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.

The microtubule cytoskeleton in axons plays key roles in intracellular transport and in defining cell shape. Despite many years of study of microtubules, many questions regarding their native architecture remain unanswered. Here, we performed cryo-electron tomography of mouse dorsal root ganglion (DRG) and Drosophila melanogaster (Dm) neurons and examined their microtubule ultrastructure in situ. We found that the microtubule minus and plus ends in DRG axons are structurally similar and frequently contact nearby components. The microtubules in DRG axons maintained a 13 protofilament (pf) architecture, even close to lattice break sites. In contrast, microtubules in Dm neurons had 12 or 13 pfs and we detected sites of pf number transition. The microtubule lumen in DRG axons is filled with globular microtubule inner proteins (MIPs). Our data suggest these have a defined structure, which is surprising given they are thought to contain the disordered protein MAP6. In summary, we reveal novel morphological and structural features of microtubules in their native environment.

The neuronal axon contains many intracellular compartments which travel between the cell body and axon tip. The nature of these cargos and the complex axonal environment through which they traverse is unclear. Here, we describe the internal components of mammalian sensory axons using cryo-electron tomography. We show that axonal endoplasmic reticulum has thin, beaded appearance and is tethered to microtubules at multiple sites. The tethers are elongated, ~7 nm long proteins which cluster in small groups. We survey the different membrane-bound cargos in axons, quantify their abundance and describe novel internal features including granules and broken membranes. We observe connecting density between membranes and microtubules which may correspond to motor proteins. In addition to membrane-bound organelles, we detect numerous proteinaceous compartments, including vaults and previously undescribed virus-like capsid particles. The abundance of these compartments suggests they undergo trafficking in axons. Our observations outline the physical characteristics of axonal cargo and provide a platform for identification of their constituents.

Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) with the small-molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin was activated in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNAi knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

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