Malan Silva

Doctoral Fellow

Research Summary

How to Specialize a Cilium: Microtubule Glutamylation Regulates Axonemal Ultrastructure

Cilia are found on most non-dividing cells in the human body and, are morphologically and functionally diverse subcellular organelles that are vital for human development and health.

All cilia are built upon a microtubule (MT) based cytoskeleton called an axoneme. Ciliary morphology can range from simple thread-shaped primary cilia to specialized retinal cilia such as rods and cones. Despite their diversity, cilia all are constructed by an evolutionarily conserved mechanism called intraflagellar transport (IFT). How IFT is modulated or built upon to generate cilia of elaborate morphology and functional specializations is not well understood. Because cilia are so ubiquitous, ciliopathies cause syndromic symptoms. However, it is clear that
particular ciliopathies affect only a subset of ciliated cells or tissues in the human body, suggesting that some ciliopathies affect the genes that specialize cilia form and/or function. However, the molecules that specialize cilia are still virtually completely unknown.

To understand ciliary specialization, I study sinusoid-shaped cilia of the cephalic male (CEM) sensory neurons in the nematode C. elegans. Unlike mammals, C. elegans do not require cilia for viability or development. Instead, C. elegans cilia function solely in a sensory capacity, making the worm an ideal model to study cilia. We used electron microscopy and tomography to compare axonemes of well-studied amphid cilia to CEM cilia and found significant ultrastructural differences between amphid and CEM cilia.

In amphid cilia, the axoneme is anchored to the ciliary membrane in the ciliary base or transition zone (TZ). The amphid TZ consists of nine MT doublets, which consist of a 13 protofilament A-tubule and 11 protofilament B-tubule, anchored to the membrane by ‘Y’ shaped structure call Y-links. The TZ immediately gives rise to the ‘middle segment’ in which nine MT doublets continue but are devoid of Y-links. The middle segment gives rise to the distal segment, which is comprised of nine MT singlets and projects to the tip of the cilium. These singlets are generated by continuing A-tubules of the MT doublets, with the B-tubule in MT doublet abruptly stopping at the end of the middle segment.

In contrast to amphid cilia, CEM cilia have a longer TZ and a shorter middle segment. In CEM cilia, nine MT doublets in the middle segment split into 18 MT singlets in the distal segment. In other words, both A- and B-tubules in middle segment doublets continue as separate singlets. This MT-doublet splitting is also observed in human spermatozoa, although the functional consequences are not known. Some of the distal MT singlets become fused at the distal tip of the CEM cilia, a feature that has been not described before in any ciliary types.

We found that ultrastructure of the CEM axoneme is modulated by the levels of a tubulin post-translational modification called polyglutamylation. CCPP-1 is a tubulin deglutamylase that we previously identified in a screen for defective PKD-2::GFP localization in CEM cilia [1]. In ccpp-1 mutants, B-tubules degenerate, suggesting that the B-tubules of CEM axonemes are sensitive to the levels of glutamylation [2]. We also looked at TTLL-11, a glutamate ligase that antagonizes CCPP-1 function in CEM neurons. In a ttll-11 mutant background, rather than splitting into singlets, the distal B-tubules in the CEM cilia are stabilized in continuing doublets. This suggests that glutamylation state regulates splitting of microtubule doublets into A- and B-like singlets in CEM cilia. Currently we are determining the mechanisms that regulate CEM axonemal specification.