Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions.

Tight junctions are cell-adhesion complexes that seal tissues and are involved in cell polarity and signaling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands are dependent on the membrane-associated scaffolding proteins ZO1 and ZO2. To understand how zona occludens (ZO) proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane-attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight-junction proteins, including adhesion receptors, cytoskeletal adapters, and transcription factors. Our results suggest that an active-phase transition of ZO proteins into a condensed membrane-bound compartment drives claudin polymerization and coalescence of a continuous tight-junction belt.

Circle scanning STED fluorescence correlation spectroscopy to quantify membrane dynamics and compartmentalization.

Quantifying molecular dynamics of cell membrane constituents is required to understand organization and function of biological membranes. Because of its complex structure unambiguous interpretation of molecular membrane dynamics requires high spatial and temporal resolution measurements. In this paper, we provide a comprehensive description of circle scanning fluorescence correlation spectroscopy and its combination with stimulated emission depletion microscopy (CS-STED-FCS). This method allows quantification of sub-diffusion processes and direct mapping of heterogeneities in membranes with high spatiotemporal resolution. We show how to use model membranes to calibrate and test the technique and how to apply it in the context of living cells to quantify membrane dynamics with high spatiotemporal resolution and good statistics.

The High Mobility Group A1 (HMGA1) Chromatin Architectural Factor Modulates Nuclear Stiffness in Breast Cancer Cells.

Plasticity is an essential condition for cancer cells to invade surrounding tissues. The nucleus is the most rigid cellular organelle and it undergoes substantial deformations to get through environmental constrictions. Nuclear stiffness mostly depends on the nuclear lamina and chromatin, which in turn might be affected by nuclear architectural proteins. Among these is the HMGA1 (High Mobility Group A1) protein, a factor that plays a causal role in neoplastic transformation and that is able to disentangle heterochromatic domains by H1 displacement. Here we made use of atomic force microscopy to analyze the stiffness of breast cancer cellular models in which we modulated HMGA1 expression to investigate its role in regulating nuclear plasticity. Since histone H1 is the main modulator of chromatin structure and HMGA1 is a well-established histone H1 competitor, we correlated HMGA1 expression and cellular stiffness with histone H1 expression level, post-translational modifications, and nuclear distribution. Our results showed that HMGA1 expression level correlates with nuclear stiffness, is associated to histone H1 phosphorylation status, and alters both histone H1 chromatin distribution and expression. These data suggest that HMGA1 might promote chromatin relaxation through a histone H1-mediated mechanism strongly impacting on the invasiveness of cancer cells.

Tight junctions control lumen morphology via hydrostatic pressure and junctional tension.

Formation of fluid-filled lumina by epithelial tissues is essential for organ development. How cells control the hydraulic and cortical forces to control lumen morphology is not well understood. Here, we quantified the mechanical role of tight junctions in lumen formation using MDCK-II cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for the hydraulic inflation of a lumen. However, the depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements of hydrostatic lumen pressure and junctional tension with modeling enabled us to explain lumen morphologies from the pressure-tension force balance. Tight junctions promote lumen inflation by decreasing cortical tension via the inhibition of myosin. In addition, our results suggest that excess apical area contributes to lumen opening. Overall, we provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape.

ZO-1 Guides Tight Junction Assembly and Epithelial Morphogenesis via Cytoskeletal Tension-Dependent and -Independent Functions.

Formation and maintenance of tissue barriers require the coordination of cell mechanics and cell-cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to investigate how tight junctions regulate cell mechanics and epithelial morphogenesis. We found that depletion of the tight junction adaptor ZO-1 disrupted junction assembly and morphogenesis in an ECM stiffness-dependent manner and led to a stiffness-dependant reorganisation of active myosin. Both junction formation and morphogenesis were rescued by inhibition of actomyosin contractility. ZO-1 depletion also impacted mechanical tension at cell-matrix and E-cadherin-based cell-cell adhesions. The effect on E-cadherin also depended on ECM stiffness and correlated with effects of ECM stiffness on actin cytoskeleton organisation. However, ZO-1 knockout also revealed tension-independent functions of ZO-1. ZO-1-deficient cells could assemble functional barriers at low tension, but their tight junctions remained corrupted with strongly reduced and discontinuous recruitment of junctional components. Our results thus reveal that reciprocal regulation between ZO-1 and cell mechanics controls tight junction assembly and epithelial morphogenesis, and that, in a second, tension-independent step, ZO-1 is required to assemble morphologically and structurally fully assembled and functionally normal tight junctions.

