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Applied Microbiology and Biotechnology May 2023Campylobacter jejuni, causing strong enteritis, is an unusual bacterium with numerous peculiarities. Chemotactically controlled motility in viscous milieu allows... (Review)
Review
Campylobacter jejuni, causing strong enteritis, is an unusual bacterium with numerous peculiarities. Chemotactically controlled motility in viscous milieu allows targeted navigation to intestinal mucus and colonization. By phase variation, quorum sensing, extensive O-and N-glycosylation and use of the flagellum as type-3-secretion system C. jejuni adapts effectively to environmental conditions. C. jejuni utilizes proteases to open cell-cell junctions and subsequently transmigrates paracellularly. Fibronectin at the basolateral side of polarized epithelial cells serves as binding site for adhesins CadF and FlpA, leading to intracellular signaling, which again triggers membrane ruffling and reduced host cell migration by focal adhesion. Cell contacts of C. jejuni results in its secretion of invasion antigens, which induce membrane ruffling by paxillin-independent pathway. In addition to fibronectin-binding proteins, other adhesins with other target structures and lectins and their corresponding sugar structures are involved in host-pathogen interaction. Invasion into the intestinal epithelial cell depends on host cell structures. Fibronectin, clathrin, and dynein influence cytoskeletal restructuring, endocytosis, and vesicular transport, through different mechanisms. C. jejuni can persist over a 72-h period in the cell. Campylobacter-containing vacuoles, avoid fusion with lysosomes and enter the perinuclear space via dynein, inducing signaling pathways. Secretion of cytolethal distending toxin directs the cell into programmed cell death, including the pyroptotic release of proinflammatory substances from the destroyed cell compartments. The immune system reacts with an inflammatory cascade by participation of numerous immune cells. The development of autoantibodies, directed not only against lipooligosaccharides, but also against endogenous gangliosides, triggers autoimmune diseases. Lesions of the epithelium result in loss of electrolytes, water, and blood, leading to diarrhea, which flushes out mucus containing C. jejuni. Together with the response of the immune system, this limits infection time. Based on the structural interactions between host cell and bacterium, the numerous virulence mechanisms, signaling, and effects that characterize the infection process of C. jejuni, a wide variety of targets for attenuation of the pathogen can be characterized. The review summarizes strategies of C. jejuni for host-pathogen interaction and should stimulate innovative research towards improved definition of targets for future drug development. KEY POINTS: • Bacterial adhesion of Campylobacter to host cells and invasion into host cells are strictly coordinated processes, which can serve as targets to prevent infection. • Reaction and signalling of host cell depend on the cell type. • Campylobacter virulence factors can be used as targets for development of antivirulence drug compounds.
Topics: Humans; Bacterial Proteins; Campylobacter jejuni; Fibronectins; Dyneins; Virulence Factors; Adhesins, Bacterial; Epithelial Cells; Bacterial Adhesion; Campylobacter Infections
PubMed: 36941439
DOI: 10.1007/s00253-023-12456-w -
Cell Dec 2022Intraflagellar transport (IFT) trains are massive molecular machines that traffic proteins between cilia and the cell body. Each IFT train is a dynamic polymer of two...
Intraflagellar transport (IFT) trains are massive molecular machines that traffic proteins between cilia and the cell body. Each IFT train is a dynamic polymer of two large complexes (IFT-A and -B) and motor proteins, posing a formidable challenge to mechanistic understanding. Here, we reconstituted the complete human IFT-A complex and obtained its structure using cryo-EM. Combined with AlphaFold prediction and genome-editing studies, our results illuminate how IFT-A polymerizes, interacts with IFT-B, and uses an array of β-propeller and TPR domains to create "carriages" of the IFT train that engage TULP adaptor proteins. We show that IFT-A⋅TULP carriages are essential for cilia localization of diverse membrane proteins, as well as ICK-the key kinase regulating IFT train turnaround. These data establish a structural link between IFT-A's distinct functions, provide a blueprint for IFT-A in the train, and shed light on how IFT evolved from a proto-coatomer ancestor.
Topics: Humans; Cilia; Biological Transport; Kinesins; Dyneins; Membrane Proteins; Protein Transport; Flagella
PubMed: 36462505
DOI: 10.1016/j.cell.2022.11.010 -
Nature Jun 2023Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans,...
Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.
Topics: Humans; Male; Artificial Intelligence; Axonemal Dyneins; Axoneme; Cilia; Cryoelectron Microscopy; Flagella; Mechanotransduction, Cellular; Microtubules; Chlamydomonas reinhardtii; Ciliary Motility Disorders; Movement; Protein Conformation
PubMed: 37258679
DOI: 10.1038/s41586-023-06140-2 -
Cell Jun 2023RNA editing is a widespread epigenetic process that can alter the amino acid sequence of proteins, termed "recoding." In cephalopods, most transcripts are recoded, and...
