AP-1 Recruits SMAP-1/SMAPs to the trans-Golgi Network to Promote Sorting in Polarized Epithelia

Coordinated AP-1 and clathrin coat assembly mediate secretory sorting on the trans-Golgi network (TGN) during conventional secretion. Here we found that SMAP-1/SMAPs deficiency caused the apical protein ERM-1 to accumulate on the basolateral side of the TGN. In contrast, the basolateral protein SLCF-1 appeared abnormally on the apical membrane. SMAP-1 colocalized with AP-1 on the TGN. The integrity of AP-1 is required for the subcellular presence of SMAP-1. Moreover, we found that the loss of SMAP-1 reduced clathrin-positive structures in the cytosol, suggesting that SMAP-1 has a regulatory role in clathrin assembly on the TGN. Functional experiments showed that overexpressing clathrin effectively alleviated exocytic defects due to the lack of SMAP-1, corroborating the role of SMAP-1 in promoting the assembly of clathrin on the TGN. Together, our results suggested that the AP-1 complex regulates the TGN localization of SMAP-1, promoting clathrin assembly to ensure polarized conventional secretion in C. elegans intestinal epithelia.

Comparative analysis of the MyTH4-FERM myosins reveals insights into the determinants of actin track selection in polarized epithelia

MyTH4-FERM (MF) myosins evolved to play a role in the creation and function of a variety of actin-based membrane protrusions that extend from cells. Here, we performed an analysis of the MF myosins, Myo7A, Myo7B, and Myo10, to gain insight into how they select for their preferred actin networks. Using enterocytes that create spatially separated actin tracks in the form of apical microvilli and basal filopodia, we show that actin track selection is principally guided by the mode of oligomerization of the myosin along with the identity of the motor domain, with little influence from the specific composition of the lever arm. Chimeric variants of Myo7A and Myo7B fused to a leucine zipper parallel dimerization sequence in place of their native tails both selected apical microvilli as their tracks, while a truncated Myo10 used its native antiparallel coiled-coil to traffic to the tips of filopodia. Swapping lever arms between the Class 7 and 10 myosins did not change actin track preference. Surprisingly, fusing the motor-neck region of Myo10 to a leucine zipper or oligomerization sequences derived from the Myo7A and Myo7B cargo proteins USH1G and ANKS4B, respectively, re-encoded the actin track usage of Myo10 to apical microvilli with significant efficiency.

Redox state and cellular uptake of copper is regulated by the N-terminus of human Copper Transporter-1

Copper(I) is essential for all life forms. Though Cu(II) is the most abundant state in environment, its reduction to Cu(I) is a prerequisite for bio-utilization, the mechanism of which is still uncharacterized. We show that in the human Copper Transporter-1, two amino-terminal methionine-histidine clusters and neighbouring aspartates distinctly binds Cu(II) to Cu(I) preceding its import. The endocytosis of hCTR1 from the basolateral membrane of polarized epithelia to Common-Recycling-Endosomes is dependent on copper reduction and coordination of Cu(I) by methionine residues. The transient binding of both Cu(II) and Cu(I) during the reduction process facilitated by aspartates also acts as another crucial determinant of hCTR1 endocytosis. Mutating 7Met-Gly-Met9 and Asp13 abrogates copper uptake and endocytosis, which was corrected by reduced and non-reoxidizable Cu(I). Histidine motifs, on the other hand, are crucial for hCTR1 functioning at limiting copper. Finally, we show that the two N-terminal His-Met-Asp clusters exhibit functional complementarity in regulating Cu(I)-induced hCTR1 endocytosis. This study proposes a model where His-Met-Asp residues of hCTR1 amino-terminal not only coordinate copper but also maintains its reduced state crucial for intracellular uptake.

