Abstract
Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organisation to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small-molecule induced, microtubule-based cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in-situ study of microtubule structure and contents.
Introduction
Understanding of the lumenal contents of cytoplasmic microtubules has been classically driven by electron microscopy based analysis in cells and tissue 1-3, and most recently by high resolution cryo-electron microscopy (cryo-EM) 4-8. Contents of cytoplasmic microtubules are thought to be restricted to globular proteins such as tubulin modifying enzymes 9, although higher order structures have been observed in sperm flagella microtubules and cilia 10,11. Unequivocal identification of lumenal proteins in a cellular context remains a challenge as the dimensions of the microtubule may confound super-resolution fluorescence imaging techniques and there are potential issues with antibody epitope accessibility in this confined environment. Cryo-EM solves the challenge of spatial resolution but can be limited by the technical requirement for very thin samples 12 and so studies have mainly focused on microtubules at the cell periphery or within neuronal processes 4-8. We recently identified a compound, 3,5-dibromo-N′-[2,5-dimethyl-1-(3-nitrophenyl)-1H-pyrrol-3-yl]methylene}-4-hydroxybenzohydrazide (named ‘kinesore’), that targets the motor protein kinesin-1, promoting extensive remodelling of the microtubule network 13. Kinesore treatment results in dynamic looping and bundling of microtubules within the cytoplasm and their extrusion from the cell body as membrane bound projections. This renders microtubules from within the cell accessible for cryo-EM studies 13,14. Here we describe the discovery of filamentous actin (F-actin) inside the microtubule lumen through in situ cryo-electron tomography (Cryo-ET) analysis of these small molecule induced projections.
Results
Formation and ultrastructure of small-molecule induced microtubule-based projections
To begin ultrastuctural analysis of projections in their native state, HAP1 cells, incubated with a fluorescent membrane stain prior to kinesore treatment, were prepared for cyro Correlative Light Electron Microscopy (Cryo-CLEM). This allowed the unambiguous identification of projections using fluorescence microscopy that could be correlated with images from the electron microscope (Figure 1A). Consistent with our earlier immunofluorescence imaging study 13, abundant projections were composed of closely aligned microtubules and we also occasionally observed vesicular structures within swollen regions. Our previous live-imaging of GFP-tubulin expressing HeLa cells suggested that the projections were initially formed though the extrusion of microtubule loops 13. Live-imaging of SiR-tubulin labelled microtubules in the HAP1 cells used here confirmed this and showed that microtubules are progressively added through additional loop extrusion events (Extended Data Fig. 1 and Movie 1), providing a rationale for the formation of extended microtubule bundles. Consistent with this live-imaging, EM analysis of regions proximal to the cell body revealed a greater number of microtubules and distinct looped bundles (Extended Data Fig. 2). Within these structures, microtubules typically maintained a consistent spacing of between 10-25 nm (blue shading) although some were also observed to traverse bundles (yellow shading).
Cryo-ET reveals actin-like filaments with the lumens of extruded microtubules
Satisfied that we could confidently identify projections in the electron microscope, additional samples were analysed without the fluorescence imaging step (avoiding ice contamination) by 3D reconstruction of tomography tilt series (Figure 1 B,C). Transverse sections through projections revealed that they could contain variable numbers of microtubules (ranging from 4 to >30) (Figure 1B, Extended Data Fig. 3). A section of a 23 microtubule projection is shown in detail. Microtubules are organised in a twisted bundle and vesicles are excluded in swellings proximal to the limiting membrane. Both transverse sections and serial images of Z-sections show that the microtubules are intact (Figure 1C, Movie 2). Notably, these microtubule structures closely resemble those observed in in vitro EM studies of kinesin-driven microtubule-microtubule cross-linking 15. Surprisingly, within the microtubule lumens, as well as globular structures we could clearly observe extended filamentous density with helical character (blue arrows). Two examples of such filaments are boxed in orange and blue on the Z-section panels in Figure 1B and are also visible as density within the microtubule lumen of transverse sections (see also, Extended Data Fig. 3). The diameter (5-9 nm) and helical appearance of these lumenal filaments is consistent with that of F-actin 16,17, that at least in principle, could reside within the ≈ 15-16 nm diameter lumenal space 18.
To assess whether kinesore-induced projections do indeed contain actin, methanol fixed cells (to optimally preserve microtubules) were stained with antibodies against actin and tubulin. This revealed patches and puncta of actin along microtubules within the projections (Extended Data Fig. 4A). Actin antibodies may not discriminate between G and F-actin and so equivalent samples were prepared using paraformaldehyde fixation and phalloidin staining for F-actin. Under these fixation conditions, projections were less well preserved, with fragmentation in β-tubulin staining but actin patches were more prominent and appeared to bridge gaps in tubulin staining (Extended Data Fig. 4B). This raises the intriguing possibility that a population of F-actin may reside within the microtubule lumen that is refractory to traditional means of detection, possibly due to limited antibody epitope or small-molecule binding site accessibility.
