-
[
EMBO Rep,
2017]
Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long-range transport processes <i>invivo</i> In all organisms studied so far, the kinesin-2 family is essential for long-range transport on axonemes. Intriguingly, in higher eukaryotes, kinesin-2 has been adapted to work on microtubules in the cytoplasm as well. Here, we show that heterodimeric kinesin-2 motors distinguish between axonemes and microtubules. Unlike canonical kinesin-1, kinesin-2 takes directional, off-axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of kinesin-2 to side-track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed here provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A- and B-tubules of the same doublet microtubule. Our results offer first mechanistic insights into why nature may have co-evolved the heterodimeric kinesin-2 with the ciliary machinery to work on the specialized axonemal surface for two-way traffic.
-
[
Proc Natl Acad Sci U S A,
2022]
Specific recognition of cellular cargo and efficient transport to its correct intracellular destination is an infrastructural challenge faced by most eukaryotic cells. This remarkable deed is accomplished by processive motor proteins that are subject to robust regulatory mechanisms. The first level of regulation entails the ability of the motor to suppress its own activity. This autoinhibition is eventually relieved by specific cargo binding. To better understand the role of the cargo during motor activation, we dissected the activation mechanism of the ciliary homodimeric kinesin-2 from Caenorhabditis elegans by its physiological cargo. In functional reconstitution assays, we identified two cargo adaptor proteins that together are necessary and sufficient to allosterically activate the autoinhibited motor. Surprisingly, the orthologous adaptor proteins from the unicellular green algae Chlamydomonas reinhardtii also fully activated the kinesin-2 from worm, even though C. reinhardtii itself lacks a homodimeric kinesin-2 motor. The latter suggested that a motor activation mechanism similar to the C. elegans model existed already well before metazoans evolved, and prompted us to scrutinize predicted homodimeric kinesin-2 orthologs in other evolutionarily distant eukaryotes. We show that the ciliate Tetrahymena thermophila not only possesses a homodimeric kinesin-2 but that it also shares the same allosteric activation mechanism that we delineated in the C. elegans model. Our results point to a much more fundamental role of homodimeric kinesin-2 in intraflagellar transport (IFT) than previously thought and warrant further scrutiny of distantly related organisms toward a comprehensive picture of the IFT process and its evolution.
-
[
Cell Calcium,
2023]
Mueller et al. [1] uncover distinct roles for CaV1 and CaV2 channels in neurotransmitter release at the C. elegans neuromuscular junction. Although nanodomain coupling occurs via clustered CaV2 channels, evidence is also presented that release of a separate vesicular pool is mediated by more peripheral, dispersed CaV1 channels, requiring obligatory coupling with RYR to amplify the Ca2+ signal.
-
[
Parasitol Today,
1988]
Ivermectin is a semi-synthetic macrocyclic lactone (Fig. I) active in single low doses against many parasites - particularly nematodes and arthropods. It has been registered for animal health use since early 1985, and was earlier this year approved for human use by the French Directorate o f Pharmacy and Drugs. Of particular interest is ivermectin's potential as a micro filaricide for treatment o f onchocerciasis. Clinical trials leave little doubt about the potential o f ivermectin as a therapeutic tool for symptomatic relief from the effects o f infection with Onchocerca volvulus, and the drug is also recognized to have potential in reducing transmission o f the parasite. The manufacturers (Merck, Sharp and Dohme) recently arranged to provide the drug free o f charge to the WHO for mass trials against onchocerciasis in 12 African and Central American countries. In this article we focus on the pharmacological properties o f ivermectin, with a brief consideration of its absorption, fate, excretion and side-effects, and a discussion o f its micro filaricidal action.
-
[
Proc Natl Acad Sci U S A,
2010]
Cilia are microtubule-based protrusions of the plasma membrane found on most eukaryotic cells. Their assembly is mediated through the conserved intraflagellar transport mechanism. One class of motor proteins involved in intraflagellar transport, kinesin-2, is unique among kinesin motors in that some of its members are composed of two distinct polypeptides. However, the biological reason for heterodimerization has remained elusive. Here we provide several interdependent reasons for the heterodimerization of the kinesin-2 motor KLP11/KLP20 of Caenorhabditis elegans cilia. One motor domain is unprocessive as a homodimer, but heterodimerization with a processive partner generates processivity. The "unprocessive" subunit is kept in this partnership as it mediates an asymmetric autoregulation of the motor activity. Finally, heterodimerization is necessary to bind KAP1, the in vivo link between motor and cargo.
-
[
Cell,
2013]
The UCS (UNC-45/CRO1/She4) chaperones play an evolutionarily conserved role in promoting myosin-dependent processes, including cytokinesis, endocytosis, RNA transport, and muscle development. To investigate the protein machinery orchestrating myosin folding and assembly, we performed a comprehensive analysis of Caenorhabditis elegans UNC-45. Our structural and biochemical data demonstrate that UNC-45 forms linear protein chains that offer multiple binding sites for cooperating chaperones and client proteins. Accordingly, Hsp70 and Hsp90, which bind to the TPR domain of UNC-45, could act in concert and with defined periodicity on captured myosin molecules. In vivo analyses reveal the elongated canyon of the UCS domain as a myosin-binding site and show that multimeric UNC-45 chains support organization of sarcomeric repeats. In fact, expression of transgenes blocking UNC-45 chain formation induces dominant-negative defects in the sarcomere structure and function of wild-type worms. Together, these findings uncover a filament assembly factor that directly couples myosin folding with myofilament formation.
-
[
Proc Natl Acad Sci U S A,
2010]
The ternary complex of cadherin, beta-catenin, and alpha-catenin regulates actin-dependent cell-cell adhesion. alpha-Catenin can bind beta-catenin and F-actin, but in mammals alpha-catenin either binds beta-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of alpha-catenin is evolutionarily conserved. The Caenorhabditis elegans alpha-catenin homolog HMP-1 is essential for actin-dependent epidermal enclosure and embryo elongation. Here we show that HMP-1 is a monomer with a functional C-terminal F-actin binding domain. However, neither full-length HMP-1 nor a ternary complex of HMP-1-HMP-2(beta-catenin)-HMR-1(cadherin) bind F-actin in vitro, suggesting that HMP-1 is auto-inhibited. Truncation of either the F-actin or HMP-2 binding domain of HMP-1 disrupts C. elegans development, indicating that HMP-1 must be able to bind F-actin and HMP-2 to function in vivo. Our study defines evolutionarily conserved properties of alpha-catenin and suggests that multiple mechanisms regulate alpha-catenin binding to F-actin.
-
[
BMC Genomics,
2021]
Background: F-box proteins represent a diverse class of adaptor proteins of the ubiquitin-proteasome system (UPS) that play critical roles in the cell cycle, signal transduction, and immune response by removing or modifying cellular regulators. Among closely related organisms of the Caenorhabditis genus, remarkable divergence in F-box gene copy numbers was caused by sizeable species-specific expansion and contraction. Although F-box gene number expansion plays a vital role in shaping genomic diversity, little is known about molecular evolutionary mechanisms responsible for substantial differences in gene number of F-box genes and their functional diversification in Caenorhabditis. Here, we performed a comprehensive evolution and underlying mechanism analysis of F-box genes in five species of Caenorhabditis genus, including C. brenneri, C. briggsae, C. elegans, C. japonica, and C. remanei.Results: Herein, we identified and characterized 594, 192, 377, 39, 1426 F-box homologs encoding putative F-box proteins in the genome of C. brenneri, C. briggsae, C. elegans, C. japonica, and C. remanei, respectively. Our work suggested that extensive species-specific tandem duplication followed by a small amount of gene loss was the primary mechanism responsible for F-box gene number divergence in Caenorhabditis genus. After F-box gene duplication events occurred, multiple mechanisms have contributed to gene structure divergence, including exon/intron gain/loss, exonization/pseudoexonization, exon/intron boundaries alteration, exon splits, and intron elongation by tandem repeats. Based on high-throughput RNA sequencing data analysis, we proposed that F-box gene functions have diversified by sub-functionalization through highly divergent stage-specific expression patterns in Caenorhabditis species.Conclusions: Massive species-specific tandem duplications and occasional gene loss drove the rapid evolution of the F-box gene family in Caenorhabditis, leading to complex gene structural variation and diversified functions affecting growth and development within and among Caenorhabditis species. In summary, our findings outline the evolution of F-box genes in the Caenorhabditis genome and lay the foundation for future functional studies.
-
[
J Biol Chem,
2001]
Actin depolymerizing factor (ADF)/cofilin changes the twist of actin filaments by binding two longitudinally associated actin subunits, In the absence of an atomic model of the ADF/cofilin-F-actin complex, we have identified residues in ADF/cofilin that are essential for filament binding. Here, we have characterized the C-terminal tail of UNC-60B (a nematode ADF/cofilin isoform) as a novel determinant for its association with F-actin, Removal of the C-terminal isoleucine (Ile(152)) by carboxypeptidase A or truncation by mutagenesis eliminated F-actin binding activity but strongly enhanced actin depolymerizing activity, Replacement of Ile(152) by Ala had a similar but less marked effect; F-actin binding was weakened and depolymerizing activity slightly enhanced. Truncation of both Arg(151) and Ile(152) or replacement of Arg(151) with Ala also abolished F-actin binding and enhanced depolymerizing activity. Loss of F-actin binding in these mutants was accompanied by loss or greatly decreased severing activity. All of the variants of UNC-60B interacted with G-actin in an indistinguishable manner from wild type. Cryoelectron microscopy showed that UNC-60B changed the twist of F-actin to a similar extent to vertebrate ADF/cofilins. Helical reconstruction and structural modeling of UNC-60B-F-actin complex reveal how the C terminus of UNC-60B might be involved in one of the two actin-binding sites.
-
Ausubel FM, Nau GJ, Kim W, Mylonakis E, Coleman JJ, Okoli I, Lee K, RajaMuthiah R, Tharmalingam N, Hernandez AM, Jayamani E
[
Antimicrob Agents Chemother,
2017]
Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole animal Caenorhabditis elegans-F. tularensis pathosystem for high throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans
p38 MAP kinase cascade is involved in the immune response to F. tularensis and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z'-factor consistently >0.5, which was then utilized to screen a library of FDA approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, 5 FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the NSAID diflunisal, at 4X MIC, blocked the replication of a F. tularensis live vaccine strain (LVS) in primary human macrophages and non-phagocytic cells. Diflunisal was non-toxic to human erythrocytes and HepG2 human liver cells at concentrations 32-g/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans - F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.