Optogenetic Control of Endoplasmic ReticulumâMitochondria

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Letter

Optogenetic control of endoplasmic reticulum-mitochondria tethering Fan Shi, Fuun Kawano, Seon-hye Emily Park, Shinji Komazaki, Yusuke Hirabayashi, Franck Polleux, and Masayuki Yazawa ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00248 • Publication Date (Web): 25 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017

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ACS Synthetic Biology

1

Optogenetic control of endoplasmic reticulum-mitochondria tethering

2 3

Fan Shi1,2, Fuun Kawano2, Seon-hye Emily Park2,7 Shinji Komazaki3, Yusuke Hirabayashi4,6

4

and Franck Polleux4 and Masayuki Yazawa2,5,*

5 6

1.

7 8

College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, China

2.

9

Department of Rehabilitation and Regenerative Medicine, Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA

10

3.

Department of Anatomy, Saitama Medical University, Saitama 350-0495, Japan

11

4.

Department of Neuroscience, Mortimer B. Zuckerman Mind Brain Behavior Institute

12

and Kavli Institute for Brain Science, Columbia University, New York, NY 10025, USA

13

5.

Department of Pharmacology, Columbia University, New York, NY 10032, USA

14

6.

Precursory Research for Embryonic Science and Technology (PRESTO), Japan

15 16 17

Science and Technology Agency, Saitama, Japan. 7.

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

18 19

*To whom correspondence should be addressed.

20

Tel: +1-212-305-1890

21

Fax: +1-212-342-3889

22

E-mail: [email protected]

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1 ACS Paragon Plus Environment

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Abstract

2

Organelle interface emerges as a dynamic platform for a variety of biological responses.

3

However, their study has been limited by the lack of tools to manipulate their occurrence in

4

live cells spatiotemporally.

5

light-inducible tethering (LIT) system allowing the induction of contacts between

6

endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of

7

light-dependent heterodimerization called iLID system.

8

iLID-based LIT approach enables control of ER-mitochondria tethering with high

9

spatiotemporal precision in various cell types including primary neurons, which will facilitate

10

Here, we report the development of a genetically-encoded

the functional study of ER-mitochondrial contacts.

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30


We demonstrate that the

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1

Keywords

2

Optogenetics, mitochondria, endoplasmic reticulum, iLID system, light-inducible tethering

3

(LIT) system, fluorescence microscopy and electron microscopy

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1

Text

2

Contacts between the ER and outer mitochondrial membranes (OMM) constitute an

3

important signaling interface that is crucial for multiple critical physiological functions

4

including local calcium (Ca2+) signaling, ATP production, mitochondrial fission and lipid

5

biogenesis(1-3).

6

forming a complex called ER-mitochondria encounter structure (ERMES)(4).

7

the ortholog of this ERMES complex has not been identified yet. However, several genes

8

have been reported as regulators for ER-mitochondria tethering: Mitofusin 2 (MFN2),

9

Presenilin2 (PS2) and Vesicle-associated membrane protein association protein B (VAPB)-

In yeast, ER-mitochondria tethering is regulated by a set of four proteins In metazoans,

10

Protein tyrosine phosphatase-interacting protein 51 (PTPIP51)(5-8).

11

genes has moderate effects on ER-mitochondria tethering in addition to mitochondria Ca2+

12

uptake, autophagosome formation and cell survival under stress(5-11). Dysfunction of

13

ER-mitochondria interaction has been also observed in various neurodegenerative

14

diseases such as Alzheimer’s disease, Parkinson’s disease and Amyotrophic Lateral

15

Sclerosis(12).

16

ER-mitochondria contacts is still limited because of the difficulty to visualize and manipulate

17

its distribution in live cells in a spatially precise and temporally dynamic way.

Disruption of these

Despite its potential significance, our understanding of the function of the

18

A chemical-induced dimerization system using rapamycin-FKBP-FRB has been

19

utilized to visualize ER-mitochondria junctions and to enhance tethering of the ER and

20

mitochondria, thereby introducing artificial mitochondria-associated ER membranes (MAM)

21

structures in mammalian cells(13, 14).

22

approach is limiting for performing subcellular manipulations and the diffusivity of chemical

23

compound limits spatial control and therefore its applicability for in vivo research.

