Biology subjects

Diz-Munoz, A.

Publications and source records attributed to Diz-Munoz, A..

At least 19 recordsLinked to original sources

bioRxiv2025

Cross-Species Biomechanical Determinants of Shape Diversity

How complex molecular mechanisms translate into diverse multicellular shapes remains unclear. By leveraging the bi-layered architecture of six cnidarian species that diverged 500 million years ago, we show that modularity in supracellular mechanics governs larval shape diversity. Using active surface theory, quantitative imaging, and an inducible genetic system, we identify species-specific variations in three biomechanical modules. Basally aligned stress fibers drive axial elongation, while oral geometry and aboral rigidity define shape polarity. Remarkably, manipulating these modules transforms one species shape into another, demonstrating the causal relationship between module variation and shape diversity. Our analysis also uncovers instances of mechanical redundancies, where distinct module combinations generate similar shapes. These findings provide a general framework for how molecular complexity funnels into mesoscale mechanical determinants shaping morphological diversity.

developmental biology↗

bioRxiv2024

Caging of membrane-to-cortex attachment proteins can trigger cellular symmetry breaking

To migrate, divide, and change shape, cells must regulate the mechanics of their periphery. The cell surface is a complex structure that consists of a thin, contractile cortical actin network tethered to the plasma membrane by specialized membrane-to-cortex attachment (MCA) proteins. This active and constantly fluctuating system maintains a delicate mechanochemical state which permits spontaneous polarization and shape change when needed. Combining in silico, in vitro, and in vivo experiments we show how membrane viscosity and MCA protein length regulate cortical dynamics. We reveal a novel mechanism whereby caging of linker proteins in the actin cortex allows for the amplification of small changes in these key parameters, leading to major alterations of cortical contractility. In cells, this mechanism alone gives rise to symmetry breaking phenomena, suggesting that local changes in lipid composition, in combination with the choice of MCA proteins, contribute to the regulation of cellular morphogenesis and function.

biophysics↗

bioRxiv2024

PIEZO-dependent mechano-sensing of the niche is essential for intestinal stem cell fate decision and maintenance

Stem cells continuously perceive and respond to various environmental signals to maintain homeostasis. In addition to biochemical factors, the stem cell niche is subjected to mechanical and physical cues. However, it remains unclear how stem cells can sense mechanical signals from their niche in vivo. Since intestinal stem cells constantly and directly face the external environment, we investigated the roles of mechano-sensing PIEZO ion channels in the gut stem cell niche. By employing mouse genetics and performing single-cell RNAseq analysis, we revealed the absolute requirement for PIEZO channels in intestinal stem cell (ISC) state dynamics and maintenance. In vivo measurement of basement membrane region stiffness demonstrated that ISCs reside in a more rigid microenvironment at the bottom of the crypt. Using 3D and 2D organoid systems combined with bioengineered substrates and a cell stretching device, we found that PIEZO channels are activated by high extracellular matrix stiffness and tissue tension to modulate ISC behavior. This study delineates the mechanistic cascade of PIEZO channel activation in ISCs from the upstream extracellular stimuli through the downstream signaling activation that coordinates stem cell fate decision and maintenance.

cell biology↗

bioRxiv2024

Intercellular adhesion boots collective cell migration through elevated membrane tension

In multicellular systems, the migration pattern of individual cells critically relies on the interactions with neighboring cells. Depending on the strength of these interactions, cells either move as a collective, as observed during morphogenesis and wound healing, or migrate individually, as it is the case for immune cells and fibroblasts. Transducers of cell-cell adhesions, such as cadherins coordinate collective dynamics by linking the cytoskeleton of neighboring cells. However, whether intercellular binding alone triggers signals that originate from within the plasma membrane itself, remains unclear. To address this question, we designed photoswitchable cell-cell adhesions that selectively connect adjacent plasma membranes without linking directly to cytoskeletal elements. We find that these intercellular adhesions are sufficient to achieve collective cell migration. Here, linking adjacent cells increases membrane tension, which activates the enzyme phospholipase D2. The resulting increase in phosphatidic acid, in turn, stimulates the mammalian target of rapamycin, a known actuator of collective cell migration. Collectively, these findings introduce a membrane-based signaling axis as promotor of collective cell dynamics, which is independent of the direct coupling of cell-cell adhesions to the cytoskeleton.

cell biology↗

bioRxiv2024

Moesin activation controls bone resorption and tunneling nanotube-dependent osteoclast fusion

