The supramolecular landscape of growing human axons

  1. Patrick C. Hoffmann
  2. Stefano L. Giandomenico
  3. Iva Ganeva
  4. Michael R. Wozny
  5. Magdalena Sutcliffe
  6. Wanda Kukulski
  7. Madeline A. Lancaster

  • Curated by eLife

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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. eLife

    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.)

  2. eLife

    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 axonal ultrastructure.

    The data presented is chiefly exploratory and observational but is generally experimentally solid and well-presented. In addition to the preliminary methodological characterisations, CLEM and tomograms presented as proof of concept, there is some quantitative work. This data is scientifically sound but is mainly confirmatory of previous findings by other groups in other systems. For example, uniform microtubule polarity in axons, the thinness of axonal ER (e.g see work by Mark Terasaki) and to some extent axonal ribosome paucity are all well-described phenomena. L1CAM-GFP or ESYT1-GFP overexpression were interesting experiments, but the authors report no differences with wild-type without presenting quantitative evidence of this conclusion. However, this data serves well as evidence of the utility of this novel human experimental system.

  3. eLife

    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 organoids can now be prepared and studied at molecular resolution will certainly allow in-depth follow-up studies. It is therefore an original proof-of-concept report that is of general interest for neuroscience and cellular structural biology. The manuscript nicely illustrates the power of tomography to visualize the intracellular landscape of a biological material previously inaccessible to this method and presents the first data on variations found in different human axon regions.

    Weaknesses:

    Although "molecular" is stated several times, the authors have only partially performed a true molecular analysis; most of it is at the ultrastructural level. All "molecular" results are either indirectly derived; lipid supply/concentration across the plasma membrane surface and local "ribosome concentration" based on identification of ribosomes on size, shape and contrast in tomograms. It would have been more compelling to at least have performed a rough subtomogram analysis here to be sure that the putative ribosomes are indeed ribosomes. I'm pretty convinced that the authors picked ribosomes, and I don't want to be hypercritical here, but there's no proof. The FLM data only support the overall impression that there are fewer ribosomes in axon shafts than in dendrites, but of course with limited spatial resolution.

    The discussion is therefore somewhat elusive, starting with "molecular features," but most of the findings are at the ultrastructural level and the authors choose " likely," "could reflect/represent," or " may explain" to account for this absence.

  4. eLife

    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 the combination of ALI-CO technique with growing axonal extensions from the organoids on EM grids and isolating them for cryo-EM sample vitrification. This eliminates the need for culturing isolated neurons from neuronal tissues for structural studies using cryo-CLEM. By uniquely labeling axonal and dendritic markers and showing their distributions by fluorescent imaging, the authors have supported their claim that the extensions from the organoids are axons. By using this technique, the authors were able to visualize the arrangement microtubules along with determining their polarity, ER morphology, membrane contacts and distribution of ribosomes. These support the claim by the authors that this technique will allow studying neurons in context of tissues or under different physiological conditions. However, the major weakness of the manuscript is that it does not demonstrate how this technique can be universally used for other cell types or tissues. The unique properly of axons to grow as thin projections from CO has made this possible for studying neurons, but how this technique can be used for other tissues is lacking in this study. This study could have been more impactful if the authors would have demonstrated more broader application of the methodology and not limited it to CO or neurons.

  5. Version published to 10.1101/2020.07.23.216622v1 on bioRxiv