3D electron microscopy reveals: twin spokes are not twins

Movement of cells has fascinated scientists for centuries. Improved handcrafted light microscopes in the late 17th century allowed observations of contracting muscle fibers, single-cell organisms gliding through water drops or cells crawling across surfaces. How cell motility is generated and regulated is an ongoing question researchers at Brandeis and many other institutions are trying to answer. The single-cell green algae Chlamydomonas reinhardtii has two eukaryotic flagella (Fig. A) and is a popular genetic model system for studying these motile organelles, which are also called cilia or undulipodia. Cilia and flagella are basically the same organelles that are highly similar from single-cell algae to humans, but when a cell has many relatively short and asymmetrically beating ones they are called cilia (e.g. on the multi-ciliated epithelial cells that line our airways and are important for mucus-clearance), while a few long ones with often symmetric waveforms are called (eukaryotic) flagella (e.g. the sperm flagellum). These should not be confused with bacterial and archaeal flagella, which are very different in structure and evolutionary origin. Eukaryotic cilia and flagella consist of a microtubule-based, cylindrical core with hundreds of similar building blocks that repeat along the length of the organelle (Fig. B-D). In a single flagellum the activity of thousands of motor proteins, dyneins, has to be coordinated to generate motility, and important regulatory complexes include the radial spokes, in Chlamydomonas two spokes per building block (RS1 and RS2) (Fig. D). Recently, Dr. Thomas Heuser, a postdoc in Dr. Daniela Nicastro’s lab at Brandeis, successfully used three-dimensional electron microscopy (electron tomography) to study the structure of rapidly frozen Chlamydomonas flagella in unprecedented detail (Heuser et al. 2009).

Erin Dymek from Dr. Elizabeth Smith’s laboratory at Dartmouth College found that the concentration of Calcium ions, a known regulatory signal modulating ciliary and flagellar motility, affects dynein activity through a conserved Calmodulin and Radial Spokes associated Complex (CSC) (Dymek and Smith, 2007). Erin Dymek and Elizabeth Smith have now teamed up with Tom Heuser and Daniela Nicastro to study the 3D location of this Calcium sensing complex in flagella. In a recent paper (Dymek et al. 2011 MBoC in press) they compared the wild type structure of Chlamydomonas flagella to several artificial microRNA-interference mutants lacking parts of the CSC. They found that in all amiRNAi mutants many of the flagellar building blocks were missing one specific radial spoke, RS2, while RS1 was always present (Fig. E-G), suggesting that the Calcium sensing CSC is located at or near RS2. Interestingly, RS1 and RS2 were previously assumed to be structurally identical, their different numbering simply referred to their proximal and distal location within the repeating building block. The current study not only indicates that the CSC is required for spoke assembly and wild type motility, but as one of the most surprising outcomes it also provides evidence for heterogeneity among the radial spokes, at least at the base where the spokes are anchored to the microtubules. The same team of biologists is now continuing to study the CSC location in the flagellar building block in greater detail by improving image processing strategies to increase resolution.

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