optogenetic regulation of ca 2+ signaling april 15, 2013 class objectives: what is optogenetics?...

Optogenetic regulation of Ca2+ signalingApril 15, 2013

Class objectives:• What is optogenetics? • Mammalian rhodopsin.• Biophysics of channelrhodopsin-2.• Opto-XR• Optically-induced dimerization

“Optogenetics is the combination of optical and molecular strategies to monitor and control designated molecular and cellular activities in living tissues and cells using genetically encoded photosensitive proteins.”

What is optogenetics?

Mammalian rhodopsin

11-cis-retinal

11-cis-retinylidene

All-trans-retinylidene

Channelrhodopsin-2 is a light-activated cation channel

Green alga Chlamydomonas reinhardtii

Nagel G et al. PNAS 2003;100:13940-13945

Nagel G et al. PNAS 2003;100:13940-13945

Action spectra of channelrhodopsins

Prigge M et al. J. Biol. Chem. 2012;287:31804-31812

ChR chimeras with different spectral properties

Prigge M et al. J. Biol. Chem. 2012;287:31804-31812

Dual light excitation and ion selectivity for blue and green light-absorbing ChRs.

RD Airan et al. Nature (2009)

Signalling specificity and in vivo functionality of Opto-XRs

RD Airan et al. Nature 000, 1-5 (2009) doi:10.1038/nature07926

OptoXR: optogenetic control of intracellular signal transduction.

Opto-1AR activation of TRPV4 channels

Activation wavelength

(nm)

Reversible Reference

Flavin-binding, kelch repeat, F box 1 (FKF1) and gigantea (GI)

Blue light Yes (hours) Yazawa et al. 2009

Phytocrome B and PIF6

Associates with red light and dissociates with far-red light

Yes (seconds)

Liu et al. 2008

CIB1 and cryptochrome 2 (CRY2), a blue light–absorbing photosensor that binds CIB1 in its photoexcited state9

Blue light Yes (minutes)

Kennedy et al. 2010

Systems for optically-induced protein interactions

Light-induced translocation of PKC

FKF1-GI system

Idevall-Hagren O et al. PNAS 2012;109:E2316-E2323

Rapid light-induced recruitment of a 5-ptase to the plasma membrane

Idevall-Hagren O et al. PNAS 2012;109:E2316-E2323

Blue light-induced recruitment of a 5-phosphatase to the plasma membrane rapidly decreases KCNQ2/3 current.

Intermission

Jia et al.(2011) Fig. 1

Jia et al.(2011) Fig. 2a-b

Jia et al.(2011) Fig. 2c-e

Jia et al.(2011) Fig. 3a-c

Jia et al.(2011) Fig. 3d-f

Jia et al.(2011) Fig. 4a-b

Jia et al.(2011) Fig. 3c-g

Jia et al.(2011) Supplemental Fig. 1a-b

Jia et al.(2011) Supplemental Fig. 1c-d

Supplemental movies

Jia et al.(2011) Supplemental Movies