“Similar differences”: radial spokes are different from each other, but conserved across species

Eukaryotic cilia and flagella are highly conserved organelles present on the surfaces of many animal cells and protists. These organelles are important for cell motility and/or sensing of the environment. Virtually all motile cilia and flagella share the same microtubule-based core structure, the axoneme, composed of nine doublet microtubules (DMTs) surrounding a central pair of singlet microtubules known as the central pair complex (CPC) (Fig. 1). Structurally, each DMT is built from many copies of a 96 nm-long unit that repeats along the longitudinal DMT axis (Fig. 1A). The proper assembly and function of the components of these repeat units are necessary for the generation of the characteristic wave form of ciliary and flagellar movement. Mutations in these components have been linked to various human diseases termed ciliopathies.

”]Important components of the 96 nm repeat unit include the dyneins – molecular motors that convert chemical energy into motion – and complexes that regulate dynein activity, such as radial spokes (RSs) and the Nexin-Dynein Regulatory Complex (N-DRC). The dyneins are arranged in two rows: the inner-dynein arms (IDAs) and the outer-dynein arms (ODAs) (Fig. 1B). All dynein motors are permanently anchored on one DMT and generate force by walking along the neighboring DMT, causing both DMTs to slide against one another. Connections between neighboring DMTs, e.g.the nexin links, restrict this sliding motion between DMTs and transform it into the bending motion typical of cilia and flagella. Dynein motors can only move in one direction: towards the minus end of the DMT. Consequently, dyneins on one side of the axoneme cause the axoneme to bend in one direction, whereas dyneins on the opposite side of the axoneme cause it to bend in the opposite direction (Fig. 1B).

Figure 1 (right): A) Model of the 96nm axonemal repeat containing important regulatory structures. B) An overview of the axoneme in cross section. C and D) Axoneme bending in opposing directions is achieved by alternating activation of dynein motors between opposing DMT subsets. [Heuser et al., 2009]

To generate the characteristic flagellar beating patterns, only subsets of dyneins must be active at any given time. If the dyneins were all active at once, this would put the cilia and flagella in a rigor-like state, as opposite sides of the axoneme would be trying to bend in opposing directions. Precise regulation of  dyneins present on subsets of DMTs is thus required to achieve a normal beating pattern. The current model for how cilia and flagella achieve such regulation is that the dyneins receive chemical and mechanical cues from the CPC, through the RSs, to the IDAs or through the N-DRC and the I1-dynein complex to the ODAs. The RSs are key players in this signaling pathway that regulates motility. Previously, classical electron microscopy studies have described RSs as T-shaped structures that are present either as pairs (termed RS1 and RS2; e.g. in Chlamydomonas) or triplets (termed RS1, RS2, and RS3; e.g. in mammalian cilia, sea urchin flagella, and Tetrahymena).  It was thought that RSs within pairs or triplets were all structurally identical. Recently, however, the Nicastro group and collaborator Elizabeth Smith’s lab at Dartmouth University demonstrated that RS1 and RS2 (green and blue in Fig. 2 bottom) of the RS pair in Chlamydomonas exhibit heterogeneity, i.e. their docking to the DMT and the structure of their bases are different, suggesting different roles in motility regulation (Barber et al., 2012; Dymek et al. 2011).

The Nicastro lab at Brandeis University aims at understanding the structure and function of cilia and flagella. Using state-of-the-art structural methods, such as cryo-electron tomography and sub-tomogram averaging, the Nicastro lab is visualizing the axonemes of different model organisms such as protists (e.g. the bi-flagellated alga Chlamydomonas or the ciliate Tetrahymena with thousands of cilia) and metazoa (e.g. sea urchin sperm flagella) at unprecedented detail (Nicastro et al. 2006; Heuser et al. 2009).

In a new study, Dr. Jianfeng Lin (a post-doc in the Nicastro lab) and his colleagues from the Nicastro lab have revealed a remarkably distinct RS heterogeneity that is highly conserved across species (Fig. 2) (Lin et al., 2012). At a resolution of 3.6 nm, it became obvious that RS3 (orange in Fig. 2 top) is structurally unique; bearing no resemblance to RS1 and RS2 (green and blue in Fig. 2 top). In comparison to RS1 & 2, RS3 is larger in mass, and has a bulkier and irregular shape, including the RS head, which consists of two rotationally symmetric halves in RS1 & 2, but is asymmetric in RS3 (Fig. 2 top).

Figure 2 (left): Top) Isosurface renderings of RS1 (green), RS2 (blue) and RS3 (orange) in sea urchin sperm flagella. Center) Cross section model of a sea urchin sperm flagellum displaying doublet specific features of the RSs. Bottom) Isosurface renderings of RS1 (green), RS2 (blue) and RS3S (orange) in Chlamydomonas flagella.

Perhaps the most intriguing structural difference observed was that RS3 exhibits features which are specific to only some of the nine DMTs, so called doublet-specific features (Fig. 2 center). For example, a structure termed the Radial Spoke Joist (RSJ) is only present on DMTs 3, 4, and 7-9 (magenta in Fig. 2 center). These features seem to act as a triad that connects three major regularory complexes, RS2, RS3, and the N-DRC. The doublet-specific features observed on RS3 suggest that RS3 plays a unique and currently unknown role in generating typical cilia and flagella beating patterns in sea urchin flagella and Tetrahymena cilia.

Although the Chlamydomonas axoneme contains only RS pairs, it does possess a structure that occupies the same site in each axonemal repeat that a third RS would occupy in organisms with RS triplets (orange in Fig. 2 bottom). This structure was termed the RS3-stand in (RS3S) by Barber et al. (2012). A comparison of the Chlamydomonas RS3S to RS3 in RS triplets revealed that the basal region of RS3 and the entire RS3S are virtually identical in structure (Fig. 2), suggesting that the RS3S is a shorter homolog of RS3 (Lin et al., 2012).

Although past proteomic studies identified the protein composition of the Chlamydomonas RS pair, i.e. of RS1 & 2, the new data suggest that the proteome of RS3 has yet to be determined. RS3 may play a novel role in regulating ciliary and flagellar motility and determining its protein composition in future studies should provide valuable clues to its function.

Reference: Nicastro et al. (2006) Science 313: 944-8; Heuser et al. (2009) J Cell Biol 187: 921-33; Dymek et al. (2011) Mol Biol Cell 22: 2520-31; Barber et al. (2012) Mol Biol Cell 23: 111-20; Lin et al. (2012) Cytoskeleton [Epub ahead of print].

Cryo-electron tomography and the structure of doublet microtubules

In a new paper in PNAS entitled “Cryo-electron tomography reveals conserved features of doublet microtubules“, Assistant Professor of Biology Daniela Nicastro and coworkers describe in striking new detail the structure and organization of the doublet microtubules (DMTs), the most conserved feature of eukaryotic cilia and flagella.

Cilia and flagella are thin, hair-like appendages on the surface of most animal and lower plant cells, which use these organelles to move, and to sense the environment. Defects in cilia and flagella are known to cause disease and developmental disorders, including polycystic kidney disease, respiratory disease, and neurological disorders. An essential feature of these organelles is the presence of nine outer DMTs (hollow protein tubes) that form the cylindrical core of the structure known as the axoneme. The doublet microtubule is formed by tubulin protofilaments and other structural proteins, which provide a scaffold for the attachment of dynein motors (that drive ciliary and flagellar motility) and regulatory components in a highly specific and ordered manner.

To address long-standing questions and controversies about the assembly, stability, and detailed structure of DMTs , the Nicastro lab used a high-resolution imaging technique, cryo-electron microscope tomography (cryo-ET), to probe the structure of DMTs from Chlamydomonas (single-celled algae) and sea urchin sperm flagella. Cryo-ET involves:

  1. rapid freezing of the sample to cryo-immobilize the molecules without forming ice crystals,
  2. tilting the specimen in the electron microscope to collect ~70 different views from +65° to –65°,
  3. computational alignment of the views to calculate a tomogram (a three-dimensional reconstruction of the imaged sample), and
  4. computational averaging of repeating structures in the tomogram to reduce noise and increase resolution.

Cryo-ET provided the necessary resolution to show that the B-tubules of DMTs are composed of 10 protofilaments, not 11, and that the inner and outer junctions between the A- and B-tubules are fundamentally different (see figure). The outer junction, crucial for the initial formation of the DMT, appears to be formed by interactions between the tubulin subunits of three protofilaments with unusual tubulin interfaces, but one of these protofilaments does not fit with the conventionally accepted orientation for tubulin protofilaments. This outer junction is important physiologically, as shown by mutations affecting the usual pattern of posttranslational modifications of tubulin. In contrast, the inner junction is not formed by direct interactions between tubulin protofilaments. Instead, a ladder-like structure that is clearly thinner than tubulin connects protofilaments of the A- and B-tubules.

The level of detail also allowed the Nicastro lab to show that the recently discovered microtubule inner proteins (MIPs) located within the A- and B-tubules are more complex than previously thought. MIPs 1 and 2 are both composed of alternating small and large subunits recurring every 16 and/or 48 nm along the inner A-tubule wall. MIP 3 forms small protein arches connecting the two B-tubule protofilaments closest to the inner junction, but does not form the inner junction itself. MIP 4 is associated with the inner surface of the A-tubule along the partition protofilaments, i.e., the five protofilaments of the A-tubule bounded by the two junctions with the B-tubule.

The Nicastro lab plans to build on this foundation in future work on the molecular assembly and stability of the doublet microtubule and axoneme, and hope to use it to elucidate molecular mechanisms of ciliary and flagellar motility and signal transduction in normal and disease states.

Other authors on the paper include Brandeis postdocs Xiaofeng Fu and Thomas Heuser, Brandeis undergrad Alan Tso (’10), and collaborators Mary Porter and Richard Linck from the University of Minnesota.

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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