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.

Alternative molecular mechanisms for force transmission at adherens junctions via ß-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. Here, we demonstrate that, in absence of α-catenin, ß-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that ß-catenin can prevent vinculin autoinhibition in the presence of α-catenin by occupying vinculin´s head-tail interaction site, thus preserving force transmission capability. Taken together, our findings suggest a multi-step force transmission process at AJs, where α-catenin and ß-catenin can alternatively and cooperatively interact with vinculin. This can explain the graded responses needed to maintain tissue mechanical homeostasis and, importantly, unveils a force-bearing mechanism involving ß-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin.

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.

Vibrator and PI4KIIIα govern neuroblast polarity by anchoring non-muscle myosin II.

A central feature of most stem cells is the ability to self-renew and undergo differentiation via asymmetric division. However, during asymmetric division the role of phosphatidylinositol (PI) lipids and their regulators is not well established. Here, we show that the sole type I PI transfer protein, Vibrator, controls asymmetric division of Drosophilaneural stem cells (NSCs) by physically anchoring myosin II regulatory light chain, Sqh, to the NSC cortex. Depletion of vib or disruption of its lipid binding and transfer activities disrupts NSC polarity. We propose that Vib stimulates PI4KIIIα to promote synthesis of a plasma membrane pool of phosphatidylinositol 4-phosphate [PI(4)P] that, in turn, binds and anchors myosin to the NSC cortex. Remarkably, Sqh also binds to PI(4)P in vitro and both Vib and Sqh mediate plasma membrane localization of PI(4)P in NSCs. Thus, reciprocal regulation between Myosin and PI(4)P likely governs asymmetric division of NSCs.