Membrane-associated Periodic Skeleton Research Articles

Cytoskeletal orchestration of glucose uptake in Sertoli cell to support efferocytosis of apoptotic germ cells

Efferocytosis of non-viable germ cells by Sertoli cells (SCs) constitutes a sentinel for testis homeostasis, yet how SCs signal for the metabolic and cytoskeletal adaption to this energetically costly process remains unexplored. Spectrin is membrane-associated periodic skeleton assembled into an actin-spectrin-based cytoskeletal structure with an interaction with glucose transporter Glut1. The contribution of spectrin to glucose uptake and efferocytosis is unknown. In this study, we identified a cross-regulation between glucose metabolism and efferocytosis in SCs. Pharmacological or genetic inhibition of glucose uptake or glycolysis compromises efferocytosis activity. We further found that βII-spectrin is a hitherto unappreciated regulator of glucose metabolism and cytoskeletal architecture. βII-spectrin deficiency impairs glucose uptake and lactate production in SCs. Moreover, a defective assembly of cytoskeleton and a loss of blood-testis barrier integrity are also featured by SCs deficient in βII-spectrin. The disruption in glucose metabolism and cytoskeletal organization synergistically lead to a defective efferocytosis. In vivo siRNA-mediated targeting of βII-spectrin in testis causes an obvious morphological aberration in seminiferous epithelium with the presence of exfoliated germ cells and multinucleated giant cells. Importantly, a decrease in expression of αII/βII-spectrin was observed in testes of Adjudin-induced infertility model. By exploring the functional relevance of βII-spectrin to the metabolic and cytoskeletal regulation of efferocytosis, our study proposes a potential link between βII-spectrin deregulation and male infertility.

Cytoskeletal assembly in axonal outgrowth and regeneration analyzed on the nanoscale

The axonal cytoskeleton is organized in a highly periodic structure, the membrane-associated periodic skeleton (MPS), which is essential to maintain the structure and function of the axon. Here, we use stimulated emission depletion microscopy of primary rat cortical neurons in microfluidic chambers to analyze the temporal and spatial sequence of MPS formation at the distal end of growing axons and during regeneration after axotomy. We demonstrate that the MPS does not extend continuously into the growing axon but develops from patches of periodic βII-spectrin arrangements that grow and coalesce into a continuous scaffold. We estimate that the underlying sequence of assembly, elongation, and subsequent coalescence of periodic βII-spectrin patches takes around 15 h. Strikingly, we find that development of the MPS occurs faster in regenerating axons after axotomy and note marked differences in the morphology of the growth cone and adjacent axonal regions between regenerating and unlesioned axons. Moreover, we find that inhibition of the spectrin-cleaving enzyme calpain accelerates MPS formation in regenerating axons and increases the number of regenerating axons after axotomy. Taken together, we provide here a detailed nanoscale analysis of MPS development in growing axons.

Proteomic and functional analyses of the periodic membrane skeleton in neurons

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. The molecular composition and functions of the MPS remain incompletely understood. Here, using co-immunoprecipitation and mass spectrometry, we identified hundreds of potential candidate MPS-interacting proteins that span diverse functional categories. We examined representative proteins in several of these categories using super-resolution imaging, including previously unknown MPS structural components, as well as motor proteins, cell adhesion molecules, ion channels, and signaling proteins, and observed periodic distributions characteristic of the MPS along the neurites for ~20 proteins. Genetic perturbations of the MPS and its interacting proteins further suggested functional roles of the MPS in axon-axon and axon-dendrite interactions and in axon diameter regulation, and implicated the involvement of MPS interactions with cell adhesion molecules and non-muscle myosin in these roles. These results provide insights into the interactome of the MPS and suggest previously unknown functions of the MPS in neurons.

Multiple layers of spatial regulation coordinate axonal cargo transport.

