismo , Espectrometría de Masas en TándemRESUMEN
Hyperosmotic stress activates in live cells numerous processes and also promotes intracellular protein/RNA aggregation and phase separation. However, the time course and the extent of these changes remain largely uncharacterized. To investigate dynamic changes in intracellular macromolecular crowding (MMC) induced by hyperosmotic stress in live cells, we used fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy (FCS) to quantify changes in the local environment by measuring the fluorescence lifetime and the diffusion of the monomeric enhanced green fluorescent protein (eGFP), respectively. Real-time monitoring of eGFP fluorescence lifetime showed that a faster response to environmental changes due to MMC is observed than when measuring the acceptor/donor emission ratio using the MMC-sensitive Förster resonance energy transfer sensor (GimRET). This suggests that eGFP molecular electronic states and/or collision frequency are affected by changes in the immediate surroundings due to MMC without requiring conformational changes as is the case for the GimRET sensor. Furthermore, eGFP diffusion assessed by FCS indicated higher intracellular viscosity due to increased MMC during hyperosmotic stress. Our findings reveal that changes in eGFP fluorescence lifetime and diffusion are early indicators of elevated intracellular MMC. Our approach can therefore be used to reveal in live cells short-lived transient states through which MMC builds over time, which could not be observed when measuring changes in other physical properties that occur at slower time scales.
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Electrónica , Transferencia Resonante de Energía de Fluorescencia , Difusión , Microscopía Fluorescente , Agregado de ProteínasRESUMEN
Multimeric abnormalities in plasma von Willebrand factor (VWF) cause bleeding or clotting disorders. Electrophoretic analysis of multimers is used to detect such abnormalities but is qualitative, slow, and difficult to standardize. Fluorescence correlation spectroscopy (FCS) is a good alternative but is affected by low selectivity and concentration bias. Here, we report the development of a homogeneous immunoassay based on dual-color fluorescence cross-correlation spectroscopy (FCCS) that overcomes these challenges. By performing a mild denaturation treatment followed by reacting with polyclonal antibodies, the concentration bias was drastically reduced. The use of a dual antibody assay improved selectivity. Diffusion times of immunolabeled VWF were measured with FCCS and standardized relative to calibrator measurements. The assay measures size changes in VWF using 1 µL of plasma and less than 10 ng of antibody per measurement and was validated over a 16-fold range of VWF antigen concentration (VWF:Ag), with a sensitivity of VWF:Ag 0.8%. Concentration bias and imprecision were less than 10%. Measurements were unaffected by hemolytic, icteric, or lipemic interference. Strong correlations were obtained with reference densitometric readouts (0.97 for calibrators, 0.85 for clinical samples), and significant differences were found between normal (n = 10), type 2A (n = 5), and type 2B (n = 5) von Willebrand's disease and acquired thrombotic thrombocytopenic purpura (n = 10) samples (p < 0.01). This FCCS based immunoassay accurately and selectively determines changes in the multimeric status of plasma VWF and may be used as a simpler, faster, and a standardizable alternative for multimer analysis, following further clinical validation in larger cohorts.
