Cx43在人胚胎早期食管上皮组织中的表达

目的:探讨间隙连接蛋白Cx43在人胚胎早期食管上皮组织中的表达规律.方法:应用免疫组织化学SABC法检测第2、3、4三个月龄段人胚胎食管上皮层Cx43蛋白的表达.结果:第2～4个月龄段,食管上皮层均有Cx43蛋白阳性的细胞分布.第2个月胚龄段,Cx43蛋白在人胚胎食管上皮基底层呈强阳性表达,由基底层向管腔面,细胞阳性强度逐渐降低;第3个月胎龄段,Cx43蛋白阳性细胞在食管上皮层广泛分布;第4个月胎龄段,Cx43蛋白在食管上皮层近基底面部分细胞呈弱阳性表达.结论:Cx43蛋白在人胚胎早期食管上皮层细胞的生长发育过程中起重要的作用.     

Apoptosis in the developing zebrafish embryo.

Apoptosis is a major part of the normal development of many organ systems and tissues. The zebrafish (Danio rerio) has become a useful model for studying early development, and recent advances in techniques used to label apoptotic cells have made it possible to visualize apoptotic cells in this model system. We have used the in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) to describe the temporal and spatial distribution of apoptotic cells during normal development of the zebrafish embryo from 12 to 96 h postfertilization. By counting labeled apoptotic cells, we have demonstrated transient high rates of cell death in various structures during development, and we have correlated these peaks with previously described developmental changes in these structures. Our analysis has focused on the nervous system and associated sensory organs including the olfactory organ, retina, lens, cornea, otic vesicle, lateral line organs, and Rohon-Beard neurons. Apoptosis is also described in other non-neural structures such as the notochord, somites, muscle, tailbud, and fins.     

Cx43/beta-gal inhibits Cx43 transport in the Golgi apparatus

A connexin construct consisting of bacterial beta-galactosidase fused to the C-terminus of connexin43 (Cx43/beta-gal) was used to examine Cx43 assembly in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool trapped by Cx43/beta-gal was retained in a compartment that co-localized with a medial Golgi apparatus marker by immunofluorescence microscopy and that was readily disassembled by treatment with brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a sub-hexameric complex, and that Cx43/beta-gal expression also inhibited Cx43 assembly into hemichannels. While this is consistent with Cx43 hemichannel assembly in the trans Golgi network (TGN), these data also suggest that the dominant negative effect of Cx43/beta-gal on Cx43 trafficking may reflect a putative sub-hexameric assembly intermediate formed in the Golgi apparatus.     

Impaired Cx43 gap junction endocytosis causes morphological and functional defects in zebrafish

Gap junctions mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting the exchange of molecules, ions, and electrical impulses. Gap junctions are assembled from connexin (Cx) proteins, with connexin 43 (Cx43) being the most ubiquitously expressed and best studied. While the molecular events that dictate the Cx43 life cycle have largely been characterized, the unusually short half-life of Cxs of only 1–5 h, resulting in constant endocytosis and biosynthetic replacement of gap junction channels, has remained puzzling. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256–289), a region known to encode key residues regulating gap junction turnover, is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43^∆256-289) and a zebrafish model (cx43^lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication. Increased Cx43 protein content in cx43^lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques, and longer Cx43 protein half-lives coincide with severely impaired development. Our findings demonstrate for the first time that continuous Cx43 gap junction endocytosis is an essential aspect of gap junction function and, when impaired, gives rise to significant physiological problems as revealed here for cardiovascular development and function.     

Heterotypic docking of Cx43 and Cx45 connexons blocks fast voltage gating of Cx43

Immunohistochemical co-localization of distinct connexins (Cxs) in junctional areas suggests the formation of heteromultimeric channels. To determine the docking effects of the heterotypic combination of Cx43 and Cx45 on the voltage-gating properties of their channels, we transfected DNA encoding Cx43 or Cx45 into N2A neuroblastoma or HeLa cells. Using a double whole-cell voltage-clamp technique, we determined macroscopic and single-channel gating properties of the intercellular channels formed. Cx43-Cx45 heterotypic channels had rectifying properties where Cx45 connexons inactivated rapidly upon hyperpolarizing voltage pulses applied to the Cx45-expressing cell. During depolarizing pulses to the Cx45-expressing cell, Cx43 connexons inactivated with substantially reduced kinetics as compared with homotypic Cx43 channels. Similar slow kinetics was observed for homotypic Cx43M257 (truncation mutant). Heterotypic channels had a main conductance whose value was predicted by the sum of corresponding homomeric connexon conductances; it was not voltage dependent and had no detectable residual conductance. The voltage-gating kinetics of heterotypic channels and their single-channel behavior implicate a role for the Cx43 carboxyl-terminal domain in the fast gating mechanism and in the establishment of residual conductance. Our results also suggest that heterotypic docking may lead to conformational changes that inhibit this action of the Cx43 carboxyl-terminal domain.     

