Spatial and Temporal Localization of Connexins in Cells Using Confocal Microscopy.

The 21-member connexin family found in humans is the building block of both single-membrane spanning channels (hemichannels) and double-membrane spanning intercellular channels. These large-pore channels are dynamic and typically have a short life span of only a few hours. Imaging connexins from the time of synthesis in the endoplasmic reticulum through to their degradation can be challenging given their distinct assembly states and transient residences in many subcellular compartments. Here, we describe how connexins can be effectively imaged on a confocal microscope in living cells when tagged with fluorescent proteins and when immunolabeled with high affinity anti-connexin antibodies in fixed cells. Temporal and spatial localization of multiple connexins and disease-linked connexin mutants at the subcellular level extensively informs on the mechanisms governing connexin regulation in health and disease.

Cx31.1 can selectively intermix with co-expressed connexins to facilitate its assembly into gap junctions.

Connexins are channel-forming proteins that function to facilitate gap junctional intercellular communication. Here, we use dual cell voltage clamp and dye transfer studies to corroborate past findings showing that Cx31.1 (encoded by GJB5) is defective in gap junction channel formation, illustrating that Cx31.1 alone does not form functional gap junction channels in connexin-deficient mammalian cells. Rather Cx31.1 transiently localizes to the secretory pathway with a subpopulation reaching the cell surface, which is rarely seen in puncta reminiscent of gap junctions. Intracellular retained Cx31.1 was subject to degradation as Cx31.1 accumulated in the presence of proteasomal inhibition, had a faster turnover when Cx43 was present and ultimately reached lysosomes. Although intracellularly retained Cx31.1 was found to interact with Cx43, this interaction did not rescue its delivery to the cell surface. Conversely, the co-expression of Cx31 dramatically rescued the assembly of Cx31.1 into gap junctions where gap junction-mediated dye transfer was enhanced. Collectively, our results indicate that the localization and functional status of Cx31.1 is altered through selective interplay with co-expressed connexins, perhaps suggesting Cx31.1 is a key regulator of intercellular signaling in keratinocytes.

Diversity in connexin biology.

Over 35 years ago the cell biology community was introduced to connexins as the subunit employed to assemble semicrystalline clusters of intercellular channels that had been well described morphologically as gap junctions. The decade that followed would see knowledge of the unexpectedly large 21-member human connexin family grow to reflect unique and overlapping expression patterns in all organ systems. While connexin biology initially focused on their role in constructing highly regulated intercellular channels, this was destined to change as discoveries revealed that connexin hemichannels at the cell surface had novel roles in many cell types, especially when considering connexin pathologies. Acceptance of connexins as having bifunctional channel properties was initially met with some resistance, which has given way in recent years to the premise that connexins have multifunctional properties. Depending on the connexin isoform and cell of origin, connexins have wide-ranging half-lives that vary from a couple of hours to the life expectancy of the cell. Diversity in connexin channel characteristics and molecular properties were further revealed by X-ray crystallography and single-particle cryo-EM. New avenues have seen connexins or connexin fragments playing roles in cell adhesion, tunneling nanotubes, extracellular vesicles, mitochondrial membranes, transcription regulation, and in other emerging cellular functions. These discoveries were largely linked to Cx43, which is prominent in most human organs. Here, we will review the evolution of knowledge on connexin expression in human adults and more recent evidence linking connexins to a highly diverse array of cellular functions.

GJB4 variants linked to skin disease exhibit a trafficking deficiency en route to gap junction formation that can be restored by co-expression of select connexins.

