Acute respiratory distress syndrome (ARDS) is a deadly clinical presentation in sepsis, COVID, and other lung disorders where vascular fluid leakage is a severe problem. Recent findings by Shadab et al. in the JBC show that a well-known player in immune function, Syk, also regulates vascular leakage in response to sepsis. An existing FDA-approved inhibitor of Syk, fostamatinib, prevents the vascular leakage and improves survival in a mouse sepsis model, providing promise for ARDS treatment in the clinic.

Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors.

The spatial sorting of cells into appropriate tissue compartments is essential for embryogenesis and tissue development. Spatial cell sorting is controlled by the interplay between cell surface affinity and intracellular mechanical properties. However, intracellular signaling that can sufficiently sort cell populations remains unexplored. In this study, we engineered chimeric cadherins by replacing the cadherin intracellular domain with cytoskeletal regulators to test their ability to induce spatial cell sorting. Using a fibroblast-based reconstitution system, we observed that Rac1 and RhoA activity in the cadherin tail induced outward and inward sorting, respectively. In particular, RhoA activity embedded cells toward the inside of E-cadherin-expressing spheroids and tumor spheroids, leading to tissue invagination. Despite the simplicity of chimeric cadherin design, our results indicate that differences in cadherin intracellular activities can determine the direction of spatial cell sorting, even when cell surface affinity is not different, and provide new molecular tools to engineer tissue architectures.

The choice of fixation method significantly impacts tissue morphology and protein visualization after immunohistochemistry (IHC). In this study, we compared the effects of paraformaldehyde (PFA) and trichloroacetic acid (TCA) fixation prior to IHC on chicken embryos. Our findings underscore the importance of validating fixation methods for accurate interpretation of IHC results, with implications for antibody validation and tissue-specific protein localization studies. We found that TCA fixation resulted in larger and more circular nuclei compared to PFA fixation. Additionally, TCA fixation altered the appearance of subcellular localization and fluorescence intensity of various proteins, including transcription factors and cytoskeletal proteins. Notably, TCA fixation revealed protein localization domains that may be inaccessible with PFA fixation. These results highlight the need for optimization of fixation protocols depending on the target epitope and model system, emphasizing the importance of methodological considerations in biological analyses.

Desmosomes are intercellular junctions that regulate mechanical integrity in epithelia and cardiac muscle. Dynamic desmosome remodeling is essential for wound healing and development, yet the mechanisms governing junction assembly remain elusive. While we and others have shown that cadherin ectodomains are highly organized, how this ordered architecture emerges during assembly is unknown. Using fluorescence polarization microscopy, we show that desmoglein 2 (Dsg2) ectodomain order gradually increases during 8 h of assembly, coinciding with increasing adhesive strength. In a scratch wound assay, we observed a similar increase in order in desmosomes assembling at the leading edge of migratory cells. Together, our findings indicate that cadherin organization is a hallmark of desmosome maturity and may play a role in conferring adhesive strength.

Sepsis, often resulting from an immune response overreaction to microorganisms and their products, can lead to acute lung injury through inflammation mediated by excessive cytokines. This study aimed to investigate the effects of regorafenib on lung injury in mice following the induction of sepsis. We divided mice into four groups (n=6 each): a sham group (undergoing laparotomy without cecal ligation and puncture [CLP]), a CLP group, a vehicle group, and a regorafenib-treated group (30 mg/kg IP, administered one hour before CLP). TNF-α, IL-1β, VEGF, MPO, caspase-11, and Ang-2 levels were significantly increased (p<0.05) in the CLP group compared to the sham group, while the regorafenib group showed significant reductions in these markers versus the CLP group (p< 0.05). In contrast, Ang-1 levels, which were reduced in the CLP group (p<0.05) compared to the sham group, were elevated in the regorafenib group compared to the CLP group. Quantitative real-time PCR revealed a significant decrease in TIE2 and VE-cadherin mRNA expression in the lung tissue of the CLP group compared to the sham group. There were no significant differences in mRNA expression of the TIE2 gene between the regorafenib and CLP group. However, VE-cadherin significantly increased after regorafenib treatment. Regorafenib demonstrated lung-protective effects through its anti-inflammatory and antiangiogenic activities and its influence on lung tissue mRNA expression of the cadherin gene.

The main characteristic of polycystic kidney disease is the development of multiple fluid-filled renal cysts. The discovery of mislocalized sodium-potassium pump (Na,K-ATPase) in the apical membrane of cyst-lining epithelia alluded to reversal of polarity as a possible explanation for the fluid secretion. The topic of apical Na,K-ATPase in cysts remains controversial. We investigated the localization of the Na,K-ATPase and assessed the apical-basolateral polarization of cyst-lining epithelia by means of immunohistochemistry in kidney tissue from six polycystic kidney disease patients undergoing nephrectomy. The Na,K-ATPase α1 subunit was conventionally situated in the basolateral membrane of all immunoreactive cysts. Proteins of the Crumbs and partitioning defective (Par) complexes were localized to the apical membrane domain in cyst epithelial cells. The apical targeting protein Syntaxin-3 also immunolocalized to the apical domain of cyst-lining epithelial cells. Proteins of the basolateral Scribble complex immunolocalized to the basolateral domain of cysts. Thus, no deviations from the typical epithelial distribution of basic cell polarity proteins were observed in the cysts from the six patients. Furthermore, we confirmed that cysts can originate from virtually any tubular segment with preserved polarity. In conclusion, we find no evidence of a reversal in apical-basolateral polarity in cyst-lining epithelia in polycystic kidney disease.

Robust linkage between adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The Drosophila multidomain protein Canoe and its mammalian homolog afadin are crucial for this, as in their absence many events of morphogenesis fail. To define the mechanism of action for Canoe, we are taking it apart. Canoe has five folded protein domains and a long intrinsically disordered region. The largest is the Dilute domain, which is shared by Canoe and myosin V. To define the roles of this domain in Canoe, we combined biochemical, genetic and cell biological assays. AlphaFold was used to predict its structure, providing similarities and contrasts with Myosin V. Biochemical data suggested one potential shared function - the ability to dimerize. We generated Canoe mutants with the Dilute domain deleted (CnoΔDIL). Surprisingly, they were viable and fertile. CnoΔDIL localized to adherens junctions and was enriched at junctions under tension. However, when its dose was reduced, CnoΔDIL did not provide fully wild-type function. Furthermore, canoeΔDIL mutants had defects in the orchestrated cell rearrangements of eye development. This reveals the robustness of junction-cytoskeletal connections during morphogenesis and highlights the power of natural selection to maintain protein structure.

The sensory epithelium of the cochlea, the organ of Corti, has complex cytoarchitecture consisting of mechanosensory hair cells intercalated by epithelial support cells. The support cells provide important trophic and structural support to the hair cells. Thus, the support cells must be stiff yet compliant enough to withstand and modulate vibrations to the hair cells. Once the sensory cells are properly patterned, the support cells undergo significant remodeling from a simple epithelium into a structurally rigid epithelium with fluid-filled spaces in the murine cochlea. Cell adhesion molecules such as cadherins are necessary for sorting and connecting cells in an intact epithelium. To create the fluid-filled spaces, cell adhesion properties of adjoining cell membranes between cells must change to allow the formation of spaces within an epithelium. However, the dynamic localization of cadherins has not been properly analyzed as these spaces are formed. There are three cadherins that are reported to be expressed during the first postnatal week of development when the tunnel of Corti forms in the cochlea. In this study, we characterize the dynamic localization of cadherins that are associated with cytoskeletal remodeling at the contacting membranes of the inner and outer pillar cells flanking the tunnel of Corti.

Desmoglein-2 (DSG2) is a calcium-binding single pass transmembrane glycoprotein and a member of the large cadherin family. Until recently, DSG2 was thought to only function as a cell adhesion protein embedded within desmosome junctions designed to enable cells to better tolerate mechanical stress. However, additional roles for DSG2 outside of desmosomes are continuing to emerge, particularly in cancer. Herein, we review the current literature on DSG2 in cancer and detail its impact on biological functions such as cell adhesion, proliferation, migration, invasion, intracellular signaling, extracellular vesicle release and vasculogenic mimicry. An increased understanding of the diverse repertoire of the biological functions of DSG2 holds promise to exploit this cell surface protein as a potential prognostic biomarker and/or target for better patient outcomes. This review explores the canonical and non-canonical functions of DSG2, as well as the context-dependent impacts of DSG2 in the realm of cancer.