In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen

Danielle M Paul, Judith Mantell, Ufuk Borucu, Jennifer Coombs, Katherine J Surridge, John Squire, Paul Verkade, Mark P Dodding

Posted on: 2 December 2019

Preprint posted on 18 November 2019

Article now published in Journal of Cell Biology at

Letting the cat out of the bag: Cytoplasmic microtubules contain actin filaments

Selected by Sina Knapp

Categories: biophysics, cell biology


The interaction between actin filaments and microtubules have been extensively studied during the last decades. This interplay between the two cytoskeletal systems is crucial during migration, polarization and division of the cell. Depending on the cellular context, the interaction of the two main cytoskeletal components occurs through either physical crosslinkers or passive interference. Although much is known about the structures of actin filaments and microtubules, a key question remains unresolved: What does the microtubule lumen consist of? In cytoplasmic microtubules, tubulin modifying enzymes have been identified and in sperm flagella microtubules, higher structures have been described. However, the content of cytoplasmic microtubules has thus far not been thoroughly revealed, as Cryo-EM analysis requires a very thin sample and super-resolution fluorescence microscopy is problematic in microtubules due to impaired antibody epitope accessibility.

Recently, the authors of this study identified a small-molecule activator (kinesore) of the microtubule motor kinesin-1, by which cytoplasmic microtubules bundle and reorganize in a nonradial network spread throughout the cytoplasm and expand in membrane-bound projections. Here the authors, by taking advantage of kinesore’s ability of inducing thin protrusive structures, set up a Cryo Electron Tomography (Cryo-ET) imaging protocol for analysis of the lumen of microtubules residing in the kinesore-induced cellular projections. Remarkably, the approach of Paul et al. revealed that filamentous actin can be found within the microtubule’s lumen.


Key findings

Kinesore treatment induces bundling of microtubules in cell projections

Cryo Correlative Light Electron Microsopy (Cryo-CLEM) after fluorescent membrane stain and kinesore treatment revealed microtubule bundles in cell projections in HAP1 cells. Live cell imaging using siR-tubulin labelled microtubules confirmed that these microtubule extensions are caused by the progressive addition of microtubules by loop extrusion events, following kinesore treatment. The cryo-EM analysis of these cell projections identified mainly aligned bundles of microtubules, containing 4-30 single ones (Fig. 1A).


The lumen of microtubules in cell projections contain actin filaments

Within the lumen of kinesore induced microtubules, cryo-ET analysis identified structures with filamentous density and helical appearance (Fig. 1B). To understand if the 5-9 nm wide filaments could be actin, kinesore treated cells were stained for tubulin and F-actin, revealing patches of F-actin linking gaps in ß-tubulin staining.

The morphology of the lumenal filaments could be grouped in two classes, differing in thickness. The filament thickness correlates with the microtubule thickness, as thinner filaments (class I filaments) were found in microtubules with smaller outer and lumenal diameters. Further, on average 27% of the total luminal length of the microtubule was filled with actin-like filaments. Thicker actin-like filaments (class II filaments) were shorter compared to thinner filaments, but the frequency of the two did not differ.

To further validate that the luminal filaments are actin fibers and to understand the difference between the two groups of filaments, layer line images after Fourier transforms of 2D projection images from 3D subvolumes were analyzed. This analysis showed that the class I filaments can be identified as F-actin by reflecting an actin layer line pattern. The class II filaments also resemble an actin layer pattern with a distinctive meridional reflection indicating affiliated proteins at the lumenal actin. This meridional reflection is speculated to resemble a formin-like encircled actin backbone.

Fig. 1: (A) Membrane bound microtubules in a 3D model are organized in tightly packed bundles. (B) Sections of microtubule lumen reveal helical filaments within. Adapted from Paul et al., 2019. Courtesy of the authors.


Why I like this preprint

By additional accumulation of cytoplasmic microtubules in cell projections, the approach of Paul et al., allows the analysis of the microtubule lumen via Cryo-ET, overcoming previous limitations in sample thickness. The identification of filamentous actin within the lumen of microtubules sheds a new light on the crosstalk of actin with microtubules. Previously, actin filaments and microtubules were thought to merely interact, but the idea of one incorporating the other gives rise to new questions in the field.



  1. Actin filament occupancy does highly vary within microtubules (ranging from 4% to 76%), what distinguishes the highly occupied from less occupied microtubules?
  2. Is filamentous actin within the lumen specific to microtubules of the cell projections? How about microtubules during mitosis or at the leading edge of a migrating cell?
  3. Do you suggest with the hypothesis of class II filaments reflecting formin-like encircling of the actin backbone a possibility for actin filament assembly and elongation within the microtubules? Why are the class II filaments shorter compared to class I filaments?


Tags: actin, cryo-et, microtubules


Read preprint (No Ratings Yet)

Author's response

Mark Dodding shared

  1. Actin filament occupancy does highly vary within microtubules (ranging from 4% to 76%), what distinguishes the highly occupied from less occupied microtubules?

This is an interesting question that we are currently exploring. One attractive hypothesis is that the projections contain microtubules originating from different parts of the cell, and that this is reflected in different actin content.

  1. Is filamentous actin within the lumen specific to microtubules of the cell projections? How about microtubules during mitosis or at the leading edge of a migrating cell?

To the best of our knowledge, F-actin in microtubules hasn’t been observed before in any context. An important question is whether this has simply been a technical limitation because it is difficult to detect without cryo-tomography of very thin samples. Advances in FIB-SEM techniques may hold the key to addressing this.

  1. Do you suggest with the hypothesis of class II filaments reflecting formin-like encircling of the actin backbone a possibility for actin filament assembly and elongation within the microtubules? Why are the class II filaments shorter compared to class I filaments?

The question of dynamics of the filaments is an important one. We currently don’t have any means to explore this. A priority going forward must be to discover a way to detect this lumenal actin using fluorescent imaging in cells and seek to reconstitute microtubule lumenal actin in vitro.


Have your say

Your email address will not be published. Required fields are marked *

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

Also in the biophysics category:

Topology changes of the regenerating Hydra define actin nematic defects as mechanical organizers of morphogenesis

Yamini Ravichandran, Matthias Vogg, Karsten Kruse, et al.

Selected by 08 May 2024

Rachel Mckeown

Developmental Biology

Structural basis of respiratory complexes adaptation to cold temperatures

Young-Cheul Shin, Pedro Latorre-Muro, Amina Djurabekova, et al.

Selected by 10 April 2024

Pamela Ornelas


Actin polymerization drives lumen formation in a human epiblast model

Dhiraj Indana, Andrei Zakharov, Youngbin Lim, et al.

Selected by 05 April 2024

Megane Rayer, Rivka Shapiro


Also in the cell biology category:

Mitochondria-derived nuclear ATP surge protects against confinement-induced proliferation defects

Ritobrata Ghose, Fabio Pezzano, Savvas Kourtis, et al.

Selected by 16 May 2024

Teodora Piskova

Cell Biology

Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery

Aslihan Terzi, Tiger Liao, Adrian Jacobo

Selected by 09 May 2024

Roberto Rodríguez-Morales


Fetal brain response to maternal inflammation requires microglia

Bridget Elaine LaMonica Ostrem, Nuria Dominguez Iturza, Jeffrey Stogsdill, et al.

Selected by 24 April 2024

Manuel Lessi


Also in the cell biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.


List by Reinier Prosee

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023


List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.


List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.


List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23


List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.


List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.


List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.


List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.


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


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


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


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

ASCB/EMBO Annual Meeting 2018

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


List by Dey Lab, Amanda Haage