Subcellular Optogenetics

 

A major goal of ours is to develop and apply subcellular optogenetic methods using light sensitive proteins. We define subcellular optogenetics as the ability to optically regulate molecular activity in selected regions within a cell. Coordinated variations in subcellular signaling play a critical role in governing cell behavior. For instance, asymmetric signaling activity across a cell governs processes such as cell migration and neuron differentiation. Signaling is also confined often to subcellular sites such as the plasma membrane, Golgi or endosomes. The ability to experimentally control signaling at this subcellular level will help decipher how networks of signaling, cytoskeletal and accessory proteins operate at various locations across a cell and direct complex cellular behaviors such as cell migration. To this end, we have developed optogenetic approaches that selectively control signaling activity at individual steps in a sequential pathway:

GPCR --> heterotrimeric G proteins --> Cdc42, RhoA or Rac.

These methods which can selectively activate a GPCR, deactivate G protein subunits or activate specific members of the Rho family allow cell migration to be directed with a light beam. 

Over the years it has been possible to visualize subcellular variation in signaling activity by generating a set of fluorescent protein tools. Similarly, developing a library of optogenetic tools to control this subcellular signaling activity can provide transformative experimental control over cell behaviors and help unravel their molecular basis. They can also be used translationally to control specific cellular behaviors therapeutically.  

 

Optical control over GPCR activity

Movie below shows immune cells expressing a GPCR, blue opsin from the human retina can be steered with a blue light beam (box). Details in PNAS 110(17):E1575-83. 

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Publications

Karunarathne, WK, Giri, L, Patel, AK, Venkatesh, KV, & Gautam, N (2013) Optical control demonstrates switch-like PIP3 dynamics underlying the initiation of immune cell migration. Proc Natl Acad Sci USA. 110(17):E1575-83. PMCID: PMC3637758

Karunarathne, WK, Giri, L, Kalyanaraman, V, & Gautam, N (2013) Optically triggering spatiotemporally confined GPCR activity in a cell and programming neurite initiation and extension. Proc Natl Acad Sci USA 110(17):E1565-74. PMCID: PMC3637763

 

Optical control over G protein activity

The movie below shows an immune cell in which a protein domain that switches off G protein activity (red) is optically recruited to one side of the cell and a chemokine receptor CXCR4 is activated globally. Inhibiting G protein activity asymmetrically in this manner leads to the generation of a PIP3 gradient and migration in the opposite direction. Details in MBoC 25(15):2305-14.

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Publication

O’Neill PR, & Gautam N. (2014) Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration. Mol Biol Cell. 25(15):2305-14.* PMCID: PMC4116304.

 *Mol Biol Cell. Highlighted article

* 24th annual MBoC Paper of the Year Award, American Society of Cell Biology.

 

Optical control over Cdc42 activity

Optical recruitment of a Cdc42 GEF to one side of a cell leads to asymmetric activation of Cdc42 and light directed cell migration.

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 Publication

O'Neill PR, Kalyanaraman V, Gautam N. (2016) Subcellular optogenetic activation of Cdc42 controls local and distal signaling to drive immune cell migration. Mol Biol Cell. 2016 Mar 3. pii: mbc.E15-12-0832 [Epub ahead of print]*

*Mol Biol Cell. Highlighted article

Other recent publications

O'Neill PR, Gautam N (2015) Optimizing optogenetic constructs for control over signaling and cell behaviours. Photochem Photobiol Sci. 14:1578-85.

Karunarathne WK, O'Neill PR, Gautam N (2015) Subcellular optogenetics - controlling signaling and single-cell behavior J Cell Sci 128:15-25.

O'Neill PR, Giri L, Karunarathne WK, Patel AK, Venkatesh KV, Gautam N. (2014) The structure of dynamic GPCR signaling networks. Wiley Interdiscip Rev Syst Biol Med. 6(1):115-23.

Giri L, Patel AK, Ajith Karunarathne WK, Kalyanaraman V, Venkatesh KV and Gautam N. (2014) A G protein betagamma subunit translocation embedded network motif underlies GPCR regulation of calcium oscillations. Biophys J. 2014 107(1):242-54.

O'Neill PR, Ajith Karunarathne WK, Kalyanaraman V, Silvius JR, and Gautam N. (2012) G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes. Proc Natl Acad Sci USA. 109 (51):E3568-77. 

Saini DK, Ajith Karunarathne WK, Angaswamy N, Saini D, Cho JH, Kalyanaraman V, Gautam N. (2010) Regulation of Golgi Structure and Secretion by Receptor induced G protein βγ Complex Translocation. Proc Natl Acad Sci. USA 107(25):11417-11422.