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. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine F-actin structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of Mrtf, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G-protein-coupled receptor (GPCR) Smog, G-protein αq subunit, Rho1 GTPase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand Fog to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, a NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.

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. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

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. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at sub-diffraction limited resolution of ~100nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in immobile population of integrin ß3 organised as nanoclusters, which (ii) in turn serve to organise nanoclusters of associated key adhesome proteins- vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signalling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct dynamics dependent on function. (iv) long-lived nanoclusters function as signalling hubs- wherein phosphorylated FAK and paxillin formed stable nanoclusters in close proximity to immobile integrin nanoclusters which are disassembled in response to inactivation signal by phosphatase PTPN12 (v) signalling takes place in response to an external signal such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. Taken together, these results demonstrate that signalling downstream of transmembrane receptors is organised as hubs of signalling proteins (FAK, paxillin, vinculin) seeded by nanoclusters of the transmembrane receptor (integrin).

TiO2 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. Here, these limitations are overcome with dielectric TiO2 nanopatterns, eliminating fluorescence quenching, thus enabling super-resolution fluorescence microscopy on nanopatterns. By dual-color super-resolution imaging, high precision and consistency among nanopatterns, bioligands, and integrin nanoclusters are observed, validating the high quality and integrity of both nanopattern functionalization and passivation. By screening TiO2 nanodiscs with various diameters, an increase in fibroblast cell adhesion, spreading area, and Yes-associated protein (YAP) nuclear localization on 100 nm diameter compared with smaller diameters was observed. Focal adhesion kinase is identified as the regulatory signal. These findings explore the optimal ligand presentation when the minimal requirements are sufficiently fulfilled in the heterogenous extracellular matrix network of isolated binding regions with abundant ligands. Integration of high-fidelity nano-biopatterning with super-resolution imaging allows precise quantitative studies to address early signaling events in response to receptor clustering and their nanoscale organization.

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 ß3 nanoclusters organized themselves with an intercluster distance of ~550 nm on uniformly coated fibronectin substrates, leading to larger focal adhesions. We determined that this spatial organization was driven by cell-intrinsic factors since there was no pre-existing pattern on the substrates. Altering this spatial organization using cyclo-RGD functionalized Titanium nanodiscs (of 100 nm, corroborating to the integrin nanocluster size) spaced at intervals of 300 nm (almost half), 600 nm (normal) or 1000 nm (almost double) resulted in abrogation in mechanotransduction, indicating that a new parameter i.e., an optimal intercluster distance is necessary for downstream function. Overexpression of α-actinin, which induces a kink in the integrin tail, disrupted the establishment of the optimal intercluster distance, while simultaneous co-overexpression of talin head with α-actinin rescued it, indicating a concentration-dependent competition, and that cytoplasmic activation of integrin by talin head is required for the optimal intercluster organization. Additionally, talin head-mediated recruitment of FHOD1 that facilitates local actin polymerization at nanoclusters, and actomyosin contractility were also crucial for establishing the optimal intercluster distance and a robust mechanotransduction response. These findings demonstrate that cell-intrinsic machinery plays a vital role in organizing integrin receptor nanoclusters within focal adhesions, encoding essential information for downstream mechanotransduction signalling.

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. Cell Biol.https://doi.org/10.1083/jcb.202110160). This major leap in detection speed enables the organization of plasma membrane and integrin-based adhesions to be probed in unprecedented detail (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202110162).

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. Screening based on deep-learning analysis of actin patterns identified actin polymerisation regulators, depletion of which suppresses chirality (mDia1) or reverses chirality direction (profilin1 and CapZß). The reversed chirality  is mDia1-independent but requires the function of actin-crosslinker α-actinin1. A robust correlation between the effects of a variety of actin assembly regulators on chirality of individual cells and cell collectives is revealed. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry.

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. Here we review how the interplay of mechanical forces, biochemical signalling and molecular self-organization determines the composition, organization, mechanosensitivity and dynamics of these adhesions. Progress in the identification of core multi-protein modules within the adhesions and characterization of rearrangements of their components in response to force, together with advanced imaging approaches, has improved understanding of adhesion maturation and turnover and the relationships between adhesion structures and functions. Perturbations of adhesion contribute to a broad range of diseases and to age-related dysfunction, thus an improved understanding of their molecular nature may facilitate therapeutic intervention in these conditions.

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. Since multi-channel fluorescence microscopy provides robust molecular specificity important for the biological interpretations of observed cellular architecture, here we develop a deep learning-based analysis pipeline for the classification of cell morphometric phenotypes from multi-channel fluorescence micrographs, termed SE-RNN (residual neural network with squeeze-and-excite blocks). We demonstrate SERNN-based classification of distinct morphological signatures observed when fibroblasts or epithelial cells are presented with different ECM. Our results underscore how cell shapes are non-random and established the framework for classifying cell shapes into distinct morphological signature in a cell-type and ECM-specific manner.