Nerve axons are shaped similar to long electric wires to quickly transmit information from one end of the body to the other. To remain healthy and functional, axons depend on a wide range of cellular cargos to be transported from the neuronal cell body to its distal processes. Because of the extended distance, a sophisticated and well-organized trafficking network is required to move cargos up and down the axon. Besides motor proteins driving cargo transport, recent data revealed that subcellular membrane specializations, including the axon initial segmentat the beginning of the axon and the membrane-associated periodic skeleton, which extends throughout the axonal length, are important spatial regulators of cargo traffic. In addition, tubulin modifications and microtubule-associated proteinspresent along the axonal cytoskeleton have been proposed to bias cargo movements. Here, we discuss the recent advances in understanding these multiple layers of regulatory mechanisms controlling axonal transport.

The role of the membrane-associated periodic skeleton in axons.

The identification of the membrane periodic skeleton (MPS), composed of a periodic lattice of actin rings interconnected by spectrin tetramers, was enabled by the development of super-resolution microscopy, and brought a new exciting perspective to our view of neuronal biology. This exquisite cytoskeleton arrangement plays an important role on mechanisms regulating neuronal (dys)function. The MPS was initially thought to provide mainly for axonal mechanical stability. Since its discovery, the importance of the MPS in multiple aspects of neuronal biology has, however, emerged. These comprise its capacity to act as a signaling platform, regulate axon diameter-with important consequences on the efficiency of axonal transport and electrophysiological properties- participate in the assembly and function of the axon initial segment, and control axon microtubule stability. Recently, MPS disassembly has also surfaced as an early player in the course of axon degeneration. Here, we will discuss the current knowledge on the role of the MPS in axonal physiology and disease.

Age-dependent ataxia and neurodegeneration caused by an \u03b1II spectrin mutation with impaired regulation of its calpain sensitivity

The neuronal membrane-associated periodic spectrin skeleton (MPS) contributes to neuronal development, remodeling, and organization. Post-translational modifications impinge on spectrin, the major component of the MPS, but their role remains poorly understood. One modification targeting spectrin is cleavage by calpains, a family of calcium-activated proteases. Spectrin cleavage is regulated by activated calpain, but also by the calcium-dependent binding of calmodulin (CaM) to spectrin. The physiologic significance of this balance between calpain activation and substrate-level regulation of spectrin cleavage is unknown. We report a strain of C57BL/6J mice harboring a single αII spectrin point mutation (Sptan1 c.3293G > A:p.R1098Q) with reduced CaM affinity and intrinsically enhanced sensitivity to calpain proteolysis. Homozygotes are embryonic lethal. Newborn heterozygotes of either gender appear normal, but soon develop a progressive ataxia characterized biochemically by accelerated calpain-mediated spectrin cleavage and morphologically by disruption of axonal and dendritic integrity and global neurodegeneration. Molecular modeling predicts unconstrained exposure of the mutant spectrin’s calpain-cleavage site. These results reveal the critical importance of substrate-level regulation of spectrin cleavage for the maintenance of neuronal integrity. Given that excessive activation of calpain proteases is a common feature of neurodegenerative disease and traumatic encephalopathy, we propose that damage to the spectrin MPS may contribute to the neuropathology of many disorders.

Super-resolution Imaging of Energy Transfer by Intensity-Based STED-FRET.

Förster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of energy transfer and (bio)molecular interaction events, though they deliver average information for an ensemble of events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy and FRET has been a challenging and elusive task. Here, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images. In addition to higher spatial resolution, STED-FRET provides a more accurate quantification of interaction and has the capacity of suppressing contributions of noninteracting partners, which are otherwise masked by averaging in conventional imaging. The method capabilities were first demonstrated on DNA-origami model systems, verified on uniformly double-labeled microtubules, and then utilized to image biomolecular interactions in the membrane-associated periodic skeleton (MPS) of neurons.

Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging

G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation.

Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy.

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodic skeleton (MPS) is conserved across animals, suggesting it is a fundamental component of neuronal extensions. The nanoscale architecture of the arrangement (190 nm) is below the resolution limit of conventional fluorescent microscopy. Fluorescent nanoscopy, on the other hand, requires costly equipment and special analysis routines, which remain inaccessible to most research groups. This report aims to resolve this issue by using protein-retention expansion microscopy (pro-ExM) to reveal the MPS of axons. ExM uses reagents and equipment that are readily accessible in most neurobiology laboratories. We first explore means to accurately estimate the expansion factors of protein structures within cells. We then describe the protocol that produces an expanded specimen that can be examined with any fluorescent microscopy allowing quantitative nanoscale characterization of the MPS. We validate ExM results by direct comparison to stimulated emission depletion (STED) nanoscopy. We conclude that ExM facilitates three-dimensional, multicolor and quantitative characterization of the MPS using accessible reagents and conventional fluorescent microscopes.

Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy

Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

Membrane-associated periodic skeleton is a signaling platform for RTK transactivation in neurons.

Actin, spectrin, and related molecules form a membrane-associated periodic skeleton (MPS) in neurons. The function of the MPS, however, remains poorly understood. Using super-resolution imaging, we observed that G protein-coupled receptors (GPCRs), cell adhesion molecules (CAMs), receptor tyrosine kinases (RTKs), and related signaling molecules were recruited to the MPS in response to extracellular stimuli, resulting in colocalization of these molecules and RTK transactivation by GPCRs and CAMs, giving rise to extracellular signal-regulated kinase (ERK) signaling. Disruption of the MPS prevented such molecular colocalizations and downstream ERK signaling. ERK signaling in turn caused calpain-dependent MPS degradation, providing a negative feedback that modulates signaling strength. These results reveal an important functional role of the MPS and establish it as a dynamically regulated platform for GPCR- and CAM-mediated RTK signaling.

A dynamic signaling scaffold

Signal Transduction In neurons, many cellular processes are regulated by receptor tyrosine kinases (RTKs), cell surface receptors whose activation can depend on other signaling pathways. Zhou et al. used super-resolution imaging to visualize colocalization of signaling proteins on the membrane-associated periodic skeleton (MPS) that is formed by actin, spectrin, and related molecules in the axons and dendrites of neurons. The colocalization of signaling proteins in different pathways leads to transactivation of RTK, which initiates intracellular signaling. In a negative feedback loop, the downstream signaling in turn leads to degradation of the MPS. Thus, the MPS is a dynamically regulated platform that coordinates signal transduction in neurons. Science , this issue p. [929][1] [1]: /lookup/doi/10.1126/science.aaw5937

βII-spectrin promotes mouse brain connectivity through stabilizing axonal plasma membranes and enabling axonal organelle transport

βII-spectrin is the generally expressed member of the β-spectrin family of elongated polypeptides that form micrometer-scale networks associated with plasma membranes. We addressed in vivo functions of βII-spectrin in neurons by knockout of βII-spectrin in mouse neural progenitors. βII-spectrin deficiency caused severe defects in long-range axonal connectivity and axonal degeneration. βII-spectrin-null neurons exhibited reduced axon growth, loss of actin-spectrin-based periodic membrane skeleton, and impaired bidirectional axonal transport of synaptic cargo. We found that βII-spectrin associates with KIF3A, KIF5B, KIF1A, and dynactin, implicating spectrin in the coupling of motors and synaptic cargo. βII-spectrin required phosphoinositide lipid binding to promote axonal transport and restore axon growth. Knockout of ankyrin-B (AnkB), a βII-spectrin partner, primarily impaired retrograde organelle transport, while double knockout of βII-spectrin and AnkB nearly eliminated transport. Thus, βII-spectrin promotes both axon growth and axon stability through establishing the actin-spectrin-based membrane-associated periodic skeleton as well as enabling axonal transport of synaptic cargo.

Structural plasticity of actin-spectrin membrane skeleton and functional role of actin and spectrin in axon degeneration.