24

rapamycin has some direct effects on mechanistic target of rapamycin (mTOR) signaling

25

pathway, a key regulator of autophagy,(15) which might have indirect but significant effects

26

on ER-mitochondria interactions. In addition, higher spatiotemporal resolution is required

27

to examine the role of ER-mitochondria interactions in soma, dendrites and axons of

28

mammalian neurons. For example, though dendritic Ca2+ dynamics play important role in

29

synaptic plasticity(16, 17), the role of ER-mitochondria interactions may be different between

30

the soma, dendrites and axons of mammalian neurons. Therefore, the development of

However, the spatial and temporal accuracy of this


Also,

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1

new technological approaches that enable the control of organelle interactions more

2

precisely with greater spatiotemporal resolution and minimize side effects is paramount for

3

cell biologists. Compared to the small organic molecule-inducible technique, optogenetic

4

approaches using genetically-encoded light-activated molecules can provide superior

5

spatial and temporal resolution.

6

homo- and hetero-dimerization have been used recently to develop a variety of optogenetic

7

tools allowing control of transcription, genome engineering and protein translocation(18-22).

8

To achieve precise control of ER-mitochondrial interaction, we developed a new

9

optogenetic tool based on optimized light-activated hetero-dimerization systems that can

10

Light-inducible protein-protein interactions including

be applied to tether ER and mitochondria in live mammalian cells.

11

To develop light-inducible ER-mitochondria tethering system, we expressed one part of

12

the light-inducible dimer pair in ER membranes by fusing with the ER anchor peptide

13

sequence from cytochrome b5 (CB5) and the other part in the outer mitochondria

14

membranes (OMM) by fusing with the OMM linker peptide sequence from ActA (Figure

15

1a)(13, 23).

16

constructs

17

hetero-dimerization leads to ER-mitochondria tethering upon illumination at the appropriate

18

wavelength.

19

ER-mitochondria tethering, we first constructed the ER and mitochondrial fusion proteins

20

using multiple light-inducible hetero-dimerization systems, Magnets (pMag/nMag), Flavin

21

Kelch-repeat F-box 1 (FKF1)/GIGANTEA (GI), Cryptochrome 2 (CRY2)/CRY-interacting

22

bHLH1 (CIB1), BphP1/Q-PAS1 and improved light induced dimer (iLID)/SspB.

23

a pair of Vivid (VVD) variants using mutagenesis that prevents their homo-dimerization and

24

induces pMag/nMag hetero-dimerization with blue light(24).

25

Oxygen and Voltage” (LOV) domain (NLOV, H105L mutant) of FKF1 were used as an

26

optimized fragment that can interact with GI upon blue light stimulation(18, 25).

27

widely used photoreceptor, which can be induced to bind its binding partner, CIB1, upon

28

blue light illumination(26). Besides blue light-inducible dimerization systems, we also took

29

advantage of a newly improved near-infrared (NIR)-activated system, BphP1/Q-PAS1.

30

BphP1 absorbs NIR (740-780 nm) causing it to switch from the ground state to active state,

To examine the expression of candidate constructs in live cells, we fused the with

fluorescent

proteins.

We

hypothesized

that

light-dependent

To examine which light-inducible systems would be optimal for inducible


Magnets is

The N-terminus and “Light,

CRY2 is a


Page 6 of 26

1

resulting in BphP1/Q-PAS1 interaction.

2

advantage of being reversible in dark conditions or with red light (660-700 nm)(27).

3

an engineered LOV2 domain of Phototropin by incorporating the SsrA peptide in the J-α

4

helix of LOV2 domain. Because SspB is the natural binding partner of SsrA, iLID leads to

5

steric occlusion of SspB binding in the dark and uncaging with blue light, resulting in a

6

light-inducible

7

hetero-dimerization pairs, we prepared the candidate constructs to enhance the ER and

8

mitochondria tethering.

hetero-dimerization(23).

The BphP1/Q-PAS1 interaction presents the

Taking

advantage

of

these

iLID is

light-inducible

9

Next, we examined the expression and function of the light-inducible constructs in

10

mammalian cell line, NIH 3T3 (Figure S1). The results using confocal microscopy suggest

11

that

12

pMag(3x)-mKate2-ActA and GI-mKate2-ActA. In addition, we found that the expression of

13

NLOV (H105L)-YFP-CB5 was low compared to the other CB5 constructs.

14

we found that the CRY2/CIB1- and BphP1/Q-PAS1-based constructs failed to induce

15

tethering of the ER and mitochondria in live cells mainly because constitutive binding

16

occurred in NIH 3T3 cells even without light-induction. To confirm that the Magnets-based

17

constructs affect mitochondria in mammalian cells, we used electron microscopy for further

18

investigation.

19

mitochondria aggregation in dark condition (Figure S2).

20

mitochondria

morphology

is

significantly

affected

in

cells

expressing

Unexpectedly,

The result reveals that the Magnets-based system induced significant

Next, we examined whether iLID/SspB could be used for light-inducible tethering (LIT)

21

system (Figure 1b).

22

expressing Venus-iLID-ActA and SspB-mKate2-CB5 and confirmed that SspB-mKate2-CB5

23

and Venus-iLID-ActA were localized in the ER and mitochondria membranes of NIH 3T3

24

cells in dark conditions, respectively.

25

SspB-mKate2-CB5 rapidly formed puncta or tubular structures and colocalized with

26

yellow/green fluorescent signal of the mitochondria (Figure 2a and Movie S1-S3) while no

27

obvious change of Venus-iLID-ActA localization was observed (Movie S2). To quantify

28

colocalization of the ER and mitochondria, we conducted optical density (OD)

29

measurements using line scan in NIH 3T3 cells expressing SspB-mKate2-CB5 and

30

Venus-iLID-ActA. The resulting plots (Figure 2a right panel) show the mKate2 and Venus

We conducted time-lapse confocal imaging of NIH 3T3 cells

Upon blue light exposure, red fluorescence of


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1

OD profiles corresponding to the yellow arrow line in the upper left image

2

(SspB-mKate2-CB5, dark).

3

increased from 0.2075 to 0.6089 after blue light stimulation (Figure 2a right panel),

4

suggesting rapid induction of colocalization of the two fusion proteins that are anchored to

5

the ER and mitochondria membranes.

The correlation coefficient of the red and green plots

6

As a negative control, we next tested the light-insensitive mutant (C450M) to confirm

7

that SspB-mKate2-CB5 translocation is dependent on blue light exposure. The C450M

8

mutation abolishes the covalent bond formation of the cysteine and cofactor flavin

9

mononucleotide, disrupting the conformational change of the iLID induced by blue light

10

stimulation(28).

11

found that the localization of SspB-mKate2-CB5 was not changed by blue light in the cells

12

expressing Venus-iLID (C450M)-ActA (Figure 2b, Figure S3 and Movie S4). The results

13

reveal that blue light exposure induces SspB-mKate2-CB5 and Venus-iLID-ActA

14

hetero-dimerization, leading to the ER-mitochondria tethering in live mammalian cells. We

15

further examined whether the expression of the photoswitch causes perturbation of ER and

16

mitochondria tethering in the dark condition.

17

cells transfected with mTFP-CB5 and SspB-mKate2-ActA, which indicate the localization of

18

mitochondria and ER.

19

system in the dark condition. We found that both groups showed similar distribution of ER

20

and mitochondria and there is no significant difference in the Pearson’s correlation

21

coefficient and Manders correlation coefficient between the groups (Figure S4).

Therefore, the C450M mutant is incapable of responding to blue light. We

As a control group, we prepared NIH 3T3

We next compared the control cells with cells expressing LIT

22

To improve the transduction of iLID-based LIT constructs in mammalian cells, we next

23

generated the internal ribosome entry site (IRES) version driven by human EF1α promoter

24

that enables us to co-express SspB-mKate2-CB5 and iLID-ActA at a 1:1 ratio using a single

25

vector in mammalian cells. We next tested the IRES version of iLID-based LIT constructs

26

in murine primary cortical neurons.

27

mitochondria-localized blue fluorescent proteins (mito-BFP), which contain Cox8

28

mitochondrial matrix-localization signal(29).

29

demonstrated that ER-targeted SspB-mKate2-CB5 became rapidly colocalized with

30

mitochondria in the axon and soma in the primary neurons expressing the LIT IRES

Neuronal mitochondria were labeled using

Confocal time-lapse fluorescent imaging



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1

constructs after blue illumination (Figure 2c and Figure S5). The results demonstrate that

2

our new optogenetic tool can be applicable in mammalian neurons.

3

To validate the LIT system further, we next used electron microscopy to examine the

4

ultrastructural

5

SspB-mKate2-CB5 and Venus-iLID-ActA in HEK 293T cells because LIT transfection

6

efficiency in these cells was significantly higher than in NIH 3T3 cells. Following blue light

7

exposure, the cells were immediately fixed in order to examine the effect of illumination on

8

the ER and mitochondria tethering using electron microscope. Micrographs obtained by

9

electron microscopy demonstrated that the cells exposed to blue light showed significant

10

expansion of ER-mitochondria contacts while control cells kept in dark showed limited

11

ER-mitochondria contacts (Figure 3).

12

induce tethering of ER and mitochondria in mammalian cells.

13

electron microscope imaging, we conclude that the LIT system induces ER-mitochondria

14

tethering

15

rapamycin-FKBP-FRB-based tethering system(14).

to

features

a

of

similar

the

ER-mitochondrial

degree

interface.

First,

we

expressed

These results confirm that our LIT system can

compared

to

Based on our confocal and

the

previously

reported

16

We next examined the spatial accuracy of our new optogenetic tool in NIH 3T3 cells.

17

By activating a small region of interest (ROI) in individual NIH 3T3 cells with blue light,

18

puncta formation of red fluorescent signal was limited in the ROI compared to global light

19

stimulation (Figure 4a and Movie S5 and S6). This result showed that our tool could

20

control this ER-mitochondria interaction in a spatially precise way.

21

To assess whether the LIT system is temporally reversible and repeatable, we tested

22

LIT reversibility by stimulating NIH 3T3 cells with blue light for 1 second, directly followed by

23

a period of time in the dark. We observed that the red fluorescent signal started to form

24

puncta and tubular structures within several seconds and then redistributed to diffuse

25

labeling in approximately 90 seconds in the dark (Movie S5 and S6, Supplementary

26

information - method). The LIT system could be switched multiple times by alternating

27

blue light and dark without significant loss in the magnitude of response (Figure 4b).

28

These results demonstrated that the LIT system constitutes a reversible and reproducible

29

way to tether ER and mitochondria in live mammalian cells.

30

The LIT system could label ER-mitochondria interfaces and also enhance the


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1

ER-mitochondria interaction to an extent qualitatively similar (Figure 1-3) to the

2

rapamycin-FKBP-FRB-based

3

ER-mitochondria tethering(14).

4

interaction first happens at the pre-existing contact sites in cells expressing the LIT system

5

with blue light exposure. Upon further illumination, lateral movement of the LIT constructs

6

could enlarge ER-mitochondria contacts.

7

expands as a ‘zipper’, this might allow the tethering of neighbor ER and mitochondria,

8

forming expanded MAM-like structures.

system,

which

suggested

a

“zippering”

model

for

This model proposes that induced ER-mitochondria

Lastly, if ER-mitochondria contact surface

9

We developed the LIT system to overcome the disadvantages of rapamycin-induced

10

ER-mitochondria tethering method in terms of less invasive, higher speed of induction and

11

better targeting effect with reversibility.

12

8.5-13.3nm (Figure S6), which is relevant to the physiological distance of ER and

13

mitochondria(14).

14

examine the role of ER-mitochondria contacts in various biological contexts, cell types and

15

pathological conditions. We also predict that mutational engineering of the LIT system to

16

modulate on/off kinetics based on the characteristics of LOV2 domain could be developed

17

to fulfill the broad requirement of various research and applications(30, 31).

18

subcellular location in cells, this tool will allow the field of the neuroscience to unveil how

19

ER-mitochondria tethering plays an important role in Ca2+ dynamics, transport, synaptic

20

integration and metabolism in neuronal axons and dendrites(12). The subcellular restriction

21

using the LIT system will be more helpful than rapamycin-based system for deciphering the

22

function of ER-mitochondria contacts in synaptic plasticity and/or dendritic integration.

23

The rapamycin-based system is not as reversible as LIT. Because LIT can be controlled

24

temporally (both illumination and extinction), it will be useful for projects that require

25

switching on-off multiple times and avoid side effects caused by continuous

26

ER-mitochondria tethering like mitochondria Ca2+ overload (11).

27

be useful as an inducible molecular linker to decipher the function of mitochondria and ER

28

tethering and rescue the cellular and molecular phenotypes that are caused by disruption of

29

ER-mitochondria interaction(32, 33).

30

which is essential for ER-mitochondria tethering in mammalian cells(34).

Estimated molecular length of the LIT constructs is

Our new approach using the LIT system could be widely applicable to

By targeting to

Our LIT system could also

Recently, we identified a new molecular linker, PDZD8,


These findings


1

reveal the critical importance of ER-mitochondria tethering for proper dendritic Ca2+

2

dynamics in mammalian neurons, opening many questions regarding the spatial

3

distribution and temporal dynamics of ER-mitochondria in neuronal dendrites. The tool

4

that we have developed here will allow for the first time to examine whether

5

ER-mitochondria contacts can regulate well-known cellular events such as branch-specific

6

Ca2+ dynamics and whether these events are causally related to synaptic plasticity.

7

Therefore, our new LIT-based approach represents a critical step towards the functional

8

characterization of ER-mitochondria contacts in mammalian neurons.

9


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Methods

2 3

Plasmid DNA preparation

4

DNA constructs were generated using conventional restriction and ligation methods and

5

PCR using 2x Phusion Master Mix (Thermo Scientific). Detailed plasmid DNA information is

6

available in Supporting Information.

