Optogenetic Control of Endoplasmic Reticulum–Mitochondria

Nov 25, 2017 - The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by t...
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Letter Cite This: ACS Synth. Biol. 2018, 7, 2−9

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Optogenetic Control of Endoplasmic Reticulum−Mitochondria Tethering Fan Shi,†,‡ Fuun Kawano,‡ Seon-hye Emily Park,‡,∇ Shinji Komazaki,§ Yusuke Hirabayashi,∥,# Franck Polleux,∥ and Masayuki Yazawa*,‡,⊥ †

College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, China Department of Rehabilitation and Regenerative Medicine, Columbia Stem Cell Initiative, Columbia University, New York, New York 10032, United States § Department of Anatomy, Saitama Medical University, Saitama 350-0495, Japan ∥ Department of Neuroscience, Mortimer B. Zuckerman Mind Brain Behavior Institute and Kavli Institute for Brain Science, Columbia University, New York, New York 10025, United States ⊥ Department of Pharmacology, Columbia University, New York, New York 10032, United States # Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama, Japan ‡

S Supporting Information *

ABSTRACT: The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by the lack of tools to manipulate their occurrence in live cells spatiotemporally. Here, we report the development of a genetically encoded light-inducible tethering (LIT) system allowing the induction of contacts between endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of light-dependent heterodimerization called an iLID system. We demonstrate that the iLID-based LIT approach enables control of ER−mitochondria tethering with high spatiotemporal precision in various cell types including primary neurons, which will facilitate the functional study of ER−mitochondrial contacts. KEYWORDS: optogenetics, mitochondria, endoplasmic reticulum, iLID system, light-inducible tethering (LIT) system, fluorescence microscopy, electron microscopy

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contacts is still limited because of the difficulty to visualize and manipulate its distribution in live cells in a spatially precise and temporally dynamic way. A chemical-induced dimerization system using rapamycinFKBP-FRB has been utilized to visualize ER−mitochondria junctions and to enhance tethering of the ER and mitochondria, thereby introducing artificial mitochondria-associated ER membranes (MAM) structures in mammalian cells.13,14 However, the spatial and temporal accuracy of this approach is limiting for performing subcellular manipulations and the diffusivity of chemical compound limits spatial control and therefore its applicability for in vivo research. Also, rapamycin has some direct effects on the mechanistic target of rapamycin (mTOR) signaling pathway, a key regulator of autophagy,15 which might have indirect but significant effects on ER− mitochondria interactions. In addition, higher spatiotemporal resolution is required to examine the role of ER−mitochondria

ontacts between the ER and outer mitochondrial membranes (OMM) constitute an important signaling interface that is crucial for multiple critical physiological functions including local calcium (Ca2+) signaling, ATP production, mitochondrial fission, and lipid biogenesis.1−3 In yeast, ER−mitochondria tethering is regulated by a set of four proteins forming a complex called ER−mitochondria encounter structure (ERMES).4 In metazoans, the ortholog of this ERMES complex has not been identified yet. However, several genes have been reported as regulators for ER−mitochondria tethering: Mitofusin 2 (MFN2), Presenilin2 (PS2), and Vesicleassociated membrane protein association protein B (VAPB) Protein tyrosine phosphatase-interacting protein 51 (PTPIP51).5−8 Disruption of these genes has moderate effects on ER−mitochondria tethering in addition to mitochondria Ca2+ uptake, autophagosome formation, and cell survival under stress.5−11 Dysfunction of the ER−mitochondria interaction has been also observed in various neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis.12 Despite its potential significance, our understanding of the function of the ER−mitochondria © 2017 American Chemical Society

Received: July 5, 2017 Published: November 25, 2017 2

DOI: 10.1021/acssynbio.7b00248 ACS Synth. Biol. 2018, 7, 2−9

Letter

ACS Synthetic Biology

Figure 1. The LIT system for optogenetic control of ER and mitochondria contacts. (a) Schematic representation of light-inducible tethering (LIT) system using light-induced heterodimerization to tether the endoplasmic reticulum (ER) and mitochondria; OMM, outer mitochondrial membrane. (b) Schematic representation of the iLID based LIT system.

Figure 2. The LIT system to tether ER and mitochondria. (a) Representative confocal fluorescent images of an individual NIH 3T3 cell expressing the LIT system in dark and with blue light. The corresponding line profiles (yellow, in upper left image) are shown on the right-hand side panels. The R value represents the correlation coefficient of the red and green fluorescent signals in the upper and lower panel, respectively. (b) Value change of Manders colocalization coefficient (MCC) M2 (mKate2 overlapping to Venus) following blue light stimulation in the NIH 3T3 cells. We observed a significant increase of M2 in cells expressing the WT LIT system while a random minor change of M2 was observed in cells expressing the C450 M mutant. (n = 15 for WT, n = 16 for C450M, three independent experiments). (c) Representative confocal fluorescent images of an axon in mouse primary cortical neuron expressing the LIT system (SspB-mKate2-CB5-IRES-iLID-ActA) and mitochondrial-localizing BFP (mito-BFP). After stimulating blue light, the mKate2 red fluorescent proteins fused to SspB became colocalized with the mitochondria (indicated by yellow arrowheads). Plot intensity profiles are drawn along the axon as reference. (d) Value change of Pearson’s correlation coefficient (PCC) after blue light stimulation of segments of axons at 12 s. (n = 29 for LIT, n = 19 for Ctrl, 2 independent experiments). *P < 0.05, ***P < 0.0001, according to Student’s t test. Scale bar, 10 μm.

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DOI: 10.1021/acssynbio.7b00248 ACS Synth. Biol. 2018, 7, 2−9

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mitochondria morphology is significantly affected in cells expressing pMag(3x)-mKate2-ActA and GI-mKate2-ActA. In addition, we found that the expression of NLOV (H105L)YFP-CB5 was low compared to the other CB5 constructs. Unexpectedly, we found that the CRY2/CIB1- and BphP1/QPAS1-based constructs failed to induce tethering of the ER and mitochondria in live cells mainly because constitutive binding occurred in NIH 3T3 cells even without light-induction. To confirm that the Magnets-based constructs affect mitochondria in mammalian cells, we used electron microscopy for further investigation. The result reveals that the Magnets-based system induced significant mitochondria aggregation in dark conditions (Figure S2). Next, we examined whether iLID/SspB could be used for a light-inducible tethering (LIT) system (Figure 1b). We conducted time-lapse confocal imaging of NIH 3T3 cells expressing Venus-iLID-ActA and SspB-mKate2-CB5 and confirmed that SspB-mKate2-CB5 and Venus-iLID-ActA were localized in the ER and mitochondria membranes of NIH 3T3 cells in dark conditions, respectively. Upon blue light exposure, red fluorescence of SspB-mKate2-CB5 rapidly formed puncta or tubular structures and colocalized with the yellow/green fluorescent signal of the mitochondria (Figure 2a and Movies S1−S3) while no obvious change of Venus-iLID-ActA localization was observed (Movie S2). To quantify colocalization of the ER and mitochondria, we conducted optical density (OD) measurements using the line scan in NIH 3T3 cells expressing SspB-mKate2-CB5 and Venus-iLID-ActA. The resulting plots (Figure 2a right panel) show the mKate2 and Venus OD profiles corresponding to the yellow arrow line in the upper left image (SspB-mKate2-CB5, dark). The correlation coefficient of the red and green plots increased from 0.2075 to 0.6089 after blue light stimulation (Figure 2a right panel), suggesting rapid induction of colocalization of the two fusion proteins that are anchored to the ER and mitochondria membranes. As a negative control, we next tested the light-insensitive mutant (C450M) to confirm that SspB-mKate2-CB5 translocation is dependent on blue light exposure. The C450M mutation abolishes the covalent bond formation of the cysteine and cofactor flavin mononucleotide, disrupting the conformational change of the iLID induced by blue light stimulation.28 Therefore, the C450M mutant is incapable of responding to blue light. We found that the localization of SspB-mKate2-CB5 was not changed by blue light in the cells expressing VenusiLID (C450M)-ActA (Figure 2b, Figure S3 and Movie S4). The results reveal that blue light exposure induces SspB-mKate2CB5 and Venus-iLID-ActA heterodimerization, leading to the ER−mitochondria tethering in live mammalian cells. We further examined whether the expression of the photoswitch causes perturbation of ER and mitochondria tethering in the dark condition. As a control group, we prepared NIH 3T3 cells transfected with mTFP-CB5 and SspB-mKate2-ActA, which indicate the localization of mitochondria and ER. We next compared the control cells with cells expressing LIT system in the dark condition. We found that both groups showed similar distribution of ER and mitochondria and there is no significant difference in the Pearson’s correlation coefficient and Manders correlation coefficient between the groups (Figure S4). To improve the transduction of iLID-based LIT constructs in mammalian cells, we next generated the internal ribosome entry site (IRES) version driven by human EF1α promoter that enables us to coexpress SspB-mKate2-CB5 and iLID-ActA at a

interactions in soma, dendrites, and axons of mammalian neurons. For example, though dendritic Ca2+ dynamics play an important role in synaptic plasticity,16,17 the role of ER− mitochondria interactions may be different between the soma, dendrites, and axons of mammalian neurons. Therefore, the development of new technological approaches that enable the control of organelle interactions more precisely with greater spatiotemporal resolution and minimize side effects is paramount for cell biologists. Compared to the small organic molecule-inducible technique, optogenetic approaches using genetically encoded light-activated molecules can provide superior spatial and temporal resolution. Light-inducible protein−protein interactions including homo- and heterodimerization have been used recently to develop a variety of optogenetic tools allowing control of transcription, genome engineering, and protein translocation.18−22 To achieve precise control of ER−mitochondrial interaction, we developed a new optogenetic tool based on optimized light-activated heterodimerization systems that can be applied to tether ER and mitochondria in live mammalian cells. To develop a light-inducible ER−mitochondria tethering system, we expressed one part of the light-inducible dimer pair in ER membranes by fusing with the ER anchor peptide sequence from cytochrome b5 (CB5) and the other part in the outer mitochondria membranes (OMM) by fusing with the OMM linker peptide sequence from ActA (Figure 1a).13,23 To examine the expression of candidate constructs in live cells, we fused the constructs with fluorescent proteins. We hypothesized that light-dependent heterodimerization leads to ER−mitochondria tethering upon illumination at the appropriate wavelength. To examine which light-inducible systems would be optimal for inducible ER−mitochondria tethering, we first constructed the ER and mitochondrial fusion proteins using multiple light-inducible heterodimerization systems, Magnets (pMag/nMag), Flavin Kelch-repeat F-box 1 (FKF1)/GIGANTEA (GI), Cryptochrome 2 (CRY2)/CRY-interacting bHLH1 (CIB1), BphP1/Q-PAS1, and improved light induced dimer (iLID)/SspB. Magnets is a pair of Vivid (VVD) variants using mutagenesis that prevents their homodimerization and induces pMag/nMag heterodimerization with blue light.24 The Nterminus and “Light, Oxygen and Voltage” (LOV) domain (NLOV, H105L mutant) of FKF1 were used as an optimized fragment that can interact with GI upon blue light stimulation.18,25 CRY2 is a widely used photoreceptor, which can be induced to bind its binding partner, CIB1, upon blue light illumination.26 Besides blue light-inducible dimerization systems, we also took advantage of a newly improved nearinfrared (NIR)-activated system, BphP1/Q-PAS1. BphP1 absorbs NIR (740−780 nm) causing it to switch from the ground state to active state, resulting in BphP1/Q-PAS1 interaction. The BphP1/Q-PAS1 interaction presents the advantage of being reversible in dark conditions or with red light (660−700 nm).27 iLID is an engineered LOV2 domain of Phototropin by incorporating the SsrA peptide in the J-α helix of LOV2 domain. Because SspB is the natural binding partner of SsrA, iLID leads to steric occlusion of SspB binding in the dark and uncaging with blue light, resulting in a light-inducible heterodimerization.23 Taking advantage of these light-inducible heterodimerization pairs, we prepared the candidate constructs to enhance the ER and mitochondria tethering. Next, we examined the expression and function of the lightinducible constructs in mammalian cell line, NIH 3T3 (Figure S1). The results using confocal microscopy suggest that 4

DOI: 10.1021/acssynbio.7b00248 ACS Synth. Biol. 2018, 7, 2−9

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Figure 3. Ultrastructural examination of ER−mitochondria tethering in HEK 293T cells induced by the LIT system. (a) Representative electron microscopic image in HEK 293T cells expressing the LIT system (SspB-mKate2-CB5 and Venus-iLID-ActA) in the dark. We could observe only few spontaneous contacts (arrowheads) between the ER and mitochondria in the HEK 293T cells where the LIT system was switched off by keeping cells in dark conditions. (b) Representative electron microscopic image in HEK 293T cells expressing the LIT system with blue light illumination. We could observe expanded tethering (arrowheads) of the ER and mitochondria in HEK 293T cells when the LIT system was switched on by stimulating with blue light for 20 s. Scale bar, 200 nm.

time in the dark. We observed that the red fluorescent signal started to form puncta and tubular structures within several seconds and then redistributed to diffuse labeling in approximately 90 s in the dark (Movies S5 and S6; Supporting Information, method). The LIT system could be switched multiple times by alternating blue light and dark without significant loss in the magnitude of response (Figure 4b). These results demonstrated that the LIT system constitutes a reversible and reproducible way to tether ER and mitochondria in live mammalian cells. The LIT system could label ER−mitochondria interfaces and also enhance the ER−mitochondria interaction to an extent qualitatively similar (Figures 1−3) to the rapamycin-FKBPFRB-based system, which suggested a “zippering” model for ER−mitochondria tethering.14 This model proposes that induced ER−mitochondria interaction first happens at the pre-existing contact sites in cells expressing the LIT system with blue light exposure. Upon further illumination, lateral movement of the LIT constructs could enlarge ER−mitochondria contacts. Lastly, if ER−mitochondria contact surface expands as a “zipper”, this might allow the tethering of neighbor ER and mitochondria, forming expanded MAM-like structures. We developed the LIT system to overcome the disadvantages of the rapamycin-induced ER−mitochondria tethering method in terms of less invasive, higher speed of induction, and better targeting effect with reversibility. Estimated molecular length of the LIT constructs is 8.5−13.3 nm (Figure S6), which is relevant to the physiological distance of ER and mitochondria.14 Our new approach using the LIT system could be widely applicable to examine the role of ER−mitochondria contacts in various biological contexts, cell types, and pathological conditions. We also predict that mutational engineering of the LIT system to modulate on/off kinetics based on the characteristics of LOV2 domain could be developed to fulfill the broad requirement of various research and applications.30,31 By targeting to subcellular location in cells, this tool will allow the field of the neuroscience to unveil how ER−mitochondria tethering plays an important role in Ca2+ dynamics, transport, synaptic integration and metabolism in neuronal axons and dendrites.12 The subcellular restriction using the LIT system will be more helpful than rapamycin-based system for deciphering the function of ER−mitochondria contacts in

1:1 ratio using a single vector in mammalian cells. We next tested the IRES version of iLID-based LIT constructs in murine primary cortical neurons. Neuronal mitochondria were labeled using mitochondria-localized blue fluorescent proteins (mitoBFP), which contain Cox8 mitochondrial matrix-localization signal.29 Confocal time-lapse fluorescent imaging demonstrated that ER-targeted SspB-mKate2-CB5 became rapidly colocalized with mitochondria in the axon and soma in the primary neurons expressing the LIT IRES constructs after blue illumination (Figure 2c and Figure S5). The results demonstrate that our new optogenetic tool can be applicable in mammalian neurons. To validate the LIT system further, we next used electron microscopy to examine the ultrastructural features of the ER− mitochondrial interface. First, we expressed SspB-mKate2-CB5 and Venus-iLID-ActA in HEK 293T cells because LIT transfection efficiency in these cells was significantly higher than in NIH 3T3 cells. Following blue light exposure, the cells were immediately fixed in order to examine the effect of illumination on the ER and mitochondria tethering using electron microscope. Micrographs obtained by electron microscopy demonstrated that the cells exposed to blue light showed significant expansion of ER−mitochondria contacts while control cells kept in the dark showed limited ER− mitochondria contacts (Figure 3). These results confirm that our LIT system can induce tethering of ER and mitochondria in mammalian cells. On the basis of our confocal and electron microscope imaging, we conclude that the LIT system induces ER−mitochondria tethering to a similar degree compared to the previously reported rapamycin-FKBP-FRB-based tethering system.14 We next examined the spatial accuracy of our new optogenetic tool in NIH 3T3 cells. By activating a small region of interest (ROI) in individual NIH 3T3 cells with blue light, the puncta formation of a red fluorescent signal was limited in the ROI compared to global light stimulation (Figure 4a and Movies S5 and S6). This result showed that our tool could control this ER−mitochondria interaction in a spatially precise way. To assess whether the LIT system is temporally reversible and repeatable, we tested LIT reversibility by stimulating NIH 3T3 cells with blue light for 1 s, directly followed by a period of 5

DOI: 10.1021/acssynbio.7b00248 ACS Synth. Biol. 2018, 7, 2−9

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Figure 4. The LIT system activation with high spatiotemporal precision. (a) Representative confocal fluorescent images of an individual NIH 3T3 cells expressing the LIT system show the distribution of the red fluorescent signal after global stimulation (top) or spatially restricted (see region of interest, ROI) stimulation (bottom). The same cells were used for global and local stimulation repeatedly (three independent experiments). The yellow rectangle in the image indicates the stimulated ROI. Scale bar, 10 μm. (b) Enlarged ROI showing ER fluorescence in area 1 and 2 after global stimulation and localized stimulation and plots of intensity profile corresponding to line 1 and 2 in part a. (c) Representative confocal images with high magnification of an individual NIH 3T3 cell expressing the LIT system that underwent two cycles of switching on and off. Scale bar, 5 μm.

here will allow for the first time examination of whether ER− mitochondria contacts can regulate well-known cellular events such as branch-specific Ca2+ dynamics and whether these events are causally related to synaptic plasticity. Therefore, our new LIT-based approach represents a critical step toward the functional characterization of ER−mitochondria contacts in mammalian neurons.

synaptic plasticity and/or dendritic integration. The rapamycinbased system is not as reversible as LIT. Because LIT can be controlled temporally (both illumination and extinction), it will be useful for projects that require switching on−off multiple times and avoiding side effects caused by continuous ER− mitochondria tethering such as mitochondria Ca2+ overload.11 Our LIT system could also be useful as an inducible molecular linker to decipher the function of mitochondria and ER tethering and rescue the cellular and molecular phenotypes that are caused by disruption of ER−mitochondria interaction.32,33 Recently, we identified a new molecular linker, PDZD8, which is essential for ER−mitochondria tethering in mammalian cells.34 These findings reveal the critical importance of ER− mitochondria tethering for proper dendritic Ca2+ dynamics in mammalian neurons, opening many questions regarding the spatial distribution and temporal dynamics of ER−mitochondria in neuronal dendrites. The tool that we have developed

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METHODS Plasmid DNA Preparation. DNA constructs were generated using conventional restriction and ligation methods and PCR using 2× Phusion Master Mix (Thermo Scientific). Detailed plasmid DNA information is available in Supporting Information. Cell Culture and Transfection. NIH 3T3 cells and HEK 293T cells (ATCC) were maintained in Dulbecco’s Modified 6

DOI: 10.1021/acssynbio.7b00248 ACS Synth. Biol. 2018, 7, 2−9

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the reversibility of light-induced ER−mitochondria tethering with 1 s blue light stimulation more accurately. Statistic Analysis. All the experiment was conducted at least twice in the same condition. Pearson’s correlation coefficient and Manders colocalization coefficient (M2, the fraction of mKate2 overlapping with Venus) were used for correlation analysis. Two-tailed students’ t-test was used for comparison as appropriate using Prism (GraphPad).

Eagle Medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 unit/mL penicillin and 100 μg/mL of streptomycin (Gibco). JetPRIME (Polyplus transfection) and Lipofectamine 2000 (Invitrogen) were used for transfecting each glass bottom dish of NIH 3T3 and HEK 293T cells for fluorescent imaging and electron microscopy, respectively. The cells were tested 24−48 h after transfection. For culturing mouse cortical neurons, embryonic heads decapitated from CD1 mouse embryos at embryonic day E15.5 were injected with plasmid DNA and 0.5% Fast Green (Sigma-Aldrich; 1:20 ratio) using the MicroInject-1000 (BTX). Ex utero electroporation was performed on the whole head (skin and skull intact) with gold-coated electrodes (GenePads 5 × 7 mm BTX) using an ECM 830 electroporator (BTX) and the following parameters. Electroporated brains were dissected in modified Hank’s Buffered Salt Solution (referred as cHBSS in the Supporting Information). Cortices were dissociated in cHBSS containing papain and DNase I (100 μg/mL, SigmaAldrich) for 15 min at 37 °C, washed three times with cHBSS supplemented with DNase I and manually triturated in Neurobasal medium supplemented with B27 (1×), FBS (2.5%), L-glutamine (2 mM). The dissociated cells were then plated at 8.0 × 104 cells in the glass part of 35 mm glass bottom dish coated with poly-D-lysine (1 mg/mL, Sigma-Aldrich) and cultured for 8 days in Neurobasal medium supplemented with B27 (1×), FBS (2.5%), and L-glutamine (2 mM). Live Cell Fluorescent Imaging. For confocal NIH 3T3 cell imaging, a Nikon A1RMP laser scanning confocal inverted microscope equipped with Apo TIRF 60× NA1.49 oil objective lens at 37 °C was used. A Venus fluorescent signal was excited with 488 nm laser line (0.5−1.5% power) and detected using standard filter set at 525/50 nm. Red fluorescent protein mKate2 was excited with 561 nm laser line (0.5−1.5% power) and detected using a standard filter set at 595/50 nm. Blue light stimulation was induced using the Stimulation panel in the imaging software (NIS, Nikon). The stimulation laser line was 488 nm (stimulation speed, 1; stimulation power, 1%). Before imaging, the culture medium was replaced with homemade normal Tyrode solution (10 mM HEPES, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 2 mM MgCl2, 10 mM D-glucose, pH 7.4). Live cell imaging of mouse cortical neurons was performed at 8 DIV in cHBSS with a 60× NA 1.4 oil objective on a Nikon Ti-E microscope equipped with a Nikon A1 confocal. BFP was excited using 405 nm laser line (0.5−1.5% power) and mKate2 (20−30% power) was excited using 561 nm laser line. Blue light stimulation was achieved using 488 nm laser line (0.5% power). Neurons that had relatively higher fluorescent protein expression were chosen for the imaging. In detail, in Figure 4c, we used an imaging time course as a follow: (1) first acquiring images (both red and green channels); (2) stimulating 1 s with 488 nm laser; (3) waiting for 5 s in dark; (4) second acquiring images (both red and green). The illumination (1 s) was sufficient to induce ER−mitochondria tethering significantly (i.e., green and red fluorescent colocalization). For acquiring each frame image of red and green fluorescent channels, it took approximately 14 s in this experiment. To observe the LIT reversibility, we took another round of fluorescent imaging 90 s after the first cycle. During this time-lapse imaging, however, 488 nm excitation could turn on the LIT system again. In Movies S5 and S6, we recorded only the red channel images (SspB-mKate2-CB5). Therefore, there was no additional exposure of the cells to 488 nm excitation. The experimental protocol enables us to investigate

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00248. (Figure S1) schematic representations of the constructions of four other ER−mito tethering pairs and the representative fluorescent images of the NIH 3T3 cells expressing the tethering systems; (Figure S2) representative electron microscopic image of HEK 293T cells expressing the Magnet-based tethering system in dark; (Figure S3) representative confocal fluorescent images of an individual NIH 3T3 cell expressing iLID lightinsensitive mutant (C450M); (Figure S4) control experiments to observe endogenous colocalization of ER and mitochondria in NIH 3T3 cells with and without the LIT system in dark; (Figure S5) light-induced mitochondria and ER tethering in primary neurons; (Figure S6) measurement between OMM and ER estimated by structure modeling of iLID, SspB-SsrA, and mKate2; (Figure S7) amino acid sequences of Venus-iLID-ActA and SspB-mKate2-CB5 constructs (PDF) Movie S1. Confocal time-lapse imaging of the ER (SspBmKate2-CB5) in an individual NIH 3T3 cell expressing the LIT system (AVI) Movie S2. Confocal time-lapse imaging of the mitochondria (Venus-iLID-ActA) in an individual NIH 3T3 cell expressing LIT system (AVI) Movie S3. Confocal time-lapse imaging of the ER (SspBmKate2-CB5) and mitochondria (Venus-iLID-ActA) in an individual NIH 3T3 cell (AVI) Movie S4. Confocal time-lapse imaging of the ER (red, SspB-mKate2-CB5) and mitochondria (green, C450 M mutant type of Venus-iLID-ActA) in an individual NIH3T3 cell (AVI) Movie S5. Confocal time-lapse imaging of the ER in an individual NIH 3T3 cell expressing the LIT system with global stimulation (AVI) Movie S6. Confocal time-lapse imaging of the ER in an individual NIH 3T3 cell expressing the LIT system with local stimulation (AVI)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1-212-305-1890. Fax: +1-212-342-3889. E-mail: [email protected]. ORCID

Masayuki Yazawa: 0000-0002-5730-6583 Present Address ∇

S.E.P.: The Graduate School of Biomedical Sciences, The University of Texas Southwestern Medical Center, Dallas, TX 75390, U.S.A.

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DOI: 10.1021/acssynbio.7b00248 ACS Synth. Biol. 2018, 7, 2−9

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ACS Synthetic Biology Author Contributions

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F.P. and M.Y. conceived the approach. F.S., F.K., Y.H.F.P., and M.Y. designed the experiments. F.S., Y.H., S.K., E.S.P., and M.Y. performed the experiments. F.S. and M.Y. analyzed the data. F.S., F.K., and M.Y. wrote the manuscript. F.P. and Y.H. edited the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank J.R.N. Quejada and R. A. Bekdash for proofreading this manuscript and giving helpful suggestions, C. Henderson for facilitating this collaborative project, H. He for research support, and B. Witover, T. Swayne, and L. E. Munteanu for help with imaging. This work was supported by grants from China Scholarship Council (CSC) to F.S., Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowships for Research Abroad to F.K., Precursory Research for Embryonic Science and Technology (PRESTO)-JST (JPMJPR16F7 to Y.H.) and ImPACT Program of Council for Science, Technology and Innovation (Cabinet Office, Government of Japan) to M.Y. as well as a grant from NIH (NS089456) and the Roger De Spoelberch Award to F.P..

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DOI: 10.1021/acssynbio.7b00248 ACS Synth. Biol. 2018, 7, 2−9

Letter

ACS Synthetic Biology and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization. Biochemistry 55, 5264−5271. (32) Gomez-Suaga, P., Paillusson, S., Stoica, R., Noble, W., Hanger, D. P., and Miller, C. C. (2017) The ER-Mitochondria Tethering Complex VAPB-PTPIP51 Regulates Autophagy. Curr. Biol. 27, 371− 385. (33) Arruda, A. P., Pers, B. M., Parlakgul, G., Guney, E., Inouye, K., and Hotamisligil, G. S. (2014) Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity. Nat. Med. 20, 1427−1435. (34) Hirabayashi, Y., Kwon, S., Paek, H., Pernice, W. M., Paul, M. A., Lee, J., Erfani, P., Rackzkowski, A., Pettrey, D. S., Pon, L. A., and Polleux, F. (2017) ER−mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons. Science 358, 623.

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DOI: 10.1021/acssynbio.7b00248 ACS Synth. Biol. 2018, 7, 2−9