Making organelles visible - in planta and in societas
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This is the presentation I gave in Salzburg at the Annual Meeting of the Society for Experimental Biology, July 2012, for receiving the President's Medal for Education and Public Affairs. http://www.sebiology.org/meetings/Salzburg2012/pres_meds.html
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Making organelles visible - in planta and in societas
- 1. Dr Anne Osterrieder SEB Salzburg July 2012 @AnneOsterrieder #seb2012
- 2. Golgi apparatus http://multimedia.mcb.harvard.edu/media.htm
- 4. Why study the Golgi apparatus? • Central role in secretory pathway • Production of cell wall material • Fundamental research - but understanding these processes necessary to understand and improve plant productivity
- 7. Fluorescent proteins © Rosario Beach Marine Laboratory http://www.tsienlab.ucsd.edu
- 8. Golgi bodies in tobacco leaf cell labelled with green fluorescent protein (GFP)
- 9. How are Golgi bodies formed? How do they maintain their structure? (...especially during movement!)
- 10. By Jack and Jason's Pancakes (www.jackandjasons.com)
- 11. Osterrieder 2012, Journal of Microscopy
- 12. Characterising golgins with big lasers at the Central Laser Facility, STFC, Harwell 10 µm
- 13. FRET (Fluorescence Resonance Energy Transfer) We use FRET to study interactions between GFP- and mRFP-labelled proteins. Energy transfer occurs if: - donor fluorophore emission spectrum overlaps significantly with acceptor absorption - Fluorophores are in close proximity (1-10 nm) GFP-donor (D) and mRFP-acceptor (A) fused to proteins of interest (A and B) Sparkes et al. 2010, J. Exp. Bot.
- 14. FLIM (Fluorescence Lifetime Imaging Microscopy) • Monitoring lifetime of GFP by measuring the nanosecond decay of the fluorophore • Lifetime is unaffected by probe expression or excitation intensity BUT GFP lifetime is decreased by FRET!
- 15. Fluorescent lifetime maps visualise interactions between trans-Golgi proteins and small regulatory proteins 10 µm Average lifetime : 2.5 ns Osterrieder et al. Traffic 2009
- 16. Fluorescent lifetime maps visualise interactions between trans-Golgi proteins and small regulatory proteins 10 µm 10 µm Average lifetime : 2.5 ns 2.4 ns Osterrieder et al. Traffic 2009
- 17. Fluorescent lifetime maps visualise interactions between trans-Golgi proteins and small regulatory proteins 10 µm 10 µm 10 µm Average lifetime : 2.5 ns 2.4 ns 2.1 ns (p= 1.14 x 10−22) Osterrieder et al. Traffic 2009
- 18. Transient interactions? 10 µm Average lifetimes • AtVPS52: 2.4 ns • AtVPS52 + AtRAb-H1b: 2.3 ns • AtVPS52 + AtRAb-H1c: 2.4 ns
- 19. Exploring the ER-Golgi interface • Testing if cis-Golgi golgins tether Golgi bodies to the ER • Express fluorescent full-length and truncated versions • Confocal microscopy and optical trapping with laser tweezers • “Grab a Golgi” – work by Imogen Sparkes and Chris Hawes
- 20. Why is this important? Understand molecular mechanisms of Golgi stack structure Understand Golgi populations in individual cells Put Golgi populations into functional context in cells Study effects on plant phenotypes
- 30. Are you ready to step out?
- 31. Start small… • Start with achievable projects • Be realistic about your skills and your time • Tie in with existing structures – Departmental newsletter – STEM Ambassador scheme – Volunteer at Science Festivals – Help with school workshops – Write article for a blog, school magazine… – Give a talk in a school, public lecture… – Sign up on social networking sites • Be specific about your content and audience
- 32. …but think big! • Build up teams with like-minded people (peers or interdisciplinary groups) and organise bigger projects. – “Brookes Science Bazaar” – university science festival – “Science writes to Life”, Pegasus Youth Theatre – collaboration with Oxford Brookes Poetry Centre and Fiona Sampson (professional poet
- 33. Make it part of your every-day work • Read something interesting? Post it to social media. • Get students to write summaries or articles about their research projects, lectures… • Upload short video clips to YouTube (microscopy movies, field trips…) • Get school science clubs to help you with your “real” research…
- 34. JUST DO IT!