Tight Junction ZO Proteins Maintain Tissue Fluidity, Ensuring Efficient Collective Cell Migration.

Tight junctions (TJs) are essential components of epithelial tissues connecting neighboring cells to provide protective barriers. While their general function to seal compartments is well understood, their role in collective cell migration is largely unexplored. Here, the importance of the TJ zonula occludens (ZO) proteins ZO1 and ZO2 for epithelial migration is investigated employing video microscopy in conjunction with velocimetry, segmentation, cell tracking, and atomic force microscopy/spectroscopy. The results indicate that ZO proteins are necessary for fast and coherent migration. In particular, ZO1 and 2 loss (dKD) induces actomyosin remodeling away from the central cortex towards the periphery of individual cells, resulting in altered viscoelastic properties. A tug-of-war emerges between two subpopulations of cells with distinct morphological and mechanical properties: 1) smaller and highly contractile cells with an outward bulging apical membrane, and 2) larger, flattened cells, which, due to tensile stress, display a higher proliferation rate. In response, the cell density increases, leading to crowding-induced jamming and more small cells over time. Co-cultures comprising wildtype and dKD cells migrate inefficiently due to phase separation based on differences in contractility rather than differential adhesion. This study shows that ZO proteins are necessary for efficient collective cell migration by maintaining tissue fluidity and controlling proliferation.

The molecular structure of mammalian primary cilia revealed by cryo-electron tomography.

Primary cilia are microtubule-based organelles that are important for signaling and sensing in eukaryotic cells. Unlike the thoroughly studied motile cilia, the three-dimensional architecture and molecular composition of primary cilia are largely unexplored. Yet, studying these aspects is necessary to understand how primary cilia function in health and disease. We developed an enabling method for investigating the structure of primary cilia isolated from MDCK-II cells at molecular resolution by cryo-electron tomography. We show that the textbook '9 + 0' arrangement of microtubule doublets is only present at the primary cilium base. A few microns out, the architecture changes into an unstructured bundle of EB1-decorated microtubules and actin filaments, putting an end to a long debate on the presence or absence of actin filaments in primary cilia. Our work provides a plethora of insights into the molecular structure of primary cilia and offers a methodological framework to study these important organelles.

How to orient cells in microcavities for high resolution imaging of cytokinesis and lumen formation.

Imaging dynamics of cellular morphogenesis with high spatial-temporal resolution in 3D is challenging, due to the low spatial resolution along the optical axis and photo-toxicity. However, some cellular structures are planar and hence 2D imaging should be sufficient, provided that the structure of interest can be oriented with respect to the optical axis of the microscope. Here, we report a 3D microfabrication method which positions and orients cell divisions very close to the microscope coverglass. We use this approach to study cytokinesis in fission yeasts and polarization to lumen formation in mammalian epithelial cells. We show that this method improves spatial resolution on range of common microscopies, including super-resolution STED. Altogether, this method could shed new lights on self-organization phenomena in single cells and 3D cell culture systems.

Expansion Stimulated Emission Depletion Microscopy (ExSTED).

Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultrastructure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides subdiffraction resolution by physically enlarging the sample before microscopy. The expansion of the fixed cells by cross-linking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complementary approaches, we combined ExM with STED (ExSTED) and demonstrated an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straightforward, we found that high-fidelity labeling via multi-epitopes is required to obtain emitter densities that allow ultrastructural details with ExSTED to be resolved. Our work provides a robust template for super-resolution microscopy of entire cells in the ten nanometer range.

SWAP70 Organizes the Actin Cytoskeleton and Is Essential for Phagocytosis.

Actin plays a critical role during the early stages of pathogenic microbe internalization by immune cells. In this study, we identified a key mechanism of actin filament tethering and stabilization to the surface of phagosomes in human dendritic cells. We found that the actin-binding protein SWAP70 is specifically recruited to nascent phagosomes by binding to the lipid phosphatidylinositol (3,4)-bisphosphate. Multi-color super-resolution stimulated emission depletion (STED) microscopy revealed that the actin cage surrounding early phagosomes is formed by multiple concentric rings containing SWAP70. SWAP70 colocalized with and stimulated activation of RAC1, a known activator of actin polymerization, on phagosomes. Genetic ablation of SWAP70 impaired actin polymerization around phagosomes and resulted in a phagocytic defect. These data show a key role for SWAP70 as a scaffold for tethering the peripheral actin cage to phagosomes.