The Caenorhabditis elegans nephrocystins act as global modifiers of cilium structure

Nephronophthisis (NPHP) is the most common genetic cause of end-stage renal disease in children and young adults. In Chlamydomonas reinhardtii, Caenorhabditis elegans, and mammals, the NPHP1 and NPHP4 gene products nephrocystin-1 and nephrocystin-4 localize to basal bodies or ciliary transition zones (TZs), but their function in this location remains unknown. We show here that loss of C. elegans NPHP-1 and NPHP-4 from TZs is tolerated in developing cilia but causes changes in localization of specific ciliary components and a broad range of subtle axonemal ultrastructural defects. In amphid channel cilia, nphp-4 mutations cause B tubule defects that further disrupt intraflagellar transport (IFT). We propose that NPHP-1 and NPHP-4 act globally at the TZ to regulate ciliary access of the IFT machinery, axonemal structural components, and signaling molecules, and that perturbing this balance results in cell type–specific phenotypes

Transorganogenesis and transdifferentiation in C. elegans are dependent on differentiated cell identity

AbstractThe differentiated cell identities and structure of fully formed organs are generally stable after their development. In contrast, we report here that development of the C. elegans proximal somatic gonad (hermaphrodite uterus and spermathecae, and male vas deferens) can be redirected into intestine-like organs by brief expression of the ELT-7 GATA transcription factor. This process converts one developing organ into another and can hence be considered “transorganogenesis.” We show that, following pulsed ELT-7 expression, cells of the uterus activate and maintain intestine-specific gene expression and are transformed at the ultrastructural level to form an epithelial tube resembling the normal intestine formed during embryogenesis. Ubiquitous ELT-7 expression activates intestinal markers in many different cell types but only cells in the somatic gonad and pharynx appear to become fully reprogrammed. We found that ectopic expression of other endoderm-promoting transcription factors, but not muscle- or ectoderm- promoting transcription factors, redirects the fate of these organs, suggesting that pharyngeal and somatic gonad cells are specifically competent to adopt intestine identity. Although the intestine, pharynx, and somatic gonad are derived from distant cell lineages, they all express the PHA-4/FoxA transcription factor. While we found that post-embryonic PHA-4 is not necessary for pharynx or uterus reprogramming and PHA-4 is not sufficient in combination with ELT-7 to induce reprogramming in other cells types, knock down of PHA-4 during embryogenesis, which abolishes normal pharynx differentiation, prevents pharyngeal precursors from being reprogrammed into intestine. These results suggest that differentiated cell identity determines susceptibility to transdifferentiation and highlight the importance of cellular context in controlling competency for reprogramming

The Tubulin Deglutamylase CCPP-1 Regulates the Function and Stability of Sensory Cilia in C. elegans

SummaryBackgroundPosttranslational modifications (PTMs) such as acetylation, detyrosination, and polyglutamylation have long been considered markers of stable microtubules and have recently been proposed to guide molecular motors to specific subcellular destinations. Microtubules can be deglutamylated by the cytosolic carboxypeptidase CCP1. Loss of CCP1 in mice causes cerebellar Purkinje cell degeneration. Cilia, which are conserved organelles that play important diverse roles in animal development and sensation, contain axonemes comprising microtubules that are especially prone to PTMs.ResultsHere, we report that a CCP1 homolog, CCPP-1, regulates the ciliary localization of the kinesin-3 KLP-6 and the polycystin PKD-2 in male-specific sensory neurons in C. elegans. In male-specific CEM (cephalic sensilla, male) cilia, ccpp-1 also controls the velocity of the kinesin-2 OSM-3/KIF17 without affecting the transport of kinesin-II cargo. In the core ciliated nervous system of both males and hermaphrodites, loss of ccpp-1 causes progressive defects in amphid and phasmid sensory cilia, suggesting that CCPP-1 activity is required for ciliary maintenance but not ciliogenesis. Affected cilia exhibit defective B-tubules. Loss of TTLL-4, a polyglutamylating enzyme of the tubulin tyrosine ligase-like family, suppresses progressive ciliary defects in ccpp-1 mutants.ConclusionsOur studies suggest that CCPP-1 acts as a tubulin deglutamylase that regulates the localization and velocity of kinesin motors and the structural integrity of microtubules in sensory cilia of a multicellular, living animal. We propose that the neuronal degeneration caused by loss of CCP1 in mammals may represent a novel ciliopathy in which cilia are formed but not maintained, depriving the cell of cilia-based signal transduction

Glutamylation Regulates Transport, Specializes Function, and Sculpts the Structure of Cilia

Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs), such as glutamylation of tubulin tails. PTMs and tubulin isotype diversity act as a “tubulin code” that regulates cytoskeletal stability and the activity of MT-associated proteins such as kinesins. We previously showed that, in C. elegans cilia, the deglutamylase CCPP-1 affects ciliary ultrastructure, localization of the TRP channel PKD-2 and the kinesin-3 KLP-6, and velocity of the kinesin-2 OSM-3/KIF17, whereas a cell-specific α-tubulin isotype regulates ciliary ultrastructure, intraflagellar transport, and ciliary functions of extracellular vesicle (EV)-releasing neurons. Here we examine the role of PTMs and the tubulin code in the ciliary specialization of EV-releasing neurons using genetics, fluorescence microscopy, kymography, electron microscopy, and sensory behavioral assays. Although the C. elegans genome encodes five tubulin tyrosine ligase-like (TTLL) glutamylases, only ttll-11 specifically regulates PKD-2 localization in EV-releasing neurons. In EV-releasing cephalic male (CEM) cilia, TTLL-11 and the deglutamylase CCPP-1 regulate remodeling of 9+0 MT doublets into 18 singlet MTs. Balanced TTLL-11 and CCPP-1 activity fine-tunes glutamylation to control the velocity of the kinesin-2 OSM-3/KIF17 and kinesin-3 KLP-6 without affecting the intraflagellar transport (IFT) kinesin-II. TTLL-11 is transported by ciliary motors. TTLL-11 and CCPP-1 are also required for the ciliary function of releasing bioactive EVs, and TTLL-11 is itself a novel EV cargo. Therefore, MT glutamylation, as part of the tubulin code, controls ciliary specialization, ciliary motor-based transport, and ciliary EV release in a living animal. We suggest that cell-specific control of MT glutamylation may be a conserved mechanism to specialize the form and function of cilia. O\u27Hagan et al. report that fine-tuning of microtubule glutamylation by the glutamylase TTLL-11 and the deglutamylase CCPP-1 regulates ciliary function by controlling ciliary receptor localization, the velocity of particular kinesin-2 and kinesin-3 motors, and the release of extracellular vesicles and sculpting a specialized axonemal ultrastructure

The Atg6/Vps30/Beclin 1 ortholog BEC-1 mediates endocytic retrograde transport in addition to autophagy in C. elegans

Autophagy and endocytosis are dynamic and tightly regulated processes that contribute to many fundamental aspects of biology including survival, longevity and development. However, the molecular links between autophagy and endocytosis are not well understood. Here, we report that BEC-1, the C. elegans ortholog of Atg6/Vps30/Beclin 1, a key regulator of the autophagic machinery, also contributes to endosome function. In particular we identified a defect in retrograde transport from endosomes to the Golgi in bec-1 mutants. MIG-14/Wntless is normally recycled from endosomes to the Golgi through the action of the retromer complex and its associated factor RME-8. Lack of retromer or RME-8 activity results in the aberrant transport of MIG-14/Wntless to the lysosome where it is degraded. Similarly, we found that lack of bec-1 also results in mislocalization and degradation of MIG-14::GFP, reduced levels of RME-8 on endosomal membranes, and the accumulation of morphologically abnormal endosomes. A similar phenotype was observed in animals treated with dsRNA against vps-34. We further identified a requirement for BEC-1 in the clearance of apoptotic corpses in the hermaphrodite gonad, suggesting a role for BEC-1 in phagosome maturation, a process that appears to depend upon retrograde transport. In addition, autophagy genes may also be required for cell corpse clearance, as we found that RNAi against atg-18 or unc-51 also results in a lack of cell corpse clearance

Genetically separable functions of the MEC-17 tubulin acetyltransferase affect microtubule organization

Background: Microtubules (MTs) are formed from the lateral association of 11-16 protofilament chains of tubulin dimers, with most cells containing 13-protofilament (13-p) MTs. How these different MTs are formed is unknown, although the number of protofilaments may depend on the nature of the \u3b1- and \u3b2-tubulins. Results: Here we show that the enzymatic activity of the Caenorhabiditis elegans \u3b1-tubulin acetyltransferase (\u3b1-TAT) MEC-17 allows the production of 15-p MTs in the touch receptor neurons (TRNs) MTs. Without MEC-17, MTs with between 11 and 15 protofilaments are seen. Loss of this enzymatic activity also changes the number and organization of the TRN MTs and affects TRN axonal morphology. In contrast, enzymatically inactive MEC-17 is sufficient for touch sensitivity and proper process outgrowth without correcting the MT defects. Thus, in addition to demonstrating that MEC-17 is required for MT structure and organization, our results suggest that the large number of 15-p MTs, normally found in the TRNs, is not essential for mechanosensation. Conclusion: These experiments reveal a specific role for \u3b1-TAT in the formation of MTs and in the production of higher order MTs arrays. In addition, our results indicate that the \u3b1-TAT protein has functions that require acetyltransferase activity (such as the determination of protofilament number) and others that do not (presence of internal MT structures). \ua9 2012 Elsevier Ltd All rights reserved

Genetic Control of Fusion Pore Expansion in the Epidermis of Caenorhabditis elegans

Developmental cell fusion is found in germlines, muscles, bones, placentae, and stem cells. In Caenorhabditis elegans 300 somatic cells fuse during development. Although there is extensive information on the early intermediates of viral-induced and intracellular membrane fusion, little is known about late stages in membrane fusion. To dissect the pathway of cell fusion in C. elegans embryos, we use genetic and kinetic analyses using live-confocal and electron microscopy. We simultaneously monitor the rates of multiple cell fusions in developing embryos and find kinetically distinct stages of initiation and completion of membrane fusion in the epidermis. The stages of cell fusion are differentially blocked or retarded in eff-1 and idf-1 mutants. We generate kinetic cell fusion maps for embryos grown at different temperatures. Different sides of the same cell differ in their fusogenicity: the left and right membrane domains are fusion-incompetent, whereas the anterior and posterior membrane domains fuse with autonomous kinetics in embryos. All but one cell pair can initiate the formation of the largest syncytium. The first cell fusion does not trigger a wave of orderly fusions in either direction. Ultrastructural studies show that epidermal syncytiogenesis require eff-1 activities to initiate and expand membrane merger