RESUMO
Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.
Assuntos
Núcleo Celular , Dineínas , Cinesinas , Proteínas Associadas aos Microtúbulos , Microtúbulos , Proteínas do Tecido Nervoso , Neurônios , Animais , Microtúbulos/metabolismo , Neurônios/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Dineínas/metabolismo , Núcleo Celular/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Transporte Ativo do Núcleo Celular , Complexo Dinactina/metabolismo , Complexo Dinactina/genética , Movimento Celular , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Cerebelo/metabolismo , Cerebelo/citologia , Sítios de Ligação , HumanosRESUMO
Respiratory syncytial virus (RSV) hijacks cholesterol or autophagy pathways to facilitate optimal replication. However, our understanding of the associated molecular mechanisms remains limited. Here, we show that RSV infection blocks cholesterol transport from lysosomes to the endoplasmic reticulum by downregulating the activity of lysosomal acid lipase, activates the SREBP2-LDLR axis, and promotes uptake and accumulation of exogenous cholesterol in lysosomes. High cholesterol levels impair the VAP-A-binding activity of ORP1L and promote the recruitment of dynein-dynactin, PLEKHM1, or HOPS VPS39 to Rab7-RILP, thereby facilitating minus-end transport of autophagosomes and autolysosome formation. Acidification inhibition and dysfunction of cholesterol-rich lysosomes impair autophagy flux by inhibiting autolysosome degradation, which promotes the accumulation of RSV fusion protein. RSV-F storage is nearly abolished after cholesterol depletion or knockdown of LDLR. Most importantly, the knockout of LDLR effectively inhibits RSV infection in vivo. These findings elucidate the molecular mechanism of how RSV co-regulates lysosomal cholesterol reprogramming and autophagy and reveal LDLR as a novel target for anti-RSV drug development.
Assuntos
Autofagia , Colesterol , Lisossomos , Receptores de LDL , Infecções por Vírus Respiratório Sincicial , Proteínas de Transporte Vesicular , Replicação Viral , Proteínas rab de Ligação ao GTP , proteínas de unión al GTP Rab7 , Lisossomos/metabolismo , Colesterol/metabolismo , Humanos , Animais , Receptores de LDL/metabolismo , Receptores de LDL/genética , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Camundongos , Complexo Dinactina/metabolismo , Retículo Endoplasmático/metabolismo , Dineínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Vírus Sincicial Respiratório Humano/fisiologia , Autofagossomos/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/genética , Células HeLa , Células A549RESUMO
The outer dynein arm (ODA) is a large, multimeric protein complex essential for ciliary motility. The composition and assembly of ODA are best characterized in the green algae Chlamydomonas reinhardtii, where individual ODA subunits are synthesized and preassembled into a mature complex in the cytosol prior to ciliary import. The single-cellular parasite Trypanosoma brucei contains a motile flagellum essential for cell locomotion and pathogenesis. Similar to human motile cilia, T. brucei flagellum contains a two-headed ODA complex arranged at 24 nm intervals along the axonemal microtubule doublets. The subunit composition and the preassembly of the ODA complex in T. brucei, however, have not been investigated. In this study, we affinity-purified the ODA complex from T. brucei cytoplasmic extract. Proteomic analyses revealed the presence of two heavy chains (ODAα and ODAß), two intermediate chains (IC1and IC2) and several light chains. We showed that both heavy chains and both intermediate chains are indispensable for flagellar ODA assembly. Our study also provided biochemical evidence supporting the presence of a cytoplasmic, preassembly pathway for T. brucei ODA.
