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1.
Biochem Biophys Res Commun ; 716: 150010, 2024 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-38704892

RESUMEN

Calcium (Ca2+) in mitochondria plays crucial roles in neurons including modulating metabolic processes. Moreover, excessive Ca2+ in mitochondria can lead to cell death. Thus, altered mitochondrial Ca2+ regulation has been implicated in several neurodegenerative diseases including Huntington's disease (HD). HD is a progressive hereditary neurodegenerative disorder that results from abnormally expanded cytosine-adenine-guanine trinucleotide repeats in the huntingtin gene. One neuropathological hallmark of HD is neuronal loss in the striatum and cortex. However, mechanisms underlying selective loss of striatal and cortical neurons in HD remain elusive. Here, we measured the basal Ca2+ levels and Ca2+ uptake in single presynaptic mitochondria during 100 external electrical stimuli using highly sensitive mitochondria-targeted Ca2+ indicators in cultured cortical and striatal neurons of a knock-in mouse model of HD (zQ175 mice). We observed elevated presynaptic mitochondrial Ca2+ uptake during 100 electrical stimuli in HD cortical neurons compared with wild-type (WT) cortical neurons. We also found the highly elevated presynaptic mitochondrial basal Ca2+ level and Ca2+ uptake during 100 stimuli in HD striatal neurons. The elevated presynaptic mitochondrial basal Ca2+ level in HD striatal neurons and Ca2+ uptake during stimulation in HD striatal and cortical neurons can disrupt neurotransmission and induce mitochondrial Ca2+ overload, eventually leading to neuronal death in the striatum and cortex of HD.


Asunto(s)
Calcio , Corteza Cerebral , Cuerpo Estriado , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Enfermedad de Huntington , Mitocondrias , Terminales Presinápticos , Animales , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Enfermedad de Huntington/genética , Calcio/metabolismo , Mitocondrias/metabolismo , Ratones , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Terminales Presinápticos/metabolismo , Células Cultivadas , Neuronas/metabolismo , Neuronas/patología , Ratones Transgénicos
2.
J Biol Chem ; 300(1): 105523, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38043799

RESUMEN

Filopodia are slender cellular protrusions containing parallel actin bundles involved in environmental sensing and signaling, cell adhesion and migration, and growth cone guidance and extension. Myosin 10 (Myo10), an unconventional actin-based motor protein, was reported to induce filopodial initiation with its motor domain. However, the roles of the multifunctional tail domain of Myo10 in filopodial formation and elongation remain elusive. Herein, we generated several constructs of Myo10-full-length Myo10, Myo10 with a truncated tail (Myo10 HMM), and Myo10 containing four mutations to disrupt its coiled-coil domain (Myo10 CC mutant). We found that the truncation of the tail domain decreased filopodial formation and filopodial length, while four mutations in the coiled-coil domain disrupted the motion of Myo10 toward filopodial tips and the elongation of filopodia. Furthermore, we found that filopodia elongated through multiple elongation cycles, which was supported by the Myo10 tail. These findings suggest that Myo10 tail is crucial for promoting long filopodia.


Asunto(s)
Miosinas , Seudópodos , Actinas/metabolismo , Adhesión Celular , Miosinas/química , Miosinas/genética , Miosinas/metabolismo , Dominios Proteicos , Seudópodos/genética , Seudópodos/metabolismo , Células COS , Animales , Chlorocebus aethiops , Humanos
3.
Biochem Biophys Res Commun ; 691: 149246, 2024 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-38029540

RESUMEN

Huntington's disease (HD) is a progressive genetic neurodegenerative disease caused by an abnormal expansion of a cytosine-adenine-guanine trinucleotide repeat in the huntingtin gene. One pathological feature of HD is neuronal loss in the striatum. Despite many efforts, mechanisms underlying neuronal loss in HD striatum remain elusive. It was suggested that the mutant huntingtin protein interacts mitochondrial proteins and causes mitochondrial dysfunction in striatal neurons. However, whether axonal transport of mitochondria is altered in HD striatal neurons remains controversial. Here, we examined axonal transport of single mitochondria labelled with Mito-DsRed2 in cultured striatal neurons of zQ175 knock-in mice (a knock-in mouse model of HD). We observed decreased anterograde axonal transport of proximal mitochondria in HD striatal neurons compared with wild-type (WT) striatal neurons. Decreased anterograde transport in HD striatal neurons was prevented by overexpressing mitochondrial Rho GTPase 1 (Miro1). Our results offer a new insight into mechanisms underlying neuronal loss in the striatum in HD.


