Actin modulates shape and mechanics of tubular membranes

International audienceThe actin cytoskeleton shapes cells and also organizes internal membranous compartments. In particular, it interacts with membranes for intracellular transport of material in mammalian cells, yeast, or plant cells. Tubular membrane intermediates, pulled along microtubule tracks, are formed during this process and destabilize into vesicles. While the role of actin in tubule destabilization through scission is suggested, literature also provides examples of actin-mediated stabilization of membranous structures. To directly address this apparent contradiction, we mimic the geometry of tubular intermediates with preformed membrane tubes. The growth of an actin sleeve at the tube surface is monitored spatiotemporally. Depending on network cohesiveness, actin is able to entirely stabilize or locally maintain membrane tubes under pulling. On a single tube, thicker portions correlate with the presence of actin. These structures relax over several minutes and may provide enough time and curvature geometries for other proteins to act on tube stability

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities

Étude de l’architecture et la dynamique du réseau d’actine in vitro and in vivo

Le changement de forme des cellules est primordial pour différents processus cellulaires tels que la motilité et division cellulaire, et certaines pathologies comme les métastases cancéreuses. La forme de la cellule est assurée par son cytosquelette. Un composant majeur du cytosquelette est l’actine. J’ai étudié le rôle de l’actine dans des systèmes in vitro et in vivo. In vitro, en utilisant un système reconstitué de l’assemblage de l’actine, j’examine le rôle de la protéine Ena/VASP. Mes résultats montrent que VASP est impliqué dans la polarisation de la croissance du réseau d’actine vers une surface en absence des protéines de coiffe, en induisant une augmentation de l’activité du complexe Arp2/3 à la surface formant un réseau d’actine polarisé. Je propose aussi un modèle de fonctionnement où la protéine VASP produit plus de filaments mère utilisés pour le branchement par Arp2/3. En utilisant le même système, j’ai identifié une nouvelle molécule qui inhibe l’activité du complexe Arp2/3 de manière contrôlée par la lumière. In vivo, j’ai commencé à explorer l’architecture de l’actine pendant la première division cellulaire de nématodes génétiquement différents de C. elegans. J’ai réduit le nombre d’outils pouvant être utilisés pour visualiser le réseau d’actine dans ces espèces. En somme, ces résultats montrent qu’en présence de Ena/VASP les protéines de coiffe ne sont pas nécessaires à la croissance polarisée du réseau, ni à sa motilité. Enfin, nous avons pu aider à identifier une molécule photoconvertible qui inhibe l’activité du complexe Arp2/3, qui peut être utilisée pour étudier le rôle du complexe dans des processus cellulaires de manière contrôlée.Cell shape changes are crucial for different cell processes such as cell motility, division, and are involved in pathologies like cancer. Cell shape is established by the cellular cytoskeleton. A key component of the cytoskeleton is actin. I studied actin network architecture and dynamics both in vitro and in vivo. For the in vitro part, I used a reconstituted system of actin assembly to examine the role of the barbed end elongation enhancement protein, Ena/VASP. I revealed the contribution of VASP in polarizing the growth of an actin network towards a surface in absence of capping protein, by promoting Arp2/3 complex activity at the surface that initiates actin network. I suggest a mode of action where VASP enhances Arp2/3 complex-based growth by providing mother filaments for Arp2/3 complex branch initiation. Using the same system and through a collaboration with chemists, we identified a new light controlled molecule based on CK-666, that inhibits Arp2/3 complex activity. In vivo, I started exploring actin architecture during the first cell division of nematode species that are genetically distant from C. elegans. I narrowed the window of tools that can be used to visualize the actin network in such nematodes. Overall these results demonstrate that capping protein was not necessary for polarized actin growth and motility in presence of VASP. VASP enhanced the activity of the Arp2/3 complex at the surface thus inducing a polarized growth of the network. I identified a photoswitchable Arp2/3 complex inhibitor, subsequent derivatives of which could be used to study the role of the Arp2/3 complex in cellular processes in a controlled manner

