eIF4F is a thermo-sensing regulatory node in the translational heat shock response.

Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.

Intra-condensate demixing of TDP-43 inside stress granules generates pathological aggregates.

Cytosolic aggregation of the nuclear protein TDP-43 is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43 enriched phase within stress granules, which subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We conclude that up-concentration inside condensates and simultaneous exposure to environmental stress could be a general pathway for protein aggregation, with intra-condensate demixing constituting a key intermediate step.

Multivalent Tau/PSD-95 interactions arrest in vitro condensates and clusters mimicking the postsynaptic density.

Alzheimer's disease begins with mild memory loss and slowly destroys memory and thinking. Cognitive impairment in Alzheimer's disease has been associated with the localization of the microtubule-associated protein Tau at the postsynapse. However, the correlation between Tau at the postsynapse and synaptic dysfunction remains unclear. Here, we show that Tau arrests liquid-like droplets formed by the four postsynaptic density proteins PSD-95, GKAP, Shank, Homer in solution, as well as NMDA (N-methyl-D-aspartate)-receptor-associated protein clusters on synthetic membranes. Tau-mediated condensate/cluster arrest critically depends on the binding of multiple interaction motifs of Tau to a canonical GMP-binding pocket in the guanylate kinase domain of PSD-95. We further reveal that competitive binding of a high-affinity phosphorylated peptide to PSD-95 rescues the diffusional dynamics of an NMDA truncated construct, which contains the last five amino acids of the NMDA receptor subunit NR2B fused to the C-terminus of the tetrameric GCN4 coiled-coil domain, in postsynaptic density-like condensates/clusters. Taken together, our findings propose a molecular mechanism where Tau modulates the dynamic properties of the postsynaptic density.

Dynamic matrices with DNA-encoded viscoelasticity for cell and organoid culture.

Three-dimensional cell and organoid cultures rely on the mechanical support of viscoelastic matrices. However, commonly used matrix materials lack control over key cell-instructive properties. Here we report on fully synthetic hydrogels based on DNA libraries that self-assemble with ultrahigh-molecular-weight polymers, forming a dynamic DNA-crosslinked matrix (DyNAtrix). DyNAtrix enables computationally predictable and systematic control over its viscoelasticity, thermodynamic and kinetic parameters by changing DNA sequence information. Adjustable heat activation allows homogeneous embedding of mammalian cells. Intriguingly, stress-relaxation times can be tuned over four orders of magnitude, recapitulating mechanical characteristics of living tissues. DyNAtrix is self-healing, printable, exhibits high stability, cyto- and haemocompatibility, and controllable degradation. DyNAtrix-based cultures of human mesenchymal stromal cells, pluripotent stem cells, canine kidney cysts and human trophoblast organoids show high viability, proliferation and morphogenesis. DyNAtrix thus represents a programmable and versatile precision matrix for advanced approaches to biomechanics, biophysics and tissue engineering.

Cellular segregation in cocultures is driven by differential adhesion and contractility on distinct timescales.

Cellular sorting and pattern formation are crucial for many biological processes such as development, tissue regeneration, and cancer progression. Prominent physical driving forces for cellular sorting are differential adhesion and contractility. Here, we studied the segregation of epithelial cocultures containing highly contractile, ZO1/2-depleted MDCKII cells (dKD) and their wild-type (WT) counterparts using multiple quantitative, high-throughput methods to monitor their dynamical and mechanical properties. We observe a time-dependent segregation process governed mainly by differential contractility on short (<5 h) and differential adhesion on long (>5 h) timescales. The overly contractile dKD cells exert strong lateral forces on their WT neighbors, thereby apically depleting their surface area. Concomitantly, the tight junction-depleted, contractile cells exhibit weaker cell-cell adhesion and lower traction force. Drug-induced contractility reduction and partial calcium depletion delay the initial segregation but cease to change the final demixed state, rendering differential adhesion the dominant segregation force at longer timescales. This well-controlled model system shows how cell sorting is accomplished through a complex interplay between differential adhesion and contractility and can be explained largely by generic physical driving forces.

Hepatocyte apical bulkheads provide a mechanical means to oppose bile pressure.

Hepatocytes grow their apical surfaces anisotropically to generate a 3D network of bile canaliculi (BC). BC elongation is ensured by apical bulkheads, membrane extensions that traverse the lumen and connect juxtaposed hepatocytes. We hypothesize that apical bulkheads are mechanical elements that shape the BC lumen in liver development but also counteract elevated biliary pressure. Here, by resolving their structure using STED microscopy, we found that they are sealed by tight junction loops, connected by adherens junctions, and contain contractile actomyosin, characteristics of mechanical function. Apical bulkheads persist at high pressure upon microinjection of fluid into the BC lumen, and laser ablation demonstrated that they are under tension. A mechanical model based on ablation results revealed that apical bulkheads double the pressure BC can hold. Apical bulkhead frequency anticorrelates with BC connectivity during mouse liver development, consistent with predicted changes in biliary pressure. Our findings demonstrate that apical bulkheads are load-bearing mechanical elements that could protect the BC network against elevated pressure.

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.

Biomolecular condensates in epithelial junctions.

Epithelial junctions are transmembrane protein complexes that regulate cell adhesion, cell polarity, tissue permeability, and tissue mechanics. Most junctional complexes contain membrane attached cytoplasmic plaques that regulate junction assembly and are composed of multivalent scaffold proteins. In this review, we discuss phase separation of multivalent proteins as a general process that drives assembly of many membrane-less cellular compartments. And we summarise recent evidence that phase separation of junctional scaffold proteins is involved in the assembly of tight junctions and focal adhesions.

Discovery of anti-inflammatory physiological peptides that promote tissue repair by reinforcing epithelial barrier formation.

Epithelial barriers that prevent dehydration and pathogen invasion are established by tight junctions (TJs), and their disruption leads to various inflammatory diseases and tissue destruction. However, a therapeutic strategy to overcome TJ disruption in diseases has not been established because of the lack of clinically applicable TJ-inducing molecules. Here, we found TJ-inducing peptides (JIPs) in mice and humans that corresponded to 35 to 42 residue peptides of the C terminus of alpha 1-antitrypsin (A1AT), an acute-phase anti-inflammatory protein. JIPs were inserted into the plasma membrane of epithelial cells, which promoted TJ formation by directly activating the heterotrimeric G protein G13. In a mouse intestinal epithelial injury model established by dextran sodium sulfate, mouse or human JIP administration restored TJ integrity and strongly prevented colitis. Our study has revealed TJ-inducing anti-inflammatory physiological peptides that play a critical role in tissue repair and proposes a previously unidentified therapeutic strategy for TJ-disrupted diseases.

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.

Transcription organizes euchromatin via microphase separation.

In eukaryotes, DNA is packed inside the cell nucleus in the form of chromatin, which consists of DNA, proteins such as histones, and RNA. Euchromatin, which is permissive for transcription, is spatially organized into transcriptionally inactive domains interspersed with pockets of transcriptional activity. While transcription and RNA have been implicated in euchromatin organization, it remains unclear how their interplay forms and maintains transcription pockets. Here we combine theory and experiment to analyze the dynamics of euchromatin organization as pluripotent zebrafish cells exit mitosis and begin transcription. We show that accumulation of RNA induces formation of transcription pockets which displace transcriptionally inactive chromatin. We propose that the accumulating RNA recruits RNA-binding proteins that together tend to separate from transcriptionally inactive euchromatin. Full phase separation is prevented because RNA remains tethered to transcribed euchromatin through RNA polymerases. Instead, smaller scale microphases emerge that do not grow further and form the typical pattern of euchromatin organization.

Expansion STED microscopy (ExSTED).

The recently developed expansion microscopy method (ExM) allows for the resolution of structures below the diffraction limit of light not by sophisticated instrumentation, but rather by physically expanding the molecular structure of cells. This happens by crosslinking the protein in the sample to a hydrogel that is polymerized in situ and subsequently expanded, tearing the proteins apart in a nearly isotropic manner. In the resulting, larger facsimile of the original sample, the fluorescence-labeled molecules of interest can be optically separated by conventional fluorescence microscopy since the intermolecular distances are enlarged by a factor ranging from ~4 to 20 depending on the chemistry used for the hydrogel. The achieved improvement in resolution thus corresponds to the expansion factor. Further increase in resolution beyond this value may be achieved by combining ExM with established super-resolution microscopy methods. Indeed, this is possible using structured illumination microscopy (SIM) (Halpern et al., 2017; Wang et al., 2018), single molecule localization microscopy (SMLM) (Zwettler et al., 2020) and stimulated emission depletion (STED), as we and others have shown recently (Gambarotto et al., 2019; Gao et al., 2018; Kim, Kim, Lee, & Shim, 2019; Unnersjö-Jess et al., 2016). Here, we provide a protocol, for our method, called ExSTED, which enabled us to achieve an increase in resolution of up to 30-fold compared to conventional microscopy, well beyond what is possible with conventional STED microscopy. Our protocol includes a strategy to achieve very high intensity fluorescence labeling, which is essential for optimal signal retention during the expansion process for ExSTED.

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.

RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation.

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.

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.

How to minimize dye-induced perturbations while studying biomembrane structure and dynamics: PEG linkers as a rational alternative.

Organic dye-tagged lipid analogs are essential for many fluorescence-based investigations of complex membrane structures, especially when using advanced microscopy approaches. However, lipid analogs may interfere with membrane structure and dynamics, and it is not obvious that the properties of lipid analogs would match those of non-labeled host lipids. In this work, we bridged atomistic simulations with super-resolution imaging experiments and biomimetic membranes to assess the performance of commonly used sphingomyelin-based lipid analogs. The objective was to compare, on equal footing, the relative strengths and weaknesses of acyl chain labeling, headgroup labeling, and labeling based on poly-ethyl-glycol (PEG) linkers in determining biomembrane properties. We observed that the most appropriate strategy to minimize dye-induced membrane perturbations and to allow consideration of Brownian-like diffusion in liquid-ordered membrane environments is to decouple the dye from a membrane by a PEG linker attached to a lipid headgroup. Yet, while the use of PEG linkers may sound a rational and even an obvious approach to explore membrane dynamics, the results also suggest that the dyes exploiting PEG linkers interfere with molecular interactions and their dynamics. Overall, the results highlight the great care needed when using fluorescent lipid analogs, in particular accurate controls.

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.