Phenylthiourea disrupts thyroid function in developing zebrafish

Thyroid hormone (T4) can be detected in thyroid follicles in wild-type zebrafish larvae from 3 days of development, when the thyroid has differentiated. In contrast, embryos or larvae treated with goitrogens (substances such as methimazole, potassium percholorate, and 6-n-propyl-2-thiouracil) are devoid of thyroid hormone immunoreactivity.Phenythiourea (PTurea; also commonly known as PTU) is widely used in zebrafish research to suppress pigmentation in developing embryos/fry. PTurea contains a thiocarbamide group that is responsible for goitrogenic activity in methimazole and 6-n-propyl-2-thiouracil. In the present study, we show that commonly used doses of 0.003% PTurea abolish T4 immunoreactivity of the thyroid follicles of zebrafish larvae. As development of the thyroid gland is not affected, these data suggest that PTurea blocks thyroid hormone production. Like other goitrogens, PTurea causes delayed hatching, retardation and malformation of embryos or larvae with increasing doses. At doses of 0.003% PTurea, however, toxic side effects seem to be at a minimum, and the maternal contribution of the hormone might compensate for compromised thyroid function during the first days of development.     

Whole-somite rotation generates muscle progenitor cell compartments in the developing zebrafish embryo

Somites are transient, mesodermally derived structures that give rise to a number of different cell types within the vertebrate embryo. To achieve this, somitic cells are partitioned into lineage-restricted domains, whose fates are determined by signals secreted from adjacent tissues. While the molecular nature of many of the inductive signals that trigger formation of different cell fates within the nascent somite has been identified, less is known about the processes that coordinate the formation of the subsomitic compartments from which these cells arise. Utilizing a combination of vital dye-staining and lineage-tracking techniques, we describe a previously uncharacterized, lineage-restricted compartment of the zebrafish somite that generates muscle progenitor cells for the growth of appendicular, hypaxial, and axial muscles during development. We also show that formation of this compartment occurs via whole-somite rotation, a process that requires the action of the Sdf family of secreted cytokines.     

Cx43/β-Gal Inhibits Cx43 Transport in the Golgi Apparatus

A connexin construct consisting of bacterial β-galactosidase fused to the C-terminus of connexin43 (Cx43/β-gal) was used to examine Cx43 assembly in NIH 3T3 cells. Cx43/β-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool trapped by Cx43/β-gal was retained in a compartment that co-localized with a medial Golgi apparatus marker by immunofluorescence microscopy and that was readily disassembled by treatment with brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/β-gal were assembled into a sub-hexameric complex, and that Cx43/β-gal expression also inhibited Cx43 assembly into hemichannels. While this is consistent with Cx43 hemichannel assembly in the trans Golgi network (TGN), these data also suggest that the dominant negative effect of Cx43/β-gal on Cx43 trafficking may reflect a putative sub-hexameric assembly intermediate formed in the Golgi apparatus.     

N-cadherin and Cx43alpha1 gap junctions modulates mouse neural crest cell motility via distinct pathways

Our previous studies showed an essential role for connexin 43 or alpha1 connexin (Cx43alpha1) gap junctions in the modulation of neural crest cell motility. Cx43alpha1 gap junctions and N-cadherin containing adherens junctions are expressed in migrating cardiac neural crest cells. Analysis of the N-cadherin knockout (KO) mouse model revealed that N-cadherin is essential for gap junction mediated dye coupling but not for expression of Cx43alpha1 gap junctions in neural crest cells. Time lapse videomicroscopy and motion analysis showed that the motility of N-cadherin KO neural crest cells were altered, but the motility changes differed compared to Cx43alpha1 KO neural crest cells. These observations suggest that the role of N-cadherin in cell motility is not simply mediated via the modulation of Cx43alpha1 mediated cell-cell communication. This was confirmed by a parallel analysis of wnt-1 deficient neural crest cells, which also showed a reduction in dye coupling, and yet no change in cell motility. Analysis of p120 catenin (p120ctn), an Amardillo family protein known to play a role in cell motility, showed that it is colocalized with N-cadherin and Cx43alpha1 in migrating neural crest cells. This subcellular distribution was altered in the N-cadherin and Cx43alpha1 KO neural crest cells. Given these results, we propose that N-cadherin and Cx43alpha1 may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.     

