Front Cover; Methods in Cell Biology: Septins ; Series Editors; Methods in Cell Biology: Septins ; Copyright; Contents; Contributors; Preface; 1 -- The nonopisthokont septins: how many there are, how little we know about them, and how we might learn more ; INTRODUCTION; 1. THE WIDE PHYLOGENETIC DISTRIBUTION AND INFERRED ANCIENT ORIGIN OF THE SEPTINS; 1.1 Search Methods; 1.2 Apparent Absence of Septins From the Amoebozoa; 1.3 Presence of Septins in Some, but Not All, Planta Lineages; 1.4 Presence of Septins in Cryptophytes and Haptophytes

1.5 Presence of Septins in At Least One Rhizarian Lineage1.6 Presence of Septins in At Least Some Alveolate Lineages; 1.7 Presence of Septins in At Least Some Heterokont Lineages; 1.8 Apparent Absence of Septins From the Excavata; 1.9 General Conclusions; 2. THE NEARLY TOTAL LACK OF FUNCTIONAL INFORMATION ABOUT THE NONOPISTHOKONT SEPTINS; 3. THE PROSPECTS FOR INSIGHTS FROM FUNCTIONAL ANALYSES OF NONOPISTHOKONT SEPTINS; Acknowledgments; REFERENCES; 2 -- Preparing recombinant yeast septins and their analysis by electron microscopy ; INTRODUCTION; 1. METHODS

1.1 Preparation of Recombinant Budding Yeast Septin Complexes2. MATERIALS; 2.1 Sample Preparation for Electron Microscopy; 2.1.1 Sample preparation for negative stain samples; 2.1.2 Sample preparation for thin film frozen hydrated samples; 3. MATERIALS; 3.1 Microscopy and Image Two-Dimensional Analysis; 4. MATERIALS; 4.1 Tagging Strategies to Label Septin Subunits Within Complexes; 4.1.1 Antibody labeling; 4.1.2 Covalent bound tags; CONCLUSION; Acknowledgments; REFERENCES; 3 -- A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins ; INTRODUCTION

1. PROTEIN PREPARATION AND LABELING1.1 Expression and Purification of Single-Cys Yeast Septin Heterooctamers; 1.2 Single-Cys Septin Subunits and Dye Labeling by Maleimide Chemistry; 1.3 Selection of Fluorophores for Förster Resonance Energy Transfer Analysis; 2. DATA COLLECTION AND ANALYSIS; 2.1 Spectroscopy Data Collection; 2.2 Semiquantitative Sensitized Emission; 2.3 Principal Component Analysis; CONCLUSIONS AND OUTLOOK; Acknowledgments; REFERENCES; 4 -- In vitro reconstitution of septin assemblies on supported lipid bilayers ; INTRODUCTION; 1. RATIONAL

1.1 TIRF-Based Assays for Visualizing Cytoskeletal Properties1.2 Supported Lipid Bilayers; 2. CLEANING OF GLASS COVERSLIPS; 2.1 Rational; 2.2 Reagents and Equipment; 2.3 Protocol for RCA Cleaning of Glass Coverslips; 3. PREPARATION OF SMALL UNILAMELLAR VESICLES; 3.1 Rational; 3.2 Reagents and Equipment; 3.3 Protocol for the Preparation of Small Unilamellar Vesicles; 4. FORMATION OF SUPPORTED LIPID BILAYERS; 4.1 Reagents and Equipment; 4.2 Fusion Protocol; 5. VISUALIZING SEPTIN ADSORPTION AND POLYMERIZATION ON SUPPORTED LIPID BILAYERS; CONCLUDING REMARKS; REFERENCES

5 -- Visualization of in vivo septin ultrastructures by platinum replica electron microscopy