首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Regulation of gap junction (GJ) organization is critical for proper function of excitable tissues such as heart and brain, yet mechanisms that govern the dynamic patterning of GJs remain poorly defined. Here, we show that zonula occludens (ZO)-1 localizes preferentially to the periphery of connexin43 (Cx43) GJ plaques. Blockade of the PDS95/dlg/ZO-1 (PDZ)-mediated interaction between ZO-1 and Cx43, by genetic tagging of Cx43 or by a membrane-permeable peptide inhibitor that contains the Cx43 PDZ-binding domain, led to a reduction of peripherally associated ZO-1 accompanied by a significant increase in plaque size. Biochemical data indicate that the size increase was due to unregulated accumulation of gap junctional channels from nonjunctional pools, rather than to increased protein expression or decreased turnover. Coexpression of native Cx43 fully rescued the aberrant tagged-connexin phenotype, but only if channels were composed predominately of untagged connexin. Confocal image analysis revealed that, subsequent to GJ nucleation, ZO-1 association with Cx43 GJs is independent of plaque size. We propose that ZO-1 controls the rate of Cx43 channel accretion at GJ peripheries, which, in conjunction with the rate of GJ turnover, regulates GJ size and distribution.  相似文献   

2.
Double-membrane–spanning gap junction (GJ) channels cluster into two-dimensional arrays, termed plaques, to provide direct cell-to-cell communication. GJ plaques often contain circular, channel-free domains (∼0.05–0.5 μm in diameter) identified >30 y ago and termed nonjunctional membrane (NM) domains. We show, by expressing the GJ protein connexin43 (Cx43) tagged with green fluorescent protein, or the novel photoconvertible fluorescent protein Dendra2, that NM domains appear to be remnants generated by the internalization of small GJ channel clusters that bud over time from central plaque areas. Channel clusters internalized within seconds forming endocytic double-membrane GJ vesicles (∼0.18–0.27 μm in diameter) that were degraded by lysosomal pathways. Surprisingly, NM domains were not repopulated by surrounding channels and instead remained mobile, fused with each other, and were expelled at plaque edges. Quantification of internalized, photoconverted Cx43-Dendra2 vesicles indicated a GJ half-life of 2.6 h that falls within the estimated half-life of 1–5 h reported for GJs. Together with previous publications that revealed continuous accrual of newly synthesized channels along plaque edges and simultaneous removal of channels from plaque centers, our data suggest how the known dynamic channel replenishment of functional GJ plaques can be achieved. Our observations may have implications for the process of endocytic vesicle budding in general.  相似文献   

3.
Direct intercellular communication mediated by gap junctions (GJs) is a hallmark of normal cell and tissue physiology. In addition, GJs significantly contribute to physical cell-cell adhesion. Clearly, these cellular functions require precise modulation. Typically, GJs represent arrays of hundreds to thousands of densely packed channels, each one assembled from two half-channels (connexons), that dock head-on in the extracellular space to form the channel arrays that link neighboring cells together. Interestingly, docked GJ channels cannot be separated into connexons under physiological conditions, posing potential challenges to GJ channel renewal and physical cell-cell separation. We described previously that cells continuously-and effectively after treatment with natural inflammatory mediators-internalize their GJs in an endo-/exocytosis process that utilizes clathrin-mediated endocytosis components, thus enabling these critical cellular functions. GJ internalization generates characteristic cytoplasmic double-membrane vesicles, described and termed earlier annular GJs (AGJs) or connexosomes. Here, using expression of the major fluorescent-tagged GJ protein, connexin 43 (Cx43-GFP/YFP/mApple) in HeLa cells, analysis of endogenously expressed Cx43, ultrastructural analyses, confocal colocalization microscopy, pharmacological and molecular biological RNAi approaches depleting cells of key-autophagic proteins, we provide compelling evidence that GJs, following internalization, are degraded by autophagy. The ubiquitin-binding protein p62/sequestosome 1 was identified in targeting internalized GJs to autophagic degradation. While previous studies identified proteasomal and endo-/lysosomal pathways in Cx43 and GJ degradation, our study provides novel molecular and mechanistic insights into an alternative GJ degradation pathway. Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general.  相似文献   