Asunto(s)
Enfermedad de Huntington , Enfermedades Neurodegenerativas , Ratones , Animales , Enfermedad de Huntington/metabolismo , Transporte Axonal , Ratones Transgénicos , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Mitocondrias/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo
4.
Heliyon ; 9(11): e22306, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-38027957

RESUMEN

Investigation of cell-to-cell variability holds critical physiological and clinical implications. Thus, numerous new techniques have been developed for studying cell-to-cell variability, and these single-cell techniques can also be used to investigate rare cells. Moreover, for studying protein-protein interactions (PPIs) in single cells, several techniques have been developed based on the principle of the single-molecule pulldown (SiMPull) assay. However, the applicability of these single-cell SiMPull (sc-SiMPull) techniques is limited because of their high technical barrier and special requirements for target cells and molecules. Here, we report a highly innovative nanobead-based approach for sc-SiMPull that is based on our recently developed microbead-based, improved version of SiMPull for cell populations. In our sc-SiMPull method, single cells are captured in microwells and lysed in situ, after which commercially available, pre-surface-functionalized magnetic nanobeads are placed in the microwells to specifically capture proteins of interest together with their binding partners from cell extracts; subsequently, the PPIs are examined under a microscope at the single-molecule level. Relative to previously published methods, nanobead-based sc-SiMPull is considerably faster, easier to use, more reproducible, and more versatile for distinct cell types and protein molecules, and yet provides similar sensitivity and signal-to-background ratio. These crucial features should enable universal application of our method to the study of PPIs in single cells.

5.
Front Mol Neurosci ; 16: 1175522, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37664244

RESUMEN

Huntington's disease (HD) is a progressive dominantly inherited neurodegenerative disease caused by the expansion of a cytosine-adenine-guanine (CAG) trinucleotide repeat in the huntingtin gene, which encodes the mutant huntingtin protein containing an expanded polyglutamine tract. One of neuropathologic hallmarks of HD is selective degeneration in the striatum. Mechanisms underlying selective neurodegeneration in the striatum of HD remain elusive. Neurodegeneration is suggested to be preceded by abnormal synaptic transmission at the early stage of HD. However, how mutant huntingtin protein affects synaptic vesicle exocytosis at single presynaptic terminals of HD striatal neurons is poorly understood. Here, we measured synaptic vesicle exocytosis at single presynaptic terminals of cultured striatal neurons (mainly inhibitory neurons) in a knock-in mouse model of HD (zQ175) during electrical field stimulation using real-time imaging of FM 1-43 (a lipophilic dye). We found a significant decrease in bouton density and exocytosis of synaptic vesicles at single presynaptic terminals in cultured striatal neurons. Real-time imaging of VGAT-CypHer5E (a pH sensitive dye conjugated to an antibody against vesicular GABA transporter (VGAT)) for inhibitory synaptic vesicles revealed a reduction in bouton density and exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of HD striatal neurons. Thus, our results suggest that the mutant huntingtin protein decreases bouton density and exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of striatal neurons, causing impaired inhibitory synaptic transmission, eventually leading to the neurodegeneration in the striatum of HD.

6.
RSC Adv ; 13(40): 28160-28164, 2023 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-37753393

RESUMEN

Understanding the precise effects of defects on the photophysical properties of quantum dots (QDs) is essential to their development with near-unity luminescence. Because of the complicated nature of defects in QDs, the origins and detailed roles of the defects still remain rarely understood. In this regard, we used detailed chemical analysis to investigate the effect of surface defects on the optical properties of InP/ZnSe/ZnS QDs by introducing shell defects through controlled trifluoroacetic acid (TFA) etching. TFA treatment on the InP/ZnSe/ZnS QDs partially removed the ZnS shell as well as ligands and reduced the quantum yield by generating energetically deep surface traps. The surface defects of QDs by TFA cause charged trap sites inducing an Auger recombination process with a rate of ca. 200 ps. Based on these results, we proposed possible trap-assisted non-radiative decay pathways between the band-edge state and surface deep traps in InP/ZnSe/ZnS QDs.