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Enfermedades de von Willebrand , Factor de von Willebrand , Humanos , Factor de von Willebrand/análisis , Factor de von Willebrand/química , Factor de von Willebrand/metabolismo , Enfermedades de von Willebrand/diagnóstico , Enfermedades de von Willebrand/tratamiento farmacológico , Plasma/química , Inmunoensayo , Análisis EspectralRESUMEN
Transactive response element DNA/RNA-binding protein 43 kDa (TDP-43) is the causative protein of amyotrophic lateral sclerosis (ALS); several ALS-associated mutants of TDP-43 have been identified. TDP-43 has several domains: an N-terminal domain, two RNA/DNA-recognition motifs, and a C-terminal intrinsically disordered region (IDR). Its structures have been partially determined, but the whole structure remains elusive. In this study, we investigate the possible end-to-end distance between the N- and C-termini of TDP-43, its alterations due to ALS-associated mutations in the IDR, and its apparent molecular shape in live cells using Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). Furthermore, the interaction between ALS-associated TDP-43 and heteronuclear ribonucleoprotein A1 (hnRNP A1) is slightly stronger than that of wild-type TDP-43. Our findings provide insights into the structure of wild-type and ALS-associated mutants of TDP-43 in a cell.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/metabolismo , Mutación , Conformación Molecular , ARNRESUMEN
Guanine (G)-rich nucleic acids are prone to assemble into four-stranded structures, so-called G-quadruplexes. Abnormal GGGGCC repeat elongations, and in particular their folding states, are associated with amyotrophic lateral sclerosis and frontotemporal dementia. Due to methodological constraints however, most studies of G quadruplex structures are restricted to in vitro conditions. Evidence of how GGGGCC repeats form into G-quadruplexes in vivo is sparse. We devised a readout strategy, exploiting the sensitivity of trans-cis isomerization of cyanine dyes to local viscosity and sterical constraints. Thereby, folding states of cyanine-labeled RNA, and in particular G-quadruplexes, can be identified in a sensitive manner. The isomerization kinetics, monitored via fluorescence blinking generated upon transitions between a fluorescent trans isomer and a non-fluorescent cis isomer, was first characterized for RNA with GGGGCC repeats in aqueous solution using fluorescence correlation spectroscopy and transient state (TRAST) monitoring. With TRAST, monitoring the isomerization kinetics from how the average fluorescence intensity varies with laser excitation modulation characteristics, we could then detect folding states of fluorescently tagged RNA introduced into live cells.
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Demencia Frontotemporal , G-Cuádruplex , Humanos , Colorantes Fluorescentes , Demencia Frontotemporal/genética , Isomerismo , ARN/químicaRESUMEN
Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.
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Enfermedades Neurodegenerativas/fisiopatología , Agregado de Proteínas/fisiología , Espectrometría de Fluorescencia/métodos , HumanosRESUMEN
Optineurin produces intracellular multi-functions involving autophagy, vesicular trafficking, and negative regulation of inflammation signaling through interaction with various proteins such as ATG8/LC3, Rab8, and polyubiquitin. Optineurin is a component of cytoplasmic inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis (ALS), and its mutation E478G, has been identified in patients with ALS. However, the mechanism by which polyubiquitin binding modulates the interaction partners of OPTN and ALS-associated IB formation is still unclear. To address this issue, we analyzed the interaction of Optineurin with Rab8 and LC3 in the absence and presence of linear polyubiquitin chains using fluorescence cross-correlation spectroscopy and IB formation efficiency of the E478G mutant of Optineurin during Rab8 depletion using fluorescence microscopy. Here, we hypothesize that linear polyubiquitin binding to Optineurin dynamically induces LC3 association and Rab8 dissociation, likely through a conformational change of Optineurin, and the dynamic conformational change may prevent the aggregate formation of mutant Optineurin.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Poliubiquitina/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proteínas de Transporte de Membrana/genética , Ratones , Modelos Biológicos , Mutación/genética , Agregado de Proteínas , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Transporte de Proteínas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Macromolecular crowding (MMC) in cells is a hot topic in biology; therefore, well-characterized measurement standards for the evaluation of the nano-environment in MMC solutions are necessary. We propose to use polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) for evaluation of macromolecular crowding in solutions. Pol-FCS can simultaneously measure the relaxation times of rotational and translational diffusion of fluorescent molecules at the same position, even in living cells with low damage. In this report, the differences in the nano-environment among solutions of small molecules, gels, and MMC solutions were evaluated by comparing their rotational and translational diffusion using Pol-FCS. Moreover, this method could distinguish the phase shift in the polyethylene glycol solution. Finally, we separately evaluated the nano-environment in the cytosol and nucleus of living cells in different cell lines and cell cycles. We expect this evaluation method to be useful in characterizing the nano-environment in MMC studies. In addition, the proposed method may be useful for other nano-environments such as liquid-liquid phase separation.