- 35. Acknowledgements Oxford Brookes University Music Chris Hawes David Mansfield (Professor Science) Imogen Sparkes Cyrus Mower Plant Cell Biology Group Lorenzo Frigerio Charlotte Carroll Central Laser Facility, STFC Stan Botchway Funding Chris Stubbs Oxford Brookes University Mark Pollard BBSRC Andy Ward …and many, many more who Wageningen University contributed to Tijs Ketelaar movies, volunteered for events, helped with Norbert de Ruijters organisation…. http://www;youtube.com/plantendomembrane
Notes de l'éditeur
- Many food proteins, storage proteins, signalling proteins go through GolgiProduction of cell wall material, transports cellulose synthase to plasmamembrane for deposition of fibersFundamental research, but understanding these processes can help in the future in plant breeding or genetic modification
- Plants are special because they do not have one big stationary Golgi, but up to hundreds of discrete mobile stack that move around over the surface of the endoplasmic reticulum (ER)
- Plants are special because they do not have one big stationary Golgi, but up to hundreds of discrete mobile stack that move around over the surface of the endoplasmic reticulum (ER)
- Movie file!!
- ARL1 – ARF-like 1Yeast arl1mutants have minor defects in protein sorting in the TGN(Rosenwald et al., 2002) and in ion homeostasis (Love et al.,2004; Munson et al., 2004a,b). Knock-down of ARL1 in HeLacells using RNAi resulted in a block in the transport ofShigatoxin B fragment from endosomes to the TGN (Luet al., 2004). These results suggest the involvement of ARL1in endosome-to-Golgi trafficking.
- FRET: there needs to be physical interactionDrawbacks of FRETNote: we use mRFP because it behaves as monomerCFP has also a lower extinction coefficient than GFP, meaning that a higher excitation intensity is needed, resulting in fasterphotobleaching (48). The spectral overlap in emission spectra between CFP and YFP can also be problematical and requires the use of narrow band pass filtersThe mechanism of fluorescence resonance energy transfer involves a donorfluorophore in an excited electronic state, which may transfer its excitation energy to a nearby acceptorchromophore in a non-radiative fashion through long-range dipole-dipole interactions. The theory supporting energy transfer is based on the concept of treating an excited fluorophore as an oscillating dipole that can undergo an energy exchange with a second dipole having a similar resonance frequency. In this regard, resonance energy transfer is analogous to the behavior of coupled oscillators, such as a pair of tuning forks vibrating at the same frequency. In contrast, radiative energy transfer requires emission and reabsorption of a photon and depends on the physical dimensions and optical properties of the specimen, as well as the geometry of the container and the wavefront pathways. Unlike radiative mechanisms, resonance energy transfer can yield a significant amount of structural information concerning the donor-acceptor pair.What are oscillators? An `oscillator' is a term for something which behaves cyclically - i.e. which repeats its behaviour at regular intervals. A differential equation may have a solution which behaves cyclically, in which case we can say that it describes an oscillator. Oscillators are useful for describing real-world objects which periodically repeat their actions - e.g neurons (sometimes), certain electrical circuits, waves, cells, etc. They can also be used as crude models of more complicated real-world objects. What are coupled oscillators? Oscillators are `coupled' if they are allowed to interact with each other in some way. For example one neuron might send a signal to another at regular intervals. Mathematically speaking, the differential equations have coupling terms which represent how one oscillator interacts with all the others. An interesting contradiction about the term `coupled oscillators' is that once you couple them they need not behave like oscillators at all in that they need not behave cyclically.
- Make sure to emphasise connection between FRET and FLIM. FRET is what lowers lifetime, thus indicates interaction between proteins. Fluorescent lifetime: average time that a molecule remains in an excited state before it decays to its ground state. Normally it gives off photons. But if acceptor molecule is close, energy is used instead to excite acceptor and the fluorophore decays quicker. Emphasise how FLIM tackles problems of FRETSuhling: We have recently shown that, in solution, the fluorescence lifetime of the green fluorescent protein (GFP) is a function of the refractive index of its environment [1]. This is in agreement with the Strickler-Berg equation which describes radiative transitions in molecules and predicts an decrease of the fluorescence lifetime as the refractive index increases [2].
- Gap over which all the proteins need to travel
- If we know how individual Golgi stacks are formed and maintained and how and where tethers function, we can start to investigate what regulates those proteins – connection to activity of cells? Looking at Golgi population of a cell? If we were able to make more or bigger Golgi stacks or with more tethering factors, or more tightly tethered, could we increase productivity of a cell? If we increase productivity, would this affect plant phenotype? Bigger, stronger plants, more harvest? Implications for crops, cell wall – biomass.
- Say what questions are – new category “Traitors of PCB”
- Social media works best if you use different platforms and connect them all
- Good way to practise and prove “soft skills” (communication, project management, team work…) – which you need for research!