Abstract 300: The Actin Cytoskeleton Confers Specificity of Cx43 Delivery to Intercalated Discs

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Shan-Shan Zhang ◽  
SoonGweon Hong ◽  
Luke P Lee ◽  
Robin M Shaw
Keyword(s):  
Connexin 43 ◽  
Single Particle Tracking ◽  
Cell Junction ◽  
Cell Border ◽  
Intercalated Discs ◽  
Biochemical Interaction ◽  
Junction Protein ◽  
Latrunculin A ◽  
Long Fibers ◽  
Cell Cell

Connexin 43 (Cx43) gap junctions (GJs) electrically couple ventricular cardiomyocytes at the intercalated disc (ID), orchestrating organized organ level contraction with each heartbeat. Disease-related disruption of the Cx43 cytoskeletal trafficking machinery is associated with mislocalization of the Cx43 gap junction protein away from the ID and lethal arrhythmias. We recently found that the majority of intracellular Cx43 cargo is associated with actin, not microtubules, and is either paused or moving slowly. It is not understood why actin is involved in Cx43 trafficking. Using micropatterned HeLa cell pairs and whole-cell automated single particle tracking algorithms, we detected that distinct actin polarity exists in the cell, including highly oriented long fibers associated with fast-moving Cx43 cargo aligned toward actively forming GJ plaques. F-actin disruption with latrunculin A (LatA) leads to a loss of Cx43 cargo directionality toward the cell-cell border, as well as a marked decrease in overall microtubule length. We also found a LatA-dependent biochemical interaction between β-actin and the microtubule plus-end-binding protein EB1, which leads growing microtubules and is a necessary component of the Cx43 forward trafficking machinery. In live cell pairs, F-actin disruption resulted in a decrease in overall EB1 activity and in the number of fully extended microtubules that reach the cell-cell border. Together, these results indicate that actin contributes to the specificity of Cx43 delivery by directing EB1-based microtubule growth toward the cell-cell junction (Please refer to attached diagram).

scholarly journals Mechanical stability of αT-catenin and its activation by force for vinculin binding

2019 ◽  
Vol 30 (16) ◽  
pp. 1930-1937 ◽  
Author(s):  
Si Ming Pang ◽  
Shimin Le ◽  
Adam V. Kwiatkowski ◽  
Jie Yan
Keyword(s):  
Cell Adhesion ◽  
Binding Site ◽  
Connexin 43 ◽  
Mechanical Stability ◽  
Critical Factor ◽  
Mechanical Stretch ◽  
Cell Junction ◽  
Unique Cell ◽  
Actomyosin Contraction ◽  
Cell Cell

αT (Testes)-catenin, a critical factor regulating cell–cell adhesion in the heart, directly couples the cadherin-catenin complex to the actin cytoskeleton at the intercalated disk (ICD), a unique cell–cell junction that couples cardiomyocytes. Loss of αT-catenin in mice reduces plakophilin2 and connexin 43 recruitment to the ICD. Since αT-catenin is subjected to mechanical stretch during actomyosin contraction in cardiomyocytes, its activity could be regulated by mechanical force. To provide insight in how force regulates αT-catenin function, we investigated the mechanical stability of the putative, force-sensing middle (M) domain of αT-catenin and determined how force impacts vinculin binding to αT-catenin. We show that 1) physiological levels of force, <15 pN, are sufficient to unfold the three M domains; 2) the M1 domain that harbors the vinculin-binding site is unfolded at ∼6 pN; and 3) unfolding of the M1 domain is necessary for high-affinity vinculin binding. In addition, we quantified the binding kinetics and affinity of vinculin to the mechanically exposed binding site in M1 and observed that αT-catenin binds vinculin with low nanomolar affinity. These results provide important new insights into the mechanosensing properties of αT-catenin and how αT-catenin regulates cell–cell adhesion at the cardiomyocyte ICD.

Abstract 307: GJA1-20k Regulates Actin Cytoskeleton and Promotes Delivery of Connexin 43 to Cardiac Intercalated Discs

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Wassim A Basheer ◽  
Shaohua Xiao ◽  
Shan-Shan Zhang ◽  
Irina Epifantseva ◽  
Ying Fu ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Targeted Delivery ◽  
Connexin 43 ◽  
Therapeutic Option ◽  
Ischemia Reperfusion ◽  
Full Length ◽  
Cell Junctions ◽  
Acute Ischemia ◽  
Intercalated Discs ◽  
Cell Cell

Rationale: Delivery of connexin 43 (Cx43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubules and actin cytoskeleton rest stops, Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has recently been shown that an internally translated isoform of Cx43, GJA1-20k, facilitates full-length Cx43 trafficking in cell lines, although the mechanism remains unknown. Objective: We explored the mechanism by which GJA1-20k regulates the trafficking of full-length Cx43 to intercalated discs. Methods and Results: In vivo overexpression of exogenous GJA1-20k, administered via AAV9-mediated gene delivery, increases the delivery of full length Cx43 to intercalated discs in mouse hearts. Using electron microscopy and fluorescence microscopy, together with biochemical co-immunoprecipitation, we found in micro-patterned HeLa cells and cardiomyocytes that GJA1-20k not only substantially increases the number and length of actin fibers, but can also rescue the effect of actin disruption. GJA1-20k complexes with actin and tubulin, improving microtubule targeting to cell-cell borders in the setting of actin disruption. Actin is also disrupted in acute ischemia-reperfusion (IR) injury. The ex vivo rescue potential of GJA1-20k was further tested in mouse hearts subjected to myocardial IR injury. As compared to control GFP and full length GAJ1-43k, only GJA1-20k gene transfer significantly improves the targeting of Cx43 to intercalated discs following IR injury. Conclusions: These results indicate that GJA1-20k positively modulates actin cytoskeleton to facilitate microtubule-based Cx43 trafficking machinery, promoting the delivery of full-length Cx43 to cardiac cell-cell junctions under normal and ischemic conditions. Therefore, up regulation of GJA1-20k is a potential therapeutic option to reverse the loss of Cx43 in IR injuries.

scholarly journals Cortactin Associates with the Cell-Cell Junction Protein ZO-1 in bothDrosophilaand Mouse

1998 ◽  
Vol 273 (45) ◽  
pp. 29672-29677 ◽  
Author(s):  
Takanori Katsube ◽  
Manabu Takahisa ◽  
Ryu Ueda ◽  
Naoko Hashimoto ◽  
Mieko Kobayashi ◽  
...  
Keyword(s):  
Cell Junction ◽  
Junction Protein ◽  
Cell Cell

scholarly journals The Herpes Simplex Virus gE-gI Complex Facilitates Cell-to-Cell Spread and Binds to Components of Cell Junctions

1998 ◽  
Vol 72 (11) ◽  
pp. 8933-8942 ◽  
Author(s):  
Kevin S. Dingwell ◽  
David C. Johnson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Junctions ◽  
Cell Junction ◽  
Cell Contact ◽  
Junction Protein ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT The herpes simplex virus (HSV) glycoprotein complex gE-gI mediates the spread of viruses between adjacent cells, and this property is especially evident for cells that form extensive cell junctions, e.g., epithelial cells, fibroblasts, and neurons. Mutants lacking gE or gI are not compromised in their ability to enter cells as extracellular viruses. Therefore, gE-gI functions specifically in the movement of virus across cell-cell contacts and, as such, provides a molecular handle on this poorly understood process. We expressed gE-gI in human epithelial cells by using replication-defective adenovirus (Ad) vectors. gE-gI accumulated at lateral surfaces of the epithelial cells, colocalizing with the adherens junction protein β-catenin but was not found on either the apical or basal plasma membranes and did not colocalize with ZO-1, a component of tight junctions. In subconfluent monolayers, gE-gI was found at cell junctions but was absent from those lateral surfaces not in contact with another cell, as was the case for β-catenin. Similar localization of gE-gI to cell junctions was observed in HSV-infected epithelial cells. By contrast, HSV glycoprotein gD, expressed using a recombinant Ad vectors, was found primarily along the apical surfaces of cells, with little or no protein found on the basal or lateral surfaces. Expression of gE-gI without other HSV polypeptides did not cause redistribution of either ZO-1 or β-catenin or alter tight-junction functions. Together these results support a model in which gE-gI accumulates at sites of cell-cell contact by interacting with junctional components. We hypothesize that gE-gI mediates transfer of HSV across cell junctions by virtue of these interactions with cell junction components.

Phosphorylation of connexin 43 induced by traumatic brain injury promotes exosome release

2018 ◽  
Vol 119 (1) ◽  
pp. 305-311 ◽  
Author(s):  
Wei Chen ◽  
Yijun Guo ◽  
Wenjin Yang ◽  
Lei Chen ◽  
Dabin Ren ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Gap Junction ◽  
Connexin 43 ◽  
Hippocampal Slices ◽  
Long Term Potentiation ◽  
Cx43 Expression ◽  
Term Potentiation ◽  
Junction Protein

Traumatic brain injury (TBI) caused by the external force leads to the neuronal dysfunction and even death. TBI has been reported to significantly increase the phosphorylation of glial gap junction protein connexin 43 (Cx43), which in turn propagates damages into surrounding brain tissues. However, the neuroprotective and anti-apoptosis effects of glia-derived exosomes have also been implicated in recent studies. Therefore, we detected whether TBI-induced phosphorylation of Cx43 would promote exosome release in rat brain. To generate TBI model, adult male Sprague-Dawley rats were subjected to lateral fluid percussion injury. Phosphorylated Cx43 protein levels and exosome activities were quantified using Western blot analysis following TBI. Long-term potentiation (LTP) was also tested in rat hippocampal slices. TBI significantly increased the phosphorylated Cx43 and exosome markers expression in rat ipsilateral hippocampus, but not cortex. Blocking the activity of Cx43 or ERK, but not JNK, significantly suppressed TBI-induced exosome release in hippocampus. Furthermore, TBI significantly inhibited the induction of LTP in hippocampal slices, which could be partially but significantly restored by pretreatment with exosomes. The results imply that TBI-activated Cx43 could mediate a nociceptive effect by propagating the brain damages, as well as a neuroprotective effect by promoting exosome release. NEW & NOTEWORTHY We have demonstrated in rat traumatic brain injury (TBI) models that both phosphorylated connexin 43 (p-Cx43) expression and exosome release were elevated in the hippocampus following TBI. The promoted exosome release depends on the phosphorylation of Cx43 and requires ERK signaling activation. Exosome treatment could partially restore the attenuated long-term potentiation. Our results provide new insight for future therapeutic direction on the functional recovery of TBI by promoting p-Cx43-dependent exosome release but limiting the gap junction-mediated bystander effect.

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