Ultrastructure of cephalic male cilia of Caenorhabditis elegans: development and specialization

Cilia are ubiquitous eukaryotic organelles. The medical importance of cilia is underscored by the growing list of diseases caused by cilia defects, called ciliopathies. In the past several decades, researchers uncovered the basic molecular machinery required to build all cilia. Despite the fact they they’re built by the same “core” set of proteins, cilia exhibit a plethoric diversity of morphology, structure and function. How this ciliary diversity is generated is not well understood. To address this question I used serial transmission electron microscopy and electron tomography to reconstruct cephalic male (CEM) cilium in wild-type Caenorhabditis elegans and mutants that altered CEM cilia shape and function. I found that CEM cilia contain a novel and specialized microtubule arrangement (Chapter 1). Inner core of most cilia contains microtubule doublets (dMT), each composed of a complete A-tubule and an incomplete B-tubule. In CEM cilia, these doublet microtubules splay to form A- and B-tubule singlet microtubules (sMT) that are attached to each other at their ends, forming a splayed doublet, a structural feature partially conserved in mammalian sperm flagella. I found that a cell specific alpha tubulin isoform TBA-6 is required for the splayed doublet microtubule architecture in CEM cilia. In tba-6 loss of function mutants, doublet A- and B- tubules remain associated with each other, thereby defaulting to their commonly observed state in other cilia types. Loss of CEM cilia-specific microtubule ultrastructure correlates with perturbation of certain ciliary proteins’ localization, of shedding rate, composition and signaling of cilia-derived extracellular vesicles, and of cilia-specific coordination of ciliary microtubule-based motors. We conclude that the splayed dMTs are a specific architectural feature of CEM cilia axoneme that regulates ciliary shape, motor-based transport and EV protein composition. In addition, formation of this CEM cilia-specific ultrastructure requires a specific isoform of alpha tubulin TBA-6. To understand how the splayed dMT is generated, I characterized CEM cilia ultrastructure undergoing development in larval males (Chapter 2). I found intact (fully fused) dMT in developing CEM cilia up to the adult stage. This suggests that the splayed dMT architecture is achieved by splaying of the intact doublets rather than coming together of separate sMTs. I also discovered that some of ciliary dMTs are formed in the absence of visible basal body microtubules. In the absence of visible basal body, CEM ciliary dMTs are established by first forming the complete A-tubule and subsequent addition of the incomplete B-tubules. I found that length and volume of CEM cilia are established at different rates: while the length of CEM cilia are established by few pioneering microtubules by larval stage 4, generation of adult CEM ciliary volume occurs later, during larval-adult transition, and coincides with fully formed ciliary microtubule core and sexual maturation of the male. Together, these findings may provide novel insights in interpreting microtubule ultrastructure phenotypes in pathological cilia. To summarize part I of this dissertation (Chapters 1 and 2), ultrastructural characterization of adult CEM cilia revealed a novel aspect of ciliary specialization – remodeling of doublet microtubules in ciliary microtubule core as well as novel features of ciliogenesis: progressive addition and asynchronous extension of microtubule doublets to the growing ciliary axoneme in the absence of visible basal body microtubules. Changes in CEM cilia-specific microtubule ultrastructure in tba-6 mutants coincide with the loss of ciliary specialization, illustrating the role of a specific tubulin isoform in determining specialized ciliary structure and function. Part II of this dissertation consists of major collaborative studies. They are arranged chronologically. In chapters three and six, I describe the discovery of posttranslational microtubule glutamylation as a second genetic mechanism to generate splayed doublet architecture and B-tubule singlets in CEM cilia. In chapter four, I reexamined the data from chapter three (O’Hagan et al. 2011) and conducted additional TEM studies to conclude that the splayed doublet architecture of CEM cilia requires a “Goldilocks” level of posttranslational glutamylation. With excessive polyglutamylation (in ccpp-1 mutants), B-tubules completely separate from the A-tubules; while without glutamylation (in ttll-11 mutants), B-tubules remain oddly attached to their A-tubule partners. These phenotypes are not CEM-specific In amphid channel cilia microtubule doublets are present in distal region of ttll-11 mutants, where only A-tubule singlets are found in wild type. Together, these results demonstrate that glutamylation-based regulation of ciliary B-tubule architecture may be a common mechanism to define specialized ciliary structural identity. In chapter four, my TEM studies extended the previous observations by Natalia Morsci to resolve polycystin accumulations at CEM cilia base. My ultrastructural data showed that PKD-2::GFP accumulations seen under light microscope were discrete vesicles trapped in the lumen outside the cilium. In wildtype CEM cilia, these vesicle-based accumulations were rare, but present. In mutants isolated in Young Bae’s screen for defects of ciliary targeting of PKD-2::GFP (Cil phenotype), these vesicles accumulated upwards of ten-fold compared to wild type. Additionally, my serial section TEM and electron tomography-based characterization of multiple cil mutants reveled the existence of two types of vesicles populations in CEM sensilla: smaller-sized EVs at the level of the axoneme that may be released to the outside environment, and larger-sized EVs that are found in the lumen surrounding the cilia base. Wang et al. 2014 established CEM cilia as a model to study extracellular vesicle (EV) biology. My TEM and tomography data provided corroborating ultrastructural evidence and insight to the otherwise vague phenotype of PKD-2::GFP accumulation in distal dendrite/cilia region observed using light microscopy. TEM results summarized in Chapter Five are one of the many mutants that I fixed, sectioned and characterized (at low resolution). These mutants were chosen based on the RNAseq experiments by Dr. Maureen Barr and included: lov-1, pkd-2, trf-1, trf-2, tag-232, and pmk-1. pmk-1 was chosen because it has the “reverse Cil” phenotype: level of PKD-2::GFP at the ciliary base is reduced (rather than increased) compared to wild type. My TEM results corroborated these observations and additionally revealed that pmk-1 mutants have less than nine dMTs in CEM transition zones. Together with TEM characterization of the developing CEM axoneme, these findings formed the empirical basis of the hypothesis that CEM cilia phenotype of pmk-1 mutants is immature (underdeveloped) cilia phenotype, rather than degeneration phenotype. Moreover these findings illustrate that cilium development could be genetically uncoupled from organism development. To summarize part II of this dissertation (Chapters 3 to 6), I discovered that balanced level of tubulin glutamylation is required for specific B-tubule architecture of ciliary axoneme in two different cilia types (CEM and amphid channel). My serial TEM and tomography-based characterization of the complete CEM sensillum revealed that it contains two different populations of extracellular vesicles based on size and location. How are cilia specialized? My thesis work based on CEM cilia revealed cilia-specific axoneme microtubule architecture and its role in contributing to cilia-specific functions. TEM studies of CEM cilia revealed that cell-specific tubulin alpha tubulin isotype six is required for splayed doublet ultrastructure in CEM cilia. Posttranslational glutamylation regulates the architecture in both CEM and Amphid cilia via regulating the stability of B-tubules. Therefore mechanisms that regulate B-tubule architecture contribute to ciliary structural and functional specialization.Ph.D.Includes bibliographical referencesby Malan Sharanga Silv