Microfluidic system for in vitro epithelial folding and calcium waves induction.

Epithelial folding is a fundamental process where initially flat monolayers transform into functional 3D structures. This protocol details fabrication steps for a polycarbonate microfluidic platform which enables triggering epithelial folds that recapitulate stereotypical cell shape changes and folding-associated mechanical stresses. We describe the steps for cell seeding to form a monolayer on the chip, and subsequent approach to trigger calcium waves in the epithelial monolayer through local epithelial deformation. Lastly, we outline quantitative analysis steps of the epithelial response. For complete details on the use and execution of this protocol, please refer to Blonski et al. (2021).

Alix is required for activity-dependent bulk endocytosis at brain synapses.

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity-dependent bulk endocytosis (ADBE). Alix (ALG-2-interacting protein X/PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrin-independent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons, we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ADBE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix leads to abnormal synaptic recovery during physiological or pathological repeated stimulations.

The Role of LIM Kinases during Development: A Lens to Get a Glimpse of Their Implication in Pathologies.

The organization of cell populations within animal tissues is essential for the morphogenesis of organs during development. Cells recognize three-dimensional positions with respect to the whole organism and regulate their cell shape, motility, migration, polarization, growth, differentiation, gene expression and cell death according to extracellular signals. Remodeling of the actin filaments is essential to achieve these cell morphological changes. Cofilin is an important binding protein for these filaments; it increases their elasticity in terms of flexion and torsion and also severs them. The activity of cofilin is spatiotemporally inhibited via phosphorylation by the LIM domain kinases 1 and 2 (LIMK1 and LIMK2). Phylogenetic analysis indicates that the phospho-regulation of cofilin has evolved as a mechanism controlling the reorganization of the actin cytoskeleton during complex multicellular processes, such as those that occur during embryogenesis. In this context, the main objective of this review is to provide an update of the respective role of each of the LIM kinases during embryonic development.

Direction of epithelial folding defines impact of mechanical forces on epithelial state.

Cell shape dynamics during development is tightly regulated and coordinated with cell fate determination. Triggered by an interplay between biochemical and mechanical signals, epithelia form complex tissues by undergoing coordinated cell shape changes, but how such spatiotemporal coordination is controlled remains an open question. To dissect biochemical signaling from purely mechanical cues, we developed a microfluidic system that experimentally triggers epithelial folding to recapitulate stereotypic deformations observed in vivo. Using this system, we observe that the apical or basal direction of folding results in strikingly different mechanical states at the fold boundary, where the balance between tissue tension and torque (arising from the imposed curvature) controls the spread of folding-induced calcium waves at a short timescale and induces spatial patterns of gene expression at longer timescales. Our work uncovers that folding-associated gradients of cell shape and their resulting mechanical stresses direct spatially distinct biochemical responses within the monolayer.

Asymmetrical Forces Dictate the Distribution and Morphology of Platelets in Blood Clots.

Primary hemostasis consists in the activation of platelets, which spread on the exposed extracellular matrix at the injured vessel surface. Secondary hemostasis, the coagulation cascade, generates a fibrin clot in which activated platelets and other blood cells get trapped. Active platelet-dependent clot retraction reduces the clot volume by extruding the serum. Thus, the clot architecture changes with time of contraction, which may have an important impact on the healing process and the dissolution of the clot, but the precise physiological role of clot retraction is still not completely understood. Since platelets are the only actors to develop force for the retraction of the clot, their distribution within the clot should influence the final clot architecture. We analyzed platelet distributions in intracoronary thrombi and observed that platelets and fibrin co-accumulate in the periphery of retracting clots in vivo. A computational mechanical model suggests that asymmetric forces are responsible for a different contractile behavior of platelets in the periphery versus the clot center, which in turn leads to an uneven distribution of platelets and fibrin fibers within the clot. We developed an in vitro clot retraction assay that reproduces the in vivo observations and follows the prediction of the computational model. Our findings suggest a new active role of platelet contraction in forming a tight fibrin- and platelet-rich boundary layer on the free surface of fibrin clots.