7.
J Phys Chem Lett ; 14(21): 4914-4922, 2023 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-37202741

RESUMEN

Myosin X forms an antiparallel dimer and moves processively on actin bundles. How the antiparallel dimer affects the stepping mechanism of myosin X remains elusive. Here, we generated several chimeras using domains of myosin V and X and performed single-molecule motility assays. We found that the chimera containing the motor domain from myosin V and the lever arm and antiparallel coiled-coil domain from myosin X has multiple forward step sizes and moves processively, similar to full-length myosin X. The chimera containing the motor domain and lever arm from myosin X and the parallel coiled-coil from myosin V takes steps of ∼40 nm at lower ATP concentrations but was nonprocessive at higher ATP concentrations. Furthermore, mutant myosin X with four mutations in the antiparallel coiled-coil domain failed to dimerize and was nonprocessive. These results imply that the antiparallel coiled-coil domain is necessary for multiple forward step sizes of myosin X.


Asunto(s)
Miosina Tipo V , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Dominios Proteicos , Dimerización , Adenosina Trifosfato
8.
Front Mol Neurosci ; 15: 1062878, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36466804

RESUMEN

The expanded use of hypothesis-free gene analysis methods in autism research has significantly increased the number of genetic risk factors associated with the pathogenesis of autism. A further examination of the implicated genes directly revealed the involvement in processes pertinent to neuronal differentiation, development, and function, with a predominant contribution from the regulators of synaptic function. Despite the importance of presynaptic function in synaptic transmission, the regulation of neuronal network activity, and the final behavioral output, there is a relative lack of understanding of the presynaptic contribution to the pathology of autism. Here, we will review the close association among autism-related mutations, autism spectrum disorders (ASD) phenotypes, and the altered presynaptic protein functions through a systematic examination of the presynaptic risk genes relating to the critical stages of synaptogenesis and neurotransmission.

9.
Curr Opin Neurobiol ; 76: 102596, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35803103

RESUMEN

Sustained neurotransmission is driven by a continuous supply of synaptic vesicles to the release sites and modulated by synaptic vesicle dynamics. However, synaptic vesicle dynamics in synapses remain elusive because of technical limitations. Recent advances in fluorescence imaging techniques have enabled the tracking of single synaptic vesicles in small central synapses in living neurons. Single vesicle tracking has uncovered a wealth of new information about synaptic vesicle dynamics both within and outside presynaptic terminals, showing that single vesicle tracking is an effective tool for studying synaptic vesicle dynamics. Particularly, single vesicle tracking with high spatiotemporal resolution has revealed the dependence of synaptic vesicle dynamics on the location, stages of recycling, and neuronal activity. This review summarizes the recent findings from single synaptic vesicle tracking in small central synapses and their implications in synaptic transmission and pathogenic mechanisms of neurodegenerative diseases.


Asunto(s)
Sinapsis , Vesículas Sinápticas , Neuronas/fisiología , Terminales Presinápticos/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/fisiología
10.
Biomolecules ; 12(4)2022 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-35454096

RESUMEN

α-synuclein (α-syn) is a presynaptic, lipid-binding protein strongly associated with the neuropathology observed in Parkinson's disease (PD), dementia with Lewy bodies (DLB), and Alzheimer's Disease (AD). In normal physiology, α-syn plays a pivotal role in facilitating endocytosis and exocytosis. Interestingly, mutations and modifications of precise α-syn domains interfere with α-syn oligomerization and nucleation that negatively affect presynaptic vesicular dynamics, protein expressions, and mitochondrial profiles. Furthermore, the integration of the α-syn oligomers into the presynaptic membrane results in pore formations, ion influx, and excitotoxicity. Targeted therapies against specific domains of α-syn, including the use of small organic molecules, monoclonal antibodies, and synthetic peptides, are being screened and developed. However, the prospect of an effective α-syn targeted therapy is still plagued by low permeability across the blood-brain barrier (BBB), and poor entry into the presynaptic axon terminals. The present review proposes a modification of current strategies, which includes the use of novel encapsulation technology, such as lipid nanoparticles, to bypass the BBB and deliver such agents into the brain.


Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Humanos , Liposomas , Nanopartículas , Enfermedad de Parkinson/metabolismo , Terminales Presinápticos/metabolismo , alfa-Sinucleína/metabolismo
11.
Chem Sci ; 13(10): 2965-2970, 2022 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-35382465

RESUMEN

Tracking mitochondrial movement in neurons is an attractive but challenging research field as dysregulation of mitochondrial motion is associated with multiple neurological diseases. To realize accurate and long-term tracking of mitochondria in neurons, we elaborately designed a novel aggregation-induced emission (AIE)-active luminogen, TPAP-C5-yne, where we selected a cationic pyridinium moiety to target mitochondria and employed an activated alkyne terminus to achieve long-term tracking through bioconjugation with amines on mitochondria. For the first time, we successfully achieved the accurate analysis of the motion of a single mitochondrion in live primary hippocampal neurons and the long-term tracking of mitochondria for up to a week in live neurons. Therefore, this new AIEgen can be used as a potential tool to study the transport of mitochondria in live neurons.

12.
Sci Rep ; 11(1): 19889, 2021 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-34615961

RESUMEN

Colloidal quantum-dots (QDs) are highly attractive materials for various optoelectronic applications owing to their easy maneuverability, high functionality, wide applicability, and low cost of mass-production. QDs usually consist of two components: the inorganic nano-crystalline particle and organic ligands that passivate the surface of the inorganic particle. The organic component is also critical for tuning electronic properties of QDs as well as solubilizing QDs in various solvents. However, despite extensive effort to understand the chemistry of ligands, it has been challenging to develop an efficient and reliable method for identifying and quantifying ligands on the QD surface. Herein, we developed a novel method of analyzing ligands in a mild yet accurate fashion. We found that oxidizing agents, as a heterogeneous catalyst in a different phase from QDs, can efficiently disrupt the interaction between the inorganic particle and organic ligands, and the subsequent simple phase fractionation step can isolate the ligand-containing phase from the oxidizer-containing phase and the insoluble precipitates. Our novel analysis procedure ensures to minimize the exposure of ligand molecules to oxidizing agents as well as to prepare homogeneous samples that can be readily analyzed by diverse analytical techniques, such as nuclear magnetic resonance spectroscopy and gas-chromatography mass-spectrometry.

13.
iScience ; 24(10): 103181, 2021 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-34703988

RESUMEN

Although defective synaptic transmission was suggested to play a role in neurodegenerative diseases, the dynamics and vesicle pools of synaptic vesicles during neurodegeneration remain elusive. Here, we performed real-time three-dimensional tracking of single synaptic vesicles in cortical neurons from a mouse model of Huntington's disease (HD). Vesicles in HD neurons had a larger net displacement and radius of gyration compared with wild-type neurons. Vesicles with high release probability (Pr) were interspersed with low-Pr vesicles in HD neurons, whereas high-Pr vesicles were closer to fusion sites than low-Pr in wild-type neurons. Non-releasing vesicles in HD neurons had an abnormally high prevalence of irregular oscillatory motion. These abnormal dynamics and vesicle pools were rescued by overexpressing Rab11, and the abnormal irregular oscillatory motion was rescued by jasplakinolide. Our studies reveal the abnormal dynamics and pools of synaptic vesicles in the early stages of HD, suggesting a possible pathogenic mechanism of neurodegenerative diseases.

14.
Nano Lett ; 21(18): 7479-7485, 2021 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-34491760

RESUMEN

Many biological processes employ mechanisms involving the locations and interactions of multiple components. Given that most biological processes occur in three dimensions, the simultaneous measurement of three-dimensional locations and interactions is necessary. However, the simultaneous three-dimensional precise localization and measurement of interactions in real time remains challenging. Here, we report a new microscopy technique to localize two spectrally distinct particles in three dimensions with an accuracy (2.35σ) of tens of nanometers with an exposure time of 100 ms and to measure their real-time interactions using fluorescence resonance energy transfer (FRET) simultaneously. Using this microscope, we tracked two distinct vesicles containing t-SNAREs or v-SNARE in three dimensions and observed FRET simultaneously during single-vesicle fusion in real time, revealing the nanoscale motion and interactions of single vesicles in vesicle fusion. Thus, this study demonstrates that our microscope can provide detailed information about real-time three-dimensional nanoscale locations, motion, and interactions in biological processes.


