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[
International Worm Meeting,
2011]
Cilia are cellular antennae; in humans, defects in ciliary form or function give rise to ciliopathies. C. elegans sensory organs, or sensilla, consist of morphologically and functionally diverse arrays of cilia that mediate sensation. The length and shape of cilia depends on axonemal microtubule (MT) architecture. In the amphid channel cilia, the ciliary axoneme is anchored to the membrane by a region known as the transition zone (TZ). In the TZ, MT doublets are made of a 13 protofilament A-tubule and an attached 11 protofilament B-tubule. MT doublets are anchored to the membrane with "Y" links of unknown composition. Beyond the TZ, amphid channel cilia display two distinct zones classified using MT architecture. The middle segment consists of nine MT doublets that lack 'Y' links. The distal segment is composed entirely of MT singlets.
Not all C. elegans cilia share this axonemal structure. For example, we have shown that cilia of the male cephalic neurons (CEMs) are composed of singlet MTs (1). We used high pressure freeze fixation and electron tomography to observe CEM cilia ultrastructure. By comparing cephalic and amphid channel cilia, we hope to understand at the molecular level how axonemes are specialized in form and function. We focused on transition from MT doublets to singlets and traced individual A- and B-tubules at the doublet-to-singlet transition. We found that the A- and B-tubules in middle segment doublets in CEM cilia separate into two sister singlets in the distal segment. These sister singlets are continuous to the distal segment of the cilium and spatially diverge from each other. Additionally we found that as compared to TZs of amphid neurons, CEM TZs span a longer distance. We are interested in determining how this spatial and structural disposition of the CEM MTs occurs, and how it affects ciliary motors. We are currently testing the hypothesis that tubulin post-translational modifications contribute to MT arrangement and stability in C. elegans sensory cilia (see Abstract by O'Hagan et al).
1. A. R. Jauregui, K. C. Q. Nguyen, D. H. Hall, M. M. Barr, The JCB 180, 973-988 (2008).
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[
International Worm Meeting,
2019]
The Tokuyasu method is room-temperature immuno-electron microscopy of cryosections. This technique is based on using sucrose as a cryoprotectant during both freezing and thawing steps [Tokuyasu 1973]. This method is optimized for preserving the antigenicity of the sample. Thus, the cocktails of heavy metals, organic solvents, and plastics that are used to stabilize, infiltrate, and stain the fine ultrastructure in plastic-section microscopy are not used. As a result, the inherent nature of the sample is minimally altered. However, compared to plastic sections, preservation of fine ultrastructure in tokuyasu sections remains inadequate and inconsistent. Despite attempts to optimize by several groups, the morphology of neurons obtained with this method remains poor still. Here we show that this method could be modified to preserve the C. elegans ventral nerve cord (VNC) at an unprecedented level of structural detail while preserving antigenicity. Osmolality driven structural collapse is a significant drawback of using sucrose in the vitrification step. When a sample block is immersed in sucrose during the process of infusion, sucrose-driven collapse of cellular turgor pressure result in deformation of cellular structures that are prone to collapse. Such tissue is the ventral nerve cord (VNC) of C. elegans. When sucrose is used, neuronal profiles of the VNC collapse inwardly, and their plasma membranes appear jagged. The critical difference between our method and previously published ones [Tokuyasu 1973, Liou et al. 1996, Bos et al. 2004, Nicolle et al. 2015], is the lack of sucrose. Removing sucrose from the sample vitrification step prevented the sucrose driven collapse of cellular turgor pressure and thus preserved the cellular structural form. This modification also necessitated compensatory changes in sample embedding, orienting and freezing steps to maintain ease of cryosectioning and sample vitrification due to lack of sucrose. To address these challenges we used high-pressure freezing to cryo-immobilize C. elegans in an enhanced copper tube. This sample carrier enables robust loading, grouping, orienting, freezing, and downstream processing to generate ribbons of hydrated cryosections of gently fixed or unfixed worms. We plan to use the Tokuyasu method of thawing cryosections as a screening step before correlative cryo fluorescence microscopy and cryo-electron tomography of C. elegans ventral nerve cord.
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[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.
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[
Dev Cell,
2014]
HORMA domain proteins are required for the careful orchestration of chromosomal organization during meiosis. Kim et al. (2014) and Silva et al. (2014) now provide structural and functional insights into the roles of C. elegans HORMA proteins, revealing parallels to the function of the HORMA protein MAD2 in mitotic checkpoint signaling.
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[
International Worm Meeting,
2009]
Tubes are a central architectural feature of many human tissues including glands, the urogenital and gastrointestinal tracts, the respiratory tract and the vascular system. Despite their importance, much remains unknown about the molecular mechanisms involved in tube development. This work explores a new role for tropomodulins in regulation of endothelial tube formation. Tropomodulins are one of the few proteins known to regulate the slow growing ends of actin filaments. The C. elegans
tmd-1 gene encodes two transcripts (
tmd-1a and
tmd-1b) that have similar domain structure to mammalian tropomodulins. We have been examining the role of TMD-1 in development of the C. elegans intestine, which is a relatively simple, transparent tube that forms by a cord-hollowing mechanism similar to that used by some capillaries and renal tubules. Antibody staining using a rabbit anti-TMD-1 antibody demonstrates that TMD-1 localizes to the luminal surface of the C. elegans intestine as it undergoes morphogenesis. Interestingly, embryos homozygous for the
tmd-1(
tm724) allele, which contains a large deletion that affects both transcripts1, exhibit areas of intestinal lumen diameter that are 65% larger than wild-type.
tmd-1(
sf20) homozygous mutants, which have a premature stop codon affecting both transcripts2, also exhibit luminal expansions (50% larger than wild-type). Currently, investigations are underway to determine the molecular mechanisms by which TMD-1 regulates intestinal lumen diameter. Possibilities include modulation of actomyosin contractility, cooperation with the actin-spectrin cytoskeleton to provide mechanical strength to the luminal surface, and/or regulation of vesicle trafficking. This work is supported by National Institutes of Health grant R15HD059952. 1 Yamashiro et al. (2008) J. Cell Sci. 121: 3867-77. 2 Stevensen et al. (2007) J. Mol. Bio. 374: 936-50.
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Hall, David, Wang, Juan, Barr, Maureen, Silva, Malan, Akella, Jyothi, Maguire, Julie
[
International Worm Meeting,
2015]
Extracellular vesicles (EVs) are membrane bound vesicles released by most cells in the body. EVs aid the exchange of cargo such as proteins, lipids, and nucleic acids between cells without requiring direct contact. EVs are proposed to play important roles in the nervous system in vitro. Under healthy conditions, EVs are neuroprotective but, may propagate and promote neurodegeneration under conditions such as injury and infection. The functions of EVs, and the factors that affect EV dynamics and composition in vivo are unknown. A subset of ciliated neurons of C. elegans release GFP-tagged EVs containing select cargo into the environment. We use the environmentally released EVs of C.elegans as a model to identify the components and conditions that affect EV dynamics in vivo i.e. cause a change in total EV content and EV composition. Our strategy includes the identification of genes and mechanisms that regulate EV biogenesis and release under normal conditions, as well as determining the functions of EVs.Using in vivo imaging of fluorescently tagged EV cargo and transmission electron microscopy, we identified proteins that regulate EV biogenesis and release including a kinesin and a myristoylated novel protein. We previously identified that purified EVs from C.elegans trigger male tail chasing behavior, which is the first example of EVs mediating animal-animal communication (Wang et al; 2014). We also found that C.elegans EVs are bactericidal. Our future studies are aimed at identifying the components important for bactericidal activity and for identifying conditions that affect the bactericidal properties of EVs. Furthermore, we will determine whether EVs purified from mutants of the known regulators of EV biogenesis and release demonstrate differences in behavioral and bactericidal assays. Our studies are expected to provide insights into the factors that regulate EV biogenesis and release, and identify factors that affect the composition of EVs under normal conditions, and under other environmental stress. This knowledge is important for our understanding of the functions of EVs in health and disease, and the factors that modulate EV properties in disease. .
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Hall, David, Morash, Margaret, Barr, Maureen, Nguyen, Ken, Bellotti, Sebastian, Silva, Malan, O'Hagan, Robert
[
International Worm Meeting,
2015]
Post-translational modifications (PTMs) added to microtubules (MTs) may act as a "Tubulin Code" that guides the activities of kinesins, dyneins, and other MT-binding proteins. Ciliary MTs are highly decorated with PTMs, but the functions of PTMs in cilia are largely unknown. We previously showed that loss of CCPP-1, a predicted MT deglutamylase, caused defective localization of the ciliary receptor PKD-2 and the kinesin-3 motor KLP-6, and abnormally fast movement of the kinesin 2 OSM-3, in male-specific B-type neuronal cilia. In amphid channel cilia,
ccpp-1 mutants displayed a progressive Dyf phenotype and deterioration of MT structure. Loss of a CCPP-1 homolog in mice also affects ciliated cells, causing degeneration of cerebellar Purkinje neurons, olfactory mitral cells, and retinal photoreceptors, and also causes sperm immotility. Therefore, MT glutamylation may play a conserved role in cilia and flagella. Here we show that select Tubulin Tyrosine Ligase-Like (TTLL) MT glutamylases oppose the activity of CCPP-1. Mutations in
ttll-4,
ttll-5, or
ttll-11 suppressed the
ccpp-1 progressive Dyf phenotype, but did not suppress PKD-2 ciliary localization defects. The
ttll-11 mutation suppressed
ccpp-1 effects on kinesin-3 KLP-6 localization and kinesin-2 OSM-3 velocity. Ciliary MT structure typically contains doublets composed of A- and B- tubules. Ultrastructural analysis revealed loss of ciliary B-tubules in
ccpp-1, while
ttll-11 mutants displayed abnormally long ciliary doublet regions. MT glutamylation reduced B-tubule stability in both neuronal types, despite differences in structural details. We hypothesize that glutamylation targets MTs for degradation by MT-severing enzymes. To elucidate the pathways by which glutamylation controls ciliary MT stability, we performed a screen for suppressors of the
ccpp-1 Dyf phenotype. Our screen should identify molecules upstream and downstream of MT glutamylation. Our data suggest that, in cilia, MT glutamylation is part of a Tubulin Code that regulates ciliary transport of molecular motors and sensory receptors and controls axonemal structure.
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Swoboda, Peter, Hall, David H., Barr, Maureen M., Piasecki, Brian P., Silva, Malan, Nguyen, Ken C.Q., Phirke, Prasad, O'Hagan, Robert
[
International Worm Meeting,
2011]
When assembled into microtubules (MTs), tubulins have protruding C-terminal tails that accumulate post-translational modifications (PTMs) such as detyrosination and polyglutamylation. PTMs have long been known to mark stable MTs. Although their cellular function and significance are not yet clear, PTMs have been proposed to act as signposts that guide various kinesin and dynein motors to transport their cargoes to specific destinations. For example, trafficking of KIF5C, a mouse kinesin-1 motor, is affected by detyrosination, and KIF1A, a mouse kinesin-3, is affected by polyglutamylation. In cilia, axonemal MTs are especially prone to PTMs, which we propose regulate MT stability as well as the function of motors that drive intraflagellar transport. We have found that perturbation of a PTM profoundly affects cilia in sensory neurons in C. elegans.
We identified a mutation in C. elegans that affects
ccpp-1, which encodes a cytosolic carboxypeptidase that deglutamylates microtubules in a subset of neuronal sensory cilia. Loss of CCPP-1 function causes defective localization and abundance of the polycystin PKD-2 and a kinesin-3, KLP-6, in male-specific neurons. Loss of CCPP-1 also causes increased velocity of motile kinesin-2 OSM-3::GFP puncta in the cilia of male-specific neurons. Loss of CCPP-1 had cell-specific effects on the level of polyglutamylation detected by a monoclonal antibody. Mutations in
ccpp-1 cause defective dye-filling of sensory neurons that becomes progressively more severe with age, suggesting that sensory neurons construct cilia that degenerate over time. Ultrastructural analysis of mutants showed that some cilia had defective MT structures and some cilia were fragmented or absent.
Others have shown that loss of CCP1, a homolog of CCPP-1, in mice causes degeneration of brain neurons such as Purkinje neurons in the cerebellum, olfactory mitral cells, and retinal photoreceptors, and also causes sperm immotility. Although all affected cells are ciliated, the impact of loss of CCP1 on cilia has not been explored in mutant mice. We propose that neurodegeneration caused by loss of CCP1 in mammals may represent a novel ciliopathy in which cilia are formed normally but degenerate, depriving the cell of cilia-based signal transduction that is necessary for maintenance of neurons.
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de Bono, Mario, Amin-Wetzel, Niko, Sengupta, Piali, Philbrook, Alison, Kazatskaya, Anna, Yuan, Lisa
[
MicroPubl Biol,
2020]
A subset of sensory neurons in C. elegans contains compartmentalized sensory structures termed cilia at their distal dendritic ends (Ward et al. 1975; Perkins et al. 1986; Doroquez et al. 2014). Cilia present on different sensory neuron types are specialized both in morphology and function, and are generated and maintained via shared and cell-specific molecules and mechanisms (Perkins et al. 1986; Evans et al. 2006; Mukhopadhyay et al. 2007; Mukhopadhyay et al. 2008; Morsci and Barr 2011; Doroquez et al. 2014; Silva et al. 2017). The bilaterally symmetric pair of URX oxygen-sensing neurons in the C. elegans head (Figure 1A) is thought to be non-ciliated (Ward et al. 1975; Doroquez et al. 2014) but nevertheless exhibits intriguing morphological similarities with ciliated sensory neurons. URX dendrites extend to the nose where they terminate in large bulb-like complex structures (Ward et al. 1975; Doroquez et al. 2014; Cebul et al. 2020) (Figure 1A). These structures concentrate oxygen-sensing signaling molecules (Gross et al. 2014; Mclachlan et al. 2018) suggesting that similar to cilia, these structures are specialized for sensory functions. Microtubule growth events similar to those observed in ciliated sensory neurons were also reported at the distal dendritic regions of URX, implying the presence of a microtubule organizer such as a remodeled basal body (Harterink et al. 2018). Moreover, a subset of ciliary genes is expressed in URX (Kunitomo et al. 2005; Harterink et al. 2018; Mclachlan et al. 2018). We tested the hypothesis that URX dendrites contain cilia at their distal ends.
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[
International C. elegans Meeting,
1991]
G proteins are a class of guanine nucleotide binding proteins that act in transmembrane signal transduction by physically coupling cell surface receptors to effector proteins. They comprise three subunits: alpha, , and gamma. The a subunit undergoes a guanine nucleotide exchange and hydrolysis cycle and, in most cells, interacts with effector molecules. G protein a subunits are members of a large family of proteins. For example, 16 G a subunit genes have been cloned from the mouse. Using PCR-based methodology, we have cloned five G a subunit genes from C. elegans. Two of them, goa-l and gqa-l are homologous to mammalian and Drosophila Galpha(o) and Galpha(q) respectively. The remaining three, gpa-l,
gpa-2, and
gpa-3, appear to be novel to C. elegans. Fino Silva and Plasterck [JMB 215, 483 (1990)], have also described the
gpa-2 gene. We have fused the 5' flanking regions of goa-l and gpa-l to the E. coli lacZ gene using Andy Fire's modular vectors [Gene 93, 189 (1990)]. The staining pattern for the goa-l-lacZ construct indicates that goa-l is expressed in a large number of neurons, both in the circumpharyngeal nerve ring and the ventral cord. This apparent expression pattern is consistent with the observation that GaO is abundant in neural tissue in other organisms. The staining pattern for the gpa-l-lacZ construct indicates that gpa-l is expressed in a small number of cells in the pharynx, four cells in the circumpharyngeal nerve ring, and what appears to be one of the phasmid neurons. In males, staining is also seen in at least one of the spicule neurons.