Nuclear envelope deformation controls cell cycle progression in response to mechanical force.

The shape of the cell nucleus can vary considerably during developmental and pathological processes; however, the impact of nuclear morphology on cell behavior is not known. Here, we observed that the nuclear envelope flattens as cells transit from G1 to S phase and inhibition of myosin II prevents nuclear flattening and impedes progression to S phase. Strikingly, we show that applying compressive force on the nucleus in the absence of myosin II-mediated tension is sufficient to restore G1 to S transition. Using a combination of tools to manipulate nuclear morphology, we observed that nuclear flattening activates a subset of transcription factors, including TEAD and AP1, leading to transcriptional induction of target genes that promote G1 to S transition. In addition, we found that nuclear flattening mediates TEAD and AP1 activation in response to ROCK-generated contractility or cell spreading. Our results reveal that the nuclear envelope can operate as a mechanical sensor whose deformation controls cell growth in response to tension.

The role of ESCRT during development and functioning of the nervous system.

The endosomal sorting complex required for transport (ESCRT) is made of subcomplexes (ESCRT 0-III), crucial to membrane remodelling at endosomes, nuclear envelope and cell surface. ESCRT-III shapes membranes and in most cases cooperates with the ATPase VPS4 to mediate fission of membrane necks from the inside. The first ESCRT complexes mainly serve to catalyse the formation of ESCRT-III but can be bypassed by accessory proteins like the Alg-2 interacting protein-X (ALIX). In the nervous system, ALIX/ESCRT controls the survival of embryonic neural progenitors and later on the outgrowth and pruning of axons and dendrites, all necessary steps to establish a functional brain. In the adult brain, ESCRTs allow the endosomal turn over of synaptic vesicle proteins while stable ESCRT complexes might serve as scaffolds for the postsynaptic parts. The necessity of ESCRT for the harmonious function of the brain has its pathological counterpart, the mutations in CHMP2B of ESCRT-III giving rise to several neurodegenerative diseases.

Alix is required during development for normal growth of the mouse brain.

Alix (ALG-2 interacting protein X) drives deformation and fission of endosomal and cell surface membranes and thereby intervenes in diverse biological processes including cell proliferation and apoptosis. Using embryonic fibroblasts of Alix knock-out mice, we recently demonstrated that Alix is required for clathrin-independent endocytosis. Here we show that mice lacking Alix suffer from severe reduction in the volume of the brain which affects equally all regions examined. The cerebral cortex of adult animals shows normal layering but is reduced in both medio-lateral length and thickness. Alix controls brain size by regulating its expansion during two distinct developmental stages. Indeed, embryonic surface expansion of the Alix ko cortex is reduced because of the loss of neural progenitors during a transient phase of apoptosis occurring between E11.5 and E12.5. Subsequent development of the Alix ko cortex occurs normally until birth, when Alix is again required for the post-natal radial expansion of the cortex through its capacity to allow proper neurite outgrowth. The need of Alix for both survival of neural progenitor cells and neurite outgrowth is correlated with its role in clathrin-independent endocytosis in neural progenitors and at growth cones. Thus Alix-dependent, clathrin independent endocytosis is essential for controlling brain size.

ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling.

The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE.

Regulation of postsynaptic function by the dementia-related ESCRT-III subunit CHMP2B.

