scholarly journals Radial contractility of Actomyosin-II rings facilitates cargo trafficking and maintains axonal structural stability following cargo-induced transient axonal expansion

2018 ◽  
Author(s):  
Tong Wang ◽  
Wei Li ◽  
Sally Martin ◽  
Andreas Papadopulos ◽  
Anmin Jiang ◽  
...  
Keyword(s):  
Axonal Degeneration ◽  
Super Resolution ◽  
Axon Diameter ◽  
Retrograde Trafficking ◽  
Local Expansion ◽  
Overall Efficiency ◽  
Axonal Trafficking ◽  
Super Resolution Microscopy ◽  
Actin Rings ◽  
Axonal Swellings

AbstractMost mammalian neurons have a narrow axon, which constrains the passage of large cargoes such as autophagosomes that can be larger than the axon diameter. Radial axonal expansion must therefore occur to ensure efficient axonal trafficking. In this study we consistently find that the trafficking speed of various large axonal cargoes is significantly slower than that of small ones, and reveal that the transit of diverse-sized cargoes causes an acute, albeit transient axonal radial expansion, which is immediately restored by constitutive contractility. Using live super-resolution microscopy, we demonstrate that actomyosin-II controls axonal radial contractility and local expansion, and that NM-II filaments associate with periodic F-actin rings via their head domains. Pharmacological inhibition of NM-II activity, significantly increases axon diameter by detaching the NM-II from F-actin, and impacts the trafficking speed, directionality, and overall efficiency of long-range retrograde trafficking. Consequently, prolonged disruption of NM-II activity leads to disruption of periodic actin rings and formation of focal axonal swellings, a hallmark of axonal degeneration.SummaryAxonal radial contractility and local expansion control the retrograde trafficking of large cargoes. The periodic actomyosin-II network comprises of NM-II filaments and F-actin rings. Loss of actomyosin-II-mediated radial contractility causes defects in axonal trafficking and stability, leading to degeneration.

scholarly journals Radial contractility of actomyosin rings facilitates axonal trafficking and structural stability

2020 ◽  
Vol 219 (5) ◽  
Author(s):  
Tong Wang ◽  
Wei Li ◽  
Sally Martin ◽  
Andreas Papadopulos ◽  
Merja Joensuu ◽  
...  
Keyword(s):  
Long Range ◽  
Axonal Degeneration ◽  
Super Resolution ◽  
Radial Expansion ◽  
Axon Diameter ◽  
Overall Efficiency ◽  
Axonal Trafficking ◽  
Super Resolution Microscopy ◽  
Actin Rings ◽  
Axonal Swellings

Most mammalian neurons have a narrow axon, which constrains the passage of large cargoes such as autophagosomes that can be larger than the axon diameter. Radial axonal expansion must therefore occur to ensure efficient axonal trafficking. In this study, we reveal that the speed of various large cargoes undergoing axonal transport is significantly slower than that of small ones and that the transit of diverse-sized cargoes causes an acute, albeit transient, axonal radial expansion, which is immediately restored by constitutive axonal contractility. Using live super-resolution microscopy, we demonstrate that actomyosin-II controls axonal radial contractility and local expansion, and that NM-II filaments associate with periodic F-actin rings via their head domains. Pharmacological inhibition of NM-II activity significantly increases axon diameter by detaching the NM-II from F-actin and impacts the trafficking speed, directionality, and overall efficiency of long-range retrograde trafficking. Consequently, prolonged NM-II inactivation leads to disruption of periodic actin rings and formation of focal axonal swellings, a hallmark of axonal degeneration.

scholarly journals The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ana Rita Costa ◽  
Sara C Sousa ◽  
Rita Pinto-Costa ◽  
José C Mateus ◽  
Cátia DF Lopes ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Myosin Ii ◽  
Super Resolution ◽  
Loss Of Function ◽  
Axon Diameter ◽  
Heavy Chains ◽  
Axonal Diameter ◽  
Muscle Myosin ◽  
Super Resolution Microscopy ◽  
Actin Rings

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII). We show that active NMII light chains are colocalized with actin rings and organized in a circular periodic manner throughout the axon shaft. In contrast, NMII heavy chains are mostly positioned along the longitudinal axonal axis, being able to crosslink adjacent rings. NMII filaments can play contractile or scaffolding roles determined by their position relative to actin rings and activation state. We also show that MPS destabilization through NMII inactivation affects axonal electrophysiology, increasing action potential conduction velocity. In summary, our findings open new perspectives on axon diameter regulation, with important implications in neuronal biology.

scholarly journals Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Stéphane Vassilopoulos ◽  
Solène Gibaud ◽  
Angélique Jimenez ◽  
Ghislaine Caillol ◽  
Christophe Leterrier
Keyword(s):  
Actin Filaments ◽  
Myosin Light Chain ◽  
Super Resolution ◽  
Ultrastructural Level ◽  
Ankyrin G ◽  
Molecular Arrangement ◽  
Resolution Electron ◽  
Super Resolution Microscopy ◽  
Actin Rings ◽  
Cortical Cytoskeleton

AbstractRecent super-resolution microscopy studies have unveiled a periodic scaffold of actin rings regularly spaced by spectrins under the plasma membrane of axons. However, ultrastructural details are unknown, limiting a molecular and mechanistic understanding of these enigmatic structures. Here, we combine platinum-replica electron and optical super-resolution microscopy to investigate the cortical cytoskeleton of axons at the ultrastructural level. Immunogold labeling and correlative super-resolution/electron microscopy allow us to unambiguously resolve actin rings as braids made of two long, intertwined actin filaments connected by a dense mesh of aligned spectrins. This molecular arrangement contrasts with the currently assumed model of actin rings made of short, capped actin filaments. Along the proximal axon, we resolved the presence of phospho-myosin light chain and the scaffold connection with microtubules via ankyrin G. We propose that braided rings explain the observed stability of the actin-spectrin scaffold and ultimately participate in preserving the axon integrity.

scholarly journals Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings

2019 ◽  
Author(s):  
Stéphane Vassilopoulos ◽  
Solène Gibaud ◽  
Angélique Jimenez ◽  
Ghislaine Caillol ◽  
Christophe Leterrier
Keyword(s):  
Actin Filaments ◽  
Myosin Light Chain ◽  
Super Resolution ◽  
Ultrastructural Level ◽  
Ankyrin G ◽  
Molecular Arrangement ◽  
Resolution Electron ◽  
Super Resolution Microscopy ◽  
Actin Rings ◽  
Cortical Cytoskeleton

Recent super-resolution microscopy studies have unveiled a periodic scaffold of actin rings regularly spaced by spectrins under the plasma membrane of axons. However, ultrastructural details are unknown, limiting a molecular and mechanistic understanding of these enigmatic structures. Here, we combine platinum-replica electron and optical super-resolution microscopy to investigate the cortical cytoskeleton of axons at the ultrastructural level. Immunogold labeling and correlative super-resolution/electron microscopy allow us to unambiguously resolve actin rings as braids made of two long, intertwined actin filaments connected by a dense mesh of aligned spectrins. This molecular arrangement contrasts with the currently assumed model of actin rings made of short, capped actin filaments. Along the proximal axon, we resolved the presence of phospho-myosin light chain and the scaffold connection with microtubules via ankyrin G. We propose that braided rings explain the observed stability of the actin-spectrin scaffold and ultimately participate in preserving the axon integrity.

scholarly journals Periodic actin structures in neuronal axons are required to maintain microtubules

2016 ◽  
Author(s):  
Yue Qu ◽  
Ines Hahn ◽  
Stephen Webb ◽  
Simon P. Pearce ◽  
Andreas Prokop
Keyword(s):  
Super Resolution ◽  
Close Correlation ◽  
Membrane Skeleton ◽  
Cortical Actin ◽  
Evolutionarily Conserved ◽  
Genes Encoding ◽  
Neuronal Processes ◽  
And Function ◽  
Super Resolution Microscopy ◽  
Actin Rings

SummaryAxons are the cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly-spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily-conserved, ubiquitous, highly-ordered feature of axons, the function of PMS is unknown. Here we studied PMS abundance, organisation and function, combining versatile Drosophila genetics with super-resolution microscopy and various functional readouts. Analyses with 11 different actin regulators and 3 actin-targeting drugs suggest PMS to contain short actin filaments which are depolymerisation resistant and sensitive to spectrin, adducin and nucleator deficiency - consistent with microscopy-derived models proposing PMS as specialised cortical actin. Upon actin removal we observed gaps in microtubule bundles, reduced microtubule polymerisation and reduced axon numbers suggesting a role of PMS in microtubule organisation. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilising protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerisation contributes to their maintenance in axons. We discuss potential implications of this novel PMS function along axon shafts for axon maintenance and regeneration.Significance statementAxons are cable-like neuronal processes that are up to a meter long in humans. These delicate structures often need to be maintained for an organism’s lifetime, i.e. up to a century in humans. Unsurprisingly, we gradually lose about 50% of axons as we age. Bundles of microtubules form the structural backbones and highways for life-sustaining transport within axons, and maintenance of these bundles is essential for axonal longevity. However, the mechanisms which actively maintain axonal microtubules are poorly understood. Here we identify cortical actin as an important factor maintaining microtubule polymerisation in axons. This finding provides potential explanations for the previously identified, but unexplained, links between mutations in genes encoding cortical actin regulators and neurodegeneration.

scholarly journals A dynamic formin-dependent deep F-actin network in axons

2015 ◽  
Vol 210 (3) ◽  
pp. 401-417 ◽  
Author(s):  
Archan Ganguly ◽  
Yong Tang ◽  
Lina Wang ◽  
Kelsey Ladt ◽  
Jonathan Loi ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Super Resolution ◽  
Growth Cones ◽  
Filamentous Actin ◽  
Actin Organization ◽  
Cytoskeletal Network ◽  
Presynaptic Boutons ◽  
Super Resolution Microscopy ◽  
Neuronal Growth Cones ◽  
Actin Rings

Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles.

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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