Golgi, 1st ed. 2023 Methods and Protocols Methods in Molecular Biology Series, Vol. 2557

This volume provides readers with a collection of new and classical methods, techniques, and applications used to address enduring questions about the structure and functions of the Golgi complex. The chapters in this volume cover diverse topics ranging from model systems; live and fixed cell imaging techniques; in vitro biochemical reconstitution systems; and specific methods developed to study Golgi formation, maintenance, and functions under physiological and pathological conditions. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and authoritative, Golgi: Methods and Protocols is a valuable tool for researchers in the field who wish to explore new areas of Golgi biology and for new investigators interested in exploring Golgi structure and function.

Preface…
Table of Contents…
Contributing Authors…

Part I Model Systems Used to Study Golgi Organization and Function

1. Live Cell Imaging of Yeast Golgi Dynamics
Giancarlo Costaguta, Gregory S. Payne, and Lydia Daboussi 

2. Quantification of Golgi Protein Mislocalization to the Budding Yeast Vacuole
Mariana Jimenez, Jordan T. Best, Swapneeta S. Date, and Todd R. Graham

3. Immunofluorescence as a Method to Study Golgi Organization in Larval Salivary Glands of Drosophila melanogaster
Tetyana Chorna and Julie A. Brill

4. Microscopy and Immunocytochemistry-Based Methods to Study Cell Wall Biosynthetic Enzymes in the Golgi
Sang-Jin Kim and Federica Brandizzi

5. Reconstitution of Golgi Biogenesis in Permeabilized Trypanosoma brucei Cells
Graham Warren 

6. Generation of GM130 Conditional Knockout Mouse
Mei Mei and Shilai Bao

7. Monitoring Effects of Membrane Traffic via Changes in Cell Polarity and Morphogenesis in Three-Dimensional Human Pluripotent Stem Cell Cysts
Maha M. Hamed, Kenichiro Taniguchi, and Mara C. Duncan 

Part II Morphological Studies of Golgi Organization and Function

8. Immunofluorescence Microscopy of the Mammalian Golgi Apparatus
Maryam Arab, Sanjeev Mudavathu, Teresa Vitali, and Martin Lowe

9. Studying the Organization of the Golgi by Super-Resolution Microscopy
Hieng Chiong Tie and Lei Lu

10. Super-Resolution Live Imaging of Cargo Traffic Through the Golgi Apparatus in Mammalian Cells
Takuro Tojima, Daisuke Miyashiro, Yasuhito Kosugi, and Akihiko Nakano

11. Studying Golgi Structure and Function by Thin Section TEM
Helena Vihinen and Eija Jokitalo

12. Algorithm for Modern Electron Microscopic Examination of the Golgi Complex
Alexander A. Mironov and Galina V. Beznoussenko

13. High-Pressure Freezing Followed by Freeze-Substitution, An Optimal Electron Microscope Technique to Study Golgi Apparatus Organization and Membrane Trafficking
Shijie Liu, Irina D. Pokrovskaya, and Brian Storrie

14. Negative Staining Electron Microscopy for Morpho-Functional Characterization of Purified Golgi Membranes from Mammalian Cells
Stefano De Tito, Gabriele Turacchio, and Carmen Valente

15. An Electron Tomographic Analysis of Giantin Deficient Golgi Proposes a New Function of the Golgin Protein Family
Ayano Satoh, Mitsuko Hayashi-Nishino, and Kunihiko Nishino

16. Modeling the Cryo-EM Structure of the Exocyst Complex
Kunrong Mei and Wei Guo

17. Quantitatively Assessing Co-Localization of Golgi Proteins by Distance Analysis Using the DiAna Software
Nina Freimuth and Michael Sauer

18. A Primer on Deep Learning-Based Cellular Image Classification of Changes in the Spatial Distribution of the Golgi Apparatus After Experimental Manipulation
Daisuke Takao, Yuki M. Kyunai, Yasushi Okada, and Ayano Satoh

Part III Biochemical and In Vitro Reconstitution Assays in Golgi Studies

19. Reconstituting Vesicle Budding from the TGN and Immunoisolation of Vesicles Enriched with a Specific Cargo Client
Mo Wang, Jinhui Wang, and Yusong Guo

20. Common Assays in Mammalian Golgi Studies
Jie Li, Jianchao Zhang, Sarah Bui, Erpan Ahat, Divya Kolli, Whitney Reid, Lijuan Xing, and Yanzhuang Wang

21. In Vitro Methods to Investigate the Disassembly of the Golgi Ribbon during the G2-M Transition of the Cell Cycle
Inmaculada Ayala and Antonino Colanzi 

