Automated quantification of protein periodic nanostructures in fluorescence nanoscopy images: abundance and regularity of neuronal spectrin membrane-associated skeleton (original) (raw)
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Proteomic and functional analyses of the periodic membrane skeleton in neurons
2020
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 candidate MPS-interacting proteins that span diverse functional categories. We validated representative proteins in several of these categories, including previously unknown MPS structural components, as well as motor proteins, cell adhesion molecules, ion channels, and signaling proteins, demonstrating periodic distributions of ∼20 proteins in neurons using super-resolution imaging. 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 new insights ...
Scientific Reports, 2020
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 wit...
Ultrastructural analysis of neuronal synapses using state-of-the-art nano-imaging techniques
Neuroscience Bulletin, 2012
Neuronal synapses are functional nodes in neural circuits. Their organization and activity define an individual's level of intelligence, emotional state and mental health. Changes in the structure and efficacy of synapses are the biological basis of learning and memory. However, investigation of the molecular architecture of synapses has been impeded by the lack of efficient techniques with sufficient resolution. Recent developments in state-of-the-art nano-imaging techniques have opened up a new window for dissecting the molecular organization of neuronal synapses with unprecedented resolution. Here, we review recent technological advances in nano-imaging techniques as well as their applications to the study of synapses, emphasizing super-resolution light microscopy and 3-dimensional electron tomography.
The Actin/Spectrin Membrane-Associated Periodic Skeleton in Neurons
Frontiers in Synaptic Neuroscience, 2018
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.
Frontiers in Neuroscience
The complex, nanoscopic scale of neuronal function, taking place at dendritic spines, axon terminals, and other minuscule structures, cannot be adequately resolved using standard, diffraction-limited imaging techniques. The last couple of decades saw a rapid evolution of imaging methods that overcome the diffraction limit imposed by Abbe’s principle. These techniques, including structured illumination microscopy (SIM), stimulated emission depletion (STED), photo-activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM), among others, have revolutionized our understanding of synapse biology. By exploiting the stochastic nature of fluorophore light/dark states or non-linearities in the interaction of fluorophores with light, by using modified illumination strategies that limit the excitation area, these methods can achieve spatial resolutions down to just a few tens of nm or less. Here, we review how these advanced imaging techniques have contr...
Quantification and its Applications in Fluorescent Microscopy Imaging
Traffic, 2009
Fluorescent microscope imaging technologies have developed at a rapid pace in recent years. Highthroughput 2D fluorescent imaging platforms are now in wide use and are being applied on a proteome wide scale. Multiple fluorophore 3D imaging of live cells is being used to give detailed localization and subcellular structure information. Further, 2D and 3D video microscopy are giving important insights into the dynamics of protein localization and transport. In parallel with these developments, significant research has gone into developing new methodologies for quantifying and extracting meaning from the imaging data. Here we outline and give entry points to the literature on approaches to quantification such as segmentation, tracking, automated classification and data visualization. Particular attention is paid to the distinction between and application of concrete quantification measures such as number of objects in a cell, and abstract measures such as texture.
2016 23rd International Conference on Pattern Recognition (ICPR), 2016
Actin-regulating proteins, such as cofilin, are essential in regulating the shape of dendritic spines, and synaptic plasticity in both neuronal functionality as well as in neurodegeneration related to aging. The analysis of the motility of cofilin in fluorescence video-microscopy allows the discovery of its effects on cell functions. However, the flow of cofilin has not been analyzed to date by automatic means. This paper presents a novel automated pattern recognition system to analyze protein trafficking in neurons. Using spatio-temporal information present in multichannel fluorescence videos, the system generates a temporal maximum intensity projection that enhances the signal-to-noise ratio of important biological structures, segments and tracks dendritic spines, and quantifies the flux and density of proteins in spines. The temporal dynamics of spines is used to generate spine energy images which are used to automatically classify the shape of dendritic spines as stubby, mushroo...
Frontiers in Synaptic Neuroscience
Neuroligins (NLGNs) form a family of cell adhesion molecules implicated in synapse development, but the mechanisms that retain these proteins at synapses are still incompletely understood. Recent studies indicate that surface-associated NLGN1 is diffusionally trapped at synapses, where it interacts with quasi-static scaffolding elements of the post-synaptic density. Whereas single molecule tracking reveals rapid diffusion and transient immobilization of NLGN1 at synapses within seconds, fluorescence recovery after photobleaching experiments indicate instead a long-term turnover of NLGN1 at synapse, in the hour time range. To gain insight into the mechanisms supporting NLGN1 anchorage at post-synapses and try to reconcile those experimental paradigms, we quantitatively analyzed here live-cell and super-resolution imaging experiments performed on NLGN1 using a newly released simulator of membrane protein dynamics for fluorescence microscopy, FluoSim. Based on a small set of parameters...