Asunto(s)
Fenómenos Biológicos , Transferencia Resonante de Energía de Fluorescencia , Fusión de Membrana , Microscopía , Proteínas SNARE
15.
Lab Chip ; 21(16): 3137-3149, 2021 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-34165117

RESUMEN

For studying protein-protein interactions (PPIs) in general, a powerful and commonly used technique is conventional coimmunoprecipitation (co-IP/pulldown) followed by western blotting. However, the technique does not provide precise information regarding the kinetics and stoichiometry of PPIs. Another drawback is that the sensitivity of conventional co-IP is not suitable for examining PPIs in rare cells such as sensory hair cells, circulating tumor cells, embryonic stem cells, and subsets of immune cells. The current single-molecule pulldown (SiMPull) assay can potentially be used for studying PPIs in rare cells but its wide application is hindered by the high technical barrier and time consumption. We report an innovative, agarose microbead-based approach for SiMPull. We used commercially available, pre-surface-functionalized agarose microbeads to capture the protein of interest together with its binding partners specifically from cell extracts and observed these interactions under a microscope at the single-molecule level. Relative to the original method, microbead-based SiMPull is considerably faster, easier to use, and more reproducible and yet provides similar sensitivity and signal-to-background ratio; specifically, with the new method, sample-preparation time is substantially decreased (from ∼10 to ∼3 h). These crucial features should facilitate wide application of the powerful and versatile SiMPull method in common biological and clinical laboratories. Notably, by exploiting the simplicity and ultrahigh sensitivity of microbead-based SiMPull, we used the method in the study of rare auditory hair cells and γδ T cells for the first time.


Asunto(s)
Proteínas , Western Blotting , Humanos , Inmunoprecipitación , Cinética , Microesferas
16.
Proc Natl Acad Sci U S A ; 118(9)2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33622785

RESUMEN

Maintaining the balance between neuronal excitation and inhibition is essential for proper function of the central nervous system. Inhibitory synaptic transmission plays an important role in maintaining this balance. Although inhibitory transmission has higher kinetic demands compared to excitatory transmission, its properties are poorly understood. In particular, the dynamics and exocytosis of single inhibitory vesicles have not been investigated, due largely to both technical and practical limitations. Using a combination of quantum dots (QDs) conjugated to antibodies against the luminal domain of the vesicular GABA transporter to selectively label GABAergic (i.e., predominantly inhibitory) vesicles together with dual-focus imaging optics, we tracked the real-time three-dimensional position of single GABAergic vesicles up to the moment of exocytosis (i.e., fusion). Using three-dimensional trajectories, we found that GABAergic synaptic vesicles traveled a shorter distance prior to fusion and had a shorter time to fusion compared to synaptotagmin-1 (Syt1)-labeled vesicles, which were mostly from excitatory neurons. Moreover, our analysis revealed that GABAergic synaptic vesicles move more straightly to their release sites than Syt1-labeled vesicles. Finally, we found that GABAergic vesicles have a higher prevalence of kiss-and-run fusion than Syt1-labeled vesicles. These results indicate that inhibitory synaptic vesicles have a unique set of dynamics and exocytosis properties to support rapid synaptic inhibition, thereby maintaining a tightly regulated coordination between excitation and inhibition in the central nervous system.


Asunto(s)
Exocitosis/fisiología , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Neuronas GABAérgicas/metabolismo , Coloración y Etiquetado/métodos , Vesículas Sinápticas/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos/química , Calcio/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Neuronas GABAérgicas/citología , Hipocampo/citología , Hipocampo/metabolismo , Imagenología Tridimensional , Inmunoconjugados/química , Transporte Iónico , Fusión de Membrana/fisiología , Cultivo Primario de Células , Puntos Cuánticos/química , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica , Sinaptotagmina I/química , Sinaptotagmina I/metabolismo
17.
J Phys Chem Lett ; 11(18): 7643-7649, 2020 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-32794712

RESUMEN

How cancer cells respond to different mechanical environments remains elusive. Here, we investigated the tension in single focal adhesions of MDA-MB-231 (metastatic breast cancer cells) and MCF-10A (normal human breast cells) cells on substrates of varying stiffness using single-cell measurements. Tension measurements in single focal adhesions using an improved FRET-based tension sensor showed that the tension in focal adhesions of MDA-MB-231 cells increased on stiffer substrates while the tension in MCF-10A cells exhibited no apparent change against the substrate stiffness. Viscoelasticity measurements using magnetic tweezers showed that the power-law exponent of MDA-MB-231 cells decreased on stiffer substrates whereas MCF-10A cells had similar exponents throughout the whole stiffness, indicating that MDA-MB-231 cells change their viscoelasticity on stiffer substrates. Such changes in tension in focal adhesions and viscoelasticity against the substrate stiffness represent an adaptability of cancer cells in mechanical environments, which can facilitate the metastasis of cancer cells to different tissues.


Asunto(s)
Fenómenos Biomecánicos , Neoplasias de la Mama/patología , Microambiente Celular , Análisis de la Célula Individual , Viscosidad , Adhesión Celular , Línea Celular , Femenino , Transferencia Resonante de Energía de Fluorescencia , Humanos
18.
Artículo en Inglés | MEDLINE | ID: mdl-32081426

RESUMEN

Myosin X (Myo10) has several unique design features including dimerization via an anti-parallel coiled coil and a long lever arm, which allow it to preferentially move on actin bundles. To understand the stepping behavior of single Myo10 on actin bundles, we labeled two heads of Myo10 dimers with different fluorophores. Unlike previously described for myosin V (Myo5) and VI (Myo6), which display alternating hand-over-hand stepping, Myo10 frequently took near simultaneous steps of both heads, and less frequently, 2-3 steps of one head before the other head stepped. We suggest that this behavior results from the unusual kinetic features of Myo10, in conjunction with the structural properties of the motor domain/lever arm, which will favor movement on actin bundles rather than on single filaments.

19.
Biophys J ; 118(1): 70-84, 2020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31818466

RESUMEN

STIM1 (a Ca2+ sensor in the endoplasmic reticulum (ER) membrane) and Orai1 (a pore-forming subunit of the Ca2+-release-activated calcium channel in the plasma membrane) diffuse in the ER membrane and plasma membrane, respectively. Upon depletion of Ca2+ stores in the ER, STIM1 translocates to the ER-plasma membrane junction and binds Orai1 to trigger store-operated Ca2+ entry. However, the motion of STIM1 and Orai1 during this process and its roles to Ca2+ entry is poorly understood. Here, we report real-time tracking of single STIM1 and Orai1 particles in the ER membrane and plasma membrane in living cells before and after Ca2+ store depletion. We found that the motion of single STIM1 and Orai1 particles exhibits anomalous diffusion both before and after store depletion, and their mobility-measured by the radius of gyration of the trajectories, mean-square displacement, and generalized diffusion coefficient-decreases drastically after store depletion. We also found that the measured displacement distribution is non-Gaussian, and the non-Gaussian parameter drastically increases after store depletion. Detailed analyses and simulations revealed that single STIM1 and Orai1 particles are confined in the compartmentalized membrane both before and after store depletion, and the changes in the motion after store depletion are explained by increased confinement and polydispersity of STIM1-Orai1 complexes formed at the ER-plasma membrane junctions. Further simulations showed that this increase in the confinement and polydispersity after store depletion localizes a rapid increase of Ca2+ influx, which can facilitate the rapid activation of local Ca2+ signaling pathways and the efficient replenishing of Ca2+ store in the ER in store-operated Ca2+ entry.


Asunto(s)
Calcio/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Células HEK293 , Humanos , Distribución Normal
20.
J Cell Sci ; 133(1)2020 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-31831524

RESUMEN

Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway that is controlled by the ER Ca2+ sensor STIM1. Abnormal activation of STIM1 directly influences Ca2+ influx, resulting in severe diseases such as Stormorken syndrome. The inactivation domain of STIM1 (IDstim) has been identified as being essential for Ca2+-dependent inactivation of STIM1 (CDI) after SOCE occurs. However, it is unknown whether IDstim is involved in keeping STIM1 inactive before CDI. Herein, we show that IDstim helps STIM1 keep inactive through intramolecular binding with the coiled-coil domain. Between IDstim and the coiled-coil domain, we found a short conserved linker whose extension or mutation leads to the constitutive activation of STIM1. We have demonstrated that IDstim needs the coiled-coil domain 1 (CC1) to inhibit the Ca2+ release-activated Ca2+ (CRAC) activation domain (CAD) activity and binds to a CC1-CAD fragment. Serial deletion of CC1 revealed that CC1α1 is a co-inhibitory domain of IDstim. CC1α1 deletion or leucine mutation, which abolishes the closed conformation, impaired the inhibitory effect and binding of IDstim. These results suggest that IDstim cooperates with CC1α1 to help STIM1 keep inactive under resting conditions.


Asunto(s)
Proteínas de Neoplasias/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Calcio/metabolismo , Células HEK293 , Humanos , Conformación Proteica , Dominios Proteicos
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