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Find video protocols related to scientific articles indexed in Pubmed.
Cilia and flagella. A molecular ruler determines the repeat length in eukaryotic cilia and flagella.
Science
PUBLISHED: 11-15-2014
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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.
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Metallothionein labeling for CLEM method for identification of protein subunits.
Microscopy (Oxf)
PUBLISHED: 11-01-2014
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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 [1]; in addition, cadmium ions and selenium ions are known to form Qdot-like nanoparticles induced by metallothionein [2]. 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.[3]. 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.
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TTC26/DYF13 is an intraflagellar transport protein required for transport of motility-related proteins into flagella.
Elife
PUBLISHED: 03-06-2014
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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.
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Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity.
J. Cell Biol.
PUBLISHED: 03-05-2014
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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.
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Identification of the outer-inner dynein linker as a hub controller for axonemal dynein activities.
Curr. Biol.
PUBLISHED: 02-14-2013
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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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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.