Enhancing MINFLUX 3D imaging precision by vortex interference.

A new acquisition framework for MINFLUX super-resolution microscopy is proposed, termed Vortex Interference MINFLUX (viMINFLUX). From our analysis, we showed that by utilizing vortex interference, the scan range and phase shift mutually modulate MINFLUX precision, resulting in a precision enhancement by a factor of two or more for the same scan range. We further showed that vortex interference can be extended to 3D imaging, whereby 3D viMINFLUX provides for nearly isotropic 3D precision, resulting in a two-fold improvement in lateral precision and a five-fold enhancement in axial precision compared to conventional 3D MINFLUX techniques.

Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling.

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. quiescent NSCs extend an actin-rich primary protrusion toward the neuropil.

Alternative molecular mechanisms for force transmission at adherens junctions via β-catenin-vinculin interaction.

Force transmission through adherens junctions (AJs) is crucial for multicellular organization, wound healing and tissue regeneration. Recent studies shed light on the molecular mechanisms of mechanotransduction at the AJs. However, the canonical model fails to explain force transmission when essential proteins of the mechanotransduction module are mutated or missing.

Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling.

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. quiescent NSCs extend an actin-rich primary protrusion toward the neuropil.

Focal adhesions are controlled by microtubules through local contractility regulation.

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly.

Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions.

Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity (increasing the signal-to-noise ratio) and robustness (signalling above a threshold rather than activation by a single receptor) of the signal compared to signalling by single receptors. Compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, if nanoclusters function as signalling hubs remains poorly understood.

TiO Nano-Biopatterning Reveals Optimal Ligand Presentation for Cell-Matrix Adhesion Formation.

Nanoscale organization of transmembrane receptors is critical for cellular functions, enabled by the nanoscale engineering of bioligand presentation. Previously, a spatial threshold of ≤60 nm for integrin binding ligands in cell-matrix adhesion is demonstrated using monoliganded gold nanoparticles. However, the ligand geometric arrangement is limited to hexagonal arrays of monoligands, while plasmonic quenching limits further investigation by fluorescence-based high-resolution imaging.

Intrinsic self-organization of integrin nanoclusters within focal adhesions is required for cellular mechanotransduction.

Upon interaction with the extracellular matrix, the integrin receptors form nanoclusters as a first biochemical response to ligand binding. Here, we uncover a critical biodesign principle where these nanoclusters are spatially self-organized, facilitating effective mechanotransduction. Mouse Embryonic Fibroblasts (MEFs) with integrin β3 nanoclusters organized themselves with an intercluster distance of ~550 nm on uniformly coated fibronectin substrates, leading to larger focal adhesions.

Ultra high-speed single-molecule fluorescence imaging.

In two articles in this issue, Fujiwara et al. developed an ultrasensitive high-speed camera capable of single-molecule fluorescence imaging at a microsecond timescale (2023. J.

Actin polymerisation and crosslinking drive left-right asymmetry in single cell and cell collectives.

Deviations from mirror symmetry in the development of bilateral organisms are common but the mechanisms of initial symmetry breaking are insufficiently understood. The actin cytoskeleton of individual cells self-organises in a chiral manner, but the molecular players involved remain essentially unidentified and the relationship between chirality of an individual cell and cell collectives is unclear. Here, we analysed self-organisation of the chiral actin cytoskeleton in individual cells on circular or elliptical patterns, and collective cell alignment in confined microcultures.

Organization, dynamics and mechanoregulation of integrin-mediated cell-ECM adhesions.

The ability of animal cells to sense, adhere to and remodel their local extracellular matrix (ECM) is central to control of cell shape, mechanical responsiveness, motility and signalling, and hence to development, tissue formation, wound healing and the immune response. Cell-ECM interactions occur at various specialized, multi-protein adhesion complexes that serve to physically link the ECM to the cytoskeleton and the intracellular signalling apparatus. This occurs predominantly via clustered transmembrane receptors of the integrin family.

Self-supervised classification of subcellular morphometric phenotypes reveals extracellular matrix-specific morphological responses.

Cell morphology is profoundly influenced by cellular interactions with microenvironmental factors such as the extracellular matrix (ECM). Upon adhesion to specific ECM, various cell types are known to exhibit different but distinctive morphologies, suggesting that ECM-dependent cell morphological responses may harbour rich information on cellular signalling states. However, the inherent morphological complexity of cellular and subcellular structures has posed an ongoing challenge for automated quantitative analysis.

BNIP-2 Activation of Cellular Contractility Inactivates YAP for H9c2 Cardiomyoblast Differentiation.

Rho GTPases and Hippo kinases are key regulators of cardiomyoblast differentiation. However, how these signaling axes are coordinated spatiotemporally remains unclear. Here, the central and multifaceted roles of the BCH domain containing protein, BNIP-2, in orchestrating the expression of two key cardiac genes (cardiac troponin T [cTnT] and cardiac myosin light chain [Myl2]) in H9c2 and human embryonic stem cell-derived cardiomyocytes are delineated.

Ligand functionalization of titanium nanopattern enables the analysis of cell-ligand interactions by super-resolution microscopy.

The spatiotemporal aspects of early signaling events during interactions between cells and their environment dictate multiple downstream outcomes. While advances in nanopatterning techniques have allowed the isolation of these signaling events, a major limitation of conventional nanopatterning methods is its dependence on gold (Au) or related materials that plasmonically quench fluorescence and, thus, are incompatible with super-resolution fluorescence microscopy. Here we describe a novel method that integrates nanopatterning with single-molecule resolution fluorescence imaging, thus enabling mechanistic dissection of molecular-scale signaling events in conjunction with nanoscale geometry manipulation.

Emerging interplay of cytoskeletal architecture, cytomechanics and pluripotency.

Pluripotent stem cells (PSCs) are capable of differentiating into all three germ layers and trophoblasts, whereas tissue-specific adult stem cells have a more limited lineage potency. Although the importance of the cytoskeletal architecture and cytomechanical properties in adult stem cell differentiation have been widely appreciated, how they contribute to mechanotransduction in PSCs is less well understood. Here, we discuss recent insights into the interplay of cellular architecture, cell mechanics and the pluripotent states of PSCs.

Junctional ER Organization Affects Mechanotransduction at Cadherin-Mediated Adhesions.

Cadherin-mediated adhesions (also known as adherens junctions) are adhesive complexes that connect neighboring cells in a tissue. While the role of the actin cytoskeleton in withstanding tension at these sites of contact is well documented, little is known about the involvement of microtubules and the associated endoplasmic reticulum (ER) network in cadherin mechanotransduction. Therefore, we investigated how the organization of ER extensions in close proximity of cadherin-mediated adhesions can affect such complexes, and vice versa.

Enhancement of Endothelialization by Topographical Features Is Mediated by PTP1B-Dependent Endothelial Adherens Junctions Remodeling.

Endothelial Cells (ECs) form cohesive cellular lining of the vasculature and play essential roles in both developmental processes and pathological conditions. Collective migration and proliferation of endothelial cells (ECs) are key processes underlying endothelialization of vessels as well as vascular graft, but the complex interplay of mechanical and biochemical signals regulating these processes are still not fully elucidated. While surface topography and biochemical modifications have been used to enhance endothelialization , thus far such single-modality modifications have met with limited success.

Label-free Single-Molecule Quantification of Rapamycin-induced FKBP-FRB Dimerization for Direct Control of Cellular Mechanotransduction.

Chemically induced dimerization (CID) has been applied to study numerous biological processes and has important pharmacological applications. However, the complex multistep interactions under various physical constraints involved in CID impose a great challenge for the quantification of the interactions. Furthermore, the mechanical stability of the ternary complexes has not been characterized; hence, their potential application in mechanotransduction studies remains unclear.

Molecular Organization of Integrin-Based Adhesion Complexes in Mouse Embryonic Stem Cells.

The mechanical microenvironment serves as an important factor influencing stem cell differentiation. Mechanobiological responses depend strongly on actomyosin contractility and integrin-based cell-extracellular matrix (ECM) interactions mediated by adhesive structures such as focal adhesions (FAs). While the roles of FAs in mechanobiology have been intensively studied in many mesenchymal and migratory cell types, recently it has been recognized that certain pluripotent stem cells (PSCs) exhibited significantly attenuated FA-mediated mechanobiological responses.

Nanoscale Architecture of the Cortical Actin Cytoskeleton in Embryonic Stem Cells.

Mechanical cues influence pluripotent stem cell differentiation, but the underlying mechanisms are not well understood. Mouse embryonic stem cells (mESCs) exhibit unusual cytomechanical properties, including low cell stiffness and attenuated responses to substrate rigidity, but the underlying structural basis remains obscure. Using super-resolution microscopy to investigate the actin cytoskeleton in mESCs, we observed that the actin cortex consists of a distinctively sparse and isotropic network.

Publisher Correction: A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesion, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin IIA filaments, augmenting focal adhesions and disrupting podosomes.

Rapid high resolution 3D imaging of expanded biological specimens with lattice light sheet microscopy.

Expansion microscopy was invented to surpass the optical diffraction limit by physically expanding biological specimens with swellable polymers. Due to the large sizes of expanded specimens, 3D imaging techniques that are capable to acquire large volumetric data rapidly at high spatial resolution are therefore required for expansion microscopy. Lattice light sheet microscopy (LLSM) was developed to image biological specimens rapidly at high 3D spatial resolution by using a thin lattice light sheet for sample illumination.

Visualizing the 'backbone' of focal adhesions.

To understand how complex machines perform their functions, it is essential to map out the 'blueprints' of how their internal components are organized. Focal adhesions (FAs) are complex mechanobiological structures involved in a plethora of cell biological processes. The application of super-resolution microscopy in concert with protein engineering offers one approach to unravel the complexity of how individual proteins are organized within FAs.