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Updated classic and highly innovative techniques essential to the study of cilia and flagella

Table of Contents

1. RNAi approaches to axonemal motor function in trypanosomes (Hill) 2. Homologous recombination to knock out dynein genes in Tetrahymena (Gaertig) 3. RNAi in Chlamydomonas (Cerutti) 4. Planarians as a model system for analysis of ciliary assembly/motility (Rompolas) 5. Role of nucleotides in dynein function (Shingyoji) 6. Protein modification to probe dynein interactions + purification and identification of crosslinked dynein products (Sakato) 7. Analysis of redox-sensitive dynein components (Wakabayashi) 8. Purification of dyneins from Chlamydomonas and analysis of dynein-dynein linkers (Kamiya/King) 9. Kinase/phosphatase-mediated control of dynein function (Sale) 10. Central pair MT complex and associated kinesins (Mitchell) 11. Calcium regulation of axonemal function In vitro motility assays (Smith) 12. Identification and analysis of dynein regulatory complex components (Porter) 13. Isolation and analysis of radial spoke proteins (Yang) 14. Purification of dyneins from other model organisms: Ciona, sea urchin, fish (Inaba) 15. Rescue of mutant phenotypes by protein electroporation (Kamiya Lab) 16. Tubulin/Dynein interactions (Raff) 17. Measurement of beat frequency and swimming velocity Cell model reactivation in vitro assays of dynein function (Kamiya) 18. Live imaging of ependymal cilia (Lechtreck/Witman) 19. Immunogold labeling of axonemal components in situ + flat embedding (Geimer) 20. CryoEM approaches to axonemal organization (Nicastro) 21. CryoEM of dynein-microtubule complexes (Oda/Kikkawa) 22. Bioinformatic approaches to dynein HC classification (Yagi) 23. Bioinformatics of non-motor dynein components (Asai) 24. Biophysical approaches to dynein mechanism (Sutoh) 25. Chlamydomonas flagellar beat analysis (Foster) 26. Biophysical measurements of motor function (step size/force production) SAXS of axonemes (Toba/Oiwa)

Cryo-Electron Microscope Tomography to Study Axonemal

Organization

Daniela Nicastro

2. Electron Microscopic Imaging and Analysis of Isolated

Dynein Particles

Anthony J. Roberts and Stan A. Burgess

3. Immunogold Labeling of Flagellar Components In Situ

Stefan Geimer

4. Scanning Electron Microscopy to Examine Cells and Organs

Jovenal T. SanAgustin, John A. Follit, Gregory Hendricks, and Gregory J. Pazour

5. X-ray Fiber Diffraction Studies on Flagellar Axonemes

Kazuhiro Oiwa, Shinji Kamimura, and Hiroyuki Iwamoto

6. Markers for Neuronal Cilia

Jacqueline S. Domire and Kirk Mykytyn

7. Immunofluorescence Staining of Ciliated Respiratory Epithelial Cells

Heymut Omran and Niki T. Loges

8. Immunoprecipitation to Examine Protein Complexes

Gregory J. Pazour

9. Tandem Affinity Purification of Ciliopathy-Associated Protein

Complexes

Karsten Boldt, Jeroen van Reeuwijk, Christian Johannes Gloeckner,

Marius Ueffing, and Ronald Roepman

10. Crosslinking Methods: Purification and Analysis of Crosslinked Dynein Products

Miho Sakato

11. Analysis of the Ciliary/Flagellar Beating of Chlamydomonas

Kenneth W. Foster

12. Assays of Cell and Axonemal Motility in Chlamydomonas reinhardtii

Ritsu Kamiya

13. High-Speed Digital Imaging of Ependymal Cilia in the Murine Brain

Karl-Ferdinand Lechtreck, Michael J. Sanderson, and George B. Witman

14. Observation of Nodal Cilia Movement and Measurement of Nodal Flow

Yasushi Okada and Nobutaka Hirokawa

15. Modification of Mouse Nodal Flow by Applying Artificial Flow

Shigenori Nonaka

16. Measuring Cilium-Induced Ca2þ Increases in Cultured Renal Epithelia

Helle A. Praetorius

About the Author

Stephen M. King is Professor of Molecular Biology and Biophysics at the University of Connecticut School of Medicine and is also director of the electron microscopy facility. He has studied the structure, function and regulation of dyneins for over 30 years using a broad array of methodologies including classical/molecular genetics, protein biochemistry, NMR structural biology and molecular modeling, combined with cell biological approaches, imaging and physiological measurements.