Lumenal actin-like filaments have two distinct morphologies
A gross survey of the morphology of lumenal filaments resulted in their classification into two pools; Class I and Class II. Class I filaments are exemplified in the right orange box in Figure 1B Z-sections and class II by the left blue box. Further examples are shown in Figure 2A and Movie 3 shows a Z-series through an extended Class I filament containing microtubule. Class II filaments appeared slightly thicker and were typically better defined than Class I. Analysis of the tomograms in our dataset that provided clearest definition of lumenal contents (n=11), containing 146 microtubules with a total length of 113 µm revealed that 27% of the total lumenal length of microtubule was occupied by actin-like filaments of either class (Figure 2B). There was considerable variation between tomograms with 76% to 4% of the lumenal length occupied, with a mean of 29%. Of the total actin-like filament length, 68% was comprised of Class I and 32% by Class II filaments (Figure 2C). Although the frequency of Class I and Class II filaments were similar, Class I filaments were longer (average 475nm) than Class II (average 274nm) (Figure 2 D,E). Class I filament containing microtubules had average outer and lumenal diameters of 25.60 ± 0.59 nm and 16.80 ± 0.76 nm respectively. Class II filaments containing microtubules were typically slightly wider at 27.29 ± 0.58 nm (outer diameter) and 17.80 ± 0.85 nm (lumenal diameter) indicating that the presence of lumenal filaments either correlates with or modifies microtubule properties (Table 1).
Layer-line analysis confirms that lumenal filaments are composed of F-actin
To further characterise microtubule cores, lumenal regions were extracted from the tomographic reconstructions as 3D subvolumes. These were then summed in Z to obtain 2D projection images. Fourier transforms of these images were then calculated and inspected. Helical filaments like actin and tubulin display distinct layer line patterns. Data from single representative microtubules containing filaments of the Class I and Class II varieties are shown in Figure 3 and measured parameters from several microtubules/filaments are shown in Table 1. Class I filament containing microtubules display an actin layer line pattern - a clear reflection at 5.94 ± 0.03 nm - the pitch of the actin genetic helix and a reflection at 29.50 ± 0.80nm corresponding to the crossover spacing of the two long pitch actin helices. This crossover spacing is shorter than for canonical actin (~35nm) 19 but greater than that reported for actin cofilin filaments (~27nm) 20 and actin is known to have a random variable twist 21. These results enable us to confidently identify the Class I lumenal filament as F-actin. A reflection at 4nm was also visible from the tubulin monomer. Class II filament containing microtubules maintain an actin-layer line pattern with reflections at 6.11 ± 0.09 nm and 27.44 ± 2.29 nm, that is augmented by a strong reflection on the meridian at 6.18 ± 0.06 nm (matching the pitch of the genetic helix of actin). This meridional reflection is indicative an additional protein(s) associated with the lumenal actin. In Extended Data Figs. 5 and 6 we show that Class I actin filaments have symmetry close to an 11 subunit in 5 turn helix of axial repeat 29.5 nm, and that Class II actin filaments have symmetry close to a 20 subunit in 9 turn helix of axial repeat 2 x 27.5 nm. We tentatively speculate that the meridional reflection from Class II filaments is consistent with a formin-like encircling of the actin backbone 22.
Discussion
The discovery of microtubule lumenal actin (ML-actin) in our cell-based extrusion system prompts the questions of how it is incorporated, its function(s) and abundance in settings that are not small-molecule modified. Two hypotheses present themselves that are not mutually exclusive. Firstly, altered microtubule dynamics caused by activation of kinesin-driven microtubule-microtubule sliding and bundling provides opportunity for actin to enter the microtubule lumen. This may occur through transient opening of the microtubule under strain when tight loops are formed (Extended Data. Fig. 1) or by kinesin-mediated disruption of the lattice 9,23,24. Put another way, F-actin incorporation may occur in response to mechanical or structural stress 25. Alternatively, microtubule extrusion has facilitated the analysis of a pool of pre-existing actin-containing microtubules with previously limited accessibility by high resolution cryo-EM. Indeed, the similarity of our images to the ‘dense core microtubules’ first observed in amphibian and rat neurons as well as platelets is striking 1-3,26,27. In either case, it will be important to understand if and how an F-actin core (of either class) alters the mechanical and dynamic properties the microtubule as well as explore this new concept as a new basis for actin-microtubule crosstalk in diverse settings 28-31.
Author contributions
Performed experiments – D.M.P, J.S., U.B., J.C., K.S., P.V., M.P.D.
Analysed data - D.M.P, J.S., P.V., J.S., P.V., M.P.D.
Wrote the draft – M.P.D.
All authors contributed to revision of the draft.
Competing Interests
The authors declare no competing interests.