The proteasome regulates the interaction between Cx43 and ZO-1

Gap junction (GJ) intercellular communication (GJIC) is vital to ensure proper cell and tissue function. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. In this study we have investigated the role of the proteasome in regulation of connexin 43 (Cx43) internalization. Although the participation of both the proteasome and lysosome has long been suggested in Cx43 degradation, the molecular mechanisms whereby proteasome contributes to regulate Cx43 internalization and intercellular communication are still unclear. The results presented in this study envision a new mechanism whereby proteasome regulates GJIC by modulating interaction between Cx43 and ZO-1. Immunoprecipitation experiments, in the presence of proteasome inhibitors, together with immunofluorescence data indicate that the proteasome regulates interaction between Cx43 and ZO-1. Overexpression of the PDZ2 domain of ZO-1 and the expression of Cx-43 fused in frame with a V5/HIS tag, suggest that interaction between the two proteins occurs through the PDZ2 domain of ZO-1 and the C-terminus of Cx43. When interaction between Cx43 and ZO-1 is reduced, as in the presence of proteasome inhibitors, Cx43 accumulates, forming large GJ plaques at plasma membrane. Data presented in this article suggest a new pathway whereby alterations in proteasome activity may impact on GJIC as well as on non-junctional communication with extracellular environment, contributing to cell and tissue dysfunction.     

Lens connexins alpha3Cx46 and alpha8Cx50 interact with zonula occludens protein-1 (ZO-1)

Connexin alpha1Cx43 has previously been shown to bind to the PDZ domain-containing protein ZO-1. The similarity of the carboxyl termini of this connexin and the lens fiber connexins alpha3Cx46 and alpha8Cx50 suggested that these connexins may also interact with ZO-1. ZO-1 was shown to be highly expressed in mouse lenses. Colocalization of ZO-1 with alpha3Cx46 and alpha8Cx50 connexins in fiber cells was demonstrated by immunofluorescence and by fracture-labeling electron microscopy but showed regional variations throughout the lens. ZO-1 was found to coimmunoprecipitate with alpha3Cx46 and alpha8Cx50, and pull-down experiments showed that the second PDZ domain of ZO-1 was involved in this interaction. Transiently expressed alpha3Cx46 and alpha8Cx50 connexins lacking the COOH-terminal residues did not bind to the second PDZ domain but still formed structures resembling gap junctions by immunofluorescence. These results indicate that ZO-1 interacts with lens fiber connexins alpha3Cx46 and alpha8Cx50 in a manner similar to that previously described for alpha1Cx43. The spatial variation in the interaction of ZO-1 with lens gap junctions is intriguing and is suggestive of multiple dynamic roles for this association.     

Aggregated DsRed-Tagged Cx43 and Over-Expressed Cx43 are Targeted to Lysosomes in Human Breast Cancer Cells

To investigate if either wild-type or aggregated Cx43 is abnormally targeted to lysosomes in human breast tumor cells, we examined the fate of DsRed-tagged Cx43 and over-expressed Cx43 in communication-deficient HBL-100 and MDA-MB-231 cells. DsRed-tagged Cx43 was assembled into gap junctions in control normal rat kidney cells that express endogenous Cx43 but not in Cx43-negative HBL-100 cells. However, when HBL-100 cells were engineered to coexpress wild-type Cx43 a population of DsRed-tagged Cx43 was rescued and assembled into gap junctions. Co-expression of wild-type Cx26 failed to rescue the assembly of DsRed-tagged Cx43 into gap junctions. Immunolocalization studies revealed that DsRed-tagged Cx43 was aggregated and partially localized to lysosomes. Interestingly, when human MDA-MB-231 breast tumor cells over-expressed wild-type Cx43, Cx43 protein primarily localized to lysosomes. Together, these studies provide evidence for Cx43 being targeted to lysosomes as a result of misfolding and aggregation, while in other cases, the delivery of wild-type Cx43 to lysosomes appears to be due to defects innate to the breast tumor cell type.     

The Noncanonical Functions of Cx43 in the Heart

There is abundant evidence showing that connexins form gap junctions. Yet this does not exclude the possibility that connexins can exert other functions, separate from that of gap junction (or even a permeable hemichannel) formation. Here, we focus on these noncanonical functions of connexin43 (Cx43), particularly in the heart. We describe two specific examples: the importance of Cx43 on intercellular adhesion, and the role of Cx43 in the function of the sodium channel. We propose that these two functions of Cx43 have important repercussions on the propagation of electrical activity in the heart, irrespective of the presence of permeable gap junction channels. Overall, the gap junction–independent functions of Cx43 in cardiac electrophysiology emerge as an exciting area of future research.     

