Biology subjects

Maraspini, R.

Publications and source records attributed to Maraspini, R..

6 recordsLinked to original sources

bioRxiv2023

Tight junctions regulate lumen morphology via hydrostatic pressure and junctional tension

Formation of fluid filled lumen by epithelial tissues is a fundamental process for organ development. How epithelial cells regulate the hydraulic and cortical forces to control lumen morphology is not completely understood. Here, we quantified the mechanical role of tight junctions in lumen formation using genetically modified MDCKII cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for hydraulic inflation of lumen. However, depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements and perturbations of hydrostatic lumen pressure and junctional tension with modelling, we were able to predict lumen morphologies from the pressure-tension force balance. We found that in MDCK tissue the tight junction promotes formation of spherical lumen by decreasing cortical tension via inhibition of myosin. In addition, we found that the apical surface area of cells is largely uncoupled from lumen volume changes, suggesting that excess apical area contributes to lumen opening in the low-pressure regime. Overall, our findings provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape.

cell biology↗

bioRxiv2022

Reciprocal regulation between cell mechanics and ZO-1 guides tight junction assembly and epithelial morphogenesis

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 regulates cytoskeletal tension at cell-matrix and cell-cell interfaces in an ECM stiffness-regulated manner, possibly via differential organisation of the actin cytoskeleton. ZO-1 depletion inhibited junction assembly and disrupted morphogenesis in an ECM stiffness-dependent manner. Both processes were rescued by inhibition of cell contractility. Although ZO-1-deficient cells could assemble functional barriers at low tension, 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 tension-independent roles of ZO-1 control proper junction organisation.

cell biology↗

bioRxiv2021

Tight junction ZO proteins maintain tissue fluidity, ensuring efficient collective cell migration

Tight junctions are essential components of epithelial tissues connecting neighboring cells to provide protective barriers. Albeit their general function to seal compartments is well understood, their role in collective cell migration is largely unexplored. Here, the importance of the tight junction 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-bulged 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 display 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.

biophysics↗

bioRxiv2020

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

Primary cilia are microtubule-based organelles involved in key signaling and sensing processes in eukaryotic cells. Unlike motile cilia, which have been thoroughly studied, the structure and the composition of primary cilia remain largely unexplored despite their fundamental role in development and homeostasis. They have for long been falsely regarded as simplified versions of motile cilia because they lack distinctive elements such as dynein arms, radial spokes, and central pair complex. However, revealing the detailed molecular composition and 3D structure of primary cilia is necessary in order to understand the mechanisms that govern their functions. Such structural investigations are so far being precluded by the challenging preparation of primary cilia for cryo-electron microscopy. Here, we developed an enabling method for investigating the structure of primary cilia at molecular resolution by cryo-electron tomography. We show that the well-known "9+0" arrangement of microtubule doublets is present only at the base of the primary cilium. A few microns away from the base the ciliary architecture changes into an unstructured bundle of EB1-decorated microtubule singlets and some actin filaments. Our results suggest the existence of a previously unobserved crosstalk between actin filaments and microtubules in the primary cilium. Our work provides unprecedented insights into the molecular structure of primary cilia and a general framework for uncovering their molecular composition and function in health and disease. This opens up new possibilities to study aspects of this important organelle that have so far been out of reach.

molecular biology↗

bioRxiv2019

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 signalling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands is dependent on the two membrane associated scaffolding proteins ZO1 and ZO2. To understand how 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.

cell biology↗

bioRxiv2018

Expansion stimulated emission depletion microscopy (ExSTED)

Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultra-structure 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 sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking 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 complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate 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 straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.

biophysics↗