mRNA localization mediates maturation of cytoplasmic cilia in Drosophila spermatogenesis

Cytoplasmic cilia, a specialized type of cilia in which the axoneme resides within the cytoplasm rather than within the ciliary compartment, are proposed to allow for the efficient assembly of very long cilia. Despite being found diversely in male gametes (e.g., Plasmodium falciparum microgametocytes and human and Drosophila melanogaster sperm), very little is known about cytoplasmic cilia assembly. Here, we show that a novel RNP granule containing the mRNAs for axonemal dynein motor proteins becomes highly polarized to the distal end of the cilia during cytoplasmic ciliogenesis in Drosophila sperm. This allows for the incorporation of these axonemal dyneins into the axoneme directly from the cytoplasm, possibly by localizing translation. We found that this RNP granule contains the proteins Reptin and Pontin, loss of which perturbs granule formation and prevents incorporation of the axonemal dyneins, leading to sterility. We propose that cytoplasmic cilia assembly requires the precise localization of mRNAs encoding key axonemal constituents, allowing these proteins to incorporate efficiently into the axoneme.

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Localized mRNA translation mediates maturation of cytoplasmic cilia in Drosophila spermatogenesis

10.1101/2020.04.21.054247 ◽

2020 ◽

Cited By ~ 2

Author(s):

Jaclyn M Fingerhut ◽

Yukiko M Yamashita

Keyword(s):

Motor Proteins ◽

Mrna Translation ◽

Local Translation ◽

Granule Formation ◽

Male Gametes ◽

Dynein Motor ◽

Cilia Formation ◽

Axonemal Dynein

AbstractCytoplasmic cilia, a specialized type of cilia in which the axoneme resides within the cytoplasm rather than within the ciliary compartment, are proposed to allow the efficient assembly of very long cilia. Despite being found diversely in male gametes (e.g. Plasmodium microgametocytes and human and Drosophila sperm), very little is known about cytoplasmic cilia assembly. Here we show that a novel RNP granule containing the mRNAs for axonemal dynein motor proteins becomes highly polarized to the distal end of the cilia during cytoplasmic ciliogenesis in Drosophila sperm. This allows for the localized translation of these axonemal dyneins and their incorporation into the axoneme directly from the cytoplasm. We found that this RNP granule contains the proteins Reptin and Pontin, loss of which perturbs granule formation and prevents incorporation of the axonemal dyneins, leading to sterility. We propose that cytoplasmic cilia require the local translation of key protein constituents such that these proteins are incorporated efficiently into the axoneme.Author SummaryCytoplasmic cilia, which are found in human and Drosophila sperm, are unique in that the axoneme is exposed to the cytoplasm. The authors show that a novel RNP granule containing axonemal dynein mRNAs facilitates localized translation of these axonemal proteins, facilitating cytoplasmic cilia formation.

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Ciliary dynein motor preassembly is regulated by Wdr92 in association with HSP90 co-chaperone, R2TP

The Journal of Cell Biology ◽

10.1083/jcb.201709026 ◽

2018 ◽

Vol 217 (7) ◽

pp. 2583-2598 ◽

Cited By ~ 21

Author(s):

Petra zur Lage ◽

Panagiota Stefanopoulou ◽

Katarzyna Styczynska-Soczka ◽

Niall Quinn ◽

Girish Mali ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Molecular Chaperones ◽

Flagellar Motility ◽

Associated Factor ◽

Dynein Motor ◽

Assembly Factors ◽

Distinct Stage ◽

Axonemal Dynein ◽

Proteomic Analyses

The massive dynein motor complexes that drive ciliary and flagellar motility require cytoplasmic preassembly, a process requiring dedicated dynein assembly factors (DNAAFs). How DNAAFs interact with molecular chaperones to control dynein assembly is not clear. By analogy with the well-known multifunctional HSP90-associated cochaperone, R2TP, several DNAAFs have been suggested to perform novel R2TP-like functions. However, the involvement of R2TP itself (canonical R2TP) in dynein assembly remains unclear. Here we show that in Drosophila melanogaster, the R2TP-associated factor, Wdr92, is required exclusively for axonemal dynein assembly, likely in association with canonical R2TP. Proteomic analyses suggest that in addition to being a regulator of R2TP chaperoning activity, Wdr92 works with the DNAAF Spag1 at a distinct stage in dynein preassembly. Wdr92/R2TP function is likely distinct from that of the DNAAFs proposed to form dynein-specific R2TP-like complexes. Our findings thus establish a connection between dynein assembly and a core multifunctional cochaperone.

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A repetitive antigen of Plasmodium falciparum that is homologous to heat shock protein 70 of Drosophila melanogaster.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.83.22.8713 ◽

1986 ◽

Vol 83 (22) ◽

pp. 8713-8717 ◽

Cited By ~ 68

Author(s):

A. E. Bianco ◽

J. M. Favaloro ◽

T. R. Burkot ◽

J. G. Culvenor ◽

P. E. Crewther ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Drosophila Melanogaster ◽

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Protein 70

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Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7–RILP–p150Glued and late endosome positioning

The Journal of Cell Biology ◽

10.1083/jcb.200811005 ◽

2009 ◽

Vol 185 (7) ◽

pp. 1209-1225 ◽

Cited By ~ 379

Author(s):

Nuno Rocha ◽

Coenraad Kuijl ◽

Rik van der Kant ◽

Lennert Janssen ◽

Diane Houben ◽

...

Keyword(s):

Motor Proteins ◽

Oxysterol Binding Protein ◽

Membrane Contact Sites ◽

Cholesterol Levels ◽

Dynein Motor ◽

Late Endosomes ◽

In Trans ◽

Membrane Contact ◽

Cholesterol Control ◽

Niemann Pick

Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150Glued bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)–LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7–RILP complex to remove p150Glued and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

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Oskar anchoring restricts pole plasm formation to the posterior of the Drosophila oocyte

Development ◽

10.1242/dev.129.15.3705 ◽

2002 ◽

Vol 129 (15) ◽

pp. 3705-3714 ◽

Cited By ~ 14

Author(s):

Nathalie F. Vanzo ◽

Anne Ephrussi

Keyword(s):

Drosophila Melanogaster ◽

Translational Control ◽

Cell Formation ◽

Positional Information ◽

Mrna Localization ◽

Posterior Pole ◽

Tight Coupling ◽

Anteroposterior Patterning ◽

Pole Cell ◽

Pole Plasm

Localization of the maternal determinant Oskar at the posterior pole of Drosophila melanogaster oocyte provides the positional information for pole plasm formation. Spatial control of Oskar expression is achieved through the tight coupling of mRNA localization to translational control, such that only posterior-localized oskar mRNA is translated, producing the two Oskar isoforms Long Osk and Short Osk. We present evidence that this coupling is not sufficient to restrict Oskar to the posterior pole of the oocyte. We show that Long Osk anchors both oskar mRNA and Short Osk, the isoform active in pole plasm assembly, at the posterior pole. In the absence of anchoring by Long Osk, Short Osk disperses into the bulk cytoplasm during late oogenesis, impairing pole cell formation in the embryo. In addition, the pool of untethered Short Osk causes anteroposterior patterning defects, owing to the dispersion of pole plasm and its abdomen-inducing activity throughout the oocyte. We show that the N-terminal extension of Long Osk is necessary but not sufficient for posterior anchoring, arguing for multiple docking elements in Oskar. This study reveals cortical anchoring of the posterior determinant Oskar as a crucial step in pole plasm assembly and restriction, required for proper development of Drosophila melanogaster.

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Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1900266116 ◽

2019 ◽

Vol 116 (35) ◽

pp. 17498-17508 ◽

Cited By ~ 3

Author(s):

Pallavi Singh ◽

Aditi Alaganan ◽

Kunal R. More ◽

Audrey Lorthiois ◽

Sabine Thiberge ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Transmission ◽

Membrane Remodeling ◽

Gene Encoding ◽

Conditional Deletion ◽

Male Gametes ◽

Complex Process ◽

Intracellular Vesicles ◽

Vesicle Secretion

Transmission of Plasmodium falciparum involves a complex process that starts with the ingestion of gametocytes by female Anopheles mosquitoes during a blood meal. Activation of gametocytes in the mosquito midgut triggers “rounding up” followed by egress of both male and female gametes. Egress requires secretion of a perforin-like protein, PfPLP2, from intracellular vesicles to the periphery, which leads to destabilization of peripheral membranes. Male gametes also develop flagella, which assist in binding female gametes for fertilization. This process of gametogenesis, which is key to malaria transmission, involves extensive membrane remodeling as well as vesicular discharge. Phospholipase A2 enzymes (PLA2) are known to mediate membrane remodeling and vesicle secretion in diverse organisms. Here, we show that a P. falciparum patatin-like phospholipase (PfPATPL1) with PLA2 activity plays a key role in gametogenesis. Conditional deletion of the gene encoding PfPATPL1 does not affect P. falciparum blood stage growth or gametocyte development but reduces efficiency of rounding up, egress, and exflagellation of gametocytes following activation. Interestingly, deletion of the PfPATPL1 gene inhibits secretion of PfPLP2, reducing the efficiency of gamete egress. Deletion of PfPATPL1 also reduces the efficiency of oocyst formation in mosquitoes. These studies demonstrate that PfPATPL1 plays a role in gametogenesis, thereby identifying PLA2 phospholipases such as PfPATPL1 as potential targets for the development of drugs to block malaria transmission.

