Here, we used the previously published phytochrome B (PhyB)/phytochrome interacting factor (PIF) system to activate and deactivate PI3K with light (Figure 1B; Levskaya et al., 2009; Toettcher et al., 2011). Uncoupling PI3K from activation of TrkA allowed us to study the effects of PI3K on membrane protein trafficking without activating other signaling pathways downstream of TrkA. To apply the PhyB/PIF system to control PI3K activity, we fused the iSH2 domain of the p85 regulatory subunit of PI3K to PIF (Figure 1C). It has previously been shown that the iSH2 domain associates with the endogenous p110 catalytic subunit of PI3K (Klippel et al., 1993). Because this heterodimer is missing the autoinhibitory domains of p85, it is constitutively active, so that PI3K phosphorylates its substrate, PI(4,5)P₂ to make PI(3,4,5)P₃ whenever the PIF-iSH2-YFP is driven to the membrane (Figure 1C; Suh et al., 2006; Idevall-Hagren et al., 2012). The iSH2 domain has been previously expressed as a fusion protein with PIF and has been shown to reversibly translocate to the PM and activate PI3K in response to light (Figure 1—figure supplement 1; Levskaya et al., 2009; Toettcher et al., 2011).

Using a cyan fluorescent protein (CFP)-labeled pleckstrin homology domain from the enzyme Akt (Akt-PH-CFP), which specifically binds the PI(3,4,5)P₃ product of PI3K (Lemmon, 2008), as a probe for PI(3,4,5)P₃, we confirmed that the PhyB/PIF-iSH2 machinery could be used to rapidly generate PI(3,4,5)P₃ in response to 650 nm light (Figure 1D and E). In response to 750 nm light, PIF-iSH2-YFP was released from the membrane and the levels of PI(3,4,5)P₃ returned to baseline, presumably due to the activity of the endogenous PI(3,4,5)P₃ phosphatase PTEN. Similar experiment using GRP1-PH-CFP, another PI(3,4,5)P₃ probe yielded the same results (Figure 1—figure supplement 2).

Activation of PI3K, without other pathways downstream of NGF/TrkA, is sufficient to drive trafficking of TRPV1 to the PM

We and others have previously demonstrated that activation of the RTK TrkA by NGF leads to increased trafficking of the ion channel TRPV1 to the PM (Zhang et al., 2005; Stein et al., 2006; Zhu and Oxford, 2007; Stratiievska et al., 2018) This increase in surface expression is associated with increased sensitivity to painful stimuli sensed by TRPV1. As shown in Figure 2A and B, the NGF-induced trafficking of TRPV1 follows the increase in PM PI(3,4,5)P₃ levels. Although TrkA couples with PLCγ and the MAP/ERK pathways in addition to PI3K, significant evidence indicates that PI3K activation is necessary to NGF-induced trafficking of TRPV1 to the PM (Zhang et al., 2005; Stein et al., 2006; Zhu and Oxford, 2007; Stratiievska et al., 2018).

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These data are reproduced from Stratiievska et al., 2018 (Figure 2A and B).

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Excel data for time courses and scatter plots (Figure 2C and D).

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We used PhyB with PIF-iSH2 to test the hypothesis that PI3K activity is sufficient to drive trafficking of TRPV1 to the PM. Together with PhyB, PIF-iSH2 (without a fluorescent tag), and Akt-PH-CFP, we expressed TRPV1 fused to yellow fluorescent protein (YFP). Stimulation of cells with 650 nm light gave the expected rise in PM-associated Akt-PH-CFP fluorescence and, importantly, a rise in PM-associated TRPV1-YFP fluorescence (Figure 2C and D). The light-induced rise in PM PI(3,4,5)P₃ and TRPV1 qualitatively resembled those induced by NGF. Because PLCγ and the MAP/ERK were not directly activated by the 650 nm light, we conclude that activation of the PI3K pathway is sufficient to cause TRPV1 trafficking to the PM.

Resolving changes in surface expression requires improved approaches

Total internal fluorescence (TIRF) microscopy was needed to resolve the NGF- and light-induced increase in PM PI(3,4,5)P₃ levels but is a poor approach for distinguishing membrane protein localization in the PM from that in intracellular compartments (Stratiievska et al., 2018). For membrane proteins with very high endoplasmic reticulum localization, like TRPV1 (Tominaga et al., 1998), this optical contamination leads to an underestimate of true changes in surface localization in TIRF experiments.

To circumvent contamination of TIRF signals on the PM with fluorescence from intracellular compartments, we implemented an inverse-electron-demand Diels-Alder cycloaddition click chemistry approach to selectively label PM localized proteins (Arsić et al., 2022). For this application, we used genetic code expansion to incorporate an ncAA at an extracellular site (Neubert et al., 2018; Bessa-Neto et al., 2021; Kuhlemann et al., 2021). We used Tet3-Bu (Figure 3A), a tetrazine-containing amino acid which can be efficiently labeled with a cyclopropane-fused strained trans-cyclooctene (sTCO) with no detectable off-target reactivity within the time scale of our experiments (see below) (Jang et al., 2020; Jana et al., 2023). Our goal was to express membrane proteins incorporating Tet3-Bu at an extracellular site and measure labeling with a membrane-impermeant sTCO-coupled dye (Figure 3B) under control conditions and then again after activation of PI3K with either NGF or 650 nm light.

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Original confocal microscopic images for TRPV1 labeling (Figure 3C).

Figure 3—source data 1_BF_TRPV1.lsm: the images contain GFP (green) and bright field (black-white). Figure 3—source data 1_GFP_Cy5_ TRPV1.lsm: the images contain GFP (green) and Cy5 (red). The last images were shown.

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Figure 3—source data 2

Original confocal microscopic images for InsR labeling (Figure 3E).

Figure 3—source data 1_BF_InsR.lsm: the images contain GFP (blue) and bright field (black-white). Figure 3—source data 1_GFP_Cy5_InsR.lsm: the images contain GFP (blue) and Cy5 (red). The last images were shown.

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Figure 3—source data 3

Excel data for scatter plots for TRPV1 and InsR labeling (Figure 3D and F).

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We tested our click chemistry/ncAA approach using TRPV1 and the InsR, a model RTK we and others have previously used in studies with different ncAAs (Nikić et al., 2015; Jones et al., 2021). For both proteins, we used amber codon suppression to site-specifically incorporate Tet3-Bu (Jang et al., 2020) into an extracellular loop (T468Tet3-Bu for TRPV1 and K676Tet3-Bu for InsR). This involved introducing a TAG stop codon at the selected site within the coding sequence that would place the ncAA on the extracellular side of the PM. We then co-expressed one plasmid encoding either TRPV1-T468TAG or InsR-K676TAG fused to GFP, a second plasmid encoding an aminoacyl tRNA synthetase evolved to incorporate the ncAA and the orthogonal tRNA, and a third plasmid encoding a dominant negative elongation release factor, which increased the efficiency of Tet3-Bu incorporation (Schmied et al., 2014). The cell culture medium was then supplemented with Tet3-Bu. As indicated by the GFP signal and in-gel fluorescence both TRPV1-T468Tet3-Bu-GFP and InsR-K676Tet3-Bu-GFP expressed at high levels only in transfected cells (Figure 3C and E and Figure 3—figure supplement 1C). To test whether incorporation of Tet3-Bu interfered with the function of TRPV1, we measured capsaicin-activated currents with whole-cell voltage clamp and found that TRPV1-T468Tet3-Bu-GFP gave robust capsaicin-activated currents (Figure 3—figure supplement 1A and B). To determine whether InsR-K676TAG protein retained its function, we measured insulin-induced endocytosis (Carpentier et al., 1992) and found endocytosis in response to insulin was maintained (Figure 3—figure supplement 2) with approximately the expected kinetics (Figure 3—video 1).

Click chemistry labeling can be used to measure NGF-induced trafficking of TRPV1 to the PM

Based on the extensive contamination of TIRF signals with fluorescence from the ER and other intracellular compartments (Stratiievska et al., 2018), we hypothesized that our previous measurements of NGF-induced trafficking of TRPV1 to the PM using TIRF represent underestimates of the true changes in PM TRPV1. If this were the case, using click chemistry to resolve NGF-induced changes in PM TRPV1 should reveal a greater increase in PM TRPV1. At the beginning of the experiment, we labeled surface TRPV1-468Tet3-Bu-GFP with a pulse of 1 µM sTCO-sulfo-Cy5 and then washed the label from the bath (Figure 4A). We used this higher concentration of sTCO dye (five times higher than in Figure 3) to label the PM channels more rapidly, as the NGF-induced trafficking of TRPV1 to the PM occurs with kinetics comparable to labeling with 200 nM sTCO-sulfo-Cy5 (compare Figure 2A to Figure 3D). We then treated the cells with NGF for 10 min and again pulse labeled the surface channels with a brief exposure to 1 µM sTCO-sulfo-Cy5. We observed an ~1.4-fold increase in PM Cy5 staining, indicating that NGF induced trafficking of TRPV1 to the surface (Figure 4B–D). In contrast, the total number of TRPV1 channels did not change, as evidenced by the total intensity of GFP fluorescence, which was not affected by NGF. This NGF-induced increase in surface expression is indeed greater than the ~1.1-fold increase in NGF-induced surface expression of TRPV1 observed in TIRF experiments (Figure 2A and B). In control experiments in which cells were exposed to the vehicle (without NGF), neither the GFP signal nor the sTCO-sulfo-Cy5 signal changed (Figure 4E), indicating that the NGF-induced increase in sTCO-sulfo-Cy5 was specific to activation of the TrkA receptor by NGF.

Figure 4

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Original confocal microscopic images for TRPV1 trafficking.

F0: 15th image, F1: 32nd image, GFP (green) and Cy5 (red) (Figure 4B and C).

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Excel data for scatter plot for TRPV1 trafficking (Figure 4D and E).

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Click chemistry labeling can be used to measure light-induced trafficking to the PM

We next asked whether click chemistry labeling could improve the resolution of PM trafficking in a system in which we have decoupled PI3K activity from other pathways downstream of NGF, i.e., the PhyB/PIF system. Results from these experiments with TRPV1-468Tet3-Bu-GFP and InsR-676Tet3-Bu-GFP are shown in Figure 5. After first labeling cells with sTCO-sulfo-Cy5, we exposed cells to an initial 10 min exposure to 750 nm light and labeled any newly delivered protein at the PM using a second treatment with sTCO-sulfo-Cy5 (Figure 5A). The GFP fluorescence remained the same under all conditions indicating that total protein expression did not change over the course of the experiments. For TRPV1-T468Tet3-Bu-GFP, the sTCO-sulfo-Cy5 labeling increased slightly in response to the initial exposure to 750 nm light (Figure 5B and D). This small increase in sTCO-sulfo-Cy5 labeling was specific to TRPV1-T468Tet3-Bu, as 750 nm light produced no change in sTCO-sulfo-Cy5 labeling of InsR-K676Tet3-Bu-GFP (Figure 5F and H). The TRPV1-specific increase in labeling after 750 nm illumination is likely due to the small, irreversible PM localization of PIF-iSH2-YFP that occurs in TRPV1-expressing cells (Figure 2—figure supplement 1). This small increase in sTCO-sulfo-Cy5 labeling of TRPV1-T468Tet3-Bu was also seen in control experiments using two sequential exposures to 750 nm light (Figure 5—figure supplement 1). Note that the increase of TRPV1-T468Tet3-Bu labeling, F₂/F₁, in experiments with 650 nm light (Figure 5) was significantly greater than in experiments with only 750 nm light (p<0.001; Figure 5—figure supplement 1).

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Original confocal microscopic images for TRPV1 trafficking (Figure 5B and C).

Figure 5—source data 1_TRPV1_Cy5_750_1.lsm, Figure 5—source data 1_TRPV1_Cy5_750_2.lsm, Figure 5—source data 1_TRPV1_Cy5_650.lsm: the mages contain GFP (green) and Cy5 (red). Figure 5—source data 1_TRPV1_ BF_PhyB.lsm: the mages contain bright field and PhyB (magenta).

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Figure 5—source data 2

Scatter plot for TRPV1 trafficking (Figure 5D and E).

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Figure 5—source data 3

Original confocal microscopic images for InsR trafficking (Figure 5F and G).

Figure 5—source data 1_InsR_750_1.lsm, Figure 5—source data 1_InsR_750_2.lsm, Figure 5—source data 1_InsR_650.lsm: the mages contain GFP (green) and Cy5 (red). Figure 5—source data 1_BF_PhyB.lsm: the images contain bright field and PhyB (magenta).

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Figure 5—source data 4

Excel data for scatter plot for InsR trafficking (Figure 5H and I).

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After the initial control period with 750 nm light, we exposed the cells to 650 nm light for 10 min to activate PI3K and then labeled newly delivered protein at the PM with sTCO-sulfo-Cy5. As shown in Figure 5B–E for TRPV1-T468Tet3-Bu and Figure 5F–I for InsR-K676Tet3-Bu-GFP, activation of PI3K with 650 nm light increased expression of protein on the PM. For TRPV1, the amplitude of the increase in surface expression, ~1.4-fold, was similar to that observed in click chemistry experiments in response to NGF (Figure 4). Surprisingly, the increase in surface expression of InsR-K676-Tet3-Bu was also about 1.4-fold. Like the NGF receptor TrkA, InsR is an RTK whose activation stimulates PI3K activity (Saltiel, 2021). Binding of insulin to InsR is a well-studied signal for InsR endocytosis (Carpentier et al., 1992), and PI3K activation downstream of InsR is key to delivery of GLUT4 transporters to the PM (Saltiel, 2021). However, to our knowledge, PI3K activity has not been previously known to stimulate InsR delivery to the PM. Although it would be exceedingly interesting if insulin binding to InsR could trigger both InsR endocytosis and exocytosis, further studies will be required to determine the physiological significance, if any, of PI3K-stimluated delivery of InsR to the PM.

Here, we present proof-of-principle experiments demonstrating the simultaneous use of an optogenetic approach for PI3K activation and genetic code expansion/click chemistry to interrogate regulation of membrane protein trafficking. This combination of approaches is especially useful for membrane proteins with significant expression in the endoplasmic reticulum and/or other intracellular compartments, which makes it very difficult to identify proteins in the PM definitively when using fusions with GFP or similar genetically encoded labels. In addition to distinguishing proteins at the surface, using a small ncAA and click chemistry is likely much less perturbing to protein structure and function than fusion with an ~20–30 kDa protein such as GFP, SNAP-tag, or HaloTag. Restricting the ncAA and fluorescent label to an extracellular region/loop is also less likely to interfere with protein-protein interactions on the intracellular side, compared with a large protein fusion to an intracellular N- or C-terminal of a protein of interest.

Successful implementation of this approach required overcoming a number of barriers: efficient incorporation of Tet3-Bu into the extracellular sites; demonstration that the Tet3-Bu-incorporating protein had the expected functional properties; development of a membrane-impermeant sTCO-coupled dye; and optimization of the PhyB/PIF and genetic code expansion machinery in the same cells. Incorporation of Tet3-Bu at the extracellular side of TRPV1 was well tolerated in the S1-S2 loop, with two of four sites yielding functional, capsaicin-activated channels. We have found incorporation efficiency to be difficult to predict. Screening through desirable sites typically is the best approach, with approximately one half to one third of selected sites showing reasonable levels of ncAA incorporation.

There is significant room for improvement of these methods, particularly in developing more rapid labeling of the ncAA. As shown in Figure 3, complete labeling with a low concentration of sTCO dye required more than 10 min. Monitoring membrane protein trafficking to the surface in real time will require a reaction that is at least 5- to 10-fold faster. Increasing the speed of labeling can be accomplished using ≥1 µM sTCO dye, but at such high concentrations of dye the background signal from dye in solution interferes with imaging. We have recently developed new ncAAs with the tetrazine ring more proximal to the amino acid beta carbon, which we term Tet4, that react with sTCO-based labels with rates as fast as 10⁶ M^–1s^–1 (Jana et al., 2023). The aminoacyl tRNA synthetases that incorporate Tet4 amino acids into mammalian cells are not as efficient as those for Tet3 amino acids. Improving the efficiency of incorporating Tet4 amino acids would allow us to express Tet4-incorporating proteins and measure delivery of proteins to the PM in real time.

Another limitation to our approach is the need to co-express five to six plasmids to use PhyB/PIF and Tet/sTCO machinery simultaneously. Developing stable cell lines for ncAA incorporation would reduce the need for multiple plasmid transfection and, perhaps, lead to more uniform expression across cell populations. The PhyB/PIF machinery requires supplementing cell medium with the phycocyanobilin (PCB) chromophore, making it incompatible with in vivo experiments. Introducing four genes, PcyA, H01, Fd, and Fnr, and knocking down/out biliverdin reductase A leads to efficient synthesis of PCB in mammalian cells (Uda et al., 2017; Uda et al., 2020). It is therefore possible that genetically modified animals might one day express the PhyB/PIF machinery and chromophore and allow optogenetic activation of PI3K in vivo. However, there is as yet no method for synthesizing the necessary ncAAs within cells, precluding in vivo use of the PhyB/PIF with tetrazine-based genetic code expansion.

PI3K is a universal signal for trafficking to the PM. In Dictyostelium, PI3K activation at the leading edge of cells underlies chemotaxis toward nutrients (Nichols et al., 2015). In macrophages, PI3K at the leading edge of cells drives motility toward the object of phagocytosis (Hawkins et al., 2006; Hawkins and Stephens, 2015). In the cases discussed here, membrane proteins constitute the cargo delivered to the PM in response to PI3K signaling. A vast literature has examined the mechanisms by which InsR is recycled from the PM in response to insulin (Knutson, 1991; Ye et al., 2017; Chen et al., 2019), but less is known about ways in which delivery of InsR to the PM is regulated. We show here, for the first time, that activation of PI3K is sufficient to increase InsR in the PM. Future studies will be needed to understand the role for this process in liver and muscle cells. For TRPV1, PI3K was identified as an important signal that enhances surface expression in response to NGF, but the steps between PI3K activation and fusion of vesicles containing TRPV1 cargo with the PM are a mystery. The tools developed here provide a means by which the cellular mechanisms underlying PI3K-mediated delivery of membrane proteins can be interrogated.

All source data are included in the manuscript and the supporting files.

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Author details

1. Duk-Su Koh

   University of Washington, Department of Physiology & Biophysics, Seattle, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Anastasiia Stratiievska

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0406-2308

2. Anastasiia Stratiievska

   University of Washington, Department of Physiology & Biophysics, Seattle, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Duk-Su Koh

   Competing interests

   No competing interests declared

3. Subhashis Jana

   Department of Biochemistry and Biophysics, Oregon State University, Corvallis, United States

   Contribution

   Resources, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

4. Shauna C Otto

   University of Washington, Department of Physiology & Biophysics, Seattle, United States

   Contribution

   Validation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

5. Teresa M Swanson

   University of Washington, Department of Physiology & Biophysics, Seattle, United States

   Contribution

   Validation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

6. Anthony Nhim

   University of Washington, Department of Physiology & Biophysics, Seattle, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

7. Sara Carlson

   University of Washington, Department of Physiology & Biophysics, Seattle, United States

   Present address

   Allen Institute for Cell Sciences, Seattle, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

8. Marium Raza

   University of Washington, Department of Physiology & Biophysics, Seattle, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

9. Ligia Araujo Naves

   University of Washington, Department of Physiology & Biophysics, Seattle, United States

   Present address

   Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

   Contribution

   Conceptualization, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

10. Eric N Senning

    Department of Neuroscience, University of Texas at Austin, Austin, United States

    Contribution

    Resources, Funding acquisition, Validation, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

11. Ryan A Mehl

    Department of Biochemistry and Biophysics, Oregon State University, Corvallis, United States

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2932-4941

12. Sharona E Gordon

    University of Washington, Department of Physiology & Biophysics, Seattle, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    seg@uw.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-0914-3361

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