The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.

The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1Crsh and Vangl2Lp mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies.

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in ScribCrc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell–cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.

BACKGROUND Lung branching morphogenesis is a fundamental developmental process, yet the cellular dynamics that occur during lung development and the molecular mechanisms underlying recent postulated branching modes are poorly understood. RESULTS Here, we implemented a time-lapse video microscopy method to study the cellular behavior and molecular mechanisms of planar bifurcation and domain branching in lung explant- and organotypic cultures. Our analysis revealed morphologically distinct stages that are shaped at least in part by a combination of localized and orientated cell divisions and by local mechanical forces. We also identified myosin light-chain kinase as an important regulator of bud bifurcation, but not domain branching in lung explants. CONCLUSION This live imaging approach provides a method to study cellular behavior during lung branching morphogenesis and suggests the importance of a mechanism primarily based on oriented cell proliferation and mechanical forces in forming and shaping the developing lung airways.

The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw) mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.

The contraction and spreading of nonmuscle cells are important phenomena in a number of cellular processes such as differentiation, morphogenesis, and tissue growth. Recent experimental work has shown that the topology and the mechanical properties of the underlying substrate play a significant role in directing the cell’s response. In this work, we introduce a multiscale model to understand the sensing, activation, and contraction of the actin cytoskeleton of nonmuscle cells based on the idea that acto-myosin cross-bridges display a catch-bond response. After investigating the respective roles of bond catchiness and acto-myosin assembly on the mechano-sensitivity of stress fibers, we present full simulations of cells laying on arrays of micropillars. Model predictions show good qualitative agreements with experimental observation, suggesting that acto-myosin catch bonds are a major mechano-sensing element in nonmuscle cells.

Background: Although the techniques to interrogate the appearance of a biomarker in tissue sections have greatly advanced, there are limitations as to how representative an analysis of a tissue section is compared with that of the entire diseased tissue. Depending on the heterogeneous expression level of a biomarker, tissue sampling can result in different interpretations of the biomarker's appearance and hence could lead to a false result regarding the biomarkers presence. Such inaccurate data, of course, could result in ineffective patient care decisions.

Immuno-oncology (IO) approaches requires an understanding of the types of immune cells present in the tumor microenvironment (TME) as well as the precise localization of these immune cells. However, the acquisition of spatial IO information is technically challenging, due to the requirement for multiplex labeling of immune cells and the need to categorize their location and biomarker content simultaneously. Additionally, the multiplex biomarker panel must be engineered in advance based on a priori assumptions about the correct marker combinations and their location (such as the tumor epithelial nests, TME, or specifically the tumor-stroma interface). This limits the ability to implement novel, scientifically driven assessments into an existing clinical trial which already has defined immunohistochemistry endpoints. To meet the needs of IO clinical trials, Flagship Biosciences has developed a proprietary image analysis platform, FACTS (Feature Analysis on Complementary Tissue Sections), to deduce both the necessary multiplex staining information and the spatial context of IO markers based on the utilization of existing monochrome or multiplex stained slides from a clinical trial. In this study, we demonstrate the utility of this approach to deliver biologically relevant endpoints important for IO clinical trials. First, we performed single IO biomarker staining for CD4, CD8 and FoxP3 in colorectal cancer patient samples and developed a novel image analysis approach that allowed for the accurate quantification of multiple IO markers within specific margins of the tumor microenvironment across multiple tissue sections. For each biomarker the positive cell populations were binned based on distance from the tumor-TME boundary (i.e.% positive cells within 100μM, 200μM or 500μM of the tumor-TME boundary). Next, we used computational alignment for evaluating the co-localization of multiple immune cell biomarkers from serial sections with single stains for CD4, CD8 or FoxP3. We demonstrate that the information extracted from multiple single stained serial sections can be used to generate co-localization information for multiple IO markers in a given patient sample. Lastly, we developed multiplex chromogenic assays for these same markers, analyzed the multiplex-stained slides with image analysis, and derived the same analysis endpoints for the multiplex slides to the digitally aligned individually labeled sections. The study found that similar interpretation of the inflammatory landscape was possible with multiplex-stained slides and digital alignment of monoplex-stained slides. In summary, this study demonstrated a novel image analysis approach that allows for quantification of IO markers utilizing clinically relevant wet assay technologies and existing IHC stained slides derived from an immune-oncology clinical trial. Citation Format: Ciara A. Martin, Joshua Black, Nathan T. Martin, Logan Cerkovnik, Jasmeet Bajwa, Kristin Wilson, Daniel Rudmann, Carsten Schnatwinkel, AJ Milici. Quantifying immune oncology markers across multiple tissue sections with digital image analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4042.