Assuntos
Axonema , Citoplasma , Dineínas , Flagelos , Proteínas de Protozoários , Trypanosoma brucei brucei , Trypanosoma brucei brucei/metabolismo , Flagelos/metabolismo , Citoplasma/metabolismo , Axonema/metabolismo , Dineínas/metabolismo , Proteínas de Protozoários/metabolismo , Microtúbulos/metabolismo , Proteômica/métodos , Cílios/metabolismoRESUMO
Ninein (Nin) is a microtubule (MT) anchor at the subdistal appendages of mother centrioles and the pericentriolar material (PCM) of centrosomes that also functions to organize MTs at noncentrosomal MT-organizing centers (ncMTOCs). In humans, the NIN gene is mutated in Seckel syndrome, an inherited developmental disorder. Here, we dissect the protein domains involved in Nin's localization and interactions with dynein and ensconsin (ens/MAP7) and show that the association with ens cooperatively regulates MT assembly in Drosophila fat body cells. We define domains of Nin responsible for its localization to the ncMTOC on the fat body cell nuclear surface, localization within the nucleus, and association with Dynein light intermediate chain (Dlic) and ens, respectively. We show that Nin's association with ens synergistically regulates MT assembly. Together, these findings reveal novel features of Nin function and its regulation of a ncMTOC.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Dineínas , Proteínas Associadas aos Microtúbulos , Centro Organizador dos Microtúbulos , Microtúbulos , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Animais , Dineínas/metabolismo , Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Centrossomo/metabolismo , Domínios Proteicos , Humanos , Corpo Adiposo/metabolismo , Drosophila/metabolismo , Núcleo Celular/metabolismo , Centríolos/metabolismo , Ligação Proteica , Proteínas de HomeodomínioRESUMO
Disease-causing bi-allelic DNA variants in CCDC39 and CCDC40 are frequent causes of the hereditary disorder of primary ciliary dyskinesia (PCD). The encoded proteins form a molecular ruler complex, crucial for maintaining the 96 nm repeat units along the ciliary axonemes. Defects of those proteins cause a stiff, rapid, and flickery ciliary beating pattern, recurrent respiratory infections, axonemal disorganization, and abnormal assembly of GAS8, CCDC39, and DNALI1. We performed molecular characterization of the defects in the 96 nm axonemal ruler due to disease-causing variants in CCDC39 and CCDC40 and analyzed the effect on additional axonemal components. We identified a cohort of 51 individuals with disease-causing variants in CCDC39 and CCDC40 via next-generation sequencing techniques and demonstrated that the IDA heavy chains DNAH1, DNAH6, and DNAH7 are conspicuously absent within the respiratory ciliary axonemes by immunofluorescence analyses. Hence, we show for the first time that the centrin2 (CETN2) containing IDAs are also affected. These findings underscore the crucial role of CCDC39 and CCDC40 in the assembly and function of IDAs in human respiratory cilia. Thus, our data improve the diagnostics of axonemal ruler defects by further characterizing the associated molecular IDA defects.
Assuntos
Axonema , Humanos , Masculino , Dineínas do Axonema/metabolismo , Dineínas do Axonema/genética , Axonema/metabolismo , Cílios/metabolismo , Cílios/patologia , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Transtornos da Motilidade Ciliar/patologia , Proteínas do Citoesqueleto , Dineínas/metabolismo , Dineínas/genética , Mutação/genética , ProteínasRESUMO
Inner dynein arms (IDAs) are formed from a protein complex that is essential for appropriate flagellar bending and beating. IDA defects have previously been linked to the incidence of asthenozoospermia (AZS) and male infertility. The testes-enriched ZMYND12 protein is homologous with an IDA component identified in Chlamydomonas. ZMYND12 deficiency has previously been tied to infertility in males, yet the underlying mechanism remains uncertain. Here, a CRISPR/Cas9 approach was employed to generate Zmynd12 knockout (Zmynd12-/-) mice. These Zmynd12-/- mice exhibited significant male subfertility, reduced sperm motile velocity, and impaired capacitation. Through a combination of co-immunoprecipitation and mass spectrometry, ZMYND12 was found to interact with TTC29 and PRKACA. Decreases in the levels of PRKACA were evident in the sperm of these Zmynd12-/- mice, suggesting that this change may account for the observed drop in male fertility. Moreover, in a cohort of patients with AZS, one patient carrying a ZMYND12 variant was identified, expanding the known AZS-related variant spectrum. Together, these findings demonstrate that ZMYND12 is essential for flagellar beating, capacitation, and male fertility.
Assuntos
Infertilidade Masculina , Camundongos Knockout , Motilidade dos Espermatozoides , Animais , Masculino , Motilidade dos Espermatozoides/genética , Camundongos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Dineínas/metabolismo , Dineínas/genética , Espermatozoides/metabolismo , Humanos , Astenozoospermia/genética , Astenozoospermia/metabolismo , Astenozoospermia/patologia , Capacitação Espermática/genética , Camundongos Endogâmicos C57BL , Sistemas CRISPR-CasRESUMO
Dynein complexes are large, multi-unit assemblies involved in many biological processes via their critical roles in protein transport and axoneme motility. Using next-generation sequencing of infertile men presenting with low or no sperm in their ejaculates, we identified damaging variants in the dynein-related gene AXDND1. We thus hypothesised that AXDND1 is a critical regulator of male fertility. To test this hypothesis, we produced a knockout mouse model. Axdnd1-/- males were sterile at all ages but presented with an evolving testis phenotype wherein they could undergo one round of histologically replete spermatogenesis followed by a rapid depletion of the seminiferous epithelium. Marker experiments identified a role for AXDND1 in maintaining the balance between differentiation-committed and self-renewing spermatogonial populations, resulting in disproportionate production of differentiating cells in the absence of AXDND1 and increased sperm production during initial spermatogenic waves. Moreover, long-term spermatogonial maintenance in the Axdnd1 knockout was compromised, ultimately leading to catastrophic germ cell loss, destruction of blood-testis barrier integrity and immune cell infiltration. In addition, sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively these data identify AXDND1 as an atypical dynein complex-related protein with a role in protein/vesicle transport of relevance to spermatogonial function and sperm tail formation in mice and humans. This study underscores the importance of studying the consequences of gene loss-of-function on both the establishment and maintenance of male fertility.
Assuntos
Camundongos Knockout , Cauda do Espermatozoide , Espermatogênese , Espermatogônias , Animais , Humanos , Masculino , Camundongos , Diferenciação Celular , Dineínas/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Camundongos Endogâmicos C57BL , Cauda do Espermatozoide/metabolismo , Espermatogênese/genética , Espermatogônias/metabolismo , Testículo/metabolismo , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismoRESUMO
Vertebrate motile cilia are classified as (9+2) or (9+0), based on the presence or absence of the central pair apparatus, respectively. Cryogenic electron microscopy analyses of (9+2) cilia have uncovered an elaborate axonemal protein composition. The extent to which these features are conserved in (9+0) cilia remains unclear. CFAP53, a key axonemal filamentous microtubule inner protein (fMIP) and a centriolar satellites component, is essential for motility of (9+0), but not (9+2) cilia. Here, we show that in (9+2) cilia, CFAP53 functions redundantly with a paralogous fMIP, MNS1. MNS1 localises to ciliary axonemes, and combined loss of both proteins in zebrafish and mice caused severe outer dynein arm loss from (9+2) cilia, significantly affecting their motility. Using immunoprecipitation, we demonstrate that, whereas MNS1 can associate with itself and CFAP53, CFAP53 is unable to self-associate. We also show that additional axonemal dynein-interacting proteins, two outer dynein arm docking (ODAD) complex members, show differential localisation between types of motile cilia. Together, our findings clarify how paralogous fMIPs, CFAP53 and MNS1, function in regulating (9+2) versus (9+0) cilia motility, and further emphasise extensive structural diversity among these organelles.
Assuntos
Axonema , Cílios , Peixe-Zebra , Animais , Cílios/metabolismo , Cílios/ultraestrutura , Peixe-Zebra/metabolismo , Camundongos , Axonema/metabolismo , Axonema/ultraestrutura , Dineínas do Axonema/metabolismo , Dineínas do Axonema/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Dineínas/metabolismoRESUMO
The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.
Assuntos
Anáfase , Proteínas de Drosophila , Drosophila melanogaster , Dineínas , Microtúbulos , Animais , Dineínas/metabolismo , Dineínas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Microtúbulos/metabolismo , Microtúbulos/genética , Simulação de Dinâmica Molecular , Mutação/genética , Fuso Acromático/metabolismo , Fuso Acromático/genética , Humanos , Mutação de Sentido IncorretoRESUMO
During entry, human papillomavirus (HPV) traffics from the endosome to the trans Golgi network (TGN) and Golgi and then the nucleus to cause infection. Although dynein is thought to play a role in HPV infection, how this host motor recruits the virus to support infection and which entry step(s) requires dynein are unclear. Here we show that the dynein cargo adaptor BICD2 binds to the HPV L2 capsid protein during entry, recruiting HPV to dynein for transport of the virus along the endosome-TGN/Golgi axis to promote infection. In the absence of BICD2 function, HPV accumulates in the endosome and TGN and infection is inhibited. Cell-based and in vitro binding studies identified a short segment near the C-terminus of L2 that can directly interact with BICD2. Our results reveal the molecular basis by which the dynein motor captures HPV to promote infection and identify this virus as a novel cargo of the BICD2 dynein adaptor.
