1. A LOV2 Domain-Based Optogenetic Tool to Control
Protein Degradation and Cellular Function
Renicke et al. (2013)
Feynman Liang
Amherst College
fliang14@amherst.edu
April 4, 2014
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 1 / 22

2. Things to keep in mind throughout the talk
Tools for studying biological systems should be...
Specific
Cryptic
Generalizable
Quantitative control
Reversible
Rapid
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 2 / 22

3. Optogenetics
Definition
Optogenetics – the synthetic approach using genetically encoded proteins
to control protein activity by light [1]
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 3 / 22

4. Pop quiz!
Without looking at the rest of the data, we can predict that one big
advantage light has over small molecules is that
Light is usually specific and does not affect endogeneous proteins
Light is not cytotoxic for most organisms so unlike small molecules its
efficacy is not limited by maximum tolerated dosage
Light-activated mechanisms are more rapid because the transport of
small molecules is limited by diffusion ( x2 ≈ 6Dτ)
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 4 / 22

5. Pop quiz!
Without looking at the rest of the data, we can predict that one big
advantage light has over small molecules is that
Light is usually specific and does not affect endogeneous proteins
Light is not cytotoxic for most organisms so unlike small molecules its
efficacy is not limited by maximum tolerated dosage
Light-activated mechanisms are more rapid because the transport of
small molecules is limited by diffusion ( x2 ≈ 6Dτ)
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 4 / 22

6. What do plants have to offer?
Figure 1: Arabidopsis thaliana [2]
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 5 / 22

7. Phototropism
Figure 2: Summary of plant phototropism [3]
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 6 / 22

8. Light-Oxygen-Voltage (LOV) photosensors
Figure 3: Left: Avena sativa (oat) phototropin1 LOV2 (AsLOV2) in dark, Right:
Partial proteolysis of AsLOV2 by chymotrypsin digestion [4]
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 7 / 22

9. A. thaliana phototropin1 LOV2 forms a coiled-coil dimer
Figure 4: Structure of A. thaliana LOV2 (AtLOV2) dimer in dark (RCSB 4HHD)
[5]
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 8 / 22

10. Ornithine decarboxylase (ODC) degron
Figure 5: Dimerization with antizyme
(AZ) causes degradation of ODC [6]
Ubiquitin-independent
degradation
Mechanism conserved across
vertebrates, yeasts, and plants
Degradation domain at
C-terminus (cODC)
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 9 / 22

11. Question time!
The last slide suggests that a light-activated cODC degron is likely to
possess which of the following qualities?
Specific
Cryptic
Generalizable
Quantitative control
Reversible
Rapid
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 10 / 22

12. Question time!
The last slide suggests that a light-activated cODC degron is likely to
possess which of the following qualities?
Specific
Cryptic
Generalizable
Quantitative control
Reversible
Rapid
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 10 / 22

13. Development of the photosensitive degron (psd) module
Figure 6: A: diagram of degradation process, B: construct screening using red
flourescent protein (RFP) indicator
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 11 / 22

14. Are you paying attention?
Which of the constructs do you think the authors christen as the psd
module and use for the remainder of the experiment?
a
b
c
d
e
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 12 / 22

15. Are you paying attention?
Which of the constructs do you think the authors christen as the psd
module and use for the remainder of the experiment?
a
b
c
d
e
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 12 / 22

16. Degradation is effective, cryptic, and reversible
Figure 7: psd module causes reversible light-activated degradation of proteins
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 13 / 22

17. Degradation can be quantitatively controlled
Figure 8: Quantitative control is achieved by varying light intensity
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 14 / 22

18. Lets stop and take stock...
Based on what we’ve seen so far, the psd module possesses (is likely
to possess) which of the following qualities?
Specific
Cryptic
Generalizable
Quantitative control
Reversible
Rapid? Depends on definition (t1/2 ≈ 25 min).
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 15 / 22

19. Lets stop and take stock...
Based on what we’ve seen so far, the psd module possesses (is likely
to possess) which of the following qualities?
Specific
Cryptic
Generalizable
Quantitative control
Reversible
Rapid? Depends on definition (t1/2 ≈ 25 min).
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 15 / 22

20. Cell cycle control
Figure 9: Photocontrol of Cdc28 regulators
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 16 / 22

21. Conditional protein inhibition
Figure 10: Light-induced degradation of an assortment of genes
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 17 / 22

22. Compartmentalized ODE modeling
Figure 11: In-silico modeling of steady state concentrations, pp1 ∝ promoter
strength
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 18 / 22

23. Is compartmentalization (i.e. forgetting all about space and only
considering time) a valid simplifying assumption or should the authors
have used a PDE model?
Yes
No
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 19 / 22

24. Is compartmentalization (i.e. forgetting all about space and only
considering time) a valid simplifying assumption or should the authors
have used a PDE model?
Yes
No
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 19 / 22

25. In summary...
Developed a photo-activated degradation technique (psd module)
that is
Specific
Cryptic
Generalizable? Only tested yeast.
Quantitative control
Reversible
Rapid? Depends on definition (t1/2 ≈ 38 min).
Demonstrated applicability of the technique to
Yeast photography
Yeast cell cycle regulation
Conditional degradation of various yeast genes
Induced yeast cell patterning
In silico modeling suggests efficacy most sensitive to cODC degron
efficiency
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 20 / 22

26. References
Christian Renicke et al.
A LOV2 Domain-Based Optogenetic Tool to Control Protein Degradation and Cellular Function
J. Chem Biol. 20(4), 619–626 (2013)
Arabidopsis thaliana
Wikipedia: The Free Encyclopedia Wikimedia Foundation, Inc. 16 Oct. 2004. Web. 3 April 2004
Phototropism
Wikipedia: The Free Encyclopedia Wikimedia Foundation, Inc. 5 Feb. 2014. Web. 3 April 2004
Shannon M. Harper et al.
Structural Basis of a Phototropin Light Switch
Science 301, 1541 (2003)
Halavaty et al.
Coiled-coil dimerization of the LOV2 domain of the blue-light photoreceptor phototropin 1 from Arabidopsis thaliana.
Acta Crystallogr. Sect.F 69:, 1316–1321 (2013)
Philip Coffino & Dieter H. Wolf
Degradation of Ornithine Decarboxylase, a Ubiquitin-Independent Proteosomal Process
Proteasomes: The World of Regulatory Proteolysis Ch. 16, 254–263 (2000)
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 21 / 22

27. The End
Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 22 / 22