Sloboda Roger D.

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Sloboda
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Roger D.
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  • Preprint
    Protein methylation in full length Chlamydomonas flagella
    ( 2009-04-28) Sloboda, Roger D. ; Howard, Louisa
    Post-translational protein modification occurs extensively in eukaryotic flagella. Here we examine protein methylation, a protein modification that has only recently been reported to occur in flagella (Schneider et al. 2008). The cobalamin (vitamin B12) independent form of the enzyme methionine synthase (MetE), which catalyzes the final step in methionine production, is localized to flagella. Here we demonstrate, using immunogold scanning electron microscopy, that MetE is bound to the outer doublets of the flagellum. Methionine can be converted to Sadenosyl methionine, which then serves as the methyl donor for protein methylation reactions. Using antibodies that recognize symmetrically or asymmetrically methylated arginine residues, we identify three highly methylated proteins in intact flagella: two symmetrically methylated proteins of about 30 and 40 kDa, and one asymmetrically methylated protein of about 75 kDa. Several other relatively less methylated proteins could also be detected. Fractionation and immunoblot analysis shows that these proteins are components of the flagellar axoneme. Immunogold thin section electron microscopy indicates that the symmetrically methylated proteins are located in the central region of the axoneme, perhaps as components of the central pair complex and the radial spokes, while the asymmetrically methylated proteins are associated with the outer doublets.
  • Article
    The Chlamydomonas flagellar membrane glycoprotein FMG-1B is necessary for expression of force at the flagellar surface
    (Company of Biologists, 2019-08-19) Bloodgood, Robert A. ; Tetreault, Joseph ; Sloboda, Roger D.
    In addition to bend propagation for swimming, Chlamydomonas cells use their flagella to glide along a surface. When polystyrene microspheres are added to cells, they attach to and move along the flagellar surface, thus serving as a proxy for gliding that can be used to assay for the flagellar components required for gliding motility. Gliding and microsphere movement are dependent on intraflagellar transport (IFT). Circumstantial evidence suggests that mechanical coupling of the IFT force-transducing machinery to a substrate is mediated by the flagellar transmembrane glycoprotein FMG-1B. Here, we show that cells carrying an insertion in the 5′-UTR of the FMG-1B gene lack FMG-1B protein, yet assemble normal-length flagella despite the loss of the major protein component of the flagellar membrane. Transmission electron microscopy shows a complete loss of the glycocalyx normally observed on the flagellar surface, suggesting it is composed of the ectodomains of FMG-1B molecules. Microsphere movements and gliding motility are also greatly reduced in the 5′-UTR mutant. Together, these data provide the first rigorous demonstration that FMG-1B is necessary for the normal expression of force at the flagellar surface in Chlamydomonas. This article has an associated First Person interview with authors from the paper.
  • Article
    Protein arginine methyltransferases interact with intraflagellar transport particles and change location during flagellar growth and resorption
    (American Society for Celll Biology, 2017-03-15) Mizuno, Katsutoshi ; Sloboda, Roger D.
    Changes in protein by posttranslational modifications comprise an important mechanism for the control of many cellular processes. Several flagellar proteins are methylated on arginine residues during flagellar resorption; however, the function is not understood. To learn more about the role of protein methylation during flagellar dynamics, we focused on protein arginine methyltransferases (PRMTs) 1, 3, 5, and 10. These PRMTs localize to the tip of flagella and in a punctate pattern along the length, very similar, but not identical, to that of intraflagellar transport (IFT) components. In addition, we found that PRMT 1 and 3 are also highly enriched at the base of the flagella, and the basal localization of these PRMTs changes during flagellar regeneration and resorption. Proteins with methyl arginine residues are also enriched at the tip and base of flagella, and their localization also changes during flagellar assembly and disassembly. PRMTs are lost from the flagella of fla10-1 cells, which carry a temperature-sensitive mutation in the anterograde motor for IFT. The data define the distribution of specific PRMTs and their target proteins in flagella and demonstrate that PRMTs are cargo for translocation within flagella by the process of IFT.
  • Article
    A protein methylation pathway in Chlamydomonas flagella is active during flagellar resorption
    (American Society for Cell Biology, 2008-08-13) Schneider, Mark J. ; Ulland, Megan ; Sloboda, Roger D.
    During intraflagellar transport (IFT), the regulation of motor proteins, the loading and unloading of cargo and the turnover of flagellar proteins all occur at the flagellar tip. To begin an analysis of the protein composition of the flagellar tip, we used difference gel electrophoresis to compare long versus short (i.e., regenerating) flagella. The concentration of tip proteins should be higher relative to that of tubulin (which is constant per unit length of the flagellum) in short compared with long flagella. One protein we have identified is the cobalamin-independent form of methionine synthase (MetE). Antibodies to MetE label flagella in a punctate pattern reminiscent of IFT particle staining, and immunoblot analysis reveals that the amount of MetE in flagella is low in full-length flagella, increased in regenerating flagella, and highest in resorbing flagella. Four methylated proteins have been identified in resorbing flagella, using antibodies specific for asymmetrically dimethylated arginine residues. These proteins are found almost exclusively in the axonemal fraction, and the methylated forms of these proteins are essentially absent in full-length and regenerating flagella. Because most cells resorb cilia/flagella before cell division, these data indicate a link between flagellar protein methylation and progression through the cell cycle.