The charged multivesicular body proteins (Chmp1-7) are an evolutionarily conserved family of cytosolic proteins that transiently assembles into helical polymers that change the curvature of cellular membrane domains. Mutations in human CHMP2B cause frontotemporal dementia, suggesting that this protein may normally control some neuron-specific process. Here, we examined the function, localization, and interactions of neuronal Chmp2b. The protein was highly expressed in mouse brain and could be readily detected in neuronal dendrites and spines. Depletion of endogenous Chmp2b reduced dendritic branching of cultured hippocampal neurons, decreased excitatory synapse density in vitro and in vivo, and abolished activity-induced spine enlargement and synaptic potentiation. To understand the synaptic effects of Chmp2b, we determined its ultrastructural distribution by quantitative immuno-electron microscopy and its biochemical interactions by coimmunoprecipitation and mass spectrometry. In the hippocampus in situ, a subset of neuronal Chmp2b was shown to concentrate beneath the perisynaptic membrane of dendritic spines. In synaptoneurosome lysates, Chmp2b was stably bound to a large complex containing other members of the Chmp family, as well as postsynaptic scaffolds. The supramolecular Chmp assembly detected here corresponds to a stable form of the endosomal sorting complex required for transport-III (ESCRT-III), a ubiquitous cytoplasmic protein complex known to play a central role in remodeling of lipid membranes. We conclude that Chmp2b-containing ESCRT-III complexes are also present at dendritic spines, where they regulate synaptic plasticity. We propose that synaptic ESCRT-III filaments may function as a novel element of the submembrane cytoskeleton of spines.

Exosomes as a novel way of interneuronal communication.

Exosomes are small extracellular vesicles which stem from endosomes fusing with the plasma membrane; they contain lipids, proteins and RNAs that are able to modify receiving cells. Functioning of the brain relies on synapses, and certain patterns of synaptic activity can change the strength of responses at sparse groups of synapses, to modulate circuits underlying associations and memory. These local changes of the synaptic physiology in one neuron driven by another have, so far, been explained by classical signal transduction modulating transcription, translation and post-translational modifications. We have accumulated in vitro evidence that exosomes released by neurons in a way depending on synaptic activity can be recaptured by other neurons. Some lipids, proteins and RNAs contained in exosomes secreted by emitting neurons could directly modify signal transduction and protein expression in receiving cells. Exosomes may be an ideal mechanism for anterograde and retrograde information transfer across synapses underlying local changes in synaptic plasticity. Exosomes might also participate in the spreading across the nervous system of pathological proteins such as PrPSc (abnormal disease-specific conformation of prion protein), APP (amyloid precursor protein) fragments, phosphorylated tau or α-synuclein.

Emerging role of neuronal exosomes in the central nervous system.

Exosomes are small extracellular vesicles, which stem from endosomes fusing with the plasma membrane, and can be recaptured by receiving cells. They contain lipids, proteins, and RNAs able to modify the physiology of receiving cells. Functioning of the brain relies on intercellular communication between neural cells. These communications can modulate the strength of responses at sparse groups of specific synapses, to modulate circuits underlying associations and memory. Expression of new genes must then follow to stabilize the long-term modifications of the synaptic response. Local changes of the physiology of synapses from one neuron driven by another, have so far been explained by classical signal transduction to modulate transcription, translation, and posttranslational modifications. In vitro evidence now demonstrates that exosomes are released by neurons in a way depending on synaptic activity; these exosomes can be retaken by other neurons suggesting a novel way for inter-neuronal communication. The efficacy of inter-neuronal transfer of biochemical information allowed by exosomes would be far superior to that of direct cell-to-cell contacts or secreted soluble factors. Indeed, lipids, proteins, and RNAs contained in exosomes secreted by emitting neurons could directly modify signal transduction and protein expression in receiving cells. Exosomes could thus represent an ideal mechanism for inter-neuronal transfer of information allowing anterograde and retrograde signaling across synapses necessary for plasticity. They might also allow spreading across the nervous system of pathological proteins like PrPsc, APP fragments, phosphorylated Tau, or Alpha-synuclein.

Alix and ALG-2 make a link between endosomes and neuronal death.

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] is a ubiquitinous adaptor protein first described for its capacity to bind to the calcium-binding protein, ALG-2. Alix regulates neuronal death in ways involving interactions with ALG-2 and with proteins of the ESCRT (endosomal sorting complex required for transport). Even though all Alix interactors characterized to date are involved in endosomal trafficking, the genuine function of the protein in this process remains unclear. We have demonstrated recently that Alix and ALG-2 form in the presence of calcium, a complex with apical caspases and with the endocytosed death receptor TNFR1 (tumour necrosis factor alpha receptor 1), thus suggesting a molecular coupling between endosomes and the cell death machinery.

Deletion of smn-1, the Caenorhabditis elegans ortholog of the spinal muscular atrophy gene, results in locomotor dysfunction and reduced lifespan.

Spinal muscular atrophy is the most common genetic cause of infant mortality and is characterized by degeneration of lower motor neurons leading to muscle wasting. The causative gene has been identified as survival motor neuron (SMN). The invertebrate model organism Caenorhabditis elegans contains smn-1, the ortholog of human SMN. Caenorhabditis elegans smn-1 is expressed in various tissues including the nervous system and body wall muscle, and knockdown of smn-1 by RNA interference is embryonic lethal. Here we show that the smn-1(ok355) deletion, which removes most of smn-1 including the translation start site, produces a pleiotropic phenotype including late larval arrest, reduced lifespan, sterility as well as impaired locomotion and pharyngeal activity. Mutant nematodes develop to late larval stages due to maternal contribution of the smn-1 gene product that allows to study SMN-1 functions beyond embryogenesis. Neuronal, but not muscle-directed, expression of smn-1 partially rescues the smn-1(ok355) phenotype. Thus, the deletion mutant smn-1(ok355) provides a useful platform for functional analysis of an invertebrate ortholog of the human SMN protein.

Alix and ALG-2 are involved in tumor necrosis factor receptor 1-induced cell death.

Alix/AIP1 regulates cell death in a way involving interactions with the calcium-binding protein ALG-2 and with proteins of ESCRT (endosomal sorting complex required for transport). Using mass spectrometry we identified caspase-8 among proteins co-immunoprecipitating with Alix in dying neurons. We next demonstrated that Alix and ALG-2 interact with pro-caspase-8 and that Alix forms a complex with the TNFalpha receptor-1 (TNF-R1), depending on its capacity to bind ESCRT proteins. Thus, Alix and ALG-2 may allow the recruitment of pro-caspase-8 onto endosomes containing TNF-R1, a step thought to be necessary for activation of the apical caspase. In line with this, expression of Alix deleted of its ALG-2-binding site (AlixDeltaALG-2) significantly reduced TNF-R1-induced cell death, without affecting endocytosis of the receptor. In a more physiological setting, we found that programmed cell death of motoneurons, which can be inhibited by AlixDeltaALG-2, is regulated by TNF-R1. Taken together, these results highlight Alix and ALG-2 as new actors of the TNF-R1 pathway.

Alix, making a link between apoptosis-linked gene-2, the endosomal sorting complexes required for transport, and neuronal death in vivo.

Alix/apoptosis-linked gene-2 (ALG-2)-interacting protein X is an adaptor protein involved in the regulation of the endolysosomal system through binding to endophilins and to endosomal sorting complexes required for transport (ESCRT) proteins, TSG101 and CHMP4b. It was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death, and several observations suggest a role for Alix in controlling cell death. We used electroporation in the chick embryo to test whether overexpressed wild-type or mutated Alix proteins influence cell death in vivo. We show that Alix overexpression is sufficient to induce cell death of neuroepithelial cells. This effect is strictly dependent on its capacity to bind to ALG-2. On the other hand, expression of Alix mutants lacking the ALG-2 or the CHMP4b binding sites prevents early programmed cell death in cervical motoneurons at day 4.5 of chick embryo development. This protection afforded by Alix mutants was abolished after deletion of the TSG101, but not of the endophilin, binding sites. Our results suggest that the interaction of the ALG-2/Alix complex with ESCRT proteins is necessary for naturally occurring death of motoneurons. Therefore, Alix represents a molecular link between the endolysosomal system and the cell death machinery.

Axon fasciculation defects and retinal dysplasias in mice lacking the immunoglobulin superfamily adhesion molecule BEN/ALCAM/SC1.

The immunoglobulin superfamily adhesion molecule BEN (other names include ALCAM, SC1, DM-GRASP, neurolin, and CD166) has been implicated in the control of numerous developmental and pathological processes, including the guidance of retinal and motor axons to their targets. To test hypotheses about BEN function, we disrupted its gene via homologous recombination and analyzed the resulting mutant mice. Mice lacking BEN are viable and fertile, and display no external morphological defects. Despite grossly normal trajectories, both motor and retinal ganglion cell axons fasciculated poorly and were occasionally misdirected. In addition, BEN mutant retinae exhibited evaginated or invaginated regions with photoreceptor ectopias that resembled the "retinal folds" observed in some human retinopathies. Together, these results demonstrate that BEN promotes fasciculation of multiple axonal populations and uncover an unexpected function for BEN in retinal histogenesis.

BEN/DM-GRASP/SC1 expression during mouse facial development: differential expression and regulation in molars and incisors.

The cell adhesion molecule BEN/DM-GRASP/SC1 is expressed in a variety of tissues during embryogenesis. Here, we studied the expression pattern of BEN/DM-GRASP/SC1 in different organs involved in facial mouse development, especially in the developing teeth. BEN/DM-GRASP/SC1 was expressed in nose, whisker, gland, and tongue epithelia, as well as in myogenic mesenchyme. In molars, BEN/DM-GRASP/SC1 was firstly expressed in the condensed mesenchyme and thereafter expression was confined to mesenchymal cells of the dental follicle. In contrast, in incisors, transient BEN/DM-GRASP/SC1 expression was restricted to epithelium. In tissue recombination experiments, BEN/DM-GRASP/SC1 expression in mesenchyme was activated by molar, but not incisor epithelium.

Expression of Alix protein during early avian development.

Alix is a cytoplasmic protein involved in both programmed cell death and endocytosis (Oncogene 21 (2002) 6801). These activities of Alix may be related to its demonstrated capacity to bind ALG-2 (Apoptosis Linked Gene-2), CIN85/SETA and endophilins (J. Biol. Chem. 275 (2000) 19275; Science 271 (1996) 521; Cell Death Differ. 6 (1999) 124; J. Biol. Chem. 274 (1999) 1533; J. Biol. Chem. 28 (2002) 29108). Here we report for the first time the developmental expression pattern of Alix protein during chick development. We show by immunochemistry that the protein appears first in the ventral part of the rostral neural tube (stage 16 HH) and then in more caudal parts, thereby following the rostro-caudal maturation of the neural tube. Later on, the protein is found in the distal part of axons as well as in the dermomyotome where it becomes restricted to the muscle progenitors. This first demonstration of Alix modulation during development suggests a role for the protein in early phases of motoneuron and muscle growth and differentiation.

Differential regulation of the chick dorsal thoracic dermal progenitors from the medial dermomyotome.

The chick dorsal feather-forming dermis originates from the dorsomedial somite and its formation depends primarily on Wnt1 from the dorsal neural tube. We investigate further the origin and specification of dermal progenitors from the medial dermomyotome. This comprises two distinct domains: the dorsomedial lip and a more central region (or intervening zone) that derives from it. We confirm that Wnt1 induces Wnt11 expression in the dorsomedial lip as previously shown, and show using DiI injections that some of these cells, which continue to express Wnt11 migrate under the ectoderm, towards the midline, to form most of the dorsal dermis. Transplantation of left somites to the right side to reverse the mediolateral axis confirms this finding and moreover suggests the presence of an attractive or permissive environment produced by the midline tissues or/and a repellent or inadequate environment by the lateral tissues. By contrast, the dorsolateral dermal cells just delaminate from the surface of the intervening space, which expresses En1. Excision of the axial organs or the ectoderm, and grafting of Wnt1-secreting cells, shows that, although the two populations of dermal progenitors both requires Wnt1 for their survival, the signalling required for their specification differs. Indeed Wnt11 expression relies on dorsal neural tube-derived Wnt1, while En1 expression depends on the presence of the ectoderm. The dorsal feather-forming dermal progenitors thus appear to be differentially regulated by dorsal signals from the neural tube and the ectoderm, and derive directly and indirectly from the dorsomedial lip. As these two dermomyotomal populations are well known to also give rise to epaxial muscles, an isolated domain of the dermomyotome that contains only dermal precursors does not exist and none of the dermomyotomal domains can be considered uniquely as a dermatome.