Correlative light and electron microscopy to study ciliogenesis

The primary cilium – a “microtubule-based organelle” (Mirvis et al. 2018) originating from a basal body – fulfills a variety of sensory functions in “most mammalian cell types” (Satir et al. 2010) (Anderson et al. 2008; Breslow & Holland 2019; Pedersen et al. 2012; Witzgall 2018a). Its formation is a complex, “multistep process” (Pitaval et al. 2017). The small GTPase RAB8 is an important molecular player for ciliogenesis in cell culture as it contributes to the extension of the ciliary vesicle at the distal end of the basal body, a process known as prerequisite for axoneme outgrowth (Lu et al. 2015; Nachury et al. 2007). However, studies investigating centrosomal RAB8 recruitment in combination with the three-dimensional visualization of membrane remodeling processes at this period of ciliogenesis are rare to date. Correlative light and electron microscopy (CLEM) techniques provide options to achieve this. We adapted two CLEM techniques to assign “fluorescently tagged proteins […] to their ultrastructural compartments” (Buerger et al. 2021). First, we extended the Kukulski protocol (Kukulski et al. 2011, 2012) and combined it with 200 kV bright-field scanning transmission electron (STEM) tomography with an electron beam with low semi-convergence angle (Rachel et al. 2020). Thereby it was possible to identify cellular structures such as mature primary cilia but also multivesicular bodies and to three-dimensionally visualize almost their entire ultrastructure in single tomograms. However, resin embedding prior to fluorescence microscopy resulted in loss of fluorescence intensity which made it impossible to preserve weak fluorescence signals of fluorescent proteins at the onset of cilia formation. For investigating centrosomal RAB8 recruitment, fluorescence microscopy was performed before sample preparation for electron microscopy. Retinal pigment epithelial (RPE1) cells showing the earliest moment detectable by fluorescence microscopy with RAB8A localizing to the centriole of a developing cilium were selected. Subsequent STEM tomography revealed a donut-like membrane structure formation during ciliogenesis which, according to our knowledge, has not been described so far: vesicles docked to the distal appendages of the basal body primarily fuse with incoming vesicles via a laterally directed process to form a donut-like structure. The central hole of this structure is closed afterwards to form the large so-called ciliary vesicle. Vesicles within the lumen of the microtubule barrel of the basal body are not enriched during these ciliogenetic processes. In contrast, fusion/budding events were observed at the distal part of docked membrane structures. RAB8A-positive membrane structures docked to the distal appendages of the basal body were present starting with the donut-like stadium of ciliogenesis. This suggests a role of RAB8A during the extension of the ciliary vesicle. By overexpressing dominant-negative mutants of RAB proteins in RPE1 cells we confirmed a role of the recycling endosome during ciliogenesis but could not unambiguously reproduce its contribution to centrosomal RAB8A recruitment. Early and late endosomes are dispensable for ciliogenesis in general. The formation of primary cilia requires incoming membrane material and a variety of proteins (Witzgall 2018b). According to the prevailing opinion, a diffusion barrier has to be passed to enter the “ciliary compartment” (Hu & Nelson 2011) (Garcia-Gonzalo & Reiter 2012; Nachury et al. 2010; Pedersen et al. 2012; Rohatgi & Snell 2010). Nature and localization of this barrier is largely obscure to date. By visualizing the ciliary membrane on an ultrastructural level, a more detailed knowledge regarding the dimension of the “ciliary compartment” (Hu & Nelson 2011) should be generated. A fusion protein of the M2 (Incardona et al. 2002; Xie et al. 1998) mutant of murine Smoothened (SmoM2) with horseradish peroxidase (HRP) and EGFP was stably expressed in Lilly Laboratories cell porcine kidney 1 (LLC-PK1) cells (Miyamoto et al. 2019) as marker for the ciliary membrane (Corbit et al. 2005); reviewed in (Hoffmeister et al. 2011). Via an HRP induced histochemical reaction (Connolly et al. 1994; Graham & Karnovsky 1966; reviewed in Witzgall 2018b) the HRP-SmoM2-EGFP fusion protein was visualized by transmission electron microscopy. Electron micrographs revealed a localization of HRP-SmoM2-EGFP not only at the membrane enclosing the ciliary axoneme but also at the membrane surrounding the base of primary cilia which is referred to as periciliary membrane in literature (Garcia et al. 2018; Garcia-Gonzalo & Reiter 2012; Long & Huang 2019; Pedersen et al. 2016). Therefore results of our work provide a hint that the composition of the membrane compartment at the ciliary base is more related to the ciliary membrane than to the plasma membrane