Epidermal keratinocytes are enriched with at least nine connexins that are key regulators of epidermal homeostasis. The role of Cx30.3 in keratinocytes and epidermal health became evident when fourteen autosomal dominant mutations in the Cx30.3-encoding GJB4 gene were linked to a rare and incurable skin disorder called erythrokeratodermia variabilis et progressiva (EKVP). While these variants are linked to EKVP, they remain largely uncharacterized hindering therapeutic options. In this study, we characterize the expression and functional status of three EKVP-linked Cx30.3 mutants (G12D, T85P, and F189Y) in tissue-relevant and differentiation-competent rat epidermal keratinocytes. We found that GFP-tagged Cx30.3 mutants were non-functional likely due to their impaired trafficking and primary entrapment within the endoplasmic reticulum (ER). However, all mutants failed to increase BiP/GRP78 levels suggesting they were not inducing an unfolded protein response. FLAG-tagged Cx30.3 mutants were also trafficking impaired yet occasionally exhibited some capacity to assemble into gap junctions. The pathological impact of these mutants may extend beyond their trafficking deficiencies as keratinocytes expressing FLAG-tagged Cx30.3 mutants exhibited increased propidium iodide uptake in the absence of divalent cations. Attempts to rescue the delivery of trafficking impaired GFP-tagged Cx30.3 mutants into gap junctions by chemical chaperone treatment were ineffective. However, co-expression of wild type Cx30.3 greatly enhanced the assembly of Cx30.3 mutants into gap junctions, although endogenous levels of Cx30.3 do not appear to prevent the skin pathology found in patients harboring these autosomal dominant mutations. In addition, a spectrum of connexin isoforms (Cx26, Cx30, and Cx43) exhibited the differential ability to trans-dominantly rescue the assembly of GFP-tagged Cx30.3 mutants into gap junctions suggesting a broad range of connexins found in keratinocytes may favourably interact with Cx30.3 mutants. We conclude that selective upregulation of compatible wild type connexins in keratinocytes may have potential therapeutic value in rescuing epidermal defects invoked by Cx30.3 EKVP-linked mutants.

Recent advances in connexin gap junction biology.

Connexins are assembled into dodecamer intercellular channels, a collection of which is termed a gap junction, and their canonical function allowing direct exchange of ions and metabolites has been unequivocally established. When initially assembled into undocked cell surface connexin hemichannels, healthy cells may also engage in cell signaling via a regulated small-molecule release. Recent advances in the field have led to an expanded view of the functional roles of intercellular channels and hemichannels in both physiology and pathology. As more of the 21-member human connexin family is intensely interrogated, mounting evidence points to the biological uniqueness of each member, and no longer can we confidently refer to all connexins engaging in the same cellular processes. Innovations in high-resolution cryo-electron microscopy have revealed important insights into the structure of functionally important domains of both hemichannels and channels. These and other studies have established a foundation of knowledge that should allow inhibitory smart drug design for situations where enhanced intercellular or hemichannel activity is at the root of a connexin-linked disease. Assessment of the connexin interactome, which varies widely for each connexin subtype, continues to provide regulatory insights into the assembly and function of connexins that exhibit a short half-life. As the most intensely studied, Cx43 is found in about 50% of all human cell types and is extensively regulated by multiple inhibitory and enhancing phosphorylation events that have direct implications on tissue function and outcomes of disease, including cancer. Here, we briefly discuss these advances and give our thoughts on where the field is headed.

Interrogation of Carboxy-Terminus Localized GJA1 Variants Associated with Erythrokeratodermia Variabilis et Progressiva.

Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell-cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.

Cellular mechanisms of connexin-based inherited diseases.

The 21-member connexin gene family exhibits distinct tissue expression patterns that can cause a diverse array of over 30 inherited connexin-linked diseases ranging from deafness to skin defects and blindness. Intriguingly, germline mutations can cause disease in one tissue while other tissues that abundantly express the mutant connexin remain disease free, highlighting the importance of the cellular context of mutant expression. Modeling connexin pathologies in genetically modified mice and tissue-relevant cells has informed extensively on no less than a dozen gain- and loss-of-function mechanisms that underpin disease. This review focuses on how a deeper molecular understanding of the over 930 mutations in 11 connexin-encoding genes is foundational for creating a framework for therapeutic interventions.

Pannexin biology and emerging linkages to cancer.

Pannexins are a family of glycoproteins that comprises three members, PANX1, PANX2, and PANX3. The widely expressed and interrogated PANX1 forms heptameric membrane channels that primarily serve to connect the cytoplasm to the extracellular milieu by being selectively permeable to small signaling molecules when activated. Apart from notable exceptions, PANX1 in many tumor cells appears to facilitate tumor growth and metastasis, suggesting that pannexin-blocking therapeutics may have utility in cancer. Attenuation of PANX1 function must also consider the fact that PANX1 is found in stromal cells of the tumor microenvironment (TME), including immune cells. This review highlights the key discoveries of the past 5 years that suggest pannexins facilitate, or in some cases inhibit, tumor cell growth and metastasis via direct protein interactions and through the regulated efflux of signaling molecules.

Connexin 43 contributes to phenotypic robustness of the mouse skull.