RNA editing is a widespread epigenetic process that can alter the amino acid sequence of proteins, termed "recoding." In cephalopods, most transcripts are recoded, and recoding is hypothesized to be an adaptive strategy to generate phenotypic plasticity. However, how animals use RNA recoding dynamically is largely unexplored. We investigated the function of cephalopod RNA recoding in the microtubule motor proteins kinesin and dynein. We found that squid rapidly employ RNA recoding in response to changes in ocean temperature, and kinesin variants generated in cold seawater displayed enhanced motile properties in single-molecule experiments conducted in the cold. We also identified tissue-specific recoded squid kinesin variants that displayed distinct motile properties. Finally, we showed that cephalopod recoding sites can guide the discovery of functional substitutions in non-cephalopod kinesin and dynein. Thus, RNA recoding is a dynamic mechanism that generates phenotypic plasticity in cephalopods and can inform the characterization of conserved non-cephalopod proteins.
Topics: Animals; Dyneins; Kinesins; RNA; Cephalopoda; Proteins; Microtubules; Microtubule Proteins; Myosins
PubMed: 37295401
DOI: 10.1016/j.cell.2023.04.032 -
Nature Cell Biology Feb 2022Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR-Cas, gene editing could cause DNA damage and trigger DNA repair processes that are...
Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR-Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9-SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9-SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9-SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering.
Topics: Actins; Aptamers, Nucleotide; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; DNA-Binding Proteins; Dyneins; Escherichia coli Proteins; Gene Editing; Gene Knock-In Techniques; HEK293 Cells; HSP90 Heat-Shock Proteins; HeLa Cells; Hep G2 Cells; Humans; Viral Proteins
PubMed: 35145221
DOI: 10.1038/s41556-021-00836-1 -
Chest May 2021Primary ciliary dyskinesia (PCD) is a heterogeneous disease with a diverse clinical and genetic spectrum among populations worldwide. Few cases of pediatric PCD have...
BACKGROUND
Primary ciliary dyskinesia (PCD) is a heterogeneous disease with a diverse clinical and genetic spectrum among populations worldwide. Few cases of pediatric PCD have been reported from China.
RESEARCH QUESTION
What are the clinical and genotypic characteristics of children with PCD in China?
STUDY DESIGN AND METHODS
Clinical characteristics, laboratory findings, and genetic results obtained for 75 patients with PCD were reviewed retrospectively at a single center in China. Genetic sequencing was conducted using whole-exome screening.
RESULTS
Patient median age at diagnosis was 7.0 years (range, 2 months-14 years). Of 75 patients, 88% (66/75) had chronic wet cough, 77% (58/75) had recurrent sinusitis, 76% (57/75) had bronchiectasis, 40% (30/75) had neonatal respiratory distress, and 28% (21/75) had coexistent asthma. Notably, postinfectious bronchiolitis obliterans (PIBO) as first presentation was found in 8% of children (6/75). Genes with the highest incidence of mutations were DNAH11 (15/51), followed by DNAH5 (9/51), CCDC39 (5/51), DNAH1 (4/51), and CCNO (3/51). Four genes (DNAI1, HEATR2, RSPH9, and DNAAF3) each were respectively found in two patients, and seven genes (CCDC40, LRRC6, SPAG1, RSPH4A, ARMC4, CCDC114, and DNAH14, a novel gene) each were mutated once. No differences in classical clinical features were observed among patients with commonly observed PCD-associated genotypes. However, three of six PIBO patients carried DNAH1 mutations.
INTERPRETATION
Besides typical clinical features, PIBO was observed as the first presentation of pediatric PCD in China. An association of the novel gene DNAH14 with PCD was observed, expanding the PCD genotypic spectrum.
Topics: Adolescent; Child; Child, Preschool; China; Ciliary Motility Disorders; Dyneins; Female; Genotype; Humans; Infant; Male; Retrospective Studies
PubMed: 33577779
DOI: 10.1016/j.chest.2021.02.006 -
Annual Review of Biophysics May 2021Dyneins make up a family of AAA+ motors that move toward the minus end of microtubules. Cytoplasmic dynein is responsible for transporting intracellular cargos in... (Review)
Review
Dyneins make up a family of AAA+ motors that move toward the minus end of microtubules. Cytoplasmic dynein is responsible for transporting intracellular cargos in interphase cells and mediating spindle assembly and chromosome positioning during cell division. Other dynein isoforms transport cargos in cilia and power ciliary beating. Dyneins were the least studied of the cytoskeletal motors due to challenges in the reconstitution of active dynein complexes in vitro and the scarcity of high-resolution methods for in-depth structural and biophysical characterization of these motors. These challenges have been recently addressed, and there have been major advances in our understanding of the activation, mechanism, and regulation of dyneins. This review synthesizes the results of structural and biophysical studies for each class of dynein motors. We highlight several outstanding questions about the regulation of bidirectional transport along microtubules and the mechanisms that sustain self-coordinated oscillations within motile cilia.
Topics: Animals; Biological Transport; Cilia; Dyneins; Humans; Intracellular Space; Microtubules
PubMed: 33957056
DOI: 10.1146/annurev-biophys-111020-101511 -
Molecular Vision 2021Cytoplasmic dyneins (dynein-1 and dynein-2) transport cargo toward the minus end of microtubules and thus, are termed the "retrograde" cellular motor. Dynein-1 cargo may... (Review)
Review
Cytoplasmic dyneins (dynein-1 and dynein-2) transport cargo toward the minus end of microtubules and thus, are termed the "retrograde" cellular motor. Dynein-1 cargo may include nuclei, mitochondria, membrane vesicles, lysosomes, phagosomes, and other organelles. For example, dynein-1 works in the cell body of eukaryotes to move cargo toward the microtubule minus end and positions the Golgi complex. Dynein-1 also participates in the movement of chromosomes and the positioning of mitotic spindles during cell division. In contrast, dynein-2 is present almost exclusively within cilia where it participates in retrograde intraflagellar transport (IFT) along the axoneme to return kinesin-2 subunits, BBSome, and IFT particles to the cell body. Cytoplasmic dyneins are hefty 1.5 MDa complexes comprised of dimers of heavy, intermediate, light intermediate, and light chains. Missense mutations of human are associated with malformations of cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMA-LED). Missense mutations in are causative of short-rib polydactyly syndrome type III and nonsyndromic retinitis pigmentosa. We review mutations of the two dynein heavy chains and their effect on postnatal retina development and discuss consequences of deletion of in the mouse retina.
Topics: Animals; Cytoplasmic Dyneins; Gene Expression; Humans; Mice; Mutation; Photoreceptor Cells, Vertebrate; Retinal Diseases
PubMed: 34526758
DOI: No ID Found -
Nature Structural & Molecular Biology Jan 2021In motile cilia, a mechanoregulatory network is responsible for converting the action of thousands of dynein motors bound to doublet microtubules into a single...
In motile cilia, a mechanoregulatory network is responsible for converting the action of thousands of dynein motors bound to doublet microtubules into a single propulsive waveform. Here, we use two complementary cryo-EM strategies to determine structures of the major mechanoregulators that bind ciliary doublet microtubules in Chlamydomonas reinhardtii. We determine structures of isolated radial spoke RS1 and the microtubule-bound RS1, RS2 and the nexin-dynein regulatory complex (N-DRC). From these structures, we identify and build atomic models for 30 proteins, including 23 radial-spoke subunits. We reveal how mechanoregulatory complexes dock to doublet microtubules with regular 96-nm periodicity and communicate with one another. Additionally, we observe a direct and dynamically coupled association between RS2 and the dynein motor inner dynein arm subform c (IDAc), providing a molecular basis for the control of motor activity by mechanical signals. These structures advance our understanding of the role of mechanoregulation in defining the ciliary waveform.
Topics: Axoneme; Biomechanical Phenomena; Chlamydomonas reinhardtii; Cilia; Cryoelectron Microscopy; Cytoskeletal Proteins; Dyneins; Flagella; Locomotion; Microtubules; Models, Molecular; Plant Proteins; Protein Structure, Tertiary; Signal Transduction; Sorting Nexins
PubMed: 33318703
DOI: 10.1038/s41594-020-00530-0 -
Journal of Cell Science Mar 2023The endosomal system orchestrates the transport of lipids, proteins and nutrients across the entire cell. Along their journey, endosomes mature, change shape via fusion... (Review)
Review
The endosomal system orchestrates the transport of lipids, proteins and nutrients across the entire cell. Along their journey, endosomes mature, change shape via fusion and fission, and communicate with other organelles. This intriguing endosomal choreography, which includes bidirectional and stop-and-go motions, is coordinated by the microtubule-based motor proteins dynein and kinesin. These motors bridge various endosomal subtypes to the microtubule tracks thanks to their cargo-binding domain interacting with endosome-associated proteins, and their motor domain interacting with microtubules and associated proteins. Together, these interactions determine the mobility of different endosomal structures. In this Review, we provide a comprehensive overview of the factors regulating the different interactions to tune the fascinating dance of endosomes along microtubules.
Topics: Dyneins; Kinesins; Endosomes; Microtubules; Microtubule-Associated Proteins
PubMed: 36382597
DOI: 10.1242/jcs.259689