Methods
Cell culture and small-molecule treatment
HAP1 cells were obtained Horizon Discovery and cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% FCS and Penicillin/Streptomycin at 37ºC in 5% CO2 incubator. For fluorescence imaging, cells were plated onto fibronectin coated cover-slips in a 6-well plate at a density of 1×105 per well the day before small-molecule treatment. Cells were prepared for electron microscopy as described below. Kinesore (3,5-dibromo-N′-[2,5-dimethyl-1-(3-nitrophenyl)-1H-pyrrol-3-yl]methylene}-4-hydroxybenzohydrazide) was obtained from Chembridge Corporation (Cat. No. 6233307) and prepared as a 50 mM stock in DMSO. In addition to manufacturer provided QC, molecular weight was verified by mass spectrometry. To stimulate projection formation. treatments were carried out in Ringer’s Buffer ([155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4, 10 mM glucose, 10 mM Hepes (pH 6.8)] (in a 37ºC incubator without CO2) containing 100 µM Kinesore (final DMSO concentration 0.2%) for one hour. Control experiments were performed with DMSO alone in Ringers buffer at 0.2%.
Live-cell imaging and analysis
To label tubulin, HAP1 cells were incubated with SiR-Tubulin (500nM) in growth media for 1 hour prior to kinesore treatment as described above, before transfer to the microscope stage. Images were acquired at 15 second intervals as single confocal sections, using a 633nm laser on a Leica SP5-II system with a 63x objective lens. Resulting data was further analysed as indicated and prepared for publication using Fiji (ImageJ) and Adobe Photoshop/Illustrator Packages.
Correlative Light Electron Microscopy
Cells were grown on Quantifoil R1.2/1.3 400 mesh gold EM grids (supplied by EM Resolutions Ltd, UK). These were plunge frozen in liquid ethane using a Leica EM GP plunge freezer and transferred to a Leica CryoCLEM stage based on the Leica DM6000FS fluorescence light microscope. In this microscope the objective is cooled and the sample is transferred to a cryo stage where its temperature can be maintained below −140 °C during observation ensuring that the sample remains vitrified. Images were collected in both bright field and in green fluorescent channel. Areas of potential interest for further study by cryoTEM were thus recorded in a manner similar to 32. The samples were recovered under liquid nitrogen, transferred to a Tecnai20 LaB6 TEM (FEI), operating at 200kV using a Gatan 626 cryotransfer holder. The areas of interest were retraced and images collected on a bottom-mounted Thermo-Fisher CETA camera.
Cryo Electron Tomography
Cryo samples were prepared as described above, clipped, and transferred into a Talos Arctica CryoTEM (FEI) operating at 200 kV. Bidirectional tilt series were collected with an angular range of +20° to −60° / +60°, angular increments of 3°, total dose of 109 e−/Å2, and a defocus range between −2 µm to −4 µm. Images were recorded in counted mode (2.21 Å/pixel) using a K2 Summit direct electron detector (Gatan) fitted to a BioQuantum energy loss spectrometer (Gatan) operating with a 20 eV slit width.
Data processing
Reconstructions, movies and microtubule/actin filament length analysis were performed using IMOD (University of Colorado, Boulder)33 and Fiji (ImageJ) software.
Layer Line analysis
Fourier transforms of 2D projections of extracted filament volumes were calculated and the layer-line positions measured using Fiji (ImageJ). The well characterized 4 nm reflection of the tubulin monomer was used as an internal calibration tool and the real space pixel size for each filament calculated. Modelling of the layer-line patterns from helices with actin-like symmetry (Supplementary Figures 5 and 6) was carried out using the HELIX program:
(Knupp & Squire: https://www.diamond.ac.uk/Instruments/Soft-Condensed-Matter/small-angle/SAXS-Software/CCP13/HELIX.html).
Supplementary Videos
Supplementary Movie 1
Movie shows HAP1 cells labelled with SiR-tubulin and treated with kinesore that is also shown in Supplementary Figure 1. Images were acquired every 15 seconds. Boxed region shows area of movie described in more detail in figure 1.
Supplementary Movie 2
Movie shows a series of images through the tomogram described in detail in Figure 1B. Microtubules in the tomogram are then identified by coloured cylinders.
Supplementary Movie 3
Movie shows Z-series through a subvolume containing two microtubules. The top microtubule contains a Class I filament, the bottom microtubule contains globular density
Acknowledgements
This work was supported by the Biotechnology and Biosciences Research Council (BBSRC) (BB/S000917/1) and a Lister Research Prize Fellowship to M.P.D.. D.M.P is supported by a British Heart Foundation Career Re-Entry Fellowship FS/14/18/3071), the Alan Turing Institute through a Turing Fellowship and the Academy of Medical Sciences by a Springboard award (SBF003\1142). We acknowledge access and support of the GW4 Facility for High-Resolution Electron Cryo-Microscopy, funded by the Wellcome Trust (202904/Z/16/Z and 206181/Z/17/Z) and BBSRC (BB/R000484/1) as well as the Wolfson Bioimaging Facility at Bristol. The Cryo-fluorescence microscope was supported by the BBSRC (BB/L014181/1). We are grateful to Professor Carolyn Moores (Birkbeck, University of London) for helpful discussions on the project and comments on the manuscript draft.