BACKGROUND: We compared skull shape and variation among genetically modified mice that exhibit different levels of connexin43 (Cx43) channel function, to determine whether Cx43 contributes to craniofacial phenotypic robustness. Specifically, we used two heterozygous mutant mouse models (G60S/+ and I130T/+) that, when compared to their wildtype counterparts, have an ~80% and ~50% reduction in Cx43 function, respectively. RESULTS: Both mutant strains showed significant differences in skull shape compared to wildtype littermates and while these differences were more severe in the G60S/+ mouse, shape differences were localized to similar regions of the skull in both mutants. However, increased skull shape variation was observed in G60S/+ mutants only. Additionally, covariation of skull structures was disrupted in the G60S/+ mutants only, indicating that while a 50% reduction in Cx43 function is sufficient to cause a shift in mean skull shape, the threshold for Cx43 function for disrupting craniofacial phenotypic robustness is lower. CONCLUSIONS: Collectively, our results indicate Cx43 can contribute to phenotypic robustness of the skull through a nonlinear relationship between Cx43 gap junctional function and phenotypic outcomes.

Mutant Cx30-A88V mice exhibit hydrocephaly and sex-dependent behavioral abnormalities, implicating a functional role for Cx30 in the brain.

Connexin 30 (Cx30; also known as Gjb6 when referring to the mouse gene) is expressed in ependymal cells of the brain ventricles, in leptomeningeal cells and in astrocytes rich in connexin 43 (Cx43), leading us to question whether patients harboring GJB6 mutations exhibit any brain anomalies. Here, we used mice harboring the human disease-associated A88V Cx30 mutation to address this gap in knowledge. Brain Cx30 levels were lower in male and female Cx30A88V/A88V mice compared with Cx30A88V/+ and Cx30+/+ mice, whereas Cx43 levels were lower only in female Cx30 mutant mice. Characterization of brain morphology revealed a disrupted ependymal cell layer, significant hydrocephalus and enlarged ventricles in 3- to 6-month-old adult male and female Cx30A88V/A88V mice compared with Cx30A88V/+ or Cx30+/+ sex-matched littermate mice. To determine the functional significance of these molecular and morphological changes, we investigated a number of behavioral activities in these mice. Interestingly, only female Cx30A88V/A88V mice exhibited abnormal behavior compared with all other groups. Cx30A88V/A88V female mice demonstrated increased locomotor and exploratory activity in both the open field and the elevated plus maze. They also exhibited dramatically reduced ability to learn the location of the escape platform during Morris water maze training, although they were able to swim as well as other genotypes. Our findings suggest that the homozygous A88V mutation in Cx30 causes major morphological changes in the brain of aging mice, possibly attributable to an abnormal ependymal cell layer. Remarkably, these changes had a more pronounced consequence for cognitive function in female mice, which is likely to be linked to the dysregulation of both Cx30 and Cx43 levels in the brain.

Palmoplantar keratoderma with deafness phenotypic variability in a patient with an inherited GJB2 frameshift variant and novel missense variant.

BACKGROUND: Variants in the GJB2 gene encoding the gap junction protein connexin-26 (Cx26) can cause autosomal recessive nonsyndromic hearing loss or a variety of phenotypically variable autosomal dominant disorders that effect skin and hearing, such as palmoplantar keratoderma (PPK) with deafness and keratitis-ichthyosis-deafness (KID) syndrome. Here, we report a patient with chronic mucocutaneous candidiasis, hyperkeratosis with resorption of the finger tips, profound bilateral sensorineural hearing loss, and normal hair and ocular examination. Exome analysis identified a novel missense variant in GJB2 (NM_004004.5:c.101T>A, p.Met34Lys) that was inherited from a mosaic unaffected parent in the setting of a well-reported GJB2 loss of function variant (NM_004004.5:c.35delG, p.Gly12Valfs*2) on the other allele. METHOD: Rat epidermal keratinocytes were transfected with cDNA encoding wildtype Cx26 and/or the Met34Lys mutant of Cx26. Fixed cells were immunolabeled in order to assess the subcellular location of the Cx26 mutant and cell images were captured. RESULTS: Expression in rat epidermal keratinocytes revealed that the Met34Lys mutant was retained in the endoplasmic reticulum, unlike wildtype Cx26, and failed to reach the plasma membrane to form gap junctions. Additionally, the Met34Lys mutant acted dominantly to wildtype Cx26, restricting its delivery to the cell surface. CONCLUSION: Overall, we show the p.Met34Lys variant is a novel dominant acting variant causing PPK with deafness. The presence of a loss a function variant on the other allele creates a more severe clinical phenotype, with some features reminiscent of KID syndrome.

Single-cell dynamics of pannexin-1-facilitated programmed ATP loss during apoptosis.

ATP is essential for all living cells. However, how dead cells lose ATP has not been well investigated. In this study, we developed new FRET biosensors for dual imaging of intracellular ATP level and caspase-3 activity in single apoptotic cultured human cells. We show that the cytosolic ATP level starts to decrease immediately after the activation of caspase-3, and this process is completed typically within 2 hr. The ATP decrease was facilitated by caspase-dependent cleavage of the plasma membrane channel pannexin-1, indicating that the intracellular decrease of the apoptotic cell is a 'programmed' process. Apoptotic cells deficient of pannexin-1 sustained the ability to produce ATP through glycolysis and to consume ATP, and did not stop wasting glucose much longer period than normal apoptotic cells. Thus, the pannexin-1 plays a role in arresting the metabolic activity of dead apoptotic cells, most likely through facilitating the loss of intracellular ATP.

Comparative Analysis of Cx31 and Cx43 in Differentiation-Competent Rodent Keratinocytes.

When considering connexin expression and regulation, the epidermis of the skin is one of the most complex tissues found in mammals even though it largely contains a single cell type, the keratinocyte. In the rodent epidermis, up to 9 connexin family members have been detected at the mRNA level. Many of these connexins are temporally and spatially regulated in coordination with keratinocyte progenitor cell differentiation and migration from the stratum basale to form the stratum spinosum and stratum granulosum layers before finally forming the stratum corneum. Cx43 is the principal connexin found in basal keratinocytes and to a lesser degree found in keratinocytes that have begun to differentiate where Cx26, Cx30 and Cx31 become prevalent. Here we show that the CRISPR-Cas9 ablation of Cx43 reduces overall gap junction coupling in monolayer cultures of rat epidermal keratinocytes (REKs) and dysregulates the differentiation of REKs when grown in organotypic cultures. Natively found in differentiated keratinocytes, Cx31 readily assembles into gap junctions when expressed in REKs where it can extensively co-assemble into the same gap junctions with co-expressed Cx30. Time-lapse imaging indicated that many Cx31 gap junctions are mobile within the plasma membrane undergoing both fusion and fission events. Finally, the persistence of pre-existing Cx31 gap junctions in the presence of the protein trafficking blocker, brefeldin A, is longer than that found for Cx43 gap junctions indicating that it has a distinctly different life expectancy in REKs. Collectively, this study highlights the importance of Cx43 in rodent keratinocyte differentiation and suggests that Cx31 acquires life-cycle properties that are distinct from Cx43.

Effects of Reduced Connexin43 Function on Mandibular Morphology and Osteogenesis in Mutant Mouse Models of Oculodentodigital Dysplasia.

Mutations in the gene encoding the gap-junctional protein connexin43 (Cx43) are the cause of the human disease oculodentodigital dysplasia (ODDD). The mandible is often affected in this disease, with clinical reports describing both mandibular overgrowth and conversely, retrognathia. These seemingly opposing observations underscore our relative lack of understanding of how ODDD affects mandibular morphology. Using two mutant mouse models that mimic the ODDD phenotype (I130T/+ and G60S/+), we sought to uncover how altered Cx43 function may affect mandibular development. Specifically, mandibles of newborn mice were imaged using micro-CT, to enable statistical comparisons of shape. Tissue-level comparisons of key regions of the mandible were conducted using histomorphology, and we quantified the mRNA expression of several cartilage and bone cell differentiation markers. Both G60S/+ and I130T/+ mutant mice had altered mandibular morphology compared to their wildtype counterparts, and the morphological effects were similarly localized for both mutants. Specifically, the biggest phenotypic differences in mutant mice were focused in regions exposed to mechanical forces, such as alveolar bone, muscular attachment sites, and articular surfaces. Histological analyses revealed differences in ossification of the intramembranous bone of the mandibles of both mutant mice compared to their wildtype littermates. However, chondrocyte organization within the secondary cartilages of the mandible was unaffected in the mutant mice. Overall, our results suggest that the morphological differences seen in G60S/+ and I130T/+ mouse mandibles are due to delayed ossification and suggest that mechanical forces may exacerbate the effects of ODDD on the skeleton.

Effects of reduced connexin43 function on skull development in the Cx43I130T/+ mutant mouse that models oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a disease caused by mutations in the GJA1 gene that encodes the gap-junctional protein connexin43 (Cx43). ODDD affects multiple organs, but craniofacial anomalies are typical. However, details on the timing of phenotypic presentation of these abnormalities and their correspondence with potential cellular changes are incomplete. Here, we perform the first assessment of the development of the ODDD craniofacial phenotype in the Cx43I130T/+ mouse model and show that the phenotypic features commonly found in patients with the disorder arise in mice between E17.5 and birth and become more profound with age. Using mice heterozygous for the I130T mutation of Gja1, we provide a detailed analysis of the craniofacial phenotype in this ODDD model using shape analyses based on micro-CT images. Results show that in addition to differences in facial bone morphology, there are significant shape differences in the cranial base. Mutant mice display delayed ossification at E17.5 and birth, particularly in bones of the face and cranial vault but ossification is normal at three months. Our immunohistochemical analyses of the palatine bone indicate that osteoblast differentiation is delayed in Cx43I130T/+ mice compared to their wildtype littermates, which likely contributes to the phenotypic variations observed in the facial bones. Our histological and immunohistochemical analyses of the synchondroses of the cranial base show no differences in molecular indicators of chondrocyte differentiation in mutant mice, suggesting that the differences to cranial base morphology displayed by Cx43I130T/+ mice are not due to differences in chondrocyte proliferation or differentiation. Together, our findings suggest that Cx43I130T/+ mice represent a surrogate model to not only inform about the craniofacial anomalies found in ODDD patients but also to show that reduced Cx43 function leads to phenotypic changes that are largely due to osteoblast defects.

GJB2 Mutations Linked to Hearing Loss Exhibit Differential Trafficking and Functional Defects as Revealed in Cochlear-Relevant Cells.

GJB2 gene (that encodes Cx26) mutations are causal of hearing loss highlighting the importance of Cx26-based channel signaling amongst the supporting cells in the organ of Corti. While the majority of these GJB2 mutations are inherited in an autosomal recessive manner, others are inherited in an autosomal dominant manner and lead to syndromic hearing loss as well as skin diseases. To assess if common or divergent mechanisms are at the root of GJB2-linked hearing loss, we expressed several mutants in cochlear-relevant HEI-OC1 cells derived from the developing organ of Corti. Since supporting cells of the mature mammalian organ of Corti have negligible Cx43, but HEI-OC1 cells are rich in Cx43, we first used CRISPR-Cas9 to ablate endogenous Cx43, thus establishing a connexin-deficient platform for controlled reintroduction of hearing-relevant connexins and Cx26 mutants. We found three distinct outcomes and cellular phenotypes when hearing loss-linked Cx26 mutants were expressed in cochlear-relevant cells. The dominant syndromic Cx26 mutant N54K had trafficking defects and did not fully prevent wild-type Cx26 gap junction plaque formation but surprisingly formed gap junctions when co-expressed with Cx30. In contrast, the dominant syndromic S183F mutant formed gap junctions incapable of transferring dye and, as expected, co-localized in the same gap junctions as wild-type Cx26 and Cx30, but also gained the capacity to intermix with Cx43 within gap junctions. Both recessive non-syndromic Cx26 mutants (R32H and R184P) were retained in intracellular vesicles including early endosomes and did not co-localize with Cx30. As might be predicted, none of the Cx26 mutants prevented Cx43 gap junction plaque formation in Cx43-rich HEI-OC1 cells while Cx43-ablation had little effect on the expression of reference genes linked to auditory cell differentiation. We conclude from our studies in cochlear-relevant cells that the selected Cx26 mutants likely evoke hearing loss via three unique connexin defects that are independent of Cx43 status.

Involvement of the Gap Junction Protein, Connexin43, in the Formation and Function of Invadopodia in the Human U251 Glioblastoma Cell Line.

The resistance of glioblastomas to treatments is mainly the consequence of their invasive capacities. Therefore, in order to better treat these tumors, it is important to understand the molecular mechanisms which are responsible for this behavior. Previous work suggested that gap junction proteins, the connexins, facilitate the aggressive nature of glioma cells. Here, we show that one of them-connexin43 (Cx43)-is implicated in the formation and function of invadopodia responsible for invasion capacity of U251 human glioblastoma cells. Immunofluorescent approaches-combined with confocal analyses-revealed that Cx43 was detected in all the formation stages of invadopodia exhibiting proteolytic activity. Clearly, Cx43 appeared to be localized in invadopodia at low cell density and less associated with the establishment of gap junctions. Accordingly, lower extracellular matrix degradation correlated with less mature invadopodia and MMP2 activity when Cx43 expression was decreased by shRNA strategies. Moreover, the kinetics of invadopodia formation could be dependent on Cx43 dynamic interactions with partners including Src and cortactin. Interestingly, it also appeared that invadopodia formation and MMP2 activity are dependent on Cx43 hemichannel activity. In conclusion, these results reveal that Cx43 might be involved in the formation and function of the invadopodia of U251 glioblastoma cells.

