Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells.

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

Lipotype acquisition during neural development is not recapitulated in stem cell-derived neurons.

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery during development, but when and how this occurs is unclear. To address this, we used high-resolution MSALL lipidomics to generate an extensive time-resolved resource of mouse brain development covering early embryonic and postnatal stages. This revealed a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. Using the resource as a reference, we next examined to which extent this can be recapitulated by commonly used protocols for in vitro neuronal differentiation of stem cells. Here, we found that the programming of the lipid metabolic machinery is incomplete and that stem cell-derived cells can only partially acquire a neural lipotype when the cell culture media is supplemented with brain-specific lipid precursors. Altogether, our work provides an extensive lipidomic resource for early mouse brain development and highlights a potential caveat when using stem cell-derived neuronal progenitors for mechanistic studies of lipid biochemistry, membrane biology and biophysics, which nonetheless can be mitigated by further optimizing in vitro differentiation protocols.

Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens.

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a three-dimensional, all-optical and noncontact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated Brillouin scattering (SBS) process; however, current implementations require high pump powers, which prohibit applications to photosensitive or live imaging of biological samples. Here we present a pulsed SBS scheme that takes advantage of the nonlinearity of the pump-probe interaction. In particular, we show that the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. We demonstrate the low phototoxicity and high specificity of our pulsed SBS approach by imaging, with subcellular detail, sensitive single cells, zebrafish larvae, mouse embryos and adult Caenorhabditis elegans. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time, opening up further possibilities for the field of mechanobiology.

Sensing their plasma membrane curvature allows migrating cells to circumvent obstacles.

To navigate through diverse tissues, migrating cells must balance persistent self-propelled motion with adaptive behaviors to circumvent obstacles. We identify a curvature-sensing mechanism underlying obstacle evasion in immune-like cells. Specifically, we propose that actin polymerization at the advancing edge of migrating cells is inhibited by the curvature-sensitive BAR domain protein Snx33 in regions with inward plasma membrane curvature. The genetic perturbation of this machinery reduces the cells' capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. Our results show how cells can read out their surface topography and utilize actin and plasma membrane biophysics to interpret their environment, allowing them to adaptively decide if they should move ahead or turn away. On the basis of our findings, we propose that the natural diversity of BAR domain proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment.

High-resolution line-scan Brillouin microscopy for live imaging of mechanical properties during embryo development.

Brillouin microscopy can assess mechanical properties of biological samples in a three-dimensional (3D), all-optical and hence non-contact fashion, but its weak signals often lead to long imaging times and require an illumination dosage harmful for living organisms. Here, we present a high-resolution line-scanning Brillouin microscope for multiplexed and hence fast 3D imaging of dynamic biological processes with low phototoxicity. The improved background suppression and resolution, in combination with fluorescence light-sheet imaging, enables the visualization of the mechanical properties of cells and tissues over space and time in living organism models such as fruit flies, ascidians and mouse embryos.

Convolutional networks for supervised mining of molecular patterns within cellular context.

Cryo-electron tomograms capture a wealth of structural information on the molecular constituents of cells and tissues. We present DeePiCt (deep picker in context), an open-source deep-learning framework for supervised segmentation and macromolecular complex localization in cryo-electron tomography. To train and benchmark DeePiCt on experimental data, we comprehensively annotated 20 tomograms of Schizosaccharomyces pombe for ribosomes, fatty acid synthases, membranes, nuclear pore complexes, organelles, and cytosol. By comparing DeePiCt to state-of-the-art approaches on this dataset, we show its unique ability to identify low-abundance and low-density complexes. We use DeePiCt to study compositionally distinct subpopulations of cellular ribosomes, with emphasis on their contextual association with mitochondria and the endoplasmic reticulum. Finally, applying pre-trained networks to a HeLa cell tomogram demonstrates that DeePiCt achieves high-quality predictions in unseen datasets from different biological species in a matter of minutes. The comprehensively annotated experimental data and pre-trained networks are provided for immediate use by the community.

Quantification of Apparent Membrane Tension and Membrane-to-Cortex Attachment in Animal Cells Using Atomic Force Microscopy-Based Force Spectroscopy.

Reliable quantification of a cell's biophysical properties is key for understanding the role of mechanics in cell biology. Plasma membrane tension, the energetic cost of increasing the surface area of the plasma membrane, has been shown to regulate a plethora of cellular processes, ranging from leading edge formation to phagocytosis and membrane trafficking. Here, we describe the measurement of this key mechanical property of the cell surface using atomic force microscopy (AFM)-based force spectroscopy. Depending on the nature of the force curve acquisition, AFM measurements can quantify various membrane tension components, such as apparent membrane tension and membrane-to-cortex attachment (MCA). We discuss the biophysical background (1), required materials (2), sample preparation (3.1), AFM-probe calibration and functionalization (3.2), force curve acquisition (3.3) and data analysis and representation (3.4).

