1 / 42

A road map for cell biology: Why studying large protein complexes is crucial at this time

Learn why studying large protein complexes is crucial in understanding cellular processes and diseases. Explore the various approaches and technologies used in these studies, including structural proteomics, mass spectrometry, light microscopy, genetics, and chemical biology.

tammyharrison
Download Presentation

A road map for cell biology: Why studying large protein complexes is crucial at this time

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. A road map for cell biology: Why studying large protein complexes is crucial at this time David Drubin, UC Berkeley

  2. Why study large complexes? • Proteins typically function in association with other proteins. • Protein complexes are important for virtually every biological process and most diseases. • Genome sequences identify tens of thousands of genes: linking these to 200-300 core biological processes will make their study manageable. • Recently developed and/or improved technologies and methodologies make studies of large complexes more feasible and informative.

  3. A brief tour of some protein complexes and their biological and biomedical importance (from the Alberts et al. textbook)

  4. Nucleosome

  5. DNA Polymerase

  6. Nuclear pores

  7. Ribosome

  8. Proteosome

  9. Cell Cycle: Short-lived complexes

  10. COPII-coated vesicle

  11. MT motor - vesicle

  12. Enzyme (Aspartate transcarbamoylase); two conformations

  13. Red blood cell skeleton

  14. Signal transduction complex

  15. Approaches to studies of protein complexes Many new and improved approaches for studies of protein complexes are now available These approaches can be used in targeted or genome-wide studies Using combinations of these approaches makes the most of their complementary features

  16. Structural Proteomics Mutant/RNAi/ Chemical phenotypes Protein structure Protein-protein interactions Biological function mechanism regulation Genetic interactions Post-translational modifications Expression studies Biochemicalactivities Protein localization

  17. Features of genome-wide, non-targeted “big science” Pros: *Efficiency *Unbiased *Patterns can emerge that only are evident when analyzing large datasets Cons: *Validation and follow-up, quality control

  18. Features of targeted studies “small science” Pros: *Science is an iterative process -much can be missed with one pass *Appreciation of vital subtleties of a system, knowing where to look and what to look for *Tools and insights for validation and quality control Cons: *Less efficient

  19. Non-Targeted “Big Science” Targeted “small science”

  20. Technologists Biologists

  21. Mass Spectrometry Mass spectrometry is having a revolutionary effect on identification of subunits of protein complexes and their post-translational modifications

  22. The Budding Yeast Kinetochore

  23. (Scott Anderson and John Yates)

  24. Molecular Analysis of Kinetochore Composition and Organization 28 kinetochore proteins purified (75% of total) 3 novel kinetochore sub-complexes defined 5 novel kinetochore proteins identified

  25. Light microscopy Light microscopy can verify the in vivo relevance of proteomically observed associations, and provide valuable information about dynamics

  26. Dad3p and Dad4p Localize to Spindles and Kinetochores Spindle (Intact Cells) Kinetochore (Chromosome Spreads)

  27. Ipl1p Targets 6 1 0 3 Tandem Mass Spectrometry Mapping of Phosphorylation Sites in vivo Phosphorylation Sites Dam1p Complex - 13 Ndc80p Complex - 1 Ctf19p Complex - 0 Ipl1p Complex - 4

  28. Genetics Genetics can verify in vivo relevance of post- translational modifications, associations, etc.

  29. dam1 (S to D) Mutants Suppress ipl1-2 Wild Type dam1 (S to D) ipl1-2 ipl1-2 dam1 (S to D)

  30. The current demand for mass spectrometry outstrips the existing capacity • Cell cycle stages • Response to stimuli and perturbation • Affects of various mutants

  31. Clone John Yates

  32. Two hybrid Arrays • Yeast Resource Center (Stan Fields) • All yeast ORFs on 16 plates

  33. Datasets from non-targeted proteomics HIGH QUALITY datasets from large-scale, genome-wide analyses such as two-hybrid screens, mass spec of complexes, etc., can greatly accelerate biological research.

  34. Yeast WASp

  35. Light microscopy is critical for determining where and when proteins associate in complexes GFP, its spectral variants, and DsRed are driving the need for advanced imaging systems to study protein complexes in vivo -High speed multi-color analysis -FRET -FRAP -Automated image acquisition and analysis

  36. Endocytic protein interactions

  37. Why doesn’t the whole cytoplasm precipitate? Interactions are regulated in space and time!

  38. Chemical Biology (Genetics) Small molecule inhibitors can be powerful tools to switch on and off associations within protein complexes in vivo

  39. Summary • Protein complexes are involved in virtually every cellular process and disease • Complete genome sequences combined with availability of new technologies make studies of protein complexes crucial at this time • The focus of all such studies should be on illuminating biological and disease mechanisms

  40. Yeast Resource Center (John Yates, Stan Fields, Trisha Davis) Kevan Shokat (UCSF) Barnes and Drubin labs (UCB) Acknowledgements

More Related