MAGI-1 PDZ2 domain blockade averts adenovirus infection via enhanced proteolysis of the apical coxsackievirus and adenovirus receptor

Adenoviruses (AdV) are etiologic agents for gastrointestinal, heart, eye, and respiratory tract infections that can be lethal for immunosuppressed people. Many AdV use the coxsackievirus and adenovirus receptor (CAR) as a primary receptor. The CAR isoform resulting from alternative splicing that includes the eighth exon, CAREx8, localizes to the apical surface of polarized epithelial cells and is responsible for the initiation of AdV infection. We have shown that the membrane level of CAREx8 is tightly regulated by two MAGI-1 PDZ domains; PDZ2, and PDZ4, resulting in increased or decreased AdV transduction, respectively. We hypothesized that targeting the interactions between the MAGI-1 PDZ2 domain and CAREx8 would decrease apical CAREx8 expression level and prevent AdV infection. Decoy peptides that target MAGI-1 PDZ2, were synthesized (TAT-E6, TAT-NET1). PDZ2 binding peptides decreased CAREx8 expression and reduced AdV transduction. CAREx8 degradation was triggered by activation of the regulated intramembrane proteolysis (RIP) pathway through the disintegrin and metalloproteinase (ADAM17) and γ-secretase. Further analysis revealed that ADAM17 interacts directly with the MAGI-1 PDZ3 domain and blocking the PDZ2 domain enhanced the accessibility of ADAM17 to the substrate (CAREx8). Finally, we validated the efficacy of TAT-PDZ2 peptides in protecting the epithelia from AdV transduction in vivo using a novel transgenic animal model. Our data suggest that TAT-PDZ2 binding peptides are novel anti-AdV molecules that act by enhanced RIP of CAREx8 and decreased AdV entry. This strategy has an additional translational potential for targeting other viral receptors which have PDZ binding domains such as the angiotensin-converting enzyme-2 receptor. IMPORTANCE Adenovirus is a common threat in immunosuppressed populations and military recruits. There are no currently approved treatments/prophylactic agents that protect from most AdV infections. Here, we developed peptide-based small molecules that can suppress AdV infection of polarized epithelia by targeting the AdV receptor, coxsackievirus, and adenovirus receptor (CAREx8). The newly discovered peptides target a specific PDZ domain of the CAREx8 interacting protein MAGI-1 and decreased AdV transduction in multiple polarized epithelia models. Peptides-induce CAREx8 degradation is triggered by extracellular domain shedding through ADAM17 followed by γ-secretase-mediated nuclear translocation of the C-terminal domain. The enhanced shedding of CAREx8 ECD further protected the epithelium from AdV infection. Taken together, these novel molecules protect the epithelium from AdV infection. This approach may be applicable to the development of novel antiviral molecules against other viruses that use a receptor with a PDZ binding domain.

An approach to the understanding of the clinical-etiopathological aspect of COVID-19 (SARS-CoV-2)

Currently, the world is facing a health and socioeconomic crisis caused by the novel coronavirus disease COVID-19. On 11 March 2020, the World Health Organization (WHO) has declared this disease as a pandemic. The condition (COVID-19) is an infectious disorder triggered by a newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2. Most of the COVID-19 infected patients will experience mild to moderate respiratory symptoms and recover without any unique therapy. Assessment of the clinical and epidemiological characteristics of SARS-CoV-2 cases suggests the infected patients will not be contagious until the onset of severe symptoms and affects the other organs. Well-differentiated cells of apical airway epithelia communicating with ACE2 were promptly infected to SARS-CoV-2 virus. But the expression of ACE 2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudo typed virus entry and it is replicated in polarized epithelia and especially exited via the apical surface. Limiting the transmission of COVID-19 infection & its prevention can be regarded as a hierarchy of controls. In this article, we briefly discuss the most recent advances in respect to aetiology, pathogenesis and clinical progression of the disease COVID-19.

A molecular atlas of proximal airway identifies subsets of known airway cell types revealing details of the unique molecular pathogenesis of Cystic Fibrosis

Introduction/AbstractCystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts in excess of 70,000 people globally. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most significant determinant of morbidity and mortality. In this study we report results from a multi-institute consortium in which single cell transcriptomics were applied to define disease-related changes to the proximal airway of CF donors (n=19) undergoing transplantation for end-stage lung disease compared to the proximal airway of previously healthy lung donors (n=19). We found that all major airway epithelial cell types were conserved between control and CF donors. Disease-dependent differences were observed, including an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subtypes coupled with an unexpected decrease in cycling basal cells. This study developed a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.

Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.

Oligomerization of the FERM-FA protein Yurt controls epithelial cell polarity

Drosophila melanogaster Yurt (Yrt) and its mammalian orthologue EPB41L5 limit apical membrane growth in polarized epithelia. EPB41L5 also supports epithelial–mesenchymal transition and metastasis. Yrt and EPB41L5 contain a four-point-one, ezrin, radixin, and moesin (FERM) domain and a FERM-adjacent (FA) domain. The former contributes to the quaternary structure of 50 human proteins, whereas the latter defines a subfamily of 14 human FERM proteins and fulfills unknown roles. In this study, we show that both Yrt and EPB41L5 oligomerize. Our data also establish that the FERM-FA unit forms an oligomeric interface and that multimerization of Yrt is crucial for its function in epithelial cell polarity regulation. Finally, we demonstrate that aPKC destabilizes the Yrt oligomer to repress its functions, thereby revealing a mechanism through which this kinase supports apical domain formation. Overall, our study highlights a conserved biochemical property of fly and human Yrt proteins, describes a novel function of the FA domain, and further characterizes the molecular mechanisms sustaining epithelial cell polarity.

Structural centrosome aberrations sensitize polarized epithelia to basal cell extrusion

Centrosome aberrations disrupt tissue architecture and may confer invasive properties to cancer cells. Here we show that structural centrosome aberrations, induced by overexpression of either Ninein-like protein (NLP) or CEP131/AZI1, sensitize polarized mammalian epithelia to basal cell extrusion. While unperturbed epithelia typically dispose of damaged cells through apical dissemination into luminal cavities, certain oncogenic mutations cause a switch in directionality towards basal cell extrusion, raising the potential for metastatic cell dissemination. Here we report that NLP-induced centrosome aberrations trigger the preferential extrusion of damaged cells towards the basal surface of epithelial monolayers. This switch in directionality from apical to basal dissemination coincides with a profound reorganization of the microtubule cytoskeleton, which in turn prevents the contractile ring repositioning that is required to support extrusion towards the apical surface. While the basal extrusion of cells harbouring NLP-induced centrosome aberrations requires exogenously induced cell damage, structural centrosome aberrations induced by excess CEP131 trigger the spontaneous dissemination of dying cells towards the basal surface from MDCK cysts. Thus, similar to oncogenic mutations, structural centrosome aberrations can favour basal extrusion of damaged cells from polarized epithelia. Assuming that additional mutations may promote cell survival, this process could sensitize epithelia to disseminate potentially metastatic cells.

Uncoupling apical constriction from tissue invagination

Apical constriction is a widely utilized cell shape change linked to folding, bending and invagination of polarized epithelia. It remains unclear how apical constriction is regulated spatiotemporally during tissue invagination and how this cellular process contributes to tube formation in different developmental contexts. Using Drosophila salivary gland (SG) invagination as a model, we show that regulation of folded gastrulation expression by the Fork head transcription factor is required for apicomedial accumulation of Rho kinase and non-muscle myosin II, which coordinate apical constriction. We demonstrate that neither loss of spatially coordinated apical constriction nor its complete blockage prevent internalization and tube formation, although such manipulations affect the geometry of invagination. When apical constriction is disrupted, compressing force generated by a tissue-level myosin cable contributes to SG invagination. We demonstrate that fully elongated polarized SGs can form outside the embryo, suggesting that tube formation and elongation are intrinsic properties of the SG.