3D tomographic analysis to reveal intermediate stages of primary cilia formation

Abstract text (incl. figure legends and references)
Introduction

The intracellular ciliogenesis pathway includes binding of vesicles to the distal appendage and their fusion to generate one ciliary vesicle. In the following, it elongates into the ciliary membrane and the axoneme grows out.

Objectives

We aim to describe these consecutive stages of the ciliogenesis pathway using electron tomographic investigations in three dimensions (3D) and also by CLEM (correlative light and electron microscopy).

Materials & methods

We use a protocol for correlative light and electron microscopy (CLEM) to analyse the timing of Rab8 recruitment to the basal body and to visualize membrane remodelling processes which take place in early stages of cilia formation. From human retinal pigment epithelial 1 (hRPE1) cells, stably expressing CENTRIN1 and RAB8A with fluorescent tags, fluorescence light microscopy images (focal stacks) are recorded after formaldehyde fixation, specifically the hRPE1 centriolar region. For electron microscopy, cells were fixed using glutardialdehyde, en-bloc contrasted (OsO4, UAc), dehydrated, resin-embedded, and sectioned (600 – 900 nm). On cells containing fluorescent signals at the centrosom, STEM tomograms were recorded, reconstructed (IMOD), and segmented. Correlation of positions of fluorescent proteins with ultrastructural features was done using the software eC-CLEM.

Results

By using fluorescence microscopy, we identified a dot-like pattern (mCherry-Rab8a) to one of the two Centrin1-GFP-positive centrioles in. In STEM tomograms of the centrosomal regions and in 3D models (by manual segmentation), novel intermediate stages of cilia formation were revealed; we named them the donut-shaped stage, and the biconcave, erythrocyte-shaped stage. Our data let us conclude that distal appendage vesicles fuse side-by-side to first build a C-shaped structure, which later employs the shape of a donut. Centripetal fusion events result in a biconcave membrane structure, later forming a cap-shaped ciliary membrane. Overlays (CLEM) showed that the earliest stage of ciliogenesis in which mCherry-Rab8a localized to the basal body in our set-up was the C-shaped ciliary membrane structure. To get insight into the cellular compartments that serve as a source for the early ciliary membrane compartment, we incubated cells with the lectin wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP), a tag helping to visualise the fusion protein by EM within its cellular context. Interestingly, we detected electron dense membrane material generated by HRP at the developing ciliary membrane during early cilia formation. This observation suggests that endocytosed membrane material contributes to the formation of the early ciliary membrane compartment.

Conclusion

We extend the stages of primary cilia formation during the intracellular cilia assembly pathway by identifying the donut-shaped and the biconcave stage, and we present evidence that endocytic trafficking routes contribute to the early ciliary membrane compartment.

Lit.: Rachel et al 2020 J Struct Biol 211: 107551

Buerger et al 2021 Meth Cell Biol 162: 171-203

Fig. 1: images show (left, middle) two xy planes (thickness 2.7 nm; 54 nm apart from each other) of a STEM tomogram (IMOD). The slice was cropped to the region of the basal body. Bar, 200 nm; (right) 3D model of the reconstructed volume after manual segmentation of the STEM tomogram; top view, to best visualize the donut shape of the centriolar vesicle