RESUMEN
Number and brightness (N&B) analysis is useful for monitoring the spatial distribution of the concentration and oligomeric state of fluorescently labeled proteins in cells. N&B analysis is based on the statistical analysis of fluorescence images by using the method of moments (MoM). Furthermore, N&B analysis can determine the particle number and particle brightness, which indicate the concentration and oligomeric state, respectively. However, the statistical accuracy and precision are limited in actual experiments with fluorescent proteins, owing to low excitation and the limited number of images. In this study, we applied maximum likelihood (ML) estimation and maximum a posteriori (MAP) estimation coupled with the empirical Bayes (EB) method (referred to as EB-MAP). In EB-MAP, we constructed a simple prior distribution for a pixel to utilize the information of the surrounding pixels. To evaluate the accuracy and precision of our method, we conducted simulations and experiments and compared the results of MoM, ML, and EB-MAP. The results showed that MoM estimated the particle number with many outliers. The outliers hampered the visibility of the spatial distribution and cellular structure. In contrast, EB-MAP suppressed the number of outliers and improved the visibility notably. The precision of EB-MAP was better by an order of magnitude in terms of particle number and 1.5 times better in terms of particle brightness compared with those of MoM. The proposed method (EB-MAP-N&B) is applicable to studies on fluorescence imaging and would aid in accurately recognizing changes in the concentration and oligomeric state in cells. Our results hold significant importance because quantifying the concentration and oligomeric state would contribute to the understanding of dynamic processes in molecular mechanism in cells.
Asunto(s)
Proyectos de Investigación , Teorema de BayesRESUMEN
Number and brightness (N&B) analysis helps to visualize protein oligomer and its localization in a living cell. N&B analysis provides apparent brightness, which reflects the oligomeric state of a fluorescently labeled protein, by analyzing the temporal intensity fluctuation at each pixel. N&B analysis is useful in understanding the dynamic oligomerization in signal transduction and neurodegenerative diseases. Furthermore, it also helps in gaining useful insights regarding the controlling mechanisms in protein function. In this chapter, we describe the basic theory and notations of N&B analysis implemented with confocal laser scanning microscopy for quantitative analyses.
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Transducción de Señal , Proteínas Fluorescentes Verdes , Microscopía Confocal , Multimerización de Proteína , Espectrometría de FluorescenciaRESUMEN
The mechanism that mediates the interaction between the contractile ring and the plasma membrane during cytokinesis remains elusive. We previously found that ERM (Ezrin/Radixin/Moesin) proteins, which usually mediate cellular pole contraction, become over-accumulated at the cell equator and support furrow ingression upon the loss of other actin-membrane associated proteins, anillin and supervillin. In this study, we addressed the molecular basis of the exchangeability between ezrin and other actin-membrane associated proteins in mediating cortical contraction during cytokinesis. We found that depletion of anillin and supervillin caused over-accumulation of the membrane-associated FERM domain and actin-binding C-terminal domain (C-term) of ezrin at the cleavage furrow, respectively. This finding suggests that ezrin differentially shares its binding sites with these proteins on the actin cytoskeleton or inner membrane surface. Using chimeric mutants, we found that ezrin C-term, but not the FERM domain, can substitute for the corresponding anillin domains in cytokinesis and cell proliferation. On the other hand, either the membrane-associated or the actin/myosin-binding domains of anillin could not substitute for the corresponding ezrin domains in controlling cortical blebbing at the cell poles. Our results highlight specific designs of actin- or membrane-associated moieties of different actin-membrane associated proteins with limited exchangeability, which enables them to support diverse cortical activities on the shared actin-membrane interface during cytokinesis.
