Antagonistic regulation controls clathrin-mediated endocytosis: AP2 adaptor facilitation vs restraint from clathrin light chains

Orchestration of a complex network of protein interactions drives clathrin-mediated endocytosis (CME). A central role for the AP2 adaptor complex beyond cargo recognition and clathrin recruitment has emerged in recent years. It is now apparent that AP2 serves as a pivotal hub for protein interactions to mediate clathrin coated pit maturation, and couples lattice formation to membrane deformation. As a key driver for clathrin assembly, AP2 complements the attenuating role of clathrin light chain subunits, which enable dynamic lattice rearrangement needed for budding. This review summarises recent insights into AP2 function with respect to CME dynamics and biophysics, and its relationship to the role of clathrin light chains in clathrin assembly

Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo

Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance

Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding

Clathrin's life beyond 40: Connecting biochemistry with physiology and disease

Understanding of the range and mechanisms of clathrin functions has developed exponentially since clathrin's discovery in 1975. Here, newly established molecular mechanisms that regulate clathrin activity and connect clathrin pathways to differentiation, disease and physiological processes such as glucose metabolism are reviewed. Diversity and commonalities of clathrin pathways across the tree of life reveal species-specific differences enabling functional plasticity in both membrane traffic and cytokinesis. New structural information on clathrin coat formation and cargo interactions emphasises the interplay between clathrin, adaptor proteins, lipids and cargo, and how this interplay regulates quality control of clathrin’s function and is compromised in infection and neurological disease. Roles for balancing clathrin-mediated cargo transport are defined in stem cell development and additional disease states

The AP2 adaptor enhances clathrin coat stiffness

Deformation of the plasma membrane into clathrin-coated vesicles is a critical step in clathrin-mediated endocytosis and requires the orchestrated assembly of clathrin and endocytic adaptors into a membrane-associated protein coat. The individual role of these membrane-bending and curvature-stabilizing factors is subject to current debate. As such, it is unclear whether the clathrin coat itself is stiff enough to impose curvature and if so, whether this could be effectively transferred to the membrane by the linking adaptor proteins. We have recently demonstrated that clathrin alone is sufficient to form membrane buds in vitro. Here, we use atomic force microscopy to assess the contributions of clathrin and its membrane adaptor protein 2 (AP2) to clathrin coat stiffness, which determines the mechanics of vesicle formation. We found that clathrin coats are less than 10-fold stiffer than the membrane they enclose, suggesting a delicate balance between the forces harnessed from clathrin coat formation and those required for membrane bending. We observed that clathrin adaptor protein AP2 increased the stiffness of coats formed from native clathrin, but did not affect less-flexible coats formed from clathrin lacking the light chain subunits. We thus propose that clathrin light chains are important for clathrin coat flexibility and that AP2 facilitates efficient cargo sequestration during coated vesicle formation by modulating clathrin coat stiffness

Évaluer la durabilité des exploitations agricoles : La méthode IDEA v4, un cadre conceptuel combinant dimensions et propriétés de la durabilité

International audienceThis article presents the fourth version of the conceptual framework used in the "IDEA" method (Indicateurs de Durabilité des Exploitations Agricoles or Farm Sustainability Indicators) (IDEA v4). It combines two approaches: one based on sustainable agriculture goals and the other applying a systemic approach focusing on the properties of sustainable agricultural systems. The IDEA v4 method includes both strong sustainability and agricultural multifunctionality paradigms. It takes into account the global challenges of sustainable agriculture. Using this framework, we were able to develop fifty-three indicators to assess the sustainability of a given farm using two complementary approaches. The first approach assesses sustainability by dividing these 53 indicators into 13 components, according to the 3 normative dimensions of sustainable development (agroecological, socio-territorial, economic). This assessment process is based on a scoring system using 100 units for each dimension. There is no compensation between the scores of the three dimensions. The second approach assesses the level of sustainability based on 5 properties of sustainable agricultural systems (Autonomy, Robustness, Ability to produce and reproduce goods and services, Territorial embeddedness and Overall responsibility); using a tree structure. This second approach is divided into 15 constituent branches of the 5 properties. The 53 indicators are aggregated with a qualitative and hierarchical approach, using the DEXi tool. This concept can be used for education, through a transdisciplinary teaching process. Following on from its three previous versions, IDEA v4 can also be used as a tool to support the process of agroecological transition.Cet article présente le nouveau cadre conceptuel d'évaluation de la durabilité de l'exploitation agricole développé dans la méthode IDEA v4. Il combine une approche évaluative basée sur les objectifs assignés à une agriculture durable et une évaluation des propriétés des systèmes agricoles durables. Il s'ancre dans le champ de la durabilité forte, de la multifonctionnalité et prend en compte les enjeux globaux d'une agriculture durable. Ce cadre conceptuel a permis de construire 53 indicateurs permettant d'analyser la durabilité de l'exploitation agricole selon ces deux approches complémentaires. La première évalue la durabilité en organisant ces 53 indicateurs selon les 3 dimensions normatives du développement durable (agroécologique, socio-territoriale, économique), structurées en 13 composantes ; l'évaluation repose sur un système de notation basé sur 100 unités de durabilités pour chacune des 3 dimensions qui ne se compensent pas entre elles. La seconde évalue la durabilité en organisant les 53 indicateurs selon les 5 propriétés des systèmes agricoles durables (autonomie, robustesse, capacité productive et reproductive de biens et services, ancrage territorial et responsabilité globale) qui sont structurées de manière arborescente en 15 branches ; l'agrégation des indicateurs y suit une démarche qualitative et hiérarchique mobilisant l'outil DEXi. Le potentiel pédagogique du concept de propriétés des systèmes favorise une approche transdisciplinaire de l'exploitation agricole. A la suite de ses trois précédentes versions, IDEA v4 renouvelle son potentiel d'usage pour accompagner la transition agroécologique