Understanding the interplay of membrane trafficking, cell surface mechanics, and stem cell differentiation.

Stem cells can generate a diversity of cell types during development, regeneration and adult tissue homeostasis. Differentiation changes not only the cell fate in terms of gene expression but also the physical properties and functions of cells, e.g. the secretory activity, cell shape, or mechanics. Conversely, these activities and properties can also regulate differentiation itself. Membrane trafficking is known to modulate signal transduction and thus has the potential to control stem cell differentiation. On the other hand, membrane trafficking, particularly from and to the plasma membrane, depends on the mechanical properties of the cell surface such as tension within the plasma membrane or the cortex. Indeed, recent findings demonstrate that cell surface mechanics can also control cell fate. Here, we review the bidirectional relationships between these three fundamental cellular functions, i.e. membrane trafficking, cell surface mechanics, and stem cell differentiation. Furthermore, we discuss commonly used methods in each field and how combining them with new tools will enhance our understanding of their interplay. Understanding how membrane trafficking and cell surface mechanics can guide stem cell fate holds great potential as these concepts could be exploited for directed differentiation of stem cells for the fields of tissue engineering and regenerative medicine.

Strength of interactions in the Notch gene regulatory network determines patterning and fate in the notochord.

Development of multicellular organisms requires the generation of gene expression patterns that determines cell fate and organ shape. Groups of genetic interactions known as Gene Regulatory Networks (GRNs) play a key role in the generation of such patterns. However, how the topology and parameters of GRNs determine patterning in vivo remains unclear due to the complexity of most experimental systems. To address this, we use the zebrafish notochord, an organ where coin-shaped precursor cells are initially arranged in a simple unidimensional geometry. These cells then differentiate into vacuolated and sheath cells. Using newly developed transgenic tools together with in vivo imaging, we identify jag1a and her6/her9 as the main components of a Notch GRN that generates a lateral inhibition pattern and determines cell fate. Making use of this experimental system and mathematical modeling we show that lateral inhibition patterning is promoted when ligand-receptor interactions are stronger within the same cell than in neighboring cells. Altogether, we establish the zebrafish notochord as an experimental system to study pattern generation, and identify and characterize how the properties of GRNs determine self-organization of gene patterning and cell fate.

Self-generated gradients steer collective migration on viscoelastic collagen networks.

Growing evidence suggests that the physical properties of the cellular microenvironment influence cell migration. However, it is not currently understood how active physical remodelling by cells affects migration dynamics. Here we report that cell clusters seeded on deformable collagen-I networks display persistent collective migration despite not showing any apparent intrinsic polarity. Clusters generate transient gradients in collagen density and alignment due to viscoelastic relaxation of the collagen networks. Combining theory and experiments, we show that crosslinking collagen networks or reducing cell cluster size results in reduced network deformation, shorter viscoelastic relaxation time and smaller gradients, leading to lower migration persistence. Traction force and Brillouin microscopy reveal asymmetries in force distributions and collagen stiffness during migration, providing evidence of mechanical cross-talk between cells and their substrate during migration. This physical model provides a mechanism for self-generated directional migration on viscoelastic substrates in the absence of internal biochemical polarity cues.

Load adaptation by endocytic actin networks.

Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly-mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feed back on assembly. By manipulating membrane tension and Arp2/3 complex activity, we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions.

A mechano-osmotic feedback couples cell volume to the rate of cell deformation.

Mechanics has been a central focus of physical biology in the past decade. In comparison, how cells manage their size is less understood. Here, we show that a parameter central to both the physics and the physiology of the cell, its volume, depends on a mechano-osmotic coupling. We found that cells change their volume depending on the rate at which they change shape, when they spontaneously spread or when they are externally deformed. Cells undergo slow deformation at constant volume, while fast deformation leads to volume loss. We propose a mechanosensitive pump and leak model to explain this phenomenon. Our model and experiments suggest that volume modulation depends on the state of the actin cortex and the coupling of ion fluxes to membrane tension. This mechano-osmotic coupling defines a membrane tension homeostasis module constantly at work in cells, causing volume fluctuations associated with fast cell shape changes, with potential consequences on cellular physiology.

Dendritic cell actin dynamics control contact duration and priming efficiency at the immunological synapse.

Dendritic cells (DCs) are crucial for the priming of naive T cells and the initiation of adaptive immunity. Priming is initiated at a heterologous cell-cell contact, the immunological synapse (IS). While it is established that F-actin dynamics regulates signaling at the T cell side of the contact, little is known about the cytoskeletal contribution on the DC side. Here, we show that the DC actin cytoskeleton is decisive for the formation of a multifocal synaptic structure, which correlates with T cell priming efficiency. DC actin at the IS appears in transient foci that are dynamized by the WAVE regulatory complex (WRC). The absence of the WRC in DCs leads to stabilized contacts with T cells, caused by an increase in ICAM1-integrin-mediated cell-cell adhesion. This results in lower numbers of activated and proliferating T cells, demonstrating an important role for DC actin in the regulation of immune synapse functionality.

Cell Surface Mechanics Gate Embryonic Stem Cell Differentiation.

Cell differentiation typically occurs with concomitant shape transitions to enable specialized functions. To adopt a different shape, cells need to change the mechanical properties of their surface. However, whether cell surface mechanics control the process of differentiation has been relatively unexplored. Here we show that membrane mechanics gate exit from naive pluripotency of mouse embryonic stem cells. By measuring membrane tension during early differentiation, we find that naive stem cells release their plasma membrane from the underlying actin cortex when transitioning to a primed state. By mechanically tethering the plasma membrane to the cortex by enhancing Ezrin activity or expressing a synthetic signaling-inert linker, we demonstrate that preventing this detachment forces stem cells to retain their naive pluripotent identity. We thus identify a decrease in membrane-to-cortex attachment as a new cell-intrinsic mechanism that is essential for stem cells to exit pluripotency.

Protocol on Tissue Preparation and Measurement of Tumor Stiffness in Primary and Metastatic Colorectal Cancer Samples with an Atomic Force Microscope.

Tumors have complex microenvironments that provide not only biochemical but also mechanical cues to the cells within. In particular, tumor stiffness has been shown to regulate tumor growth, invasion, and metastasis. Understanding how the mechanical microenvironment controls cell behavior could be key to unraveling new therapeutic approaches. Here, we describe a protocol to prepare primary and metastatic human colorectal cancer samples and measure tumor stiffness at the tissue level using an atomic force microscope in spectroscopy mode. For complete details on the use and execution of this protocol, please refer to Shen et al. (2020).

Using migrating cells as probes to illuminate features in live embryonic tissues.

The biophysical and biochemical properties of live tissues are important in the context of development and disease. Methods for evaluating these properties typically involve destroying the tissue or require specialized technology and complicated analyses. Here, we present a novel, noninvasive methodology for determining the spatial distribution of tissue features within embryos, making use of nondirectionally migrating cells and software we termed "Landscape," which performs automatized high-throughput three-dimensional image registration. Using the live migrating cells as bioprobes, we identified structures within the zebrafish embryo that affect the distribution of the cells and studied one such structure constituting a physical barrier, which, in turn, influences amoeboid cell polarity. Overall, this work provides a unique approach for detecting tissue properties without interfering with animal's development. In addition, Landscape allows for integrating data from multiple samples, providing detailed and reliable quantitative evaluation of variable biological phenotypes in different organisms.

Reduction of Liver Metastasis Stiffness Improves Response to Bevacizumab in Metastatic Colorectal Cancer.

Tumors are influenced by the mechanical properties of their microenvironment. Using patient samples and atomic force microscopy, we found that tissue stiffness is higher in liver metastases than in primary colorectal tumors. Highly activated metastasis-associated fibroblasts increase tissue stiffness, which enhances angiogenesis and anti-angiogenic therapy resistance. Drugs targeting the renin-angiotensin system, normally prescribed to treat hypertension, inhibit fibroblast contraction and extracellular matrix deposition, thereby reducing liver metastases stiffening and increasing the anti-angiogenic effects of bevacizumab. Patients treated with bevacizumab showed prolonged survival when concomitantly treated with renin-angiotensin inhibitors, highlighting the importance of modulating the mechanical microenvironment for therapeutic regimens.

Pay attention to membrane tension: Mechanobiology of the cell surface.

The cell surface is a mechanobiological unit that encompasses the plasma membrane, its interacting proteins, and the complex underlying cytoskeleton. Recently, attention has been directed to the mechanics of the plasma membrane, and in particular membrane tension, which has been linked to diverse cellular processes such as cell migration and membrane trafficking. However, how tension across the plasma membrane is regulated and propagated is still not completely understood. Here, we review recent efforts to study the interplay between membrane tension and the cytoskeletal machinery and how they control cell form and function. We focus on factors that have been proposed to affect the propagation of membrane tension and as such could determine whether it can act as a global or local regulator of cell behavior. Finally, we discuss the limitations of the available tool kit as new approaches that reveal its dynamics in cells are needed to decipher how membrane tension regulates diverse cellular processes.

A 3D Brillouin microscopy dataset of the in-vivo zebrafish eye.

In this work we present three-dimensional (3D) measurements of Brillouin scattering spectra of the in-vivo zebrafish larvae eye. This dataset was obtained by Brillouin microscopy, an emerging all-optical and non-contact technique that gives access to material properties through the process of Brillouin scattering. Herein, we share a representative 3D dataset of spectral properties of 48-52 h post-fertilization (hpf) zebrafish embryos. These spectral properties can be related to a complex longitudinal modulus and thus elastic and viscous properties given knowledge of refractive index and material density. The dataset encompasses the crystalline lens as well as several different retinal layers. This data provides a valuable resource as well as a starting point for researchers interested in the mechanobiology of vertebrate eye development.