A cryo-ET survey of microtubules and intracellular compartments in mammalian axons

Abstract

The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the internal components of mammalian sensory axons. We determined the polarity of the axonal microtubules (MTs) by combining subtomogram classification and visual inspection, finding MT plus and minus ends are structurally similar. Subtomogram averaging of globular densities in the MT lumen suggests they have a defined structure, which is surprising given they likely contain the disordered protein MAP6. We found the endoplasmic reticulum in axons is tethered to MTs through multiple short linkers. We surveyed membrane-bound cargos and describe unexpected internal features such as granules and broken membranes. In addition, we detected proteinaceous compartments, including numerous virus-like capsid particles. Our observations outline novel features of axonal cargos and MTs, providing a platform for identification of their constituents.

Neurons as a model system for cryo-electron tomography

“…This is in agreement with previous studies showing that microtubules have the same polarity in axons and mixed polarity in dendrites, which serves to separate axonal and dendritic cargo trafficking, as kinesins move towards the plus end dyneins towards the minus end ( van Beuningen and Hoogenraad, 2016 ). While this was confirmed in axons of cultured mouse neurons, it was reported that 32% of axons in Drosophila primary culture contained microtubules of mixed polarity ( Foster et al, 2022 ). Furthermore, whereas virtually all microtubules were formed of 13 protofilaments in mouse neurons, about half of the microtubules in Drosophila consisted of 12 protofilaments and the other half of 13 protofilaments.…”

“…Such a level of detail had never been visualized in plastic-embedded samples. Recently, loosely packed cytoskeletal arrays were revealed in mammalian dorsal root ganglion axons, containing intermediate filaments, microtubules, actin, and an unknown type of thin filaments, which the authors hypothesized were made by spectrins ( Foster et al, 2022 ).…”

“…This quest started in Prof. Baumeister’s department in thin slime mold Dyctiostelium discoideum ( Medalia et al, 2002 ). The arrangement of actin was then characterized in neuronal axons, the pre- and the postsynapse, as well as in fibroblasts ( Koning et al, 2008 , Fernández-Busnadiego et al, 2010 , Fernández-Busnadiego et al, 2011 , Fukuda et al, 2015 , Schrod et al, 2018 , Hoffmann et al, 2021 , Foster et al, 2022 ). Interestingly, actin filaments in axons were in all cases reported to run along the axon, parallel to microtubules.…”

“…The understanding of the architecture of organelles has advanced thanks to the application of cryo-ET to intact neurons. In addition to synaptic vesicles and endocytic vesicles, mitochondria, endosomes, and the endoplasmic reticulum (ER) have been characterized in detail ( Fernández-Busnadiego et al, 2015 , Schrod et al, 2018 , Fischer et al, 2018 , Hoffmann et al, 2021 , Foster et al, 2022 ). In particular cryo-ET showed that in the native state, the mitochondria and ER in axons and dendrites can adopt a very thin shape ( Fig.…”

“…1 D, E) ( Fernández-Busnadiego et al, 2015 , Schrod et al, 2018 ). Cryo-ET has revealed in numerous instances the pleomorphic linker-mediated interactions between microtubules and organelles being transported in neurons, such as transport vesicles, synaptic vesicle packets and ER ( Schrod et al, 2018 , Sartori-Rupp et al, 2019 , Hoffmann et al, 2021 , Foster et al, 2022 ). Thanks to the recent instrumentation improvements, a sufficient resolution was reached to visually identify the dynein/dynactin complex as the linker connecting a microtubule to a transport vesicle, the transport vesicle-bound V-ATPase, as well as the 26S proteasome ( Asano et al, 2015 , Foster et al, 2022 ).…”