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Two ZBP1 KH domains facilitate β-actin mRNA localization, granule formation, and cytoskeletal attachment

The Journal of Cell Biology ◽

10.1083/jcb.200206003 ◽

2002 ◽

Vol 160 (1) ◽

pp. 77-87 ◽

Cited By ~ 144

Author(s):

Kim L. Farina ◽

Stefan Hüttelmaier ◽

Kiran Musunuru ◽

Robert Darnell ◽

Robert H. Singer

Keyword(s):

Rna Binding ◽

Leading Edge ◽

Mrna Localization ◽

Binding Studies ◽

Granule Formation ◽

Rna Recognition Motifs ◽

Hnrnp K ◽

Actin Mrna ◽

Recognition Motifs ◽

Localization Element

Chicken embryo fibroblasts (CEFs) localize β-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of β-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of β-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized β-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both β-actin mRNA localization and cell motility.

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Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor

Genes & Development ◽

10.1101/gad.531009 ◽

2009 ◽

Vol 23 (13) ◽

pp. 1546-1558 ◽

Cited By ~ 124

Author(s):

M. Dienstbier ◽

F. Boehl ◽

X. Li ◽

S. L. Bullock

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Mrna Localization ◽

Dynein Motor

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How signals of calcium ions initiate the beats of cilia and flagella

10.1101/585034 ◽

2019 ◽

Author(s):

M. V. Satarić ◽

T. Nemeš ◽

D. Sekulić ◽

J. A. Tuszynski

Keyword(s):

Atp Hydrolysis ◽

Cellular Motility ◽

Cell Organelles ◽

Ciliary Movement ◽

Ciliary Function ◽

Dynein Motor ◽

Combined Action ◽

Axonemal Dynein ◽

Cilia And Flagella

ABSTRACTCilia and flagella are cell organelles serving basic roles in cellular motility. Ciliary movement is performed by a sweeping-like repeated bending motion, which gives rise to a self-propagating “ciliary beat”. The hallmark structure in cilia is the axoneme, a stable architecture of microtubule doublets. The motion of axoneme is powered by the axonemal dynein motor family powered by ATP hydrolysis. It is still unclear how the organized beat of cilium and flagella emerges from the combined action of hundreds of dynein molecules. It has been hypothesized that such coordination is mediated by mechanical stress due to transverse, radial or sliding deformations. The beating asymmetry is crucial for airway ciliary function and it requires tubulin glutamination a unique posttranslational modification of C-termini of constituent microtubules that is highly abundant in cilia and flagella. The exact role of tubulin glutamination in ciliary or flagellar function is still unclear. Here we examine the role of calcium (Ca2+) ions based on the experimental evidences that the flagellar asymmetry can be increased due to the entry of extracellular Ca2+through, for example, nimodipine-sensitive pathway located in the flagella. We propose a new scenario based on the polyelectrolyte properties of cellular microtubules (MTs) such that dynamic influx of Ca2+ions provides the initiation and synchronization of dynein sliding along microtubules. We also point out the possible interplay between tubulin polyglutaminated C-termini and localized pulses of Ca2+ions along microtubules.

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The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly

10.1101/2021.05.11.443597 ◽

2021 ◽

Author(s):

Petra zur Lage ◽

Zhiyan Xi ◽

Jennifer Lennon ◽

Iain Hunter ◽

Wai Kit Chan ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

Cell Types ◽

Drosophila Cell ◽

Heavy Chains ◽

Neuron Response ◽

Dynein Motor ◽

Assembly Factor ◽

Axonemal Dynein ◽

Dynein Arms ◽

Ciliary Dyskinesia

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila homologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and spermatocytes, the only two Drosophila cell types bearing motile cilia/flagella. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly.

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