The supramolecular landscape of growing human axons

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    Evaluation Summary:

    The authors have elegantly combined two techniques, air-liquid interface cerebral organoid (ALI-CO) with correlative light and electron cryo-microscopy (cryo-CLEM), to study the ultrastructure of developing human axons. The technique presented is useful and the data is of high quality and well presented. With a somewhat stronger demonstration of the molecular resolution achieved and a description of how this technique can be expanded to study other organoids or cellular structures in non-neuronal cells and tissues, this paper will be of broad interest to neuroscientists and those developing cryo-electron tomography methods.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

During brain development, human 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 inaccessible to imaging at molecular resolution in a near-native context. Here we apply cryo-correlative light microscopy and electron tomography to growing axonal tracts from human cerebral organoids. 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 data provide an unprecedented resource and reveal a molecular architecture that helps explain the unique biology of growing human axons.

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  1. Evaluation Summary:

    The authors have elegantly combined two techniques, air-liquid interface cerebral organoid (ALI-CO) with correlative light and electron cryo-microscopy (cryo-CLEM), to study the ultrastructure of developing human axons. The technique presented is useful and the data is of high quality and well presented. With a somewhat stronger demonstration of the molecular resolution achieved and a description of how this technique can be expanded to study other organoids or cellular structures in non-neuronal cells and tissues, this paper will be of broad interest to neuroscientists and those developing cryo-electron tomography methods.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers …

  2. Reviewer #1 (Public Review):

    The authors here present a useful new technique for investigating human neuronal axons by cryo-electron tomography. Axons are extended from organoids onto EM grids, which are then vitrified and studied by CLEM and cryo-tomography. The authors also present some observational results derived from the cryo-tomography, with some support from light microscopy.

    The technique is an exciting one, with the important benefit of isolating human axons on EM grids, compared to previous cryo-ET methods of studying of whole rodent neurons. This is the first cryo-tomography experiment on human axons to my knowledge, however, once free of the organoid context, the axons cannot really be described as benefiting from a more physiological 3D organoid environment. The technique will be of good use to neuroscientists working on …

  3. Reviewer #2 (Public Review):

    The use of cerebral air-liquid interface organoids (i.e., ALI-COs recently introduced by Lancaster Lab) to study growing axons in combination with cryoCLEM/cryoET has been convincingly demonstrated in this manuscript. The authors show filaments, microtubules, mitochondria, vesicles, ER, contact sites and ribosome-like particles in the tomograms obtained. They quantitatively analysed the polarity of individual microtubules, the plasma membrane surface area to deduce the lipid supply and the local ribosome concentration in growing human axon shafts. Although argumentatively sound, most of the results are at the ultrastructural level, some are conjectural, and a comparison to previously known findings obtained by other methods is not present in the present version.

    Strengths:

    The fact that such cerebral …

  4. Reviewer #3 (Public Review):

    Determining cellular ultrastructure by cryo-electron tomography (cryo-ET) or by correlative light and electron cryo-microscopy (cryo-CLEM) have so far been on isolated cells. These have limited the ability to study cellular ultrastructure in context of tissues or in near-native conditions. For the first time, Hoffman et al. have elegantly utilized cerebral organoid technology to study the ultrastructure of growing axons by cryo-CLEM. In this manuscript the authors describe the procedure of growing cerebral organoids at air-liquid interface, along with uniquely labeling various cellular components like cytoskeleton elements and organelles for studying their distribution over time by live cell imaging and fluorescent microscopy. Although cryo-CLEM studies of axons are not new, but the novelty of this work is …