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1.
Yi Chuan ; 43(11): 1038-1049, 2021 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-34815207

RESUMO

Eukaryotic cilia and flagella are evolutionarily conserved organelles that protrude from the cell surface. The unique location and properties of cilia allow them to function in vital processes such as motility and signaling. Ciliary assembly and maintenance rely on intraflagellar transport (IFT). Bidirectional movement of IFT particles composed of IFT-A and IFT-B complexes is powered by kinesin-2 and dynein-2 motors. IFT delivers building blocks between their site of synthesis in the cell body and the ciliary assembly site at the tip of the cilium. The integrity of the flagellum, a specialized organelle of mammalian sperm to generate the motility, is critical for normal sperm function. Recent findings suggest that IFT is indispensable for sperm flagellum formation and male fertility in mice and human. In this review, we summarize the role and mechanisms of IFT proteins during enflagellation in spermiogenesis, thereby discussing the pathological mechanisms of male infertility and providing theoretical basis for the diagnosis and treatment of male infertility.


Assuntos
Flagelos , Cinesina , Animais , Transporte Biológico , Cílios/metabolismo , Flagelos/metabolismo , Cinesina/metabolismo , Masculino , Camundongos , Espermatogênese
2.
PLoS One ; 16(10): e0258497, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34624068

RESUMO

CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK.


Assuntos
Proteínas Hedgehog , Cílios , Flagelos/metabolismo , Transporte Proteico , Receptor Smoothened
3.
J Cell Sci ; 134(20)2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34585727

RESUMO

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction based on microtubules and some attached projections. Radial spokes (RSs) facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 radial spoke proteins (RSP1-RSP23), although the roles of some radial spoke proteins remain unknown. Recently, RSP15 has been reported to be bound to the stalk of RS2, but its homolog in mammals has not been identified. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. We found that LRRC23 localizes to the RS complex within murine sperm flagella and interacts with RSPH3A and RSPH3B. The knockout of Lrrc23 resulted in male infertility due to RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was not significantly affected. These data indicate that LRRC23 is a key regulator that underpins the integrity of the RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia. This article has an associated First Person interview with the first author of the paper.


Assuntos
Axonema , Motilidade Espermática , Animais , Axonema/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Fertilidade/genética , Flagelos/metabolismo , Masculino , Camundongos , Motilidade Espermática/genética
4.
Proteomics ; 21(20): e2100004, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34558204

RESUMO

All eukaryotic flagella are made of microtubules and driven by dynein motor proteins. However, every organism is unique in terms of its flagellar waveform, beat frequency, and its general motility pattern. With recent research, it is becoming clear that despite overall conservation in flagellar structure, the pattern of tubulin post-translational modifications within the flagella are diverse and may contribute to variations in their patterns of motility. In this study, we have analyzed the tubulin post-translational modification in the protozoan parasites Giardia lamblia and Trichomonas vaginalis using global, untargeted mass spectrometry. We show that tubulin monoglycylation is a modification localized to the flagella present in G. lamblia but absent in T. vaginalis. We also show the presence of glutamylated tubulin in both G. lamblia and T. vaginalis. Using MS/MS, we were also able to identify the previously unknown sites of monoglycylation in ß-tubulin at E438 and E439 in G. lamblia. Using isolated flagella, we also characterized the flagellar proteome in G. lamblia and T. vaginalis and identified 475 proteins in G. lamblia and 386 proteins in T. vaginalis flagella. Altogether, the flagellar proteomes as well as the sites of tubulin PTMs in these organisms, reveal potential mechanisms in regulating flagellar motilities in these neglected protozoan parasites.


Assuntos
Giardia lamblia , Trichomonas vaginalis , Flagelos/metabolismo , Giardia lamblia/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Espectrometria de Massas em Tandem , Trichomonas vaginalis/metabolismo , Tubulina (Proteína)
5.
Nat Commun ; 12(1): 5442, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521846

RESUMO

Reversible switching of the bacterial flagellar motor between clockwise (CW) and counterclockwise (CCW) rotation is necessary for chemotaxis, which enables cells to swim towards favorable chemical habitats. Increase in the viscous resistance to the rotation of the motor (mechanical load) inhibits switching. However, cells must maintain homeostasis in switching to navigate within environments of different viscosities. The mechanism by which the cell maintains optimal chemotactic function under varying loads is not understood. Here, we show that the flagellar motor allosterically controls the binding affinity of the chemotaxis response regulator, CheY-P, to the flagellar switch complex by modulating the mechanical forces acting on the rotor. Mechanosensitive CheY-P binding compensates for the load-induced loss of switching by precisely adapting the switch response to a mechanical stimulus. The interplay between mechanical forces and CheY-P binding tunes the chemotactic function to match the load. This adaptive response of the chemotaxis output to mechanical stimuli resembles the proprioceptive feedback in the neuromuscular systems of insects and vertebrates.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Flagelos/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Regulação Alostérica , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Mimetismo Biológico , Fenômenos Biomecânicos , Quimiotaxia/genética , Escherichia coli/genética , Escherichia coli/ultraestrutura , Retroalimentação Sensorial/fisiologia , Flagelos/genética , Flagelos/ultraestrutura , Expressão Gênica , Insetos/fisiologia , Proteínas Quimiotáticas Aceptoras de Metil/química , Proteínas Quimiotáticas Aceptoras de Metil/genética , Pinças Ópticas , Ligação Proteica , Vertebrados/fisiologia , Viscosidade
6.
J Cell Sci ; 134(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34342348

RESUMO

Axonemal dyneins power the beating of motile cilia and flagella. These massive multimeric motor complexes are assembled in the cytoplasm, and subsequently trafficked to cilia and incorporated into the axonemal superstructure. Numerous cytoplasmic factors are required for the dynein assembly process, and, in mammals, defects lead to primary ciliary dyskinesia, which results in infertility, bronchial problems and failure to set up the left-right body axis correctly. Liquid-liquid phase separation (LLPS) has been proposed to underlie the formation of numerous membrane-less intracellular assemblies or condensates. In multiciliated cells, cytoplasmic assembly of axonemal dyneins also occurs in condensates that exhibit liquid-like properties, including fusion, fission and rapid exchange of components both within condensates and with bulk cytoplasm. However, a recent extensive meta-analysis suggests that the general methods used to define LLPS systems in vivo may not readily distinguish LLPS from other mechanisms. Here, I consider the time and length scales of axonemal dynein heavy chain synthesis, and the possibility that during translation of dynein heavy chain mRNAs, polysomes are crosslinked via partially assembled proteins. I propose that axonemal dynein factory formation in the cytoplasm may be a direct consequence of the sheer scale and complexity of the assembly process itself.


Assuntos
Dineínas do Axonema , Axonema , Animais , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismo , Axonema/metabolismo , Cílios/metabolismo , Citoplasma/metabolismo , Dineínas/genética , Dineínas/metabolismo , Flagelos/metabolismo
7.
J Cell Sci ; 134(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34415027

RESUMO

Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation. However, the degree of train disassembly at the tip remains unknown. Here, we performed two-color imaging of fluorescent protein-tagged IFT components, which indicates that IFT-A and IFT-B proteins from a given anterograde train usually return in the same set of retrograde trains. Similarly, concurrent turnaround was typical for IFT-B proteins and the IFT dynein subunit D1bLIC-GFP but severance was observed as well. Our data support a simple model of IFT turnaround, in which IFT-A, IFT-B and IFT dynein typically remain associated at the tip and segments of the anterograde trains convert directly into retrograde trains. Continuous association of IFT-A, IFT-B and IFT dynein during tip remodeling could balance protein entry and exit, preventing the build-up of IFT material in flagella.


Assuntos
Chlamydomonas , Dineínas , Transporte Biológico , Chlamydomonas/metabolismo , Cílios/metabolismo , Dineínas/genética , Dineínas/metabolismo , Flagelos/metabolismo , Transporte Proteico
9.
PLoS Pathog ; 17(8): e1009329, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34339455

RESUMO

The flagellar pocket (FP) is the only endo- and exocytic organelle in most trypanosomes and, as such, is essential throughout the life cycle of the parasite. The neck of the FP is maintained enclosed around the flagellum via the flagellar pocket collar (FPC). The FPC is a macromolecular cytoskeletal structure and is essential for the formation of the FP and cytokinesis. FPC biogenesis and structure are poorly understood, mainly due to the lack of information on FPC composition. To date, only two FPC proteins, BILBO1 and FPC4, have been characterized. BILBO1 forms a molecular skeleton upon which other FPC proteins can, theoretically, dock onto. We previously identified FPC4 as the first BILBO1 interacting partner and demonstrated that its C-terminal domain interacts with the BILBO1 N-terminal domain (NTD). Here, we report by yeast two-hybrid, bioinformatics, functional and structural studies the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240). Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD and that both domains interact with FPC4. We have determined a 1.9 Å resolution crystal structure of the BILBO2 NTD in complex with the FPC4 BILBO1-binding domain. Together with mutational analyses, our studies reveal key residues for the function of the BILBO2 NTD and its interaction with FPC4 and evidenced a tripartite interaction between BILBO1, BILBO2, and FPC4. Our work sheds light on the first atomic structure of an FPC protein complex and represents a significant step in deciphering the FPC function in Trypanosoma brucei and other pathogenic kinetoplastids.


Assuntos
Citocinese , Citoesqueleto/metabolismo , Flagelos/metabolismo , Organelas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência , Técnicas do Sistema de Duplo-Híbrido
10.
J Mol Biol ; 433(21): 167188, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34454944

RESUMO

Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante-chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E. coli (EPEC). In both EPEC and E. coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV9 forms a tripartite particle of ∼22 nm with a N-terminal domain connected by a short linker to a C-domain ring structure with a ∼5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 Å and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with local conformational changes at the pore surface and ring entry mouth that may also be modulated by the ATPase inner stalk. We propose that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion.


Assuntos
Adenosina Trifosfatases/química , Escherichia coli Enteropatogênica/ultraestrutura , Proteínas de Escherichia coli/química , Flagelos/ultraestrutura , Canais de Translocação SEC/química , Sistemas de Secreção Tipo III/ultraestrutura , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Regulação Alostérica , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Medição da Troca de Deutério , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/genética , Flagelos/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Espectrometria de Massas , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Especificidade por Substrato , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
11.
Nat Commun ; 12(1): 4223, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244518

RESUMO

The bacterial flagellar MS ring is a transmembrane complex acting as the core of the flagellar motor and template for flagellar assembly. The C ring attached to the MS ring is involved in torque generation and rotation switch, and a large symmetry mismatch between these two rings has been a long puzzle, especially with respect to their role in motor function. Here, using cryoEM structural analysis of the flagellar basal body and the MS ring formed by full-length FliF from Salmonella enterica, we show that the native MS ring is formed by 34 FliF subunits with no symmetry variation. Symmetry analysis of the C ring shows a variation with a peak at 34-fold, suggesting flexibility in C ring assembly. Finally, our data also indicate that FliF subunits assume two different conformations, contributing differentially to the inner and middle parts of the M ring and thus resulting in 23- and 11-fold subsymmetries in the inner and middle M ring, respectively. The internal core of the M ring, formed by 23 subunits, forms a hole of the right size to accommodate the protein export gate.


Assuntos
Proteínas de Bactérias/ultraestrutura , Flagelos/ultraestrutura , Proteínas de Membrana/ultraestrutura , Sistemas de Secreção Tipo III/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Fracionamento Celular , Microscopia Crioeletrônica , Flagelos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/ultraestrutura , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
12.
J Bacteriol ; 203(16): e0015921, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34096782

RESUMO

Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA (similar to MotA in Escherichia coli) and PomB (similar to MotB in E. coli), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after cross-linking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner. IMPORTANCE The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by cross-linking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Flagelos/metabolismo , Vibrio alginolyticus/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos/química , Flagelos/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Vibrio alginolyticus/química , Vibrio alginolyticus/genética , Vibrio alginolyticus/crescimento & desenvolvimento
13.
Commun Biol ; 4(1): 646, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059784

RESUMO

The flagellar protein export apparatus switches substrate specificity from hook-type to filament-type upon hook assembly completion, thereby initiating filament assembly at the hook tip. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for flagellar chaperones in complex with their cognate filament-type substrates. Interactions of the flexible linker of FlhA (FlhAL) with its nearest FlhAC subunit in the FlhAC ring is required for the substrate specificity switching. To address how FlhAL brings the order to flagellar assembly, we analyzed the flhA(E351A/W354A/D356A) ΔflgM mutant and found that this triple mutation in FlhAL increased the secretion level of hook protein by 5-fold, thereby increasing hook length. The crystal structure of FlhAC(E351A/D356A) showed that FlhAL bound to the chaperone-binding site of its neighboring subunit. We propose that the interaction of FlhAL with the chaperon-binding site of FlhAC suppresses filament-type protein export and facilitates hook-type protein export during hook assembly.


Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Proteínas de Membrana/metabolismo , Salmonella enterica/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Flagelos/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Chaperonas Moleculares/genética , Mutação/genética , Ligação Proteica , Transporte Proteico/genética , Especificidade por Substrato
14.
Nat Commun ; 12(1): 3999, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183670

RESUMO

Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigate how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Flagelos/metabolismo , Salmonella typhimurium/genética
15.
PLoS Pathog ; 17(6): e1009666, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34143858

RESUMO

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.


Assuntos
Flagelos/metabolismo , Cinesina/metabolismo , Leishmania mexicana/patogenicidade , Leishmaniose/metabolismo , Virulência/fisiologia , Animais , Proliferação de Células , Leishmania mexicana/fisiologia , Camundongos , Morfogênese , Proteínas de Protozoários/metabolismo , Psychodidae
16.
J Exp Bot ; 72(15): 5312-5335, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34077536

RESUMO

Calcium (Ca2+)-dependent signalling plays a well-characterized role in the response to different environmental stimuli, in both plant and animal cells. In the model organism for green algae, Chlamydomonas reinhardtii, Ca2+ signals were reported to have a crucial role in different physiological processes, such as stress responses, photosynthesis, and flagella functions. Recent reports identified the underlying components of the Ca2+ signalling machinery at the level of specific subcellular compartments and reported in vivo imaging of cytosolic Ca2+ concentration in response to environmental stimuli. The characterization of these Ca2+-related mechanisms and proteins in C. reinhardtii is providing knowledge on how microalgae can perceive and respond to environmental stimuli, but also on how this Ca2+ signalling machinery has evolved. Here, we review current knowledge on the cellular mechanisms underlying the generation, shaping, and decoding of Ca2+ signals in C. reinhardtii, providing an overview of the known and possible molecular players involved in the Ca2+ signalling of its different subcellular compartments. The advanced toolkits recently developed to measure time-resolved Ca2+ signalling in living C. reinhardtii cells are also discussed, suggesting how they can improve the study of the role of Ca2+ signals in the cellular response of microalgae to environmental stimuli.


Assuntos
Chlamydomonas reinhardtii , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Chlamydomonas reinhardtii/metabolismo , Citosol/metabolismo , Flagelos/metabolismo
17.
J Cell Sci ; 134(12)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34137439

RESUMO

The intraflagellar transport (IFT) system is a remarkable molecular machine used by cells to assemble and maintain the cilium, a long organelle extending from eukaryotic cells that gives rise to motility, sensing and signaling. IFT plays a critical role in building the cilium by shuttling structural components and signaling receptors between the ciliary base and tip. To provide effective transport, IFT-A and IFT-B adaptor protein complexes assemble into highly repetitive polymers, called IFT trains, that are powered by the motors kinesin-2 and IFT-dynein to move bidirectionally along the microtubules. This dynamic system must be precisely regulated to shuttle different cargo proteins between the ciliary tip and base. In this Cell Science at a Glance article and the accompanying poster, we discuss the current structural and mechanistic understanding of IFT trains and how they function as macromolecular machines to assemble the structure of the cilium.


Assuntos
Cílios , Dineínas , Transporte Biológico , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Cinesina/genética , Cinesina/metabolismo , Microtúbulos/metabolismo , Transporte Proteico
18.
J Bacteriol ; 203(17): e0022721, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34124944

RESUMO

Swarming motility is flagellum-mediated movement over a solid surface, and Bacillus subtilis cells require an increase in flagellar density to swarm. SwrB is a protein of unknown function required for swarming that is necessary to increase the number of flagellar hooks but not basal bodies. Previous work suggested that SwrB activates flagellar type III secretion, but the mechanism by which it might perform this function is unknown. Here, we show that SwrB likely acts substoichiometrically as it localizes as puncta at the membrane in numbers fewer than those of flagellar basal bodies. Moreover, the action of SwrB is likely transient as puncta of SwrB were not dependent on the presence of the basal bodies and rarely colocalized with flagellar hooks. Random mutagenesis of the SwrB sequence found that a histidine within the transmembrane segment was conditionally required for activity and punctate localization. Finally, three hydrophobic residues that precede a cytoplasmic domain of poor conservation abolished SwrB activity when mutated and caused aberrant migration during electrophoresis. Our data are consistent with a model in which SwrB interacts with the flagellum, changes conformation to activate type III secretion, and departs. IMPORTANCE Type III secretion systems (T3SSs) are elaborate nanomachines that form the core of the bacterial flagellum and injectisome of pathogens. The machines not only secrete proteins like virulence factors but also secrete the structural components for their own assembly. Moreover, proper construction requires complex regulation to ensure that the parts are roughly secreted in the order in which they are assembled. Here, we explore a poorly understood activator of the flagellar T3SS activation in Bacillus subtilis called SwrB. To aid mechanistic understanding, we determine the rules for subcellular punctate localization, the topology with respect to the membrane, and critical residues required for SwrB function.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flagelos/química , Flagelos/genética , Flagelos/metabolismo , Regulação Bacteriana da Expressão Gênica , Domínios Proteicos , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
19.
PLoS One ; 16(6): e0252800, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34143799

RESUMO

Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomer's cytoplasmic domain, which stabilises the nonameric ring assembly.


Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Proteínas de Membrana/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Microscopia Crioeletrônica/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia de Fluorescência/métodos , Modelos Moleculares , Conformação Proteica , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/ultraestrutura , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo
20.
FASEB J ; 35(6): e21646, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33993568

RESUMO

Axonemal I1 dynein (dynein f) is the largest inner dynein arm in cilia and a key regulator of ciliary beating. It consists of two dynein heavy chains, and an intermediate chain/light chain (ICLC) complex. However, the structural organization of the nine ICLC subunits remains largely unknown. Here, we used biochemical and genetic approaches, and cryo-electron tomography imaging in Chlamydomonas to dissect the molecular architecture of the I1 dynein ICLC complex. Using a strain expressing SNAP-tagged IC140, tomography revealed the location of the IC140 N-terminus at the proximal apex of the ICLC structure. Mass spectrometry of a tctex2b mutant showed that TCTEX2B dynein light chain is required for the stable assembly of TCTEX1 and inner dynein arm interacting proteins IC97 and FAP120. The structural defects observed in tctex2b located these 4 subunits in the center and bottom regions of the ICLC structure, which overlaps with the location of the IC138 regulatory subcomplex, which contains IC138, IC97, FAP120, and LC7b. These results reveal the three-dimensional organization of the native ICLC complex and indicate potential protein-protein interactions that are involved in the pathway by which I1 regulates ciliary motility.


Assuntos
Axonema/metabolismo , Chlamydomonas/metabolismo , Cílios/metabolismo , Dineínas/química , Mutação , Proteínas de Plantas/química , Chlamydomonas/crescimento & desenvolvimento , Dineínas/genética , Dineínas/metabolismo , Flagelos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformação Proteica
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