Asunto(s)
Actinas/genética , Citocinesis/genética , Proteínas del Citoesqueleto/genética , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Sitios de Unión , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proliferación Celular , Proteínas del Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
Membranes are ubiquitous structures in cells. The effects of membranes on various functional molecules have been reported, but their behaviors under macromolecular crowding and cell-sized confinement have not fully been understood. In this study, we model an intracellular environment by crowding micrometer-sized droplets and investigate the effects of membrane properties on molecular diffusion. The molecular diffusion inside small droplets covered with a lipid layer of phosphatidylcholine (PC) becomes slower compared with that of the corresponding bulk solutions under a crowding condition of polysaccharide dextran but not of its monomer unit, glucose. The addition of a poly(ethylene glycol) conjugated lipid (PEGylated lipid) to the PC membrane significantly alters the degree of slow diffusion observed inside small droplets of concentrated dextran. Interestingly, the change is not monotonic against dextran concentration; that is, the PEGylated membrane increases and decreases the degree of slow diffusion with increasing dextran concentration. We explain the nonmonotonic alternation from the increase in effective dextran concentration and the hindered temporal adsorption of dextran to the membrane. Because diffusion alteration by adding PEGylated lipid is observed for condensed small droplets of linear polymer PEG and hydrophilic protein bovine serum albumin, the phenomenon is general for other polymer systems as well. Furthermore, our findings may facilitate the understanding of intracellular molecular behaviors based on membrane effects as well as the development of numerous applications using polymer droplets.
RESUMEN
Molecular behaviors in small liquid droplets (picoliter scale), such as phase transitions and chemical reactions, are essential for the industrial application of small droplets and their use as artificial cells. However, the droplets often differ from those in bulk solutions (milliliter scale). Since the droplet size is much larger than the molecular size, the so-called size effect that draws these differences has attracted attention as a target to be solved. Although the small volume and the membrane surface surrounding the droplet are thought to be the origin of the size effect, there were little attempts to separate and quantify them. To solve the problem, we develop a series of systems for the evaluation. Using these systems, we have evaluated the size effect of concentrated polymer solutions on molecular diffusion by dividing it into small volume and membrane surface contributions. Our results demonstrate that the size effect on the molecular diffusion originates from the long-range interaction with the surface enhanced with decreasing volume. The quantitative size effect revealed by the systems provides novel insights in the biophysical understanding of molecular behaviors in cells and to the regulation and design of micrometer-sized materials.
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Polietilenglicoles/análisis , Animales , Bovinos , Difusión , Fluorescencia , Proteínas Fluorescentes Verdes/química , Tamaño de la Partícula , Albúmina Sérica Bovina/química , Propiedades de SuperficieRESUMEN
Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 µs/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 × 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and cross-correlation between first- and second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.
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Proteínas de Drosophila/genética , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Receptores Opioides mu/genética , Factores de Transcripción/genética , Animales , Línea Celular Tumoral , Dexametasona/farmacología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal/instrumentación , Microscopía Fluorescente/instrumentación , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/ultraestructura , Células PC12 , Transporte de Proteínas/efectos de los fármacos , Puntos Cuánticos , Ratas , Receptores Opioides mu/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/ultraestructura , Factores de Transcripción/metabolismoRESUMEN
A full fiber-optic fluorescence correlation spectroscopy (FF-FCS) technique has been developed without the use of objectives and dichroic mirrors. To achieve this, an excitation laser has been focused onto a sample by a lensed optical fiber or a gradient index lens attached on the terminal surface of the optical fiber. The FF-FCS system does not exhibit a higher sensitivity than the conventional FCS system; however, it is much simpler and smaller. This work demonstrates the feasibility of FF-FCS by measuring fluorescent beads. In the future, we expect FF-FCS to be widely used as a laboratory tool and an embedded tool for quality-control systems, such as cytometers.
RESUMEN
Molecular crowding creates a unique environment in cells and imposes physical constraints such as the excluded volume effect, water activity, and dielectric constant that can affect the structure and function of biomolecules. It is therefore important to develop a method for quantifying the effects of molecular crowding in cells. In this study, we developed a Förster resonance energy transfer (FRET) probe based on a guanine-quadruplex (G4) DNA motif that shows distinct FRET signals in response to crowding conditions in the presence of salt and poly(ethylene glycol). FRET efficiencies varied in different solutions, reflecting the dependence of G4 stability and topology on salt concentration and water activity. In living cells, FRET signals in the nucleus were higher than those in the cytosol; the signals in membraneless nuclear compartments (i.e., nucleolus) were especially high, suggesting that a decrease in water activity is important for the crowding effect in the nucleus. Thus, the use of DNA sensors with variable structures can elucidate the local effects of molecular crowding in cells.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , ADN/química , G-Cuádruplex , Animales , Carbocianinas/química , Bovinos , ADN/genética , Sondas de ADN/química , Sondas de ADN/genética , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Células HeLa , Humanos , Espacio Intracelular/metabolismo , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Polietilenglicoles/química , Potasio/química , Albúmina Sérica Bovina/química , Sodio/químicaRESUMEN
Encapsulation of guest molecules into the hollow spaces of crystals has been applied for a variety of purposes such as structure determination, separation, and catalysis of the guest. Although host-guest studies have been developed mainly in crystals of small molecules, those of biomacromolecules have recently been applied. In those reports, a huge hollow space in the protein crystal is commonly used for encapsulation of the guest. Our previous study revealed that cylindrical hemocyanins stack inside the crystal as a linear hollow structure. The diameter of the linear hollow is approximately 110â¯Å, which is large enough for most proteins to pass through. In the present study, we evaluated the potential of hemocyanin crystals as a host to encapsulate biomacromolecules. Confocal microscopy revealed that hemocyanin crystals encapsulate proteins of molecular mass up to 250â¯kDa, i.e., 27â¯kDa green fluorescence protein, 105â¯kDa allophycocyanin, 220â¯kDa C-phycocyanin, and 250â¯kDa phycoerythrin, and DNAs up to 200-bp long, whereas 440â¯kDa ferritin not. Further analysis revealed that hemocyanin crystals prefer a negatively charged guest rather than a positive charge to encapsulate. Moreover, a photobleaching experiment showed that the guest does not move once entrapped. This knowledge of the host-guest study using the hollow hemocyanin crystal should be of significance for further application of hollow proteinaceous crystals as a host.
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Cristalización/métodos , Decapodiformes/química , Hemocianinas/química , Animales , Proteínas Fluorescentes Verdes/química , Modelos Moleculares , Ficocianina/química , Ficoeritrina/química , PorosidadRESUMEN
Transactive response DNA/RNA-binding protein 43-kDa (TDP-43) C-terminal fragments, such as a 25-kDa fragment (TDP-25), have been identified as a ubiquitinated and phosphorylated components of inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis patients. Cells contain proteins that function as molecular chaperones and prevent aggregate formation of misfolded and aggregation-prone proteins. Recently, we reported that heat shock protein (HSP)70, an abundant molecular chaperone, binds to TDP-25 in an ATP-dependent manner; however, whether HSP70 can prevent the formation of TDP-25-related IBs remains unknown. Here, we showed that HSP70 prevented TDP-25 aggregation according to green fluorescent protein-tagged TDP-25 (G-TDP-25) colocalization in the cytoplasm with mCherry-tagged HSP70 (HSP70-R). The mobile fraction of HSP70-R in the cytoplasmic IBs associated with G-TDP-25 increased relative to that of G-TDP-25, suggesting that HSP70 strongly bound to G-TDP-25 in the IBs, whereas a portion remained dissociated from the IBs. Importantly, the proportion of G-TDP-25 IBs was significantly decreased by HSP70-R overexpression; however, G-TDP-25 levels in the insoluble fraction remained unchanged by HSP70-R overexpression, suggesting that G-TDP-25 formed aggregated species that cannot be dissolved, even in the presence of strong detergents. These results indicated that HSP70 prevented the accumulation of G-TDP-25 aggregates in cytoplasmic IBs, but was insufficient for G-TDP-25 disassembly and solubilization.