Axon degeneration sculpts neuronal connectivity patterns during development and is an early hallmark of several adult-onset neurodegenerative disorders. Substantial progress has been made in identifying effector mechanisms driving axon fragmentation, but less is known about the upstream signaling pathways that initiate this process. Here, we investigate the behavior of the actin-spectrin-based Membrane-associated Periodic Skeleton (MPS), and effects of actin and spectrin manipulations in sensory axon degeneration. We show that trophic deprivation (TD) of mouse sensory neurons causes a rapid disassembly of the axonal MPS, which occurs prior to protein loss and independently of caspase activation. Actin destabilization initiates TD-related retrograde signaling needed for degeneration; actin stabilization prevents MPS disassembly and retrograde signaling during TD. Depletion of βII-spectrin, a key component of the MPS, suppresses retrograde signaling and protects axons against degeneration. These data demonstrate structural plasticity of the MPS and suggest its potential role in early steps of axon degeneration.

The Actin/Spectrin Membrane-Associated Periodic Skeleton in Neurons.

Neurons are the most asymmetric cell types, with their axons commonly extending over lengths that are thousand times longer than the diameter of the cell soma. Fluorescence nanoscopy has recently unveiled that actin, spectrin and accompanying proteins form a membrane-associated periodic skeleton (MPS) that is ubiquitously present in mature axons from all neuronal types evaluated so far. The MPS is a regular supramolecular protein structure consisting of actin “rings” separated by spectrin tetramer “spacers”. Although the MPS is best organized in axons, it is also present in dendrites, dendritic spine necks and thin cellular extensions of non-neuronal cells such as oligodendrocytes and microglia. The unique organization of the actin/spectrin skeleton has raised the hypothesis that it might serve to support the extreme physical and structural conditions that axons must resist during the lifespan of an organism. Another plausible function of the MPS consists of membrane compartmentalization and subsequent organization of protein domains. This review focuses on what we know so far about the structure of the MPS in different neuronal subdomains, its dynamics and the emerging evidence of its impact in axonal biology.

Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.

Automated quantification of protein periodic nanostructures in fluorescence nanoscopy images: abundance and regularity of neuronal spectrin membrane-associated skeleton

Fluorescence nanoscopy imaging permits the observation of periodic supramolecular protein structures in their natural environment, as well as the unveiling of previously unknown protein periodic structures. Deciphering the biological functions of such protein nanostructures requires systematic and quantitative analysis of large number of images under different experimental conditions and specific stimuli. Here we present a method and an open source software for the automated quantification of protein periodic structures in super-resolved images. Its performance is demonstrated by analyzing the abundance and regularity of the spectrin membrane-associated periodic skeleton (MPS) in hippocampal neurons of 2 to 40 days in vitro, imaged by STED and STORM nanoscopy. The automated analysis reveals that both the abundance and the regularity of the MPS increase over time and reach maximum plateau values after 14 DIV. A detailed analysis of the distributions of correlation coefficients provides indication of dynamical assembly and disassembly of the MPS.

Structural organization of the actin-spectrin–based membrane skeleton in dendrites and soma of neurons

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. In the MPS, short actin filaments, capped by actin-capping proteins, form ring-like structures that wrap around the circumference of neurites, and these rings are periodically spaced along the neurite by spectrin tetramers, forming a quasi-1D lattice structure. This 1D MPS structure was initially observed in axons and exists extensively in axons, spanning nearly the entire axonal shaft of mature neurons. Such 1D MPS was also observed in dendrites, but the extent to which it exists and how it develops in dendrites remain unclear. It is also unclear whether other structural forms of the membrane skeleton are present in neurons. Here, we investigated the spatial organizations of spectrin, actin, and adducin, an actin-capping protein, in the dendrites and soma of cultured hippocampal neurons at different developmental stages, and compared results with those obtained in axons, using superresolution imaging. We observed that the 1D MPS exists in a substantial fraction of dendritic regions in relatively mature neurons, but this structure develops slower and forms with a lower propensity in dendrites than in axons. In addition, we observed that spectrin, actin, and adducin also form a 2D polygonal lattice structure, resembling the expanded erythrocyte membrane skeleton structure, in the somatodendritic compartment. This 2D lattice structure also develops substantially more slowly in the soma and dendrites than the development of the 1D MPS in axons. These results suggest membrane skeleton structures are differentially regulated across different subcompartments of neurons.

Prevalent presence of periodic actin–spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species

Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens.