The functional architecture of axonal actin

International audienceThe cytoskeleton builds and supports the complex architecture of neurons. It orchestrates the specification, growth, and compartmentation of the axon: axon initial segment, axonal shaft, presynapses. The cytoskeleton must then maintain this intricate architecture for the whole life of its host, but also drive its adaptation to new network demands and changing physiological conditions. Microtubules are readily visible inside axon shafts by electron microscopy, whereas axonal actin study has long been focused on dynamic structures of the axon such as growth cones. Super-resolution microscopy and live-cell imaging have recently revealed new actin-based structures in mature axons: rings, hotspots and trails. This has caused renewed interest for axonal actin, with efforts underway to understand the precise organization and cellular functions of these assemblies. Actin is also present in presynapses, where its arrangement is still poorly defined, and its functions vigorously debated. Here we review the organization of axonal actin, focusing on recent advances and current questions in this rejuvenated field

Presynapses contain distinct actin nanostructures

The architecture of the actin cytoskeleton that concentrates at presynapses remains poorly known, hindering our understanding of its roles in synaptic physiology. In this work, we measure and visualize presynaptic actin by diffraction-limited and super-resolution microscopy, thanks to a validated model of bead-induced presynapses in cultured neurons. We identify a major population of actin-enriched presynapses that concentrates more presynaptic components and shows higher synaptic vesicle cycling than their non-enriched counterparts. Pharmacological perturbations point to an optimal actin amount and the presence of distinct actin structures within presynapses. We directly visualize these nanostructures using Single Molecule Localization Microscopy (SMLM), defining three distinct types: an actin mesh at the active zone, actin rails between the active zone and deeper reserve pools, and actin corrals around the whole presynaptic compartment. Finally, CRISPR-tagging of endogenous actin allows us to validate our results in natural synapses between cultured neurons, confirming the role of actin enrichment and the presence of three types of presynaptic actin nanostructures

Mapping of domains on HIV envelope protein mediating association with calnexin and protein-disulfide isomerase.

The cell catalysts calnexin (CNX) and protein-disulfide isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope (Env) glycoprotein. Following HIV binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX, whereas only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in vitro conditions, which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in cellulo with CNX or in vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2, and the C terminus of V3 did not bind CNX-associated Env, whereas those against C1, V1/V2, and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discrete, largely nonoverlapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents

Damage and repair of the axolemmal membrane: From neural development to axonal trauma and restoration

Integrity of the plasma membrane is essential for the maintenance of physiological conditions, metabolic activity and the shape of eukaryotic cells. In neurons, the plasma membrane surrounding the axon—the axolemma—fulfills all these functions plus those inherent to the specific function of the neuron: maintaining the membrane potential by the regulated and concerted operation of ion-selective channels. Membrane expansion and neurite growth are directly linked through intricate cellular signaling mechanisms during the early stages of embryonic development. During axonal development there is an increase in the surface area of the axolemma which provokes an increase in membrane tension. Membrane insertion involved in axonal growth reduces the membrane tension, and this in turn allows distal membrane expansion and axonal extension. Under certain pathological conditions, such as spinal cord and traumatic brain injuries, the axolemmal damage results in different degrees of neuronal degeneration due to unregulated ionic influx, followed by oxidative damage, finally triggering neuronal apoptosis. Neurons possess counteractive mechanisms to arrest these degenerative processes which involve sealing the axolemma as a first step toward membrane repair, followed by attempts at axonal extension. In this review we address the main molecular actors and mechanisms involved in axonal growth during embryonic development and the recapitulation of these mechanisms during the post-traumatic regeneration process. We also discuss the efficacy of some classical and novel therapeutic approaches to axolemmal sealing and repair in different pathologies.Fil: Quintá, Héctor Ramiro. Hospital Aleman. Laboratorio de Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin