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

Abstract

17Most mammalian neurons have a narrow axon, which constrains the passage of large 18 cargoes such as autophagosome as they can be larger than the axon diameter. Variations 19 in tension must therefore occur radially to facilitate changes in axonal diameter and 20 ensure efficient axoplasmic trafficking. Here, we reveal that the transit of diverse large 21 membrane-bound cargoes causes an acute, albeit transient, radial expansion of the 22 axonal diameter, which is immediately restored by constricting forces. We demonstrate 23 that non-muscle myosin II (NM-II) forms ~200 nm periodic structures, which associate 24 with axonal F-actin rings. Inhibition of NM-II activity with blebbistatin significantly 25 increases axon diameter without affecting the periodicity of either the F-actin rings or 26 NM-II. This sustained radial expansion significantly affects the trafficking speed, 27 directionality, and reduces the overall efficiency of long-range retrograde axonal 28 cargoes, eventually leading to focal axon swelling and cargo accumulation, which are 29 hallmarks of axonal degeneration. 30 31 between 0.64 m and 0.74 m 3 . In contrast, the size of axonal cargoes is highly variable, 42 encompassing autophagosomes (0.5-1.5 m) 5 , mitochondria (0.75-3 m) 6 and 43 endosomes (50 nm-1 m) 7 . Thus, the range of cargo sizes is comparable to, or 44 surprisingly even larger than some of the CNS axons themselves. This advocates for 45 the existence of radial contractility in the axons, which would allow the transient 46 expansion of axon calibre and facilitate the passage of large cargoes. Indeed, the 47 expansion of axonal diameter surrounding large cargoes, i.e. autophagosomes 8 or 48 mitochondria 9 , has been observed by super-resolution microscopy and 2D-electron 49 microscopy (EM) in both normal and degenerating axons 10, 11 . Considering the spatial 50 limitation exerted by the rigid axon membrane 12 , the trafficking of large cargoes is 51 likely to be affected. In fact, a simulation study based on axon structure and intra-axonal 52 microfluidic dynamics predicted that cargo trafficking was impeded by the friction from 53 the axonal walls in small-calibre axons 13 . In line with this prediction, a correlation 54 between axon diameter and axon trafficking was recently reported in Drosophila 14, 15 55 and rodent neurons 16, 17 . However, direct evidence showing whether and how axonal 56 radial contractility affects cargo trafficking is still lacking. 57 58We hypothesized that the underlying structural basis for axonal radial contractility is 59 the subcortical actomyosin network, which is organized into specialized structures 60 called membrane-associated periodic cytoskeletal structures (MPS), as revealed with 61 super-resolution microscopy along the shafts of mature axons 18 . F-actin, together with 62 adducin and spectrin, forms a subcortical lattice with a ~190 nm periodic interval 63 covering the majority of the axon length 18, 19 . The disrupting of axonal F-actin leads to 64 loss of MPS 20, 21 , whereas the dep...

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

“…Although we were not able to directly visualize adducin at the ultrastructural level, our results suggest that this lateral binding role is dominant to enhance actin rings interaction with spectrins. Further studies are needed to clarify the precise localization and role of adducin as well as to confirm the localization of myosin filaments associated with the MPS, in order to explain how actin-associated proteins can regulate radial and longitudinal tension along the axon as well as axonal diameter Wang et al, 2018).…”

“…3e, brackets). These filaments could be myosins associated with the MPS, and suggest that myosins can crosslink neighboring rings (Wang et al, 2018).…”

Graphene Promotes Axon Elongation through Local Stall of Nerve Growth Factor Signaling Endosomes

“…This effect may also provide another cause for the observed stall of NGF signaling endosomes ( Figure 2). Indeed, transient radial expansions of axonal diameter were recently described to facilitate the processivity of the fast-moving retrograde carriers 44 . Accordingly, the inhibition of these expansions prompted by graphene (Figure 4h), along with the reduced MT interdistance (Figure 4j), might create a more crowded space that inhibits cargo mobility and reduces the retrograde transport of NGF-loaded endosomes, ultimately favoring their local action on axon outgrowth.…”

Crowd Control: Effects of Physical Crowding on Cargo Movement in Healthy and Diseased Neurons

“…Cargos that are transported in axons may occasionally contact the axonal cortex (Topalidou et al, 2012; Narayanareddy et al, 2014; Wortman et al, 2014). For instance, in axons, retrogradely moving lysotracker-marked vesicles induce local stretching of the plasma membrane of the axon (Wang et al, 2018). This could lead to increased drag, and may cause moving organelles to encounter greater stiffness from the proximate axonal cortex compared to stiffness encountered within the cytosol.…”

“…Cytosolic diffusion is affected by the viscosity of the medium where the cytoskeletal filaments themselves can act as molecular sieves (Lewis et al, 2009; Song et al, 2009). On the other hand, active transport by molecular motors on MT tracks is affected by crowding on MT tracks (Hagiwara et al, 1994; Akhmanova and Steinmetz, 2015; Gumy et al, 2017), by the axonal plasma membrane (Narayanareddy et al, 2014; Leite et al, 2016; Wang et al, 2018), organelles, and cytoskeletal filaments near the tracks (Figures 1B,D,E) (Sood et al, 2018; Bommel et al, 2019). Both diffusion and motor-dependent movement face physical constraints that cargo must circumvent for steady transport.…”