Close

A cryo-ET study of microtubules in axons

H E Foster, C Ventura Santos, A P Carter

Preprint posted on 29 March 2021 https://www.biorxiv.org/content/10.1101/2021.03.29.437471v1

CryoET of Dm and DRG neurons provides exciting new insights into microtubule architecture, highlighting the importance of studying cellular components in their native environment.

Selected by Ashleigh Davey

Categories: cell biology, neuroscience

Background:

Inside neurons, microtubules work as “molecular roads”, allowing the transport of cellular cargo across great distances. This is especially important in neuronal axons, which require motor proteins to transport cargo from the cell body to the presynapse and vice versa – a process known as axonal transport. Additionally, microtubules play a vital role in cellular structural support, making them an essential cytoskeletal component.

Microtubules are asymmetric, dynamic structures which grow and collapse depending on the requirements of the cell. Composed of tubulin dimers, microtubules have two distinct ends – the plus end (the location of microtubule growth and shrinkage) and a more stable minus end. The orientation of plus and minus ends is important for neuronal polarisation, with the minus ends in axons being predominantly orientated towards the cell body (Heidemann et al. 1981). Recent advances in microtubule research have utilised CryoET, a microscopy technique which can resolve cellular structures without sample fixation. Historically, these studies typically used reconstituted microtubule networks formed through microtubule polymerisation in vitro. Although this research has provided major insights into microtubule structure, data from in vitro polymerized microtubules may not be representative of neuronal microtubules in their native environment.

Here, Foster et al. investigate the structure and interactions of axonal microtubules using CryoET, providing new insights into how axonal microtubule environments can differ between model species (Foster et al. 2021). The authors analyse Drosophila melanogaster (Dm) and mouse dorsal root ganglia (DMG) primary neurons at their thinnest sections, overcoming the sample depth limitations of CryoET (Rice et al. 2018).  Their work validates previous findings of microtubule structure in vitro, whilst also discovering exciting new microtubule structures visible within the cellular environment, providing new in-depth analysis of neuronal microtubule architecture in a cellular context.

 

Findings:

Foster et al. begin their CryoET analysis by investigating the polarity of neuronal microtubules in two model species. Using subtomogram averaging, whereby structures in multiple tomograms are aligned and averaged to increase resolution, the authors were able to determine the polarity of individual microtubules within Dm and DMG neurites by analysing the ends of microtubule projections (Figure 1A). Projections with tubulin subunits rotated clockwise are determined to be a minus end, with plus ends showing an anti-clockwise slant (Figure 1B). From this, Foster et al. investigated the orientation of microtubules in both Dm neurons, determining that 68% of Dm microtubules had uniform orientation and 32% had mixed orientation. In contrast, when the same analysis was performed on DMG axons, 95% of microtubules had uniform orientation, which supports previous findings (Heidemann et al. 1981) (Kleele et al. 2014).

 

 

Figure 1: CryoET Analysis of Dm and DRG neurites to determine microtubule polarity. A) An example tomogram of microtubules visible in a Dm neurite. B) Z-projections of two microtubules from A. The tubulin subunit slew indicates the orientation of the microtubule, with a clockwise and anticlockwise slew indicating a minus and plus end respectively.

 

The protofilaments at the end of 22 DRG microtubules were studied in detail. Microtubule minus ends showed a straight or slightly curved appearance, and strikingly these ends were “naked” – meaning that no visible γ-TuRC caps (a common feature in the minus ends in mitotic spindles) were present. The plus ends also had a similar morphology to the minus ends. Subsequently, the length between the shortest and longest protofilament (the taper length) was also determined. Foster et al. report that the majority of microtubules in this study have a taper length of 15-40nm, however, 2 microtubules were identified as having a taper length of over 100nm. This indicates that sheet-like structures can be present at the ends of axonal microtubules. One 340nm sheet-like end was observed to be attached via multiple short tethers to the outside of an adjacent microtubule. The authors note that this novel plus-end architecture may allow for protein interactions involved with plus end stabilisation or directing plus ends towards the axon tip. Additionally, 14 out of 28 plus and minus ends made contact with other microtubules, membranes, or intermediate filaments. Although these contacts may be non-specific, Foster et al. propose that they may be involved in microtubule dynamic regulation.

Using the same subtomogram averaging as before, the numbers of protofilaments within microtubules was determined. Although protofilament numbers can vary (Chaaban and Brouhard 2017), there is evidence that mammalian neurons typically have 13 protofilaments per microtubule (Unger et al. 1990). Here, the authors show that 96% of the DRG neuron dataset had 13 protofilament structure, whereas only 39% of Dm microtubules had 13 protofilaments. 52% of the Dm dataset were observed to have 12 protofilaments, with some neurites containing both 12 and 13 protofilament microtubules. Additionally, using multireference alignment, Foster et al. identified 6 microtubules which contain both 12 and 13 protofilament regions, with observable transition sites. This indicates that protofilament number transitions may be cell-type specific.

Lastly, the density and interactions of microtubule inner proteins (MIPs) was studied. MIPS are globular proteins that form connections with the luminal face of the microtubule. The authors report that DRG neurons had 40-100 MIPs per micron length, with no reduction in MIP density near or within microtubule ends and lattice breaks. A possible explanation for this is that the MIPs are anchored to the microtubules. Further analysis of DRG MIP structure using subtomogram averaging details a ring-like structure with a defined ~9nm inner pore. In contrast, Dm neurons do not contain MIPs with a regular structure, which may be due to the lack of MAP6 protein within invertebrate MIPs.

 

Why I chose this preprint:

Foster et al. use a novel cryoET methodology to provide insights into neuronal microtubule architecture, including identification of an unreported plus-end structure. This work highlights the need to observe cellular components in their native environment and illustrates the structural differences in microtubule architecture between species. This is an exciting paper in the field of neurobiology, and further CryoET studies will provide many more insights into neuronal composition and interactions.

 

Questions for the authors:

  • What role do you expect the protofilament sheet structures to play within the microtubule, if any?
  • What other microtubule features could be explored using CryoET?
  • What was the most technically demanding part of this project?

 

References:

Foster HE, Ventura Santos C and Carter AP (2021) A cryo-ET study of microtubules in axons. bioRxiv. DOI: 10.1101/2021.03.29.437471.

Heidemann SR, Landers JM and Hamborg MA (1981) Polarity orientation of axonal microtubules. The Journal of cell biology 91(3 Pt 1): 661–665. DOI: 10.1083/jcb.91.3.661.

Rice WJ, Cheng A, Noble AJ, et al. (2018) Routine determination of ice thickness for cryo-EM grids. Journal of structural biology 204(1): 38–44. DOI: 10.1016/j.jsb.2018.06.007.

Kleele T, Marinković P, Williams PR, et al. (2014) An assay to image neuronal microtubule dynamics in mice. Nature communications 5: 4827. DOI: 10.1038/ncomms5827.

Chaaban S and Brouhard GJ (2017) A microtubule bestiary: structural diversity in tubulin polymers. Molecular biology of the cell 28(22): 2924–2931. DOI: 10.1091/mbc.E16-05-0271.

Unger E, Böhm KJ and Vater W (1990) Structural diversity and dynamics of microtubules and polymorphic tubulin assemblies. Electron microscopy reviews 3(2): 355–395. DOI: 10.1016/0892-0354(90)90007-f.

Tags: axon, axons, cryoet, map6, microtubule, neuron, neurons, protofilament, structure, subtomogram averaging

Posted on: 1 June 2021

doi: https://doi.org/10.1242/prelights.29153

Read preprint (No Ratings Yet)

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage
Close