Existence of cellular structures with specific size raises a fundamental question in biology: How do cells measure length? One conceptual answer to this question is by a molecular ruler, but examples of such rulers in eukaryotes are lacking. In this work, we identified a molecular ruler in eukaryotic cilia and flagella. Using cryo-electron tomography, we found that FAP59 and FAP172 form a 96-nanometer (nm)-long complex in Chlamydomonas flagella and that the absence of the complex disrupted 96-nm repeats of axonemes. Furthermore, lengthening of the FAP59/172 complex by domain duplication resulted in extension of the repeats up to 128 nm, as well as duplication of specific axonemal components. Thus, the FAP59/172 complex is the molecular ruler that determines the 96-nm repeat length and arrangements of components in cilia and flagella.
CLEM (correlative light and electron microscopy) is one of the powerful techniques to elucidate the localization and structure of the target proteins or their complexes in cell. First, target proteins labeled fluorescently can be searched using a fluorescence microscope, i.e., due to its low resolution (200nm), it is used as rough searching of target proteins. After rough detection of the localization of target proteins, they can be easily observed by electron microscopy with a high resolution and processed into fine structure, especially 3D structure. On the other hand, in the case of only electron microscopy, it is difficult for researchers to detect their localization due to a narrow range of views and no labeling of them.Thus, CLEM normally needs fluorescent labels for fluorescence microscopy but a label for electron microscopy is also expectedly for easier detection. Thus we focused on metallothionein. Metallothionein binds to cadmium ions, i.e., heavy atoms with strong density in electron microscopy ; in addition, cadmium ions and selenium ions are known to form Qdot-like nanoparticles induced by metallothionein . These are 2 ? 5nm in size, fluorescent wavelength changes depending on the size of nanoparticles. Thus, target proteins fused with metallothionein could be observed by both of fluorescence microscopy and electron microscopy.We here used Chlamydomonas reinhardtii, single cell green algae with two flagella. Flagella are used for bending motion and motility. Flagella contain FAP20 (Flagellar Asociate Protein 20) and PACRG (PArkin Co-Regulated gene), which are related to composing axoneme architecture. If Chlamydomonas reinhardtii doesn't have FAP20 or PACRG, they can't generate bending motion. It is considered that FAP20 and PACRG locate on the root of the radial spoke. Recently the location of FAP20 was reported by Yanagisawa et al.. First, we also focus on detecting localization of FAP20 and then will do so on that of PACKRG.We could observe fluorescence of metallothionein fused with FAP20 to form nanoparticle. We are now trying to observe larger electron density from metallothionein with cadmium for CLEM.
Cilia/flagella are assembled and maintained by the process of intraflagellar transport (IFT), a highly conserved mechanism involving more than 20 IFT proteins. However, the functions of individual IFT proteins are mostly unclear. To help address this issue, we focused on a putative IFT protein TTC26/DYF13. Using live imaging and biochemical approaches we show that TTC26/DYF13 is an IFT complex B protein in mammalian cells and Chlamydomonas reinhardtii. Knockdown of TTC26/DYF13 in zebrafish embryos or mutation of TTC26/DYF13 in C. reinhardtii, produced short cilia with abnormal motility. Surprisingly, IFT particle assembly and speed were normal in dyf13 mutant flagella, unlike in other IFT complex B mutants. Proteomic and biochemical analyses indicated a particular set of proteins involved in motility was specifically depleted in the dyf13 mutant. These results support the concept that different IFT proteins are responsible for different cargo subsets, providing a possible explanation for the complexity of the IFT machinery. DOI: http://dx.doi.org/10.7554/eLife.01566.001.
Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella.
In flagella, the outer dynein arm (ODA) and inner dynein arm (IDA) play distinct roles in generating beating motion. However, functional communications between the two dyneins have not been investigated.
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