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. This study shows that BNIP-2 mRNA and protein expression increase with the onset of cTnT and Myl2 and promote the alignment of H9c2 cardiomyocytes. Mechanistically, BNIP-2 is required for the inactivation of YAP through YAP phosphorylation and its cytosolic retention. Turbo-ID proximity labeling corroborated by super-resolution analyses and biochemical pulldown data reveals a scaffolding role of BNIP-2 for LATS1 to phosphorylate and inactivate YAP in a process that requires BNIP-2 activation of cellular contractility. The findings identify BNIP-2 as a pivotal signaling scaffold that spatiotemporally integrates RhoA/Myosin II and LATS1/YAP mechanotransduction signaling to drive cardiomyoblast differentiation, by switching the genetic programming from YAP-dependent growth to YAP-silenced differentiation. These findings offer insights into the importance of scaffolding proteins in bridging the gap between mechanical and biochemical signals in cell growth and differentiation and the prospects in translational applications.

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. Our method exploits nanofabricated titanium (Ti) whose oxide (TiO2) is a dielectric material with no plasmonic effects. We describe the surface chemistry for decorating specific ligands such as cyclo-RGD (arginine, glycine and aspartate: a ligand for fibronectin-binding integrins) on TiO2 nanoline and nanodot substrates, and demonstrate the ability to perform dual-color super-resolution imaging on these patterns. Ti nanofabrication is similar to other metallic materials like Au, while the functionalization of TiO2 is relatively fast, safe, economical, easy to set up with commonly available reagents, and robust against environmental parameters such as humidity. Fabrication of nanopatterns takes ~2-3 d, preparation for functionalization ~1.5-2 d, and functionalization 3 h, after which cell culture and imaging experiments can be performed. We suggest that this method may facilitate the interrogation of nanoscale geometry and force at single-molecule resolution, and should find ready applications in early detection and interpretation of physiochemical signaling events at the cell membrane in the fields of cell biology, immunology, regenerative medicine, and related fields.

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. Notably, the distinctive cytomechanical and morphodynamic profiles of PSCs are accompanied by a number of unique molecular mechanisms. The extent to which such mechanobiological signatures are intertwined with pluripotency regulation remains an open question that may have important implications in developmental morphogenesis and regenerative medicine.

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. Here, we show that the extension of the ER to cadherin-mediated adhesions is tension dependent and appears to be cadherin-type specific. Furthermore, the different structural organization of the ER/microtubule network seems to affect the localization of ER-bound PTP1B at cadherin-mediated adhesions. This phosphatase is involved in the modulation of vinculin, a molecular clutch which enables differential engagement of the cadherin-catenin layer with the actomyosin cytoskeleton in response to tension. This suggests a link between structural organization of the ER/microtubule network around cadherin-specific adhesions, to control the mechanotransduction of adherens junctions by modulation of vinculin conformational state.

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 in vitro, thus far such single-modality modifications have met with limited success. As combination therapy that utilizes multiple modalities has shown improvement in addressing various intractable and complex biomedical conditions, here, we explore a combined strategy that utilizes topographical features in conjunction with pharmacological perturbations. We characterized EC behaviors in response to micrometer-scale grating topography in concert with pharmacological perturbations of endothelial adherens junctions (EAJ) regulators. We found that the protein tyrosine phosphatase, PTP1B, serves as a potent regulator of EAJ stability, with PTP1B inhibition synergizing with grating topographies to modulate EAJ rearrangement, thereby augmenting global EC monolayer sheet orientation, proliferation, connectivity, and collective cell migration. Our data delineates the crosstalk between cell-ECM topography sensing and cell-cell junction integrity maintenance and suggests that the combined use of grating topography and PTP1B inhibitor could be a promising strategy for promoting collective EC migration and proliferation.

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. However, due to the current limitations of LLSM mechanism and the optical design of LLS microscopes, it is challenging to image large expanded specimens at isotropic high spatial resolution using LLSM. To address the problem, we first optimized the sample preparation and expansion procedure for LLSM. Then, we implement a tiling lattice light sheet method to minimize sample translation during imaging and achieve much faster 3D imaging speed at high spatial resolution with more isotropic performance. Taken together, we report a general and improved 3D super-resolution imaging method for expanded samples.

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. Here, we report a single-molecule detector that can accurately quantify almost all key interactions involved in CID and the mechanical stability of the ternary complex, in a label-free manner. Its application is demonstrated using rapamycin-induced heterodimerization of FRB and FKBP as an example. We revealed the sufficient mechanical stability of the FKBP/rapamycin/FRB ternary complex and demonstrated its utility in the precise switching of talin-mediated force transmission in integrin-based cell adhesions.

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. Surprisingly, the architecture and mechanics of the mESC actin cortex appear to be largely myosin II-independent. The network density can be modulated by perturbing Arp2/3 and formin, whereas capping protein (CP) negatively regulates cell stiffness. Transient Arp2/3-containing aster-like structures are implicated in the organization and mechanical homeostasis of the cortical network. By generating a low-density network that physically excludes myosin II, the interplay between Arp2/3, formin, and CP governs the nanoscale architecture of the actin cortex and prescribes the cytomechanical properties of mESCs.

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. Myosin IIA filaments are indispensable effectors in the microtubule-driven regulation of integrin-mediated adhesions. Myosin IIA filament assembly depends on Rho activation by the RhoGEF GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capture by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the assembly of myosin IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.