Aggregated DsRed-tagged Cx43 and over-expressed Cx43 are targeted to lysosomes in human breast cancer cells

To investigate if either wild-type or aggregated Cx43 is abnormally targeted to lysosomes in human breast tumor cells, we examined the fate of DsRed-tagged Cx43 and over-expressed Cx43 in communication-deficient HBL-100 and MDA-MB-231 cells. DsRed-tagged Cx43 was assembled into gap junctions in control normal rat kidney cells that express endogenous Cx43 but not in Cx43-negative HBL-100 cells. However, when HBL-100 cells were engineered to coexpress wild-type Cx43 a population of DsRed-tagged Cx43 was rescued and assembled into gap junctions. Co-expression of wild-type Cx26 failed to rescue the assembly of DsRed-tagged Cx43 into gap junctions. Immunolocalization studies revealed that DsRed-tagged Cx43 was aggregated and partially localized to lysosomes. Interestingly, when human MDA-MB-231 breast tumor cells over-expressed wild-type Cx43, Cx43 protein primarily localized to lysosomes. Together, these studies provide evidence for Cx43 being targeted to lysosomes as a result of misfolding and aggregation, while in other cases, the delivery of wild-type Cx43 to lysosomes appears to be due to defects innate to the breast tumor cell type.     

Inhibition of protocadherin-alpha function results in neuronal death in the developing zebrafish

The pcdhα/CNR gene comprises a diverse array of neuronal cell-surface proteins of the cadherin superfamily, although very little is known about their role in neural development. Here we provide the first in-depth characterization of pcdh1α in zebrafish. Whole-mount immunocytochemistry demonstrates that a large proportion of endogenous cytoplasmic domain immunoreactivity is present in the nucleus, suggesting that endoproteolytic cleavage and nuclear translocation of the intracellular domain are important aspects of pcdh1α activity in vivo. Using whole-mount immunocytochemistry and BAC-based expression of Pcdh1α-GFP fusion proteins, we find that Pcdh1α does not appear to form stable, synaptic puncta at early stages of synaptogenesis. We also demonstrate that the presence of the Pcdh1α cytoplasmic domain is essential for normal function. Truncation of Pcdh1α proteins, using splice-blocking antisense morpholinos to prevent the addition of the common intracellular domain to the entire pcdh1α cluster, results in neuronal apoptosis throughout the developing brain and spinal cord, demonstrating an essential role for pcdh1α in early neural development. This cell death phenotype can be attenuated by the expression of a soluble Pcdh1α cytoplasmic domain.     

不同类型幽门螺杆菌对GES-1细胞Cx32、Cx43表达的影响

目的观察不同类型幽门螺杆菌（Helicobacter pylori,H.pylori）对人胃黏膜上皮细胞系GES-1细胞间隙连接蛋白（Connexin,Cx）32和43表达的影响,探讨与Cx32、Cx43表达异常有关的H.pylori菌株类型。方法将临床培养分离的不同H.pylori菌株类型包括东亚型CagA＋H.pylori、西方型CagA＋H.pylori及CagA-H.pylori与GES-1细胞共培养24 h及48 h,对照组不加H.pylori培养24 h及48 h。采用间接免疫荧光方法（IIF）及计算机图像分析技术检测GES-1细胞Cx32、Cx43表达。结果对照组24 h和48 h及加H.pylori各组24 h GES-1细胞Cx32、Cx43表达阳性率均为100%,东亚型CagA＋H.pylori组48 h Cx32、Cx43表达阳性率均低于对照组、CagA-H.pylori组和西方型CagA＋H.pylori组（P〈0.05）;对照组24 h和48 h Cx32、Cx43绿色荧光位于细胞膜,西方型CagA＋H.pylori组和东亚型CagA＋H.pylori组24 h和48 h Cx32绿色荧光大部分位于细胞膜,少部分位于细胞浆,Cx43绿色荧光大部分位于细胞浆,少部分位于细胞膜;东亚型CagA＋H.pylori组和西方型CagA＋H.pylori组24 h及48 hCx32、Cx43表达强度低于对照组和CagA-H.pylori组（P〈0.05）,且东亚型CagA＋H.pylori组较西方型CagA＋H.py-lori组减弱更明显（P〈0.05）。结论 H.pylori下调GES-1细胞Cx32、Cx43表达,以CagA＋H.pylori菌株特别是东亚型CagA＋H.pylori菌株作用更明显。