Cell-cell fusion is an evolutionarily conserved process that is essential for many functions, including fertilisation and the formation of placenta, muscle and osteoclasts, multinucleated cells that are unique in their ability to resorb bone. The mechanisms of osteoclast multinucleation involve dynamic interactions between the actin cytoskeleton and the plasma membrane that are still poorly characterized. Here, we found that moesin, a cytoskeletal linker protein member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role in both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin inhibition favors their ability to fuse into multinucleated osteoclasts. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances the formation of tunneling nanotubes (TNTs), F-actin-based intercellular bridges that we reveal here to trigger cell-cell fusion. Moesin also controls HIV-1- and inflammation-induced cell fusion. In addition, moesin regulates the formation of the sealing zone, the adhesive structure determining osteoclast bone resorption area, and thus controls bone degradation, via a {beta}3-integrin/RhoA/SLK pathway. Supporting our results, moesin-deficient mice present a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of cell-cell fusion and osteoclast biology, opening new opportunities to specifically target osteoclast activity in bone disease therapy.

cell biology↗

bioRxiv2023

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

Colorectal cancer 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 unknown. Using CRC patient derived organoids (PDOs), we found 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 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.

cancer biology↗

bioRxiv2023

The membrane-actin linkers ezrin, radixin, and moesin are dispensable for macrophage migration and cortex mechanics.

The cellular actin cortex provides crucial mechanical support and plays critical roles in numerous functions, including cell division and migration. The proteins of the ERM family, ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the individual contributions of these three proteins to leukocyte migration, we generated single and triple ERM knock-out macrophages. Surprisingly, we found that even in the absence of ERMs, macrophages can still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore we discovered that, unlike every other cell type previously investigated, the single or triple knock-out of ERMs does not affect macrophage migration in a large diversity of contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanics of their actin cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their adaptive cortical plasticity.

cell biology↗

bioRxiv2023

The distance between the plasma membrane and the actomyosin cortex acts as a nanogate to control cell surface mechanics

Animal cell shape changes are controlled by the actomyosin cortex, a peripheral actin network tethered to the plasma membrane by membrane-to-cortex attachment (MCA) proteins. Previous studies have focused on how myosin motors or actin turnover can generate the local deformations required for morphogenesis. However, how the cell controls local actin nucleation remains poorly understood. By combining molecular engineering with biophysical approaches and in situ characterization of cortical actin network architecture, we show that membrane-to-cortex tethering determines the distance between the plasma membrane and the actomyosin cortex at the nanoscale of single actin nucleators. In turn, the size of this gap dictates actin filament production and the mechanical properties of the cell surface. Specifically, it tunes formin activity, controlling actin bundling and cortical tension. Our study defines the membrane-to-cortex distance as a nanogate that cells can open or close by MCA proteins to control the activity of key molecules at the cell surface.

biophysics↗

bioRxiv2022

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 3D, all-optical and hence non-contact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated photon-phonon process, which improves the signal-to-background ratio (SBR) as well as provides higher spectral resolution. However, current implementations of stimulated Brillouin spectroscopy (SBS) require high pump powers, which prohibit applications in many areas of biology, especially when studying photosensitive samples, or when live-imaging in 3D and/or over extended time periods. Here, we present a pulsed SBS scheme that takes full advantage of the non-linearity of the pump-probe interaction in SBS. In particular, we show that through quasi-pulsing and diligent optimization of signal detection parameters, the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. Moreover, we devise a custom analysis approach that facilitates the analysis of complex, multi-peaked Brillouin spectra in order to harness the high spectral resolution of SBS for the specific identification of biomechanical components inside the point-spread function of the microscope. We then demonstrate the low-phototoxicity and high-specificity of our pulsed SBS approach by imaging sensitive single cells, zebrafish larvae, and mouse embryos as well as adult C. elegans with sub-cellular detail. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time. We expect that the substantially lower photo-burden and improved SBR of pulsed SBS will facilitate studying biomechanics in 3D at high spatio-temporal resolution in living biological specimens in a non-invasive manner, opening up exciting new possibilities for the field of mechanobiology.

