Molecular tension microscopy of the LINC complex in live cells.

We present a protocol to measure the effect of pharmacological treatments on the mechanical tension experienced by nesprins at the cytoplasmic surface of the nuclear envelope of mammalian cells in culture. We apply this protocol to MDCK epithelial cells exposed to the actin depolymerization agent cytochalasin D. To do so, we perform confocal spectral imaging of transiently expressed molecular tension sensors of mini-nesprin 2G and analyze the FRET signal from the sensors with a custom-made Fiji script. For complete details on the use and execution of this protocol, please refer to Déjardin et al. (2020).

Quantitative proteomic analysis of MDCK cell adhesion.

MDCK cells are a key reagent in modern vaccine production. As MDCK cells are normally adherent, creation of suspension cells for vaccine production using genetic engineering approaches is highly desirable. However, little is known regarding the mechanisms and effectors underlying MDCK cell adhesion. In this study, we performed a comparative analysis of whole protein levels between MDCK adhesion and suspension cells using an iTRAQ-based (isobaric tags for relative and absolute quantitation) proteomics approach. We found that expression of several proteins involved in cell adhesion exhibit reduced expression in suspension cells, including at the mRNA level. Proteins whose expression was reduced in suspension cells include cadherin 1 (CDH1), catenin beta-1 (CTNNB1), and catenin alpha-1 (CTNNA1), which are involved in intercellular adhesion; junction plakoglobin (JUP), desmoplakin (DSP), and desmoglein 3 (DSG3), which are desmosome components; and transglutaminase 2 (TGM2) and alpha-actinin-1 (ACTN1), which regulate the adhesion between cells and the extracellular matrix. A functional verification experiment showed that inhibition of E-cadherin significantly reduced intercellular adhesion of MDCK cells. E-Cadherin did not significantly affect the proliferation of MDCK cells and the replication of influenza virus. These findings reveal possible mechanisms underlying adhesion of MDCK cells and will guide the creation of MDCK suspension cells by genetic engineering.

Cholesterol is enriched in the sphingolipid patches on the substrate near nonpolarized MDCK cells, but not in the sphingolipid domains in their plasma membranes.

Information about the distributions of cholesterol and sphingolipids within the plasma membranes of mammalian cells provides insight into the roles of these molecules in membrane function. In this report, high-resolution secondary ion mass spectrometry was used to image the distributions of metabolically incorporated rare isotope-labeled sphingolipids and cholesterol on the surfaces of nonpolarized epithelial cells. Sphingolipid domains that were not enriched with cholesterol were detected in the plasma membranes of subconfluent Madin-Darby canine kidney cells. Surprisingly, cholesterol-enriched sphingolipid patches were observed on the substrate adjacent to these cells. Based on the shapes of these cholesterol-enriched sphingolipid patches on the substrate and their proximity to cellular projections, we hypothesize that they are deposits of membranous particles released by the cell.

Three-dimensional morphogenesis of MDCK cells induced by cellular contractile forces on a viscous substrate.

Substrate physical properties are essential for many physiological events such as embryonic development and 3D tissue formation. Physical properties of the extracellular matrix such as viscoelasticity and geometrical constraints are understood as factors that affect cell behaviour. In this study, we focused on the relationship between epithelial cell 3D morphogenesis and the substrate viscosity. We observed that Madin-Darby Canine Kidney (MDCK) cells formed 3D structures on a viscous substrate (Matrigel). The structures appear as a tulip hat. We then changed the substrate viscosity by genipin (GP) treatment. GP is a cross-linker of amino groups. Cells cultured on GP-treated-matrigel changed their 3D morphology in a substrate viscosity-dependent manner. Furthermore, to elucidate the spatial distribution of the cellular contractile force, localization of mono-phosphorylated and di-phosphorylated myosin regulatory light chain (P-MRLCs) was visualized by immunofluorescence. P-MRLCs localized along the periphery of epithelial sheets. Treatment with Y-27632, a Rho-kinase inhibitor, blocked the P-MRLCs localization at the edge of epithelial sheets and halted 3D morphogenesis. Our results indicate that the substrate viscosity, the substrate deformation, and the cellular contractile forces induced by P-MRLCs play crucial roles in 3D morphogenesis.

Protrusive activity guides changes in cell-cell tension during epithelial cell scattering.

Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering.

Novel growth regime of MDCK II model tissues on soft substrates.

It is well established that MDCK II cells grow in circular colonies that densify until contact inhibition takes place. Here, we show that this behavior is only typical for colonies developing on hard substrates and report a new growth phase of MDCK II cells on soft gels. At the onset, the new phase is characterized by small, three-dimensional droplets of cells attached to the substrate. When the contact area between the agglomerate and the substrate becomes sufficiently large, a very dense monolayer nucleates in the center of the colony. This monolayer, surrounded by a belt of three-dimensionally packed cells, has a well-defined structure, independent of time and cluster size, as well as a density that is twice the steady-state density found on hard substrates. To release stress in such dense packing, extrusions of viable cells take place several days after seeding. The extruded cells create second-generation clusters, as evidenced by an archipelago of aggregates found in a vicinity of mother colonies, which points to a mechanically regulated migratory behavior.

Micropatterning cells on permeable membrane filters.

Epithelium is abundantly present in the human body as it lines most major organs. Therefore, ensuring the proper function of epithelium is pivotal for successfully engineering whole organ replacements. An important characteristic of mature epithelium is apical-basal polarization which can be obtained using the air-liquid interface (ALI) culture system. Micropatterning is a widely used bioengineering strategy to spatially control the location and organization of cells on tissue culture substrates. Micropatterning is therefore an interesting method for generating patterned epithelium. Enabling micropatterning of epithelial cells however requires micropatterning methods that are designed to (i) be compatible with permeable membranes substrates and (ii) allow prolonged culture of patterned cells, both of which are required for appropriate epithelial apical-basal polarization. Here, we describe a number of methods we have developed for generating monoculture as well as coculture of epithelial cells that are compatible with ALI culture.

Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line.

Cell culture-based manufacturing of influenza vaccines is ideally based on easily scalable platforms using suspension cells that grow in chemically defined media. Consequently, different adherent cell lines selected for high virus yields were adapted to grow in suspension culture. This includes the MDCK suspension cell line MDCK.SUS2, which was shown to be a suitable substrate for influenza virus propagation in previous studies. In this study, we investigated options for further improvement of influenza A/PR/8 (H1N1) virus titres and cell-specific virus yields. Best results were achieved by performing a 1:2 dilution with fresh medium at time of infection. In shake flask cultivations, even for multiplicities of infection as low as 10⁻5, all cells were infected at 36 h post infection as determined by flow cytometry. In addition, these cells showed a better viability than cells infected without previous washing steps, which was reflected by a reduced level of apoptotic cells, and virus yields exceeding 3 log10 HAU/100 µL. Comparison of bioreactor infections of MDCK.SUS2 cells to the parental adherent MDCK cells showed similar HA titres of 2.94 and 3.15 log10 HAU/100 µL and TCID50 of 1 × 109 and 2.37 × 109 infectious virions/mL. Surprisingly, virus-induced apoptosis differed between the two cell lines, with the MDCK.SUS2 cells showing a much stronger apoptosis induction than the adherent MDCK cells. Obviously, despite their resistance to anoikis, the suspension cells were more susceptible to virus-induced apoptosis. Whether this is related to the adaptation process itself and/or to changes in cell survival pathways influenced by adhesion molecules or influenza virus proteins needs to be clarified in additional studies.

Lkb1 regulates organogenesis and early oncogenesis along AMPK-dependent and -independent pathways.

The tumor suppressor Lkb1/STK11/Par-4 is a key regulator of cellular energy, proliferation, and polarity, yet its mechanisms of action remain poorly defined. We generated mice harboring a mutant Lkb1 knockin allele that allows for rapid inhibition of Lkb1 kinase. Culturing embryonic tissues, we show that acute loss of kinase activity perturbs epithelial morphogenesis without affecting cell polarity. In pancreas, cystic structures developed rapidly after Lkb1 inhibition. In lung, inhibition resulted in cell-autonomous branching defects. Although the lung phenotype was rescued by an activator of the Lkb1 target adenosine monophosphate-activated kinase (AMPK), pancreatic cyst development was independent of AMPK signaling. Remarkably, the pancreatic phenotype evolved to resemble precancerous lesions, demonstrating that loss of Lkb1 was sufficient to drive the initial steps of carcinogenesis ex vivo. A similar phenotype was induced by expression of mutant K-Ras with p16/p19 deletion. Combining culture of embryonic tissues with genetic manipulation and chemical genetics thus provides a powerful approach to unraveling developmental programs and understanding cancer initiation.