The tubulin code specializes neuronal cilia for extracellular vesicle release

Abstract

Cilia are microtubule-based organelles that display diversity in morphology, ultrastructure, protein composition, and function. The ciliary microtubules of C. elegans sensory neurons exemplify this diversity and provide a paradigm to understand mechanisms driving ciliary specialization. Only a subset of ciliated neurons in C. elegans are specialized to make and release bioactive extracellular vesicles (EVs) into the environment. The cilia of extracellular vesicle releasing neurons have distinct axonemal features and specialized intraflagellar transport that are important for releasing EVs. In this review, we discuss the role of the tubulin code in the specialization of microtubules in cilia of EV releasing neurons.

Keywords: C. elegans; cilia; ciliopathy; extracellular vesicle; glutamylation; intraflagellar transport; kinesin; microtubule; post-translational modifications; tubulin code.

Figure 1.. Cilia are ubiquitous organelles with diverse microtubule arrangements

A. Cartoon of a primary cilium. Dashed lines represent the ciliary segment shown in cross section on the right. Microtubule numbers, lengths, and their arrangements are different between cilia types and even within the cilium. B. Schematic of a doublet microtubule with a 13 protofilament ‘A’ tubule, and a 10 protofilament ‘B’ tubule. C. Transmission electron microscopy (TEM) cross-sections of motile and primary cilia. Tetrahymena thermophila cilia and C. elegans amphid channel cilia are shown to represent motile and primary cilia respectively. Motile cilia contain a central pair of microtubules, among several other distinct structural features. Primary cilia do not contain central pair microtubules and other structures important for ciliary motility. Images have been reproduced with permission from (Yee et al., 2015; Osinka et al., 2019). D. Examples of motile and primary cilia in different cellular contexts in the human body: Motile cilia on airway epithelial cells move mucus in the respiratory tract, whereas motile cilia on ependymal cells in the brain contribute to cerebrospinal fluid flow. Primary cilia on renal tubules are important for kidney function, while primary cilia on cells in the midbrain are important for neurogenesis during embryonic stages. Images reproduced with permission from (Banizs et al., 2005; Shah et al., 2008; Gazea et al., 2016; Silva et al., 2019).

Figure 2.. Ciliary diversity in C. elegans

A. Illustration of ciliated nervous system of hermaphrodite and male C. elegans. The cell bodies and dendrites of sensory neurons, and cell bodies of the glial cells surrounding the neurons are depicted. Cilia are present at dendritic tips (area marked by red rectangle). Dashed lines on hermaphrodite body indicate the region containing male-specific neurons in the head and the tail. Image reproduced from (Altun and Hall, 2003) B. Illustration of a typical sensillum in C. elegans. The amphid sensillum is shown here. Ciliated sensory endings are surrounded by the sheath and socket glia, and extend into a pore in the cuticle to access the environment. Some cilia remain embedded in the sheath cell and are not open to the environment. Image reproduced from (Altun and Hall, 2003). C. Examples of ciliary morphological, ultrastructural, and functional diversity in C. elegans. C. elegans cilia assume simple to elaborate forms and have different arrangement of microtubules within cilia. TEM of middle segments of ASI, AWC, and CEP cilia: all three cilia have differences in their microtubule arrangements and numbers. Scale bars are 100nm for ASI and AWC and 200nm for CEP cilium. Fluorescent micrographs reproduced from (Altun and Hall, 2003) and TEM cross-sections have been reproduced from (Doroquez et al., 2014).

Figure 3.. Only a subset of C. elegans cilia release extracellular vesicles into the environment

A. Cartoon of extracellular vesicle releasing neurons (EVNs) in the hermaphrodite and male C. elegans. There are six EV releasing IL2 neurons that are present in both sexes; males have an additional 21 EVNs that include the four CEM neurons in the head, 16 RnB neurons and 1 HOB neuron in the tail. Cartoon reproduced from (Wang et al., 2015), with permission. B. Male C. elegans expressing PKD-2::GFP (expressed only in male-specific EVNs) and CWP-1::GFP (expressed in both sex-shared IL2 and male-specific EVNs). Insets show EVs (red arrows) in higher magnification. Green arrowheads point to CEM cilium and yellow arrows point to the cuticular pore of RnB neurons. Scale bar is 10μm. Reproduced with permission from (Wang et al., 2014). C. Illustrations of the cephalic sensory organ/sensillum in males and the inner labial sensory organ/sensillum in both males and hermaphrodite C. elegans. The plan of EVN sensilla is similar to that of the amphid sensilla described in Figure 2B. The cephalic sheath, cephalic socket, and cuticle surround the CEM cilia, and the inner labial sheath, inner labial socket, subcuticle, and the cuticle surround the IL2 cilia. EVs are ‘shed’ into the extracellular space shared between the EVNs and surrounding glia (lumen). EVN cilia and the contents of the lumen of EVN sensilla are open to the environment. EVs observed outside the animal are ‘released’ EVs and visualized by fluorescent protein tagging. Illustrations reproduced with permission from (Wang et al., 2015) (cephalic sensilla in male) and (Altun and Hall, 2003) (inner labial sensilla in hermaphrodite).

Figure 4.. The ciliary tubulin code of C. elegans

A. List of α and β tubulins in C. elegans. Axonemal tubulins are underlined and in bold letters. B. Alignment of C-terminal region of α-tubulins in C. elegans. In human α-tubulins, a serine residue (S439) precedes the glutamates that are substrates for microtubule glutamylases (Janke, 2014). The serine in C. elegans α-tubulins that corresponds to a S439 in human α-tubulins is marked in bold letters. Glutamates that are strong candidates for sites of microtubule glutamylation are marked in red. TBA-6, TBA-8, and TBA-9 do not have a serine that corresponds to S439, and the glutamates in these tubulins that could be sites of microtubule glutamylation are marked in blue to separate them from the strong candidates marked in red. The C terminal tail of TBA-6 is longer than other α-tubulins, and has a lower number of glutamates. C. Cartoon representations of tubulin post-translational modifications in C. elegans cilia. All EVN specific molecules are marked in red. TTLL-4, TTLL-5, and TTLL-11 (A isoform) glutamylate microtubules in most cilia whereas EVNs employ a cell-specific tubulin glutamylase TTLL-11 (B isoform) to glutamylate axonemal microtubules composed of a cell-specific α-tubulin TBA-6. CCPP-1 and CCPP-6 may reduce microtubule glutamylation in most cilia. CCPP-1 reduces microtubule glutamylation in the EVN cilia, and the role of CCPP-6 in EVNs is not identified. Molecular motors KAP-1 (accessory subunit of heterotrimeric kinesin-2) and OSM-3 read ciliary microtubule glutamylation in most ciliated neurons, whereas OSM-3 and cell-specific kinesin-3 KLP-6 read the distinct microtubule glutamylation code of EVN cilia. D Δ2 tubulin is observed in phasmid cilia and cilia on unidentified cilia in the nose. The known deglutamylases CCPP-1 and CCPP-6 may have a role in generating Δ2 tubulin. E. The tubulin acetyltransferase ATAT-2 acetylates MTs in CEP and OLQ neurons.

Figure 5.. The tubulin code regulates EVN specific ultrastructure and IFT

A. TEM images of IL2 cilia TZ in cross-section at L2 and young adult age animals. Scale bar is 100nm. Cartoon on right depicts that IL2 ciliary microtubules develop from centriolar singlet MTs similar to other ciliated neurons but, change in number during animal development. Images reproduced from (Akella et al., 2019), with permission. B. TEM images of different segments of CEM cilia in cross-section in adult wild-type male C. elegans. The ciliary segments are represented from proximal to distal regions. CEM ciliary doublet MTs split into individual A-tubule and B-tubule singlets that rejoin to form modified doublets that terminate before the distal region, which has only A-tubule singlets. Scale bar is 250nm. Images are reproduced from (Silva et al., 2017) with permission. C. Cartoon representation of the specialized ultrastructure and IFT on CEM cilia, and their control by tubulin code regulators. In wild-type CEM cilia, IFT is carried out by heterotrimeric kinesin-2 with very little contribution from OSM-3. A small population of OSM-3 moves independently carrying unidentified cargo. Cell-specific kinesin-3 KLP-6 also moves independently of the two IFT motors and carries unidentified cargo. In tba-6 mutants, the characteristic CEM doublet MT split is lost, and OSM-3 is aberrantly recruited to the IFT machinery, IFT-A is carried by heterotrimeric kinesin-2, and IFT-B is carried by OSM-3. In ttll-11 mutants, the doublet MTs do not split and remain joined, and KLP-6 moves slower. In ccpp-1 mutants, the doublet MTs split but do not rejoin, and B-tubules degenerate (marked with dashed lines). OSM-3 and KLP-6 move faster on hyperglutamylated microtubules. Some elements of the cartoon are reproduced from (Morsci and Barr, 2011), with permission.

Figure 6.. EV defects in tubulin code mutants

Cartoon of cephalic sensory organs of wild-type adult males and males of mutants of tubulin code regulators. In the tubulin code mutants tba-6, ttll-11, ccpp-1 and klp-6, fluorescent protein-tagged EVs are not released into the environment and excessively accumulate within the cephalic lumen, the latter determined by electron microscopy and tomography. Figure modified from (Wang et al., 2015) with permission.