Cryo Electron Microscopy Structural Characterization Of Outer Dynein Arm And The Role Of Post Translational Modification In Eukaryotic Cilia
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Cryo-electron Microscopy Structural Characterization of Outer Dynein Arm and the Role of Post-translational Modification in Eukaryotic Cilia
"Cilia are microtubules-based organelles that protrude outside of almost every eukaryotic cells. Cilia are the main players in many cellular functions, such as motility, signal transduction, and embryonic development. The building and maintenance of cilia are achieved by many proteins working together in a process called intraflagellar transport (IFT). During IFT, tubulins, the building block of cilia, are carried into the cilium to construct doublet microtubules. Big subcomplex such as axonemal outer dynein arm (ODA) complex is pre-packed in the cytoplasm before being transported into the cilia for assembly. ODA comprises dynein heavy chains (HCs), intermediate chains, and light chains responsible for ATP-dependent force production that leads to cilia bending. Recently, it was shown in Tetrahymena thermophila that a regulatory protein, Shulin, packs together three dyneins HCs of ODA to keep the HC heads in an inactive pre-powerstroke conformation during IFT. To uncover how the inactive form of ODA becomes activated, we obtained the cryo-EM structure of the active ODA bound onto the doublet microtubule via the docking complex. Using molecular dynamic simulation, we propose a model of ODA attachment to doublet microtubule to induce remodelling and activation of the ODA. The motility rate of ODA could be regulated by post-translational modification (PTM) within the cilia, such as acetylation of the doublet microtubules (DMT). PTMs of ciliary proteins, such as acetylation, polyglutamylation, and phosphorylation, maintain and regulate ciliary functions. Tubulin becomes acetylated by [alpha]-tubulin acetyltransferase-1 ([alpha]TAT1) after being assembled into the doublet microtubules (DMT). One of the main sites for acetylation is within the lumen of DMT, on lysine 40 (K40) of [alpha]-tubulin. The K40 loop is structurally flexible and disordered during in vitro reconstitution. In Tetrahymena thermophila, cells carrying a K40R substitution in [alpha]-tubulin show loss of acetyl-[alpha]-tubulin, while [alpha]TAT1 knock-out cells (MEC-17) show complete loss of acetyl-[alpha]-tubulin in cilia that led to short and labile cilia. To understand the effect of acetylation in the cilia, we obtained 3.5 Angstrom resolution cryo-EM structures of doublet microtubules from wild type (WT) and mutants (K40R and MEC-17) that have un-acetylated tubulins. The K40 loops were visible in both mutants and the WT. Using molecular dynamics, the inner junction region of the WT structure shows lower energy and better stability when acetylated, suggesting that acetylation may play a role in microtubule inner protein interaction and stability. Our mass spectrometry results identified kinase, phosphatase, and polyglutamylation-related enzymes that are downregulated in the acetylation mutants. We propose a model that, shifting the balance of one type of PTM may initiate a compensatory effect by another type of PTM to maintain cilia function"--
The Chlamydomonas Sourcebook
The Chlamydomonas Sourcebook, 3rd Edition Introduction to Chlamydomonas and Its Laboratory Use (Volume 1) The gold-standard reference covering the basic biology of the Chlamydomonas alga and techniques for its laboratory analysis Originally published as the standalone Chlamydomonas Sourcebook, then expanded as the first volume in a three-part comprehensive gold-standard reference, The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use has been fully revised and updated to include a wealth of new resources for the Chlamydomonas community. Early chapters cover current understandings of its taxonomy, ultrastructure, cell and life cycles, and nuclear and organelle genomes, followed by technique-oriented chapters covering such topics as cell culture, mutagenesis, genetic analysis, construction of mutant libraries, and protein localization using immunofluorescence. This volume presents the latest in research and best practices, making it a must-have resource for researchers and students working in plant science and photosynthesis, fertility, mammalian vision, and biochemistry; crop scientists; plant physiologists; and plant, molecular, and human disease biologists. - Remains the only complete reference to provide both the historical background and the most up-to-date information and applications on Chlamydomonas - Includes best practices for applications in research, including methods for culture, genetic analysis, genomic and transcriptomic analysis, and mutant screening - Helps researchers solve common laboratory problems, provides details on the properties of particular strains, and offers a comprehensive survey of molecular approaches - Provides a broad perspective for studies in cell and molecular biology, genetics, plant physiology, and related fields
Dyneins
Research on dyneins has a direct impact on human diseases, such as viruses and cancer. With an accompanying website showing over 100 streaming videos of cell dynamic behavior for best comprehension of material, Dynein: Structure, Biology and Disease is the only reference covering the structure, biology and application of dynein research to human disease. From bench to bedside, Dynein: Structure, Biology and Disease offers research on fundamental cellular processes to researchers and clinicians across developmental biology, cell biology, molecular biology, biophysics, biomedicine, genetics and medicine. Broad-based up-to-date resource for the dynein class of molecular motors Chapters written by world experts in their topics Numerous well-illustrated figures and tables included to complement the text, imparting comprehensive information on dynein composition, interactions, and other fundamental features