Nanoscopic compartmentalization of membrane protein motion at the axon initial segment

Albrecht, David; Winterflood, Christian M.; Sadeghi, Mohsen; Tschager, Thomas; Noé, Frank; Ewers, Helge

doi:10.1083/jcb.201603108

Cited by 98 publications

(98 citation statements)

References 39 publications

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“…This zone contains a high density of proteins that are anchored to cortical actin and act as a picket fence that restrains the diffusion of unanchored constituents (Winckler et al, 1999), such as GPI-anchored proteins like TAG-1. The limited diffusion of GPI-anchored proteins at the AIS may be also related to the periodic organization of the actin cytoskeleton recently revealed by super-resolution microscopy (Albrecht et al, 2016). In addition, the AIS is the site of a selective barrier for vesicles carrying dendritic cargoes that only enter into the base of the axon, before stopping and returning to the soma.…”

Section: Discussionmentioning

confidence: 99%

The Kv1-associated molecules TAG-1 and Caspr2 are selectively targeted to the axon initial segment in hippocampal neurons

Pinatel

Hivert

Saint‐Martin

et al. 2017

Journal of Cell Science

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Caspr2 and TAG-1 (also known as CNTNAP2 and CNTN2, respectively) are cell adhesion molecules (CAMs) associated with the voltage-gated potassium channels Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) at regions controlling axonal excitability, namely, the axon initial segment (AIS) and juxtaparanodes of myelinated axons. The distribution of Kv1 at juxtaparanodes requires axo-glial contacts mediated by Caspr2 and TAG-1. In the present study, we found that TAG-1 strongly colocalizes with Kv1.2 at the AIS of cultured hippocampal neurons, whereas Caspr2 is uniformly expressed along the axolemma. Live-cell imaging revealed that Caspr2 and TAG-1 are sorted together in axonal transport vesicles. Therefore, their differential distribution may result from diffusion and trapping mechanisms induced by selective partnerships. By using deletion constructs, we identified two molecular determinants of Caspr2 that regulate its axonal positioning. First, the LNG2-EGF1 modules in the ectodomain of Caspr2, which are involved in its axonal distribution. Deletion of these modules promotes AIS localization and association with TAG-1. Second, the cytoplasmic PDZ-binding site of Caspr2, which could elicit AIS enrichment and recruitment of the membrane-associated guanylate kinase (MAGuK) protein MPP2. Hence, the selective distribution of Caspr2 and TAG-1 may be regulated, allowing them to modulate the strategic function of the Kv1 complex along axons.

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Section: Discussionmentioning

confidence: 99%

The Kv1-associated molecules TAG-1 and Caspr2 are selectively targeted to the axon initial segment in hippocampal neurons

Pinatel

Hivert

Saint‐Martin

et al. 2017

Journal of Cell Science

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“…Notably, the MPS is able to organize other associated molecules, such as ankyrin, ion channels, and adhesion molecules, into a periodic distribution along axons (1,(9)(10)(11)14), potentially affect a variety of signaling pathways in axons (1). The MPS can also act as a diffusion barrier and contribute to filtering membrane proteins at the axon initial segment (21). In addition, the MPS appears to affect the stability of microtubules in axons (22).…”

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confidence: 99%

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

Han

Zhou

Xia

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Actin, spectrin, and associated molecules form a membraneassociated 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.actin | spectrin | adducin | super-resolution imaging | STORM I t was recently discovered that actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) structure in neurons (1). As revealed by superresolution stochastic optical reconstruction microscopy (STORM) (1), this structure contains molecules homologous to those present in the erythrocyte membrane skeleton, including spectrin (2-4), actin, and actincapping proteins such as adducin (5, 6), but adopts a structural organization that is distinct from the polygonal lattice structure of the erythrocyte membrane skeleton (7,8). In this structure, short actin filaments capped by adducin are organized into repetitive, ring-like structures that wrap around the circumference of the axon underneath the axonal membrane, and adjacent actin rings are connected by spectrin tetramers, forming a long-range quasi-1D periodic structure with a periodicity of ∼190 nm (1). This periodic structure is formed extensively throughout the axonal shaft, including the axon initial segment and the unmyelinated distal axons, as well as the nodes of Ranvier and internodal segments in myelinated axons (1, 9-14), but appears to be perturbed at synaptic sites (13,15,16). It is a highly prevalent structure observed in many different types of neurons, including both excitatory and inhibitory neuro...

