Menu

Close

Revealing the nanoscale morphology of the primary cilium using super-resolution fluorescence microscopy

Joshua Yoon, Colin J. Comerci, Lucien E. Weiss, Ljiljana Milenkovic, Tim Stearns, W. E. Moerner

Preprint posted on October 08, 2018 https://www.biorxiv.org/content/early/2018/10/08/437640

Article now published in Biophysical Journal at http://dx.doi.org/10.1016/j.bpj.2018.11.3136

Lighting up the cell’s antenna: beyond the diffraction limit, the primary cilium doesn’t look quite like a cylinder

Selected by Gautam Dey

Context

The mammalian primary cilium, non-motile cousin to motile cilia and flagella1, is a 200-300 nm thick antenna-like structure that projects approximately 5 microns from the cell surface. The primary cilium serves as a sensory organelle and signaling hub2,3, feeding into several critical signaling pathways and cell cycle progression mechanisms. A range of human diseases (“ciliopathies”) are caused by ciliary dysfunction, in turn often linked to defects in intraflagellar transport (IFT)4. Since transport is required to construct the cilium in the first place, IFT loss-of-function could either affect signaling directly or indirectly through the ensuing structural defects- highlighting the need for a way to quantitatively investigate ciliary structure in 3D.

 

Major findings 

The authors apply 3D STORM (later 2-color STED), to the structural interrogation of primary cilia in mouse embryonic fibroblasts (MEFs). In particular, they leverage recent developments in tweaking the point spread function (PSF) produced by single molecule fluorescent emitters- careful engineering of the light path in their in-house ‘4f’system5 produces a double helical PSF that encodes depth information within the angle between the twinned spots representing each emitter.

The transmembrane Hedgehog (Hh) receptor Smoothened (SMO) localizes to the primary cilium upon activation of the pathway.  The authors image primary cilia using a SNAP-tagged SMO that is click-labeled with an Alexa647 dye after Hh activation and fixation, producing single emitter point clouds with a precision of 25 nm or better (Figure 1). These reconstructions reveal a wide heterogeneity in cilia shapes, even in wild type cells, produced by morphological features markedly absent from textbook representations- such as kinking, bulging, and even possible budding (Figure 1).

Figure 1: Reproduced in full from Figure 1 of Yoon et al. 2018 under a Creative Commons CC-BY-NC-ND 4.0 International License. Labeling and 3D SR imaging of the ciliary membrane. (a) SNAP-SMO proteins, where the SNAP protein is on the extracellular side, are covalently labeled with BG-Alexa647 along the ciliary membrane, which are usually found near the coverslip surface. PACT-YFP indicates the base of the cilium and a nearby bright fiducial is used to correct for spatial drift. (b) Overlaid diffraction-limited images of the SNAP-SMO and PACT-YFP in chemically fixed control MEF cells that were treated with SAG. 3D SR microscopy using the double-helix point spread function (DH-PSF) was performed to obtain a localization map of SNAP-SMO molecules along one primary cilium, reconstructed as a (c) 2D histogram and (d) 3D scatterplot. For SNAP-SMO distributions of other primary cilia, there is evidence of (e) kinking, (f) bulging, and budding within the same control MEF cells. Scale bar = 1 μm.

 

The authors go on to develop a quantitative framework for ciliary shape analysis, first converting the point clouds to a 2D mesh and then using the local Gaussian curvature of the ciliary surface as a way to identify morphological features. Applying these analyses to IFT25 mutants (not thought to play a role in ciliogenesis), they nevertheless discover significant structural defects in the cilium that would be invisible to conventional imaging techniques, and go on to show that these defects can be phenocopied by a drug-induced transport block. They argue that the bulging in IFT25 mutants is therefore a likely consequence of aberrant cargo accumulation at the tips, including and not limited to monomeric tubulin (a claim bolstered by 2-color STED imaging of alpha-tubulin and SMO at a resolution of 50-100 nm).

 

What next?

The images in this paper are stunning and have certainly changed the way I view the primary cilium! I imagine the transport-coupled changes in cilium structure could usefully feed back into existing models of IFT transport. The authors also do a great job of outlining the paper’s methods in great technical detail without sacrificing clarity or readability. As the authors do, I believe the entire pipeline holds great promise for imaging subcellular morphology at unprecedented detail (as long as dense and uniform surface labelling with blinking fluorophores is possible).

I’d be curious to know whether the structural defects in the IFT25 mutants or transport-inhibited cells are also visible by EM tomography- the only data I found in the literature were from the testis, where IFT25 is highly abundant and the defects much more pronounced6.

 

References:

  1. Khan, S. & Scholey, J. M. Assembly, Functions and Evolution of Archaella, Flagella and Cilia. Curr. Biol. 28, R278–R292 (2018).
  2. Singla, V. & Reiter, J. F. The primary cilium as the cell’s antenna: signaling at a sensory organelle. Science 313, 629–33 (2006).
  3. Seeley, E. S. & Nachury, M. V. The perennial organelle: assembly and disassembly of the primary cilium. J. Cell Sci. 123, 511–518 (2010).
  4. Ishikawa, H. & Marshall, W. F. Intraflagellar Transport and Ciliary Dynamics. Cold Spring Harb. Perspect. Biol. 9, a021998 (2017).
  5. Gahlmann, A. et al. Quantitative Multicolor Subdiffraction Imaging of Bacterial Protein Ultrastructures in Three Dimensions. Nano Lett. 13, 987–993 (2013).
  6. Liu, H. et al. IFT25, an intraflagellar transporter protein dispensable for ciliogenesis in somatic cells, is essential for sperm flagella formation†. Biol. Reprod. 96, 993–1006 (2017).

Tags: 3d storm, primary cilium, sted, super-resolution microscopy

Posted on: 19th October 2018

Read preprint (No Ratings Yet)




  • 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
    Close