bioengineering↗

bioRxiv2022

Lipotype acquisition during neural development in vivo 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 tissue-specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery, but when and how this occurs is unclear. To address this, we used high-resolution mass spectrometry-based (MSALL) lipidomics to perform a quantitative and comprehensive analysis of mouse brain development covering early embryonic and postnatal stages. We discovered a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical brain lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. In contrast, when profiling rodent and human stem cell-derived neurons, we observed that these do not acquire a brain lipotype per se. However, upon probing the lipid metabolic wiring by supplementing brain lipid precursors, we found that the stem cell-derived neurons were partially able to establish a brain-like lipotype, demonstrating that the cells are partially metabolically committed. Altogether, our report provides an extensive lipidomic resource for brain development and highlights a potential challenge in using stem cell-derived neurons for mechanistic studies of lipid biochemistry, membrane biology and biophysics that can be mitigated by further optimizing in vitro differentiation protocols. Significance StatementWe report an extensive time-resolved resource of lipid molecule abundances across mouse brain development, starting as early as 10 days post-fertilization. The resource reveals a bifurcation in the establishment of the neural lipotype where the canonical 22:6-glycerophospholipid and 18:0-sphingolipid biomarkers are attained in utero, whereas cholesterol is attained after birth. Furthermore, we uncover that the neural lipotype is not established in rodent and human stem cell-derived neurons in vitro.

developmental biology↗

bioRxiv2022

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

Brillouin microscopy (BM) can be used to assess the mechanical properties of biological samples in a 3D, all-optical, and hence non-contact fashion, but its weak signals require long imaging times and illumination dosages harmful to living organisms. Here, we present a line-scanning Brillouin microscope optimized for fast and high-resolution live-imaging of dynamic biological processes with low photo-toxicity. In combination with fluorescence light-sheet imaging, we demonstrate the capabilities of our microscope to visualize the mechanical properties of cells and tissues over space and time in living model organisms such as fruit flies, ascidians, and mouse embryos.

bioengineering↗

bioRxiv2021

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, the osmotic and electric properties of cells are 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 spread, migrate or are externally deformed. Cells undergo slow deformation at constant volume, while fast deformation leads to volume loss. We propose a mechano-sensitive 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.

biophysics↗

bioRxiv2021

Curvature sensing steers actin-driven cell migration

Cell migration is a hallmark out-of-equilibrium process in biology. In addition to persistent self-propelled motion, many cells display remarkable adaptive behaviors when they navigate complex environments within the body. Combining theory and experiments, we identify a curvature-sensing mechanism underlying obstacle avoidance in immune-like cells. The genetic perturbation of this machinery leads to a reduced capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. We propose that the active polymerization of the actin cytoskeleton at the advancing edge of migrating cells is locally inhibited by the curvature-sensitive BAR protein Snx33 in regions with inward plasma membrane curvature. This coupling between actin and membrane dynamics leads to a mechanochemical instability that generates complex protrusive patterns at the cellular front. Adaptive motility thus arises from two simultaneous curvature-dependent effects, i) the specific reduction of propulsion in regions where external objects deform the plasma membrane and ii) the intrinsic patterning capacity due to the membrane-actin coupling that promotes spontaneous changes in the cells protrusions. Our results show how cells utilize actin- and plasma membrane biophysics to sense 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 proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment.

cell biology↗

bioRxiv2021

Strength of interactions in the Notch gene regulatory network determine lateral inhibition patterning

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 requires that 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.

developmental biology↗

bioRxiv2020

Adaptive actin organization counteracts elevated membrane tension to ensure robust endocytosis

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, as has been observed in lamellipodia, 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 feedback 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.

cell biology↗

bioRxiv2019

Cell surface mechanics gate 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 conversely cell surface mechanics control the process of differentiation has been relatively unexplored. Here, we show that membrane mechanics gate the exit from naive pluripotency of mouse embryonic stem cells. By measuring membrane tension during 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 with a synthetic signalling-inert linker, we demonstrate that preventing this detachment forces stem cells to retain their naive pluripotent state. 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.

biophysics↗

bioRxiv2018

Imaging mechanical properties of sub-micron ECM in live zebrafish using Brillouin microscopy

In this work, we quantify the mechanical properties of extra-cellular matrix (ECM) in live zebrafish using Brillouin microscopy. Optimization of the imaging conditions and parameters, combined with careful spectral analysis, allows us to resolve the thin ECM and distinguish its Brillouin frequency shift from the surrounding tissue. High-resolution mechanical mapping further enables to directly measure the thickness of the ECM label-free and in-vivo. We find the ECM to be ~400nm thick, in excellent agreement with electron microscopy quantification. Our results open the door for future studies that aim to investigate the role of ECM mechanics for zebrafish morphogenesis and axis elongation.

bioengineering↗

bioRxiv2018

Cell-extrinsic mechanical forces restore neutrophil polarization in the absence of branched actin assembly

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted Arp2/3 and WAVE complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.

cell biology↗