7 8

Cell culture and transfection

9

NIH 3T3 cells and HEK 293T cells (ATCC) were maintained in Dulbecco’s Modified Eagle

10

Medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone),

11

100 unit/ml penicillin and 100 µg/ml of streptomycin (Gibco).

12

transfection) and Lipofectamine 2000 (Invitrogen) were used for transfecting each glass

13

bottom dish of NIH 3T3 and HEK 293T cells for fluorescent imaging and electron

14

microscopy, respectively.

15

culturing mouse cortical neurons, embryonic heads decapitated from CD1 mouse embryos

16

at embryonic day E15.5 were injected with plasmid DNA and 0.5% Fast Green

17

(Sigma-Aldrich; 1:20 ratio) using the MicroInject-1000 (BTX).

18

was performed on the whole head (skin and skull intact) with gold-coated electrodes

19

(GenePads 5 × 7 mm BTX) using an ECM 830 electroporator (BTX) and the following

20

parameters. Electroporated brains were dissected in modified Hank's Buffered Salt Solution

21

(referred as cHBSS in Supporting Information). Cortices were dissociated in cHBSS

22

containing papain and DNase I (100 µg/ml, Sigma-Aldrich) for 15 min at 37°C, washed

23

three times with cHBSS supplemented with DNase I and manually triturated in Neurobasal

24

medium supplemented with B27 (1x), FBS (2.5%), L-glutamine (2 mM).

25

cells were then plated at 8.0 x 104 cells in the glass part of 35mm glass bottom dish coated

26

with poly-D-lysine (1 mg/ml, Sigma-Aldrich) and cultured for 8 days in Neurobasal medium

27

supplemented with B27 (1x), FBS (2.5%) and L-glutamine (2 mM).

JetPRIME (Polyplus

The cells were tested 24-48 hours after transfection.

For

Ex utero electroporation

The dissociated

28 29

Live Cell fluorescent imaging

30

For confocal NIH 3T3 cell imaging, a Nikon A1RMP laser scanning confocal inverted



1

microscope equipped with Apo TIRF 60x NA1.49 oil objective lens at 37°C was used.

2

Venus fluorescent signal was excited with 488nm laser line (0.5-1.5% power) and detected

3

using standard filter set at 525/50nm. Red fluorescent protein mKates was excited with

4

561nm laser line (0.5-1.5% power) and detected using standard filter set at 595/50nm.

5

Blue light stimulation was induced using the Stimulation panel in the imaging software (NIS,

6

Nikon). The stimulation laser line was 488nm (stimulation speed, 1; stimulation power, 1%).

7

Before imaging, the culture medium was replaced with home-made normal Tyrode solution

8

(10mM HEPES, 140mM NaCl, 5.4mM KCl, 1.8mM CaCl2, 2mM MgCl2, 10mM D-glucose,

9

pH 7.4). Live cell imaging of mouse cortical neurons was performed at 8 DIV in cHBSS with

10

a 60x NA 1.4 oil objective on a Nikon Ti-E microscope equipped with a Nikon A1 confocal.

11

BFP was excited using 405 nm laser line (0.5-1.5% power) and mKate2 (20-30% power)

12

was excited using 561 nm laser line. Blue light stimulation was achieved using 488 nm laser

13

line (0.5% power). Neurons that had relatively higher fluorescent protein expression were

14

chosen for the imaging. In details, in Fig. 4c, we used an imaging time course as a follow: 1)

15

1st acquiring images (both red and green channels); 2) stimulating 1s with 488 nm laser; 3)

16

waiting for 5s in dark; 4) 2nd acquiring images (both red and green). The illumination (1s)

17

was sufficient to induce ER-mitochondria tethering significantly (i.e. green and ref

18

fluorescent co-localization). For acquiring each frame image of red and green fluorescent

19

channels, it took approximately 14s in this experiment. To observe the LIT reversibility, we

20

took another round of fluorescent imaging 90s after the first cycle. During this time-lapse

21

imaging, however, 488-nm excitation could turn on LIT system again. In Supplementary

22

Movie 5 and 6, we recorded only the red channel images (SspB-mKate2-CB5). Therefore,

23

there was no additional exposure of the cells to 488-nm excitation. The experimental

24

protocol enables us to investigate the reversibility of light-induced ER-mitochondria

25

tethering with 1s blue light stimulation more accurately.

26 27

Statistic analysis

28

All the experiment was conducted at least twice in the same condition. Pearson’s

29

correlation coