Generation and LED light photoactivation of Opto-receptors

Generation and LED Light-induced Photoactivation of Opto-Receptors

Anassuya Ramachandran and Caroline S Hill – The Francis Crick Institute

General considerations

1. In our experience, plating healthy cells at the right confluence is a critical determinant of the success of these experiments. We have predominantly used NIH-3T3 cells for these assays. Use cells that are in the exponential phase of growth for plating for transfections and ensure that cells are around 70% confluent on the day of transfection. Low cell numbers result in increased toxicity after transfection and affect the basal state of signalling as determined by Western blotting.
    
2. Titration of levels of receptors and SMADs is essential for a successful experiment. High levels of receptors will result in light-independent activation of receptors and subsequent SMAD phosphorylation. Similarly, high levels of SMAD expression will result in a proportion that is phosphorylated in a light-independent manner. Both parameters will impact on experimental outcomes and must be actively addressed. Efficient transfection complexes are formed at a fixed DNA amount:transfection reagent ratio; if the amount of plasmid DNA for receptors etc is lowered, the remaining quantity must be made up with empty vector (see below).
    
3. Where possible, it is highly recommended that all constructs of interest are cloned into the same plasmid backbone to minimise competition between different constructs for the transcription machinery.
    
4. The general design of the Opto-Receptors was as described by Sako et al., 2016. In general, from N -> C terminus, each Opto-receptor consisted of a myristoylation domain, a glycine-serine rich linker, intracellular domain of a type I receptor (including the GS domain and the kinase domain), a glycine/serine-rich linker, LOV domain and HA-tag. Please refer to Supplementary Files 1 and 2 in Ramachandran et al., 2018 for detailed nucleotides sequences. Opto-receptors were generated by overlapping PCR.
    
5. Blue light photoactivation was achieved with a 14W Grow Panel Blue Hydroponic Light Board (Amazon). Unpublished data from our group suggests that lower power blue light sources would be as effective in achieving photoactivation of the Opto-Receptors.

Reagents

1. Plasmids:

a. pCS2+ (empty vector) (https://www.addgene.org/vector-database/2295/)

b. pCS2+ – Opto-TGFBR1*, hereafter called Opto-TGFBR1*. Contains the constitutively-active human TGFBR1 GS and kinase domains and is cloned into pCS2+. The mutation T204D renders the kinase constitutively active.

c. pCS2+ – Opto-ACVR1, hereafter called Opto-ACVR1. Contains the wildtype human ACVR1 GS and kinase domains, and is cloned into pCS2+. 

d. pCMV2–Flag-SMAD1, hereafter called Flag-SMAD1. N-terminal Flag-tagged full length human SMAD1 (Lechleider et al., 2001).

e. pGFP-C1-SMAD3, hereafter called GFP-SMAD3. N-terminal GFP-tagged full length human SMAD3 (Nicolas et al., 2004).

2. NIH-3T3 cells

3. Culture media:

1. Routine culture of NIH-3T3 was in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Cat. No: 41966029) supplemented with 10% foetal calf serum (FCS) and 1% penicillin/streptomycin (hereafter called complete media).
2. Serum starvation of NIH-3T3 was in DMEM supplemented with 0.5% FCS and 1% penicillin/streptomycin (hereafter called starvation media).
3. OptiMEM (Thermo Fisher Scientific, Cat. No: 31985070) was used for making transfection complexes. 

4. Fugene HD (Promega, Cat. No: E2312)

Protocol

1.   Plate 200,000 NIH-3T3 cells per well of a 6-well plate in full media. 

2.    The following day, cells were transfected as required. For each well:

1. Dilute 2 µg of plasmid DNA mix (see point 6 below) in 200 µl of OptiMEM.
2. Add 8 µl of Fugene HD.
3. Allow complexes to form at room temperature for 15 minutes.
4. Add DNA:Fugene HD complexes dropwise to cells.

3.   Twenty four hours after transfection, starve the cells in starvation media.

4.    The following day, expose cells to blue light for 1 hr in a humidified, tissue culture incubator. The control plates should be wrapped in foil (dark) and placed in the same incubator.

5.    After light induction, cells were washed twice with cold phosphate buffered saline (PBS) and whole cell extracts were prepared for analysis by Western blotting (Germain et al., 2000).

6.    Plasmid DNA mixes are as follows:

a. Transfection controls:

i. Empty vector transfected. One well, to be exposed to blue light.

Per well:

                     pCS2+ (empty vector)                     2 µg

                     OptiMEM                                        To 200 µl

ii. SMAD alone transfected. One well each for Flag-SMAD1 and GFP-SMAD3, both to be exposed to blue light.

Per well:

SMAD1 alone:

                     pCS2+ (empty vector)                     2 µg

                     Flag-SMAD1                                    25 ng

                     OptiMEM                                         To 200 µl

SMAD3 alone:

                     pCS2+ (empty vector)                     2 µg

                     GFP-SMAD3                                    5 ng
                     OptiMEM                                        To 200 µl

b. Experimental controls - Opto-TGFBR1* and GFP-SMAD3. Two wells, one well in the dark and the other to be exposed to blue light. This is done to ensure that the Opto-TGFBR1* construct is working.

Per well (remember to make enough for two wells):

                     pCS2+ (empty vector)                     2 µg

Opto-TGFBR1*                                 25 ng

                     GFP-SMAD3                                    5 ng

                     OptiMEM                                        To 200 µl

c. Experimental samples:

i. Opto-TGFBR1* and Flag-SMAD1. Two wells, one well in the dark and the other to be exposed to blue light. This is done to determine whether Opto-TGFBR1* can phosphorylate Flag-SMAD1.

Per well (remember to make enough for two wells)

pCS2+ (empty vector)                    2 µg

Opto-TGFBR1*                               25 ng

                     Flag-SMAD1                                 25 ng

                     OptiMEM                                      To 200 µl

ii. Opto-ACVR1 and Flag-SMAD1. Two wells, one well in the dark and the other to be exposed to blue light. This is done to determine whether Opto-ACVR1 can phosphorylate Flag-SMAD1.

Per well (remember to make enough for two wells)

      pCS2+ (empty vector)                  2 µg

Opto-ACVR1                                 50 ng

                     Flag-SMAD1                                 25 ng

                     OptiMEM                                     To 200 µl

iii. Opto-TGFBR1*, Opto-ACVR1 and SMAD1. Two wells, one well in the dark and the other to be exposed to blue light. This is done to determine whether the combination of Opto-TGFBR1* and Opto-ACVR1 can phosphorylate Flag-SMAD1.

     Per well (remember to make enough for two wells)

                     pCS2+ (empty vector)                 2 µg

Opto-TGFBR1*                             25 ng
Opto-ACVR1                                50 ng

                     Flag-SMAD1                               25 ng

                     OptiMEM                                    To 200 µl

References

Germain, S., Howell, M., Esslemont, G.M., and Hill, C.S. (2000). Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif. Genes Dev 14, 435-451.

Lechleider, R.J., Ryan, J.L., Garrett, L., Eng, C., Deng, C., Wynshaw-Boris, A., and Roberts, A.B. (2001). Targeted mutagenesis of Smad1 reveals an essential role in chorioallantoic fusion. Dev Biol 240, 157-167.

Nicolas, F.J., De Bosscher, K., Schmierer, B., and Hill, C.S. (2004). Analysis of Smad nucleocytoplasmic shuttling in living cells. J Cell Sci 117, 4113-4125.

Ramachandran, A., Vizan, P., Das, D., Chakravarty, P., Vogt, J., Rogers, K.W., Muller, P., Hinck, A.P., Sapkota, G.P., and Hill, C.S. (2018). TGF-b uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition. Elife 7, e31756.

Sako, K., Pradhan, S.J., Barone, V., Ingles-Prieto, A., Muller, P., Ruprecht, V., Capek, D., Galande, S., Janovjak, H., and Heisenberg, C.P. (2016). Optogenetic Control of Nodal Signaling Reveals a Temporal Pattern of Nodal Signaling Regulating Cell Fate Specification during Gastrulation. Cell Rep 16, 866-877.

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