22. Generation and Analysis of hTERT-RPE1 VPS54 Knock-Out and Rescued Cell Lines
Amrita Khakurel, Tetyana Kudlyk, and Vladimir V. Lupashin

23. Rapid COG Depletion in Mammalian Cell by Auxin-Inducible Degradation System
Farhana Taher Sumya, Irina D. Pokrovskaya, and Vladimir V. Lupashin

24. Generation of Stable Cell Lines Expressing Golgi Reassembly Stacking Proteins (GRASPs) by Viral Transduction
Sarah Bui, Jie Li, Drew Stark, Ali Houmani, and Yanzhuang Wang

25. Quantitative Proteomics Analysis of Purified Rat Liver Golgi
Xuequn Chen and Yanzhuang Wang

26. Comparative Genomics for Evolutionary Cell Biology using AMOEBAE: Understanding the Golgi and Beyond
Lael D. Barlow, William Maciejowski, Kiran More, Kara Terry, Romana Vargová, Kristína Záhonová, and Joel B. Dacks

27. Common Markers and Small Molecule Inhibitors in Golgi Studies
Sarah Bui, Drew Stark, Jie Li, Jianchao Zhang, and Yanzhuang Wang

Part IV Mechanism of Golgi Structure Formation and Membrane Trafficking

28. Visualizing Reversible Cisternal Stacking in Budding Yeast Pichia pastoris
Roma Dahara, Bhawik Jain, and Dibyendu Bhattacharyya

29. Methods for Studying Membrane-Proximal GAP Activity on Prenylated Rab GTPase Substrates
Carolyn M. Highland, Laura L. Thomas, and J. Christopher Fromme

30. Studying the Role of Lipid Geometry in COPI Vesicle Formation
Hyewon Choi, Kunyou Park, Victor W. Hsu, and Seung-Yeol Park

31. Unbiased Quantification of Golgi Scattering and Golgi-Centrosome Association
Keyada B. Frye, Xiaodong Zhu, Alexey Khodjakov, and Irina Kaverina

32. Detection and Analysis of Microtubule Nucleator γ-Tubulin Ring Complex
Franco K.C. Au, Khoi T.D. Le, and Robert Z. Qi

33. Quantification of Golgi Entry and Exit Kinetics of Protein Cargoes
Jingqi Wang, Ellie Hyun-Jung Cho, Paul A. Gleeson, and Lou Fourriere

34. CARTS Formation Assay
Yuichi Wakana and Mitsuo Tagaya

35. Quantification of Protein Exit at the Trans-Golgi Network
Mai Ly Tran, Yeongho Kim, and Julia von Blume

36. Endosomal Transport to Lysosomes and the Trans-Golgi Network in Neurons and Other Cells: Visualizing Maturational Flux
Ryan J. Mulligan, Chan Choo Yap, and Bettina Winckler

37. Immunoprecipitation and Western Blot Analysis of AP-1 Clathrin-Coated Vesicles
Margaret Kell, Abby Halpern, and Heike Fölsch

38. Live imaging of Golgi Outposts in Drosophila Dendritic Arbors
Josephine W. Mitchell and Jill Wildonger

39. Targeted Protein Unfolding at the Golgi Apparatus
Jasmin Schillinger and Doris Hellerschmied

Part V Golgi Functions in Physiology and Pathology

40. Modeling N-Glycosylation: A Systems Biology Approach for Evaluating Changes in the Steady State Organization of Golgi Resident Proteins
Rebecca Morgan, Ben West, A. Jamie Wood, and Daniel Ungar

41. Simple N-Glycan Profile Analysis Using Lectin Staining, Mass Spectrometry, and GlycoMaple
Wei-Wei Ren and Morihisa Fujita

42. In Vitro Methods to Study the Golgi Apparatus Role in Proteoglycan and Glycosaminoglycan Synthesis
Kristian Prydz

43. Analysis of Golgi Protein Acetylation using In Vitro Assays and Parallel Reaction Monitoring Mass Spectrometry
Dea Slade and Markus Hartl

44. Identification and Characterization of GRASP55 O-GlcNAcylation
Xiaoyan Zhang

45. Assaying Sterol-Regulated ER-to-Golgi Transport of SREBP Cleavage-Activating Protein Using Immunofluorescence Microscopy
Chiaki T. Ishida, Wei Shao, and Peter J. Espenshade

46. Analysis of Golgi Morphology Using Immunofluorescence and CellProfiler Software
Isabel Mejia, Yu-Chuan Chen, and Begoña Díaz

47. Analysis of Golgi Secretory Functions in Cancer
Priyam Banerjee, Xiaochao Tan, William K. Russell, and Jonathan M. Kurie

Subject Index List…

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts