Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids

Abstract

During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments.

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

“…2 D ). Similar to previous cryo-ET studies of mammalian axons ( Hoffmann et al, 2021 ; Lučić et al, 2007 ; Schrod et al, 2018 ), the observed actin filaments ran roughly parallel to the MT network rather than forming rings around the axon shaft. This actin may correspond to “deep” actin, which is dynamic and transported slowly through axons ( Ganguly et al, 2015 ).…”

“…We did not observe the actin/spectrin membrane-associated periodic skeleton, which forms a sheath under the axonal plasma membrane (He et al, 2016;Xu et al, 2013). The membrane-associated periodic skeleton was also not detected in previous cryo-ET studies of mammalian neurons (Fischer et al, 2018;Hoffmann et al, 2021;Lučić et al, 2007;Schrod et al, 2018;Tao et al, 2018a). We speculate it may be excluded from the very thin regions targeted in this study.…”

“…It is a key site for lipid biosynthesis and distribution (Vance et al, 1991), an effector of calcium homeostasis (de Juan-Sanz et al, 2017) and a source of autophagy membranes (Maday and Holzbaur, 2014). The ER in mammalian axons was previously observed by cryo-ET (Fernández-Busnadiego et al, 2015;Fischer et al, 2018;Hoffmann et al, 2021;Lučić et al, 2007;Schrod et al, 2018). Similar to these studies, the ER in DRG axons predominantly had a "beaded" appearance, where the two lipid bilayers periodically came in contact, while other stretches had open morphology (Fig.…”

High-content analysis of neuronal morphology in human iPSC-derived neurons

“…We use neuronal markers SMI312 to identify axons and MAP2 to identify dendrites ( Hoffmann et al., 2021 ). However, other markers may also be used.…”

Quantitative Cryo-Electron Tomography

“…Other examples of such cryo-ET approaches are studies concerning axon growth. Quantification of the endoplasmic reticulum in the growing shaft correlates with scarcity of ribosomes suggesting the high demand for lipid biosynthesis while low translational competence during growth ( Hoffmann et al, 2021 ). Furthermore, the quantitative analysis of cytoskeletal elements found that the endoplasmic reticulum is tethered to microtubules through short linkers in the axons ( Foster et al, 2021 ).…”