4.
Direct intercellular communication mediated by gap junctions (GJs) is a hallmark of normal cell and tissue physiology. In addition, GJs significantly contribute to physical cell-cell adhesion. Clearly, these cellular functions require precise modulation. Typically, GJs represent arrays of hundreds to thousands of densely packed channels, each one assembled from two half-channels (connexons), that dock head-on in the extracellular space to form the channel arrays that link neighboring cells together. Interestingly, docked GJ channels cannot be separated into connexons under physiological conditions, posing potential challenges to GJ channel renewal and physical cell-cell separation. We described previously that cells continuously—and effectively after treatment with natural inflammatory mediators—internalize their GJs in an endo-/exocytosis process that utilizes clathrin-mediated endocytosis components, thus enabling these critical cellular functions. GJ internalization generates characteristic cytoplasmic double-membrane vesicles, described and termed earlier annular GJs (AGJs) or connexosomes. Here, using expression of the major fluorescent-tagged GJ protein, connexin 43 (Cx43-GFP/YFP/mApple) in HeLa cells, analysis of endogenously expressed Cx43, ultrastructural analyses, confocal colocalization microscopy, pharmacological and molecular biological RNAi approaches depleting cells of key-autophagic proteins, we provide compelling evidence that GJs, following internalization, are degraded by autophagy. The ubiquitin-binding protein p62/sequestosome 1 was identified in targeting internalized GJs to autophagic degradation. While previous studies identified proteasomal and endo-/lysosomal pathways in Cx43 and GJ degradation, our study provides novel molecular and mechanistic insights into an alternative GJ degradation pathway. Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general.  相似文献   

5.
Gap junctions (GJs) traverse apposing membranes of neighboring cells to mediate intercellular communication by passive diffusion of signaling molecules. We have shown previously that cells endocytose GJs utilizing the clathrin machinery. Endocytosis generates cytoplasmic double-membrane vesicles termed annular gap junctions or connexosomes. However, the signaling pathways and protein modifications that trigger GJ endocytosis are largely unknown. Treating mouse embryonic stem cell colonies – endogenously expressing the GJ protein connexin43 (Cx43) – with epidermal growth factor (EGF) inhibited intercellular communication by 64% and activated both, MAPK and PKC signaling cascades to phosphorylate Cx43 on serines 262, 279/282, and 368. Upon EGF treatment Cx43 phosphorylation transiently increased up to 4-fold and induced efficient (66.4%) GJ endocytosis as evidenced by a 5.9-fold increase in Cx43/clathrin co-precipitation.  相似文献   

6.
Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca(2+) responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell-T cell signaling machinery that regulates T cell activation.  相似文献   

7.
Globally 360 million people have disabling hearing loss and, of these, 32 million are children. Human hearing relies on 15,000 hair cells that transduce mechanical vibrations to electrical signals in the auditory nerve. The process is powered by the endo-cochlear potential, which is produced by a vascularized epithelium that actively transports ions in conjunction with a gap junction (GJ) system. This “battery” is located “off-site” in the lateral wall of the cochlea. The GJ syncytium contains the GJ protein genes beta 2 (GJB2/connexin26 (Cx26)) and 6 (GJB6/connexin30 (Cx30)), which are commonly involved in hereditary deafness. Because the molecular arrangement of these proteins is obscure, we analyze GJ protein expression (Cx26/30) in human cochleae by using super-resolution structured illumination microscopy. At this resolution, the Cx26 and Cx30 proteins were visible as separate plaques, rather than being co-localized in heterotypic channels, as previously suggested. The Cx26 and Cx30 proteins thus seem not to be co-expressed but to form closely associated assemblies of GJ plaques. These results could assist in the development of strategies to treat genetic hearing loss in the future.  相似文献   

8.
9.
In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC. No heterologous GJIC was observed between Cx26- and Cx43-transfected TMEC suggesting that heterotypic GJs do not form or that Cx26/Cx43 channels do not permit dye transfer.  相似文献   

10.
Gap junctions (GJs) are aggregates of channels that provide for direct cytoplasmic connection between cells. Importantly, this connection is thought responsible for cell-to-cell transfer of the cardiac action potential. The GJ channels of ventricular myocytes are composed of connexin43 (Cx43). Interaction of Cx43 with zonula occludens-1 (ZO-1) is localized not only at the GJ plaque, but also to the region surrounding the GJ, the perinexus. Cx43 in the perinexus is not detectable by immunofluorescence, yet localization of Cx43/ZO-1 interaction to this region indicated the presence of Cx43. Therefore, we hypothesized that Cx43 occurs in the perinexus at a lower concentration per unit membrane than in the GJ itself, making it difficult to visualize. To overcome this, the Duolink protein–protein interaction assay was used to detect Cx43. Duolink labeling of cardiomyocytes localized Cx43 to the perinexus. Quantification demonstrated that signal in the perinexus was lower than in the GJ but significantly higher than in nonjunctional regions. Additionally, Duolink of Triton X-100-extracted cultures suggested that perinexal Cx43 is nonjunctional. Importantly, the voltage gated sodium channel Nav1.5, which is responsible for initiation of the action potential, was found to interact with perinexal Cx43 but not with ZO-1. This work provides a detailed characterization of the structure of the perinexus at the GJ edge and indicates that one of its potential functions in the heart may be in facilitating conduction of action potential.  相似文献   

11.
Follicle-stimulating hormone is the major regulator of growth and development of antral follicles in the ovary. Granulosa cells (GCs) in these follicles are coupled via gap junctions (GJs) consisting of connexin 43 (Cx 43). Because we and others have found that Cx 43 and GJs, respectively, are more abundant in large antral follicles compared with small antral and preantral follicles, we hypothesized that FSH may control Cx 43 gene expression, GJ formation, and intercellular communication. To directly address these points, we chose a rat GC line (GFSHR-17) expressing the FSH receptor and the Cx 43 gene. The functionality of FSH receptors was shown by the effects of porcine FSH, namely cell rounding, reduced cellular proliferation, and stimulation of progesterone production of GFSHR-17 cells, which are effects that were detectable within hours. Treatment with FSH also statistically significantly increased Cx 43 mRNA levels, as shown after 6 to 9 h in Northern blots. These effects were antedated by altered GJ communication, which was observed within seconds. Using a single-cell/whole-cell patch clamp technique, we showed that FSH rapidly and reversibly enhanced electrical cell coupling of GFSHR-17 cells. Increased GJ communication was associated with statistically significantly decreased phosphorylation of Cx 43, which was observed within 10 min after FSH addition, during immunoprecipitation experiments. Our results demonstrate, to our knowledge for the first time, that the gonadotropin FSH acutely and directly stimulates intercellular communication of GFSHR-17 cells through existing GJs. Moreover, FSH also increases levels of Cx 43 mRNA. These changes are associated with reduced proliferation and enhanced differentiation of GFSHR-17 cells. In vivo factors in addition to FSH may be involved in the regulation of GJ/GJ communication between GCs in the follicle, but our results suggest that improved cell-to-cell coupling, enhanced Cx 43 gene expression, and possibly, formation of new GJs are direct consequences of FSH receptor activation and may antedate and/or initiate the pivotal effects of FSH on GCs.  相似文献   

12.
Connexin 43 (Cx43) is a major gap junction (GJ) protein found in many mammalian cell types. The C-terminal (CT) domain of Cx43 has unique characteristics in terms of amino acid (aa) sequence and its length differs from other connexins. This CT domain can be associated with protein partners to regulate GJ assembly and degradation, which results in the direct control of gap junction intercellular communication (GJIC). However, the essential roles of the CT regions involved in these mechanisms have not been fully elucidated. In this study, we aimed to investigate the specific regions of Cx43CT involved in GJ formation and internalization. Wild type Cx43((382aa)) and 10 CT truncated mutants were stably expressed in HeLa cells as GFP or DsRed tagged proteins. First, we found that the deletion of 235-382aa from Cx43 resulted in failure to make GJ and establish GJIC. Second, the Cx43 with 242-382aa CT deletion could form functional GJs and be internalized as annular gap junctions (AGJs). However, the plaques consisting of Cx43 with CT deletions (Δ242-382aa to Δ271-382aa) were longer than the plaques consisting of Cx43 with CT deletions (Δ302-382aa). Third, co-culture experiments of cells expressing wild type Cx43((382)) with cells expressing Cx43CT mutants revealed that the directions of GJ internalization were dependent on the length of the respective CT. Moreover, a specific region, 325-342aa residues of Cx43, played an important role in the direction of GJ internalization. These results showed the important roles of the Cx43 C-terminus in GJ expression and its turnover.  相似文献   

13.
Gap junction (GJ) channels are formed by two hemichannels (connexons), each contributed by the cells taking part in this direct cell-cell communication conduit. Hemichannels that do not interact with their counterparts on neighboring cells feature as a release pathway for small paracrine messengers such as nucleotides, glutamate, and prostaglandins. Connexins are phosphorylated by various kinases, and we compared the effect of various kinase-activating stimuli on GJ channels and hemichannels. Using peptides identical to a short connexin (Cx) amino acid sequence to specifically block hemichannels, we found that protein kinase C, Src, and lysophosphatidic acid (LPA) inhibited GJs and hemichannel-mediated ATP release in Cx43-expressing C6 glioma cells (C6-Cx43). Lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) inhibited GJs, but they stimulated ATP release via hemichannels in C6-Cx43. LPS and bFGF inhibited hemichannel-mediated ATP release in HeLa-Cx43 cells, but they stimulated it in HeLa-Cx43 with a truncated carboxy-terminal (CT) domain or in HeLa-Cx26, which has a very short CT. Hemichannel potentiation by LPS was inhibited by blockers of the arachidonic acid metabolism, and arachidonic acid had a potentiating effect like LPS and bFGF. We conclude that GJ channels and hemichannels display similar or oppositely directed responses to modulatory influences, depending on the balance between kinase activity and the activity of the arachidonic acid pathway. Distinctive hemichannel responses to pathological stimulation with LPS or bFGF may serve to optimize the cell response, directed at strictly controlling cellular ATP release, switching from direct GJ communication to indirect paracrine signaling, or maximizing cell-protective strategies.  相似文献   

14.
Gap junctions (GJs) exhibit a complex modus of assembly and degradation to maintain balanced intercellular communication (GJIC). Several growth factors, including vascular endothelial growth factor (VEGF), have been reported to disrupt cell–cell junctions and abolish GJIC. VEGF directly stimulates VEGF-receptor tyrosine kinases on endothelial cell surfaces. Exposing primary porcine pulmonary artery endothelial cells (PAECs) to VEGF for 15 min resulted in a rapid and almost complete loss of connexin43 (Cx43) GJs at cell–cell appositions and a concomitant increase in cytoplasmic, vesicular Cx43. After prolonged incubation periods (60 min), Cx43 GJs reformed and intracellular Cx43 were restored to levels observed before treatment. GJ internalization correlated with efficient inhibition of GJIC, up to 2.8-fold increased phosphorylation of Cx43 serine residues 255, 262, 279/282, and 368, and appeared to be clathrin driven. Phosphorylation of serines 255, 262, and 279/282 was mediated by MAPK, whereas serine 368 phosphorylation was mediated by PKC. Pharmacological inhibition of both signaling pathways significantly reduced Cx43 phosphorylation and GJ internalization. Together, our results indicate that growth factors such as VEGF activate a hierarchical kinase program—including PKC and MAPK—that induces GJ internalization via phosphorylation of well-known regulatory amino acid residues located in the Cx43 C-terminal tail.  相似文献   

15.
Human spiral ganglion (SG) neurons show remarkable survival properties and maintain electric excitability for a long time after complete deafness and even separation from the organ of Corti, features essential for cochlear implantation. Here, we analyze and compare the localization and distribution of gap junction (GJ) intercellular channels and connexin 43 (Cx43) in cells surrounding SG cell bodies in man and guinea pig by using transmission electron microscopy and confocal immunohistochemistry. GJs and Cx43 expression has been recognized in satellite glial cells (SGCs) in non-myelinating sensory ganglia including the human SG. In man, SG neurons can survive as mono-polar or “amputated” cells with unbroken central projections following dendrite degeneration and consolidation of the dendrite pole. Cx43-mediated GJ signaling between SGCs is believed to play a key role in this “healing” process and could explain the unique preservation of human SG neurons and the persistence of cochlear implant function.  相似文献   

16.
Cells within the vast majority of human tissues communicate directly through clustered arrays of intercellular channels called gap junctions. Gene ablation studies in mouse models have revealed that these intercellular channels are necessary for a variety of organ functions and that some of these genes are essential for survival. Molecular genetics has uncovered that germ line mutations in nearly half of the genes that encode the 21-member connexin family of gap junction proteins are linked to one or more human diseases. Frequently, these mutations are autosomal recessive, whereas in other cases, autosomal dominant mutations manifest as disease. Given the broad and overlapping distribution of connexins in a wide arrangement of tissues, it is hard to predict where connexin-linked diseases will clinically manifest. For instance, the most prevalent connexin in the human body is connexin-43 (Cx43), yet autosomal dominant mutations in the GJA1 gene, which encodes Cx43, exhibit modest developmental disorders resulting in a disease termed oculodentodigital dysplasia. Autosomal recessive mutations in the gene encoding Cx26 result in moderate to severe sensorineural hearing loss, whereas autosomal dominant mutations produce hearing loss and a wide range of skin diseases, including palmoplantar keratoderma. Here, we will focus on autosomal dominant mutations of the genes encoding Cx26 and Cx43 in relation to models that link genotypes to phenotypic outcomes with particular reference to how these approaches provide insight into human disease.  相似文献   

17.
Gap junction (GJ) channels assembled from connexin (Cx) proteins provide a structural basis for direct electrical and metabolic cell-cell communication. Here, we focus on gating and permeability properties of Cx43/Cx45 heterotypic GJs exhibiting asymmetries of both voltage-gating and transjunctional flux (J(j)) of fluorescent dyes depending on transjunctional voltage (V(j)). Relatively small differences in the resting potential of communicating cells can substantially reduce or enhance this flux at relative negativity or positivity on Cx45 side, respectively. Similarly, series of V(j) pulses resembling bursts of action potentials (APs) reduce J(j) when APs initiate in the cell expressing Cx43 and increase J(j) when APs initiate in the cell expressing Cx45. J(j) of charged fluorescent dyes is affected by ionophoresis and V(j)-gating and the asymmetry of J(j)-V(j) dependence in heterotypic GJs is enhanced or reduced when ionophoresis and V(j)-gating work in a synergistic or antagonistic manner, respectively. Modulation of cell-to-cell transfer of metabolites and signaling molecules by V(j) may occur in excitable as well as non-excitable tissues and may be more expressed in the border between normal and pathological regions where intercellular gradients of membrane potential and concentration of ions are substantially altered. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.  相似文献   

18.
Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.  相似文献   

19.
Connexin 43 (Cx43) is a gap junction (GJ) protein widely expressed in mammalian tissues that mediates cell-to-cell coupling. Intercellular channels comprising GJ aggregates form from docking of paired connexons, with one each contributed by apposing cells. Zonula occludens-1 (ZO-1) binds the carboxy terminus of Cx43, and we have previously shown that inhibition of the Cx43/ZO-1 interaction increases GJ size by 48 h. Here we demonstrated that increases in GJ aggregation occur within 2 h (~Cx43 half-life) following disruption of Cx43/ZO-1. Immunoprecipitation and Duolink protein-protein interaction assays indicated that inhibition targets ZO-1 binding with Cx43 in GJs as well as connexons in an adjacent domain that we term the "perinexus." Consistent with GJ size increases being matched by decreases in connexons, inhibition of Cx43/ZO-1 reduced the extent of perinexal interaction, increased the proportion of connexons docked in GJs relative to undocked connexons in the plasma membrane, and increased GJ intercellular communication while concomitantly decreasing hemichannel-mediated membrane permeance in contacting, but not noncontacting, cells. ZO-1 small interfering RNA and overexpression experiments verified that loss and gain of ZO-1 function govern the transition of connexons into GJs. It is concluded that ZO-1 regulates the rate of undocked connexon aggregation into GJs, enabling dynamic partitioning of Cx43 channel function between junctional and proximal nonjunctional domains of plasma membrane.  相似文献   

20.
Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号