Étude de l’architecture et la dynamique du réseau d’actine in vitro and in vivo

Cell shape changes are crucial for different cell processes such as cell motility, division, and are involved in pathologies like cancer. Cell shape is established by the cellular cytoskeleton. A key component of the cytoskeleton is actin. I studied actin network architecture and dynamics both in vitro and in vivo. For the in vitro part, I used a reconstituted system of actin assembly to examine the role of the barbed end elongation enhancement protein, Ena/VASP. I revealed the contribution of VASP in polarizing the growth of an actin network towards a surface in absence of capping protein, by promoting Arp2/3 complex activity at the surface that initiates actin network. I suggest a mode of action where VASP enhances Arp2/3 complex-based growth by providing mother filaments for Arp2/3 complex branch initiation. Using the same system and through a collaboration with chemists, we identified a new light controlled molecule based on CK-666, that inhibits Arp2/3 complex activity. In vivo, I started exploring actin architecture during the first cell division of nematode species that are genetically distant from C. elegans. I narrowed the window of tools that can be used to visualize the actin network in such nematodes. Overall these results demonstrate that capping protein was not necessary for polarized actin growth and motility in presence of VASP. VASP enhanced the activity of the Arp2/3 complex at the surface thus inducing a polarized growth of the network. I identified a photoswitchable Arp2/3 complex inhibitor, subsequent derivatives of which could be used to study the role of the Arp2/3 complex in cellular processes in a controlled manner.Le changement de forme des cellules est primordial pour différents processus cellulaires tels que la motilité et division cellulaire, et certaines pathologies comme les métastases cancéreuses. La forme de la cellule est assurée par son cytosquelette. Un composant majeur du cytosquelette est l’actine. J’ai étudié le rôle de l’actine dans des systèmes in vitro et in vivo. In vitro, en utilisant un système reconstitué de l’assemblage de l’actine, j’examine le rôle de la protéine Ena/VASP. Mes résultats montrent que VASP est impliqué dans la polarisation de la croissance du réseau d’actine vers une surface en absence des protéines de coiffe, en induisant une augmentation de l’activité du complexe Arp2/3 à la surface formant un réseau d’actine polarisé. Je propose aussi un modèle de fonctionnement où la protéine VASP produit plus de filaments mère utilisés pour le branchement par Arp2/3. En utilisant le même système, j’ai identifié une nouvelle molécule qui inhibe l’activité du complexe Arp2/3 de manière contrôlée par la lumière. In vivo, j’ai commencé à explorer l’architecture de l’actine pendant la première division cellulaire de nématodes génétiquement différents de C. elegans. J’ai réduit le nombre d’outils pouvant être utilisés pour visualiser le réseau d’actine dans ces espèces. En somme, ces résultats montrent qu’en présence de Ena/VASP les protéines de coiffe ne sont pas nécessaires à la croissance polarisée du réseau, ni à sa motilité. Enfin, nous avons pu aider à identifier une molécule photoconvertible qui inhibe l’activité du complexe Arp2/3, qui peut être utilisée pour étudier le rôle du complexe dans des processus cellulaires de manière contrôlée

PML Nuclear bodies: the cancer connection and beyond

ABSTRACTPromyelocytic leukemia (PML) nuclear bodies, membrane-less organelles in the nucleus, play a crucial role in cellular homeostasis. These dynamic structures result from the assembly of scaffolding PML proteins and various partners. Recent crystal structure analyses revealed essential self-interacting domains, while liquid–liquid phase separation contributes to their formation. PML bodies orchestrate post-translational modifications, particularly stress-induced SUMOylation, impacting target protein functions. Serving as hubs in multiple signaling pathways, they influence cellular processes like senescence. Dysregulation of PML expression contributes to diseases, including cancer, highlighting their significance. Therapeutically, PML bodies are promising targets, exemplified by successful acute promyelocytic leukemia treatment with arsenic trioxide and retinoic acid restoring PML bodies. Understanding their functions illuminates both normal and pathological cellular physiology, guiding potential therapies. This review explores recent advancements in PML body biogenesis, biochemical activity, and their evolving biological roles

WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly.

International audienceThe WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex-based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly

Experimental and Computational Analysis of High-Intensity Focused Ultrasound Thermal Ablation in Breast Cancer Cells: Monolayers vs. Spheroids

High-intensity focused ultrasound (HIFU) is a non-invasive therapeutic modality that uses precise acoustic energy to ablate cancerous tissues through coagulative necrosis. In this context, we investigate the efficacy of HIFU ablation in two distinct cellular configurations, namely 2D monolayers and 3D spheroids of epithelial breast cancer cell lines (MDA-MB 231 and MCF7). The primary objective is to compare the response of these two in vitro models to HIFU while measuring their ablation percentages and temperature elevation levels. HIFU was systematically applied to the cell cultures, varying ultrasound intensity and duty cycle during different sonication sessions. The results indicate that the degree of ablation is highly influenced by the duty cycle, with higher duty cycles resulting in greater ablation percentages, while sonication duration has a minimal impact. Numerical simulations validate experimental observations, highlighting a significant disparity in the response of 2D monolayers and 3D spheroids to HIFU treatment. Specifically, tumor spheroids require lower temperature elevations for effective ablation, and their ablation percentage significantly increases with elevated duty cycles. This study contributes to a comprehensive understanding of acoustic energy conversion within the biological system during HIFU treatment for 2D versus 3D ablation targets, holding potential implications for refining and personalizing breast cancer therapeutic strategies

Actin dynamics drive cell-like membrane deformation

International audienceCell membrane deformations are crucial for proper cell function. Specialized protein assemblies initiate inward or outward membrane deformations that the cell uses respectively to uptake external substances or probe the environment. The assembly and dynamics of the actin cytoskeleton are involved in this process, although their detailed role remains controversial. We show here that a dynamic, branched actin network is sufficient to initiate both inward and outward membrane deformation. The polymerization of a dense actin network at the membrane of liposomes produces inward membrane bending at low tension , while outward deformations are robustly generated regardless of tension. Our results shed light on the mechanism cells use to internalize material , both in mammalian cells, where actin polymerization forces are required when membrane tension is increased, and in yeast, where those forces are necessary to overcome the opposing turgor pressure. By combining experimental observations with physical modelling, we propose a mechanism that explains how membrane tension and the architecture of the actin network regulate cell-like membrane deformations