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“…19 Numerous reports highlight the existence of a periodic cortical actinspectrin network in axons 15,[20][21][22] and possibly dendrites 23,24 , and we found the periodic arrangement of membrane compartments to be alternating with such actin rings in our fluorescence imaging studies. 19 We thus asked whether we could find evidence for the same 180 -200 nm periodicity in our iSCAT trajectory data. We applied a moving window of 5 ms over the frame-to-frame displacement of the paricles and plotted the 20% of positions with the lowest standard deviation over this window in blue (Fig 3a).…”

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confidence: 56%

“…27 This notion is supported by recent observations in our laboratory revealing confinement of GPI-GFP motion between 200 nm spaced actin rings in the axon initial segment. 19 Using iSCAT, we did not observe clearly defined compartments, yet still find signatures of subdiffusive motion on the tens of ms time scale. Spatially-resolved analysis of protein motion suggests a periodicity consistent with actin ring spacings, although we cannot confirm axon initial segment localisation.…”

Section: Cc-by-nc-nd 40 International License Not Peer-reviewed) Is mentioning

confidence: 99%

“…This observation agrees with our previous description of a periodic pattern in localisations from SPT with quantum dots (QDs) on live neurons. 19 We remark that Owing to the interferometric nature of iSCAT, the contrast of AuNPs varied with its height above the coverglass (Fig 4a). For 40 nm AuNPs, the contrast ranged from -50% to +50%, yielding a maximum SNR of 50%/2% = 25 at 635 nm.…”

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Revealing compartmentalised membrane diffusion in living cells with interferometric scattering microscopy

Wit

Albrecht

Ewers

et al. 2016

Preprint

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Abstract:Single-particle tracking is a powerful tool for studying single molecule behaviour involving plasma membrane-associated events in cells. Here, we show that interferometric scattering microscopy (iSCAT) combined with gold nanoparticle labeling can be used to follow the motion of membrane proteins in the plasma membrane of live cultured mammalian cell lines and hippocampal neurons. The unique combination of microsecond temporal resolution and nanometer spatial precision reveals signatures of a compartmentalised plasma membrane in neurons.. CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/091736 doi: bioRxiv preprint first posted online Dec. 5, 2016;Single-particle tracking (SPT) generally involves tagging a single molecule with a probe, and detecting its position in a series of images, using computational image processing to achieve sub-pixel localisation precision.1 These trajectories contain information on the diffusion coefficient of the molecule, its momentum and possible underlying forces controlling its motion. [2][3][4][5] Furthermore, the trajectory can be correlated to features of the cell or the motion of interacting partners, providing a means to analyse dynamic changes in the behaviour of single molecules. While single particle tracking was first established using gold beads 6 , current implementations also make use of quantum dots (QDs) or single fluorescent dyes as probes, which have lead to the development of high-density methods. 7-9The enormous advantage of fluorescence emission as a contrast mechanism in terms of maximal background suppression is somewhat offset by restrictions in terms of the achievable simultaneous imaging speed and precision. Scattering labels, such as gold nanoparticles, are not subject to the photophysical and photochemical limitations of fluorescent dyes and can thus in principle achieve much higher spatiotemporal precision. Gold particles have been used extensively in the past for tracking fast nanoscale dynamics, 10 including SPT in living cells with up to 20 µs temporal resolution and about 15 nm spatial precision revealing signatures of hop diffusion. 11More recently, interferometric scattering microscopy (iSCAT) 12 has demonstrated even higher spatiotemporal capabilities down to few nm precision 13 and a few µs temporal resolution in vitro. 14 Here, we use gold nanoparticles (AuNPs) coupled to GFP-tagged or YFP-tagged membrane proteins to demonstrate iSCAT-based SPT of membrane proteins with nanometer precision at kHz speeds in live epithelial cells and cultured hippocampal neurons. We use this approach to study if the increase in spatiotemporal precision allows for the detection of anomalous diffusion at the nanoscale. Such effects could arise from interactions between the . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (whic...

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Nanoscopic compartmentalization of membrane protein motion at the axon initial segment

Cited by 98 publications

References 39 publications

The Kv1-associated molecules TAG-1 and Caspr2 are selectively targeted to the axon initial segment in hippocampal neurons

The Kv1-associated molecules TAG-1 and Caspr2 are selectively targeted to the axon initial segment in hippocampal neurons

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

Revealing compartmentalised membrane diffusion in living cells with interferometric scattering microscopy

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