Assuntos
Proteínas do Capsídeo , Papillomavirus Humano 16 , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Humanos , Proteínas do Capsídeo/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Dineínas/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia , Internalização do Vírus , Ligação Proteica , Células HeLa , Proteínas Associadas aos Microtúbulos/metabolismo , Dineínas do Citoplasma/metabolismoRESUMO
Mechanical stress significantly affects the physiological functions of cells, including tissue homeostasis, cytoskeletal alterations, and intracellular transport. As a major cytoskeletal component, microtubules respond to mechanical stimulation by altering their alignment and polymerization dynamics. Previously, we reported that microtubules may modulate cargo transport by one of the microtubule-associated motor proteins, dynein, under compressive mechanical stress. Despite the critical role of tensile stress in many biological functions, how tensile stress on microtubules regulates cargo transport is yet to be unveiled. The present study demonstrates that the low-level tensile stress-induced microtubule deformation facilitates dynein-driven transport. We validate our experimental findings using all-atom molecular dynamics simulation. Our study may provide important implications for developing new therapies for diseases that involve impaired intracellular transport.
Assuntos
Dineínas , Microtúbulos , Simulação de Dinâmica Molecular , Estresse Mecânico , Microtúbulos/metabolismo , Microtúbulos/química , Dineínas/metabolismo , Dineínas/química , Resistência à Tração , Transporte BiológicoRESUMO
At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are together required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.
Assuntos
Anáfase , Cinesinas , Microtúbulos , Fuso Acromático , Humanos , Fuso Acromático/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Dineínas/metabolismo , Dineínas/genética , Torque , Segregação de Cromossomos , Citoesqueleto de Actina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
Calcium is critical for regulating the waveform of motile cilia and flagella. Calaxin is currently the only known molecule involved in the calcium-dependent regulation in ascidians. We have recently shown that Calaxin stabilizes outer arm dynein (OAD), and the knockout of Calaxin results in primary ciliary dyskinesia phenotypes in vertebrates. However, from the knockout experiments, it was not clear which functions depend on calcium and how Calaxin regulates the waveform. To address this question, here, we generated transgenic zebrafish expressing a mutant E130A-Calaxin deficient in calcium binding. E130A-Calaxin restored the OAD reduction of calaxin -/- sperm and the abnormal movement of calaxin -/- left-right organizer cilia, showing that Calaxin's stabilization of OADs is calcium-independent. In contrast, our quantitative analysis of E130A-Calaxin sperms showed that the calcium-induced asymmetric beating was not restored, linking Calaxin's calcium-binding ability with an asymmetric flagellar beating for the first time. Our data show that Calaxin is a calcium-dependent regulator of the ciliary beating and a calcium-independent OAD stabilizer.
Assuntos
Proteínas de Ligação ao Cálcio , Espermatozoides , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Masculino , Animais Geneticamente Modificados , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Cílios/metabolismo , Dineínas/metabolismo , Dineínas/genética , Flagelos/metabolismo , Flagelos/fisiologia , Motilidade dos Espermatozoides/genética , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas do Citoesqueleto/metabolismoRESUMO
Herpesvirus assembly requires the cytoplasmic association of large macromolecular and membrane structures that derive from both the nucleus and cytoplasmic membrane systems. Results from the study of human cytomegalovirus (HCMV) in cells where it organizes a perinuclear cytoplasmic virus assembly compartment (cVAC) show a clear requirement for the minus-end-directed microtubule motor, dynein, for virus assembly. In contrast, the assembly of herpes simplex virus -1 (HSV-1) in epithelial cells where it forms multiple dispersed, peripheral assembly sites is only mildly inhibited by the microtubule-depolymerizing agent, nocodazole. Here, we make use of a neuronal cell line system in which HSV-1 forms a single cVAC and show that dynein and its co-factor dynactin localize to the cVAC, and dynactin is associated with membranes that contain the virion tegument protein pUL11. We also show that the virus membrane-associated structural proteins pUL51 and the viral envelope glycoprotein gE arrive at the cVAC by different routes. Specifically, gE arrives at the cVAC after retrieval from the plasma membrane, suggesting the need for an intact retrograde transport system. Finally, we demonstrate that inhibition of dynactin function profoundly inhibits cVAC formation and virus production during the cytoplasmic assembly phase of infection.IMPORTANCEMany viruses reorganize cytoplasmic membrane systems and macromolecular transport systems to promote the production of progeny virions. Clarifying the mechanisms by which they accomplish this may reveal novel therapeutic strategies and illustrate mechanisms that are critical for normal cellular organization. Here, we explore the mechanism by which HSV-1 moves macromolecular and membrane cargo to generate a virus assembly compartment in the infected cell. We find that the virus makes use of a well-characterized, microtubule-based transport system that is stabilized against drugs that disrupt microtubules.