Dynamic regulation of connexins in stem cell pluripotency.

Characterization of the pluripotent "ground state" has led to a greater understanding of species-specific stem cell differences and has imparted an appreciation of the pluripotency continuum that exists in stem cells in vitro. Pluripotent stem cells are functionally coupled via connexins that serve in gap junctional intercellular communication (GJIC) and here we report that the level of connexin expression in pluripotent stem cells depends upon the state in which stem cells exist in vitro. Human and mouse pluripotent stem cells stabilized in a developmentally primitive or "naïve" state exhibit significantly less connexin expression compared with stem cells which are "primed" for differentiation. This dynamic connexin expression pattern may be governed, in part, by differential regulation by pluripotency transcription factors expressed in each cell state. Species-specific differences do exist, however, with mouse stem cells expressing several additional connexin transcripts not found in human pluripotent stem cells. Moreover, pharmacological inhibition of GJIC shows limited impact on naïve human stem cell survival, self-renewal, and pluripotency but plays a more significant role in primed human pluripotent stem cells. However, CRISPR-Cas9 gene ablation of Cx43 in human and mouse primed and naïve pluripotent stem cells reveals that Cx43 is dispensable in each of these four pluripotent stem cell types.

Connexin43 is Dispensable for Early Stage Human Mesenchymal Stem Cell Adipogenic Differentiation But is Protective against Cell Senescence.

In the last couple of decades, there has been a growing optimism surrounding the potential transformative use of human mesenchymal stem cells (MSCs) and human-induced pluripotent stem cells (iPSCs) for regenerative medicine and disease treatment. In order for this to occur, it is first essential to understand the mechanisms underpinning their cell-fate specification, which includes cell signaling via gap junctional intercellular communication. Here, we investigated the role of the prototypical gap junction protein, connexin43 (Cx43), in governing the differentiation of iPSCs into MSCs and MSC differentiation along the adipogenic lineage. We found that control iPSCs, as well as iPSCs derived from oculodentodigital dysplasia patient fibroblasts harboring a GJA1 (Cx43) gene mutation, successfully and efficiently differentiated into LipidTox and perilipin-positive cells, indicating cell differentiation along the adipogenic lineage. Furthermore, the complete CRISPR-Cas9 ablation of Cx43 from iPSCs did not prevent their differentiation into bona fide MSCs or pre-adipocytes, strongly suggesting that even though Cx43 expression is upregulated during adipogenesis, it is expendable. Interestingly, late passage Cx43-ablated MSCs senesced more quickly than control cells, resulting in failure to properly differentiate in vitro. We conclude that despite being upregulated during adipogenesis, Cx43 plays no detectable role in the early stages of human iPSC-derived MSC adipogenic differentiation. However, Cx43 may play a more impactful role in protecting MSCs from premature senescence.

Destination and consequences of Panx1 and mutant expression in polarized MDCK cells.

The channel-forming membrane glycoprotein pannexin 1 (Panx1) is best characterized as an ATP release channel. To investigate the trafficking and sorting of Panx1, we used polarized MDCK cells and non-polarized BICR-M1Rk cells to track the fate of GFP-tagged Panx1. In non-polarized cells, Panx1 was found throughout the plasma membrane, including the lamellipodia of the tumor cells and the cell surface-targeting domain was mapped to residues 307-379. Panx1 was preferentially enriched at the apical membrane domain of polarized MDCK cells grown as monolayer sheets or as spheroids. Residual Panx1 localized within basolateral membranes of polarized MDCK cells was independent of a putative dileucine sorting motif LL365/6 found within the C-terminal of Panx1. Unexpectedly, stable expression of a Panx1 mutant, where a putative tyrosine-based basolateral sorting motif (YxxØ) was mutated (Y308F), or a truncated Δ379 Panx1 mutant, caused MDCK cells to lose cell-cell contacts and their ability to polarize as they underwent a switch to a more fibroblast-like phenotype. We conclude that Panx1 is preferentially delivered to the apical domain of polarized epithelial cells, and Panx1 mutants drive phenotypic changes to MDCK cells preventing their polarization.