Assuntos
Membrana Celular , Complexo Dinactina , Dineínas , Herpesvirus Humano 1 , Proteínas Associadas aos Microtúbulos , Neurônios , Proteínas do Envelope Viral , Montagem de Vírus , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/metabolismo , Dineínas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , Humanos , Neurônios/virologia , Neurônios/metabolismo , Complexo Dinactina/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Linhagem Celular , Animais , Compartimentos de Replicação Viral/metabolismo , Microtúbulos/metabolismoRESUMO
The material transport system, facilitated by motor proteins, plays a vital role in maintaining a non-equilibrium cellular state. However, understanding the temporal coordination of motor protein activity requires an advanced imaging technique capable of measuring 3D angular displacement in real-time. In this study, a Fourier transform-based plasmonic dark-field microscope has been developed using anisotropic nanoparticles, enabling the prolonged and simultaneous observation of endosomal lateral and rotational motion. A sequence of discontinuous 3D angular displacements has been observed during the pause and run phases of transport. Notably, a serially correlated temporal pattern in the intermittent rotational events has been demonstrated during the tug-of-war mechanism, indicating Markovian switching between the exploitational and explorational modes of motor protein exchange prior to resuming movement. Alterations in transition frequency and the exploitation-to-exploration ratio upon dynein inhibitor treatment highlight the relationship between disrupted motor coordination and reduced endosomal transport efficiency. Collectively, these results suggest the importance of orchestrated temporal motor protein patterns for efficient cellular transport.
Assuntos
Endossomos , Endossomos/metabolismo , Humanos , Microscopia/métodos , Dineínas/metabolismo , Transporte Biológico/fisiologia , Proteínas Motores Moleculares/metabolismoRESUMO
Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding of adjacent axoneme microtubule doublets to periodically bend the flagellum for beating, while intraflagellar transport (IFT) kinesins and dyneins carry IFT trains bidirectionally along the axoneme. Despite assembling motile cilia and flagella, IFT train speeds have only previously been quantified in immobilized flagella-mechanical immobilization or genetic paralysis. This has limited investigation of the interaction between IFT and flagellar beating. Here, in uniflagellate Leishmania parasites, we use high-frequency, dual-color fluorescence microscopy to visualize IFT train movement in beating flagella. We discovered that adhesion of flagella to a microscope slide is detrimental, reducing IFT train speed and increasing train stalling. In flagella free to move, IFT train speed is not strongly dependent on flagella beat type; however, permanent disruption of flagella beating by deletion of genes necessary for formation or regulation of beating showed an inverse correlation of beat frequency and IFT train speed.
Assuntos
Flagelos , Leishmania , Microtúbulos , Axonema/metabolismo , Axonema/genética , Transporte Biológico , Cílios/metabolismo , Cílios/genética , Dineínas/metabolismo , Dineínas/genética , Flagelos/metabolismo , Flagelos/genética , Cinesinas/metabolismo , Cinesinas/genética , Leishmania/citologia , Leishmania/genética , Leishmania/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Microtúbulos/metabolismoRESUMO
Outer dynein arms (ODAs) are responsible for ciliary beating in eukaryotes. They are assembled in the cytoplasm and shipped by intraflagellar transport (IFT) before attachment to microtubule doublets via the docking complex. The LRRC56 protein has been proposed to contribute to ODAs maturation. Mutations or deletion of the LRRC56 gene lead to reduced ciliary motility in all species investigated so far, but with variable impact on dynein arm presence. Here, we investigated the role of LRRC56 in the protist Trypanosoma brucei, where its absence results in distal loss of ODAs, mostly in growing flagella. We show that LRRC56 is a transient cargo of IFT trains during flagellum construction and surprisingly, is required for efficient attachment of a subset of docking complex proteins present in the distal portion of the organelle. This relation is interdependent since the knockdown of the distal docking complex prevents LRRC56's association with the flagellum. Intriguingly, lrrc56-/- cells display shorter flagella whose maturation is delayed. Inhibition of cell division compensates for the distal ODAs absence thanks to the redistribution of the proximal docking complex, restoring ODAs attachment but not the flagellum length phenotype. This work reveals an unexpected connection between LRRC56 and the docking complex.
Assuntos
Dineínas , Flagelos , Proteínas de Protozoários , Trypanosoma brucei brucei , Trypanosoma brucei brucei/metabolismo , Flagelos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Dineínas/metabolismo , Microtúbulos/metabolismo , Cílios/metabolismo , Transporte Biológico/fisiologia , Axonema/metabolismoRESUMO
Intraflagellar transport (IFT) is required for ciliary assembly. The IFT machinery comprises the IFT motors kinesin-2 and IFT dynein plus IFT-A and IFT-B complexes, which assemble into IFT trains in cilia. To gain mechanistic understanding of IFT and ciliary assembly, here, we performed an absolute quantification of IFT machinery in Chlamydomonas reinhardtii cilium. There are â¼756, â¼532, â¼276 and â¼350 molecules of IFT-B, IFT-A, IFT dynein and kinesin-2, respectively, per cilium. The amount of IFT-B is sufficient to sustain rapid ciliary growth in terms of tubulin delivery. The stoichiometric ratio of IFT-B:IFT-A:dynein is â¼3:2:1 whereas the IFT-B:IFT-A ratio in an IFT dynein mutant is 2:1, suggesting that there is a plastic interaction between IFT-A and IFT-B that can be influenced by IFT dynein. Considering diffusion of kinesin-2 during retrograde IFT, it is estimated that one kinesin-2 molecule drives eight molecules of IFT-B during anterograde IFT. These data provide new insights into the assembly of IFT trains and ciliary assembly.
Assuntos
Chlamydomonas reinhardtii , Cílios , Dineínas , Flagelos , Cinesinas , Proteômica , Cílios/metabolismo , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/genética , Proteômica/métodos , Cinesinas/metabolismo , Cinesinas/genética , Dineínas/metabolismo , Flagelos/metabolismo , Transporte BiológicoRESUMO
Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Actinas , Membrana Celular , Centrossomo , Proteínas de Drosophila , Drosophila melanogaster , Animais , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/metabolismo , Membrana Celular/metabolismo , Centrossomo/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Dineínas/metabolismo , Exocitose , Microtúbulos/metabolismoRESUMO
Human infertility affects 10-15% of couples. Asthenozoospermia accounts for 18% of men with infertility and is a common male infertility phenotype. The nexin-dynein regulatory complex (N-DRC) is a large protein complex in the sperm flagellum that connects adjacent doublets of microtubules. Defects in the N-DRC can disrupt cilia/flagellum movement, resulting in primary ciliary dyskinesia and male infertility. Using whole-exome sequencing, we identified a pathological homozygous variant of the dynein regulatory complex subunit 3 (DRC3) gene, which expresses leucine-rich repeat-containing protein 48, a component of the N-DRC, in a patient with asthenozoospermia. The variant ENST00000313838.12: c.644dup (p. Glu216GlyfsTer36) causes premature translational arrest of DRC3, resulting in a dysfunctional DRC3 protein. The patient's semen count, color, and pH were normal according to the reference values of the World Health Organization guidelines; however, sperm motility and progressive motility were reduced. DRC3 protein was not detected in the patient's sperm and the ultrastructure of the patient's sperm flagella was destroyed. More importantly, the DRC3 variant reduced its interaction with other components of the N-DRC, including dynein regulatory complex subunits 1, 2, 4, 5, 7, and 8. Our data not only revealed the essential biological functions of DRC3 in sperm flagellum movement and structure but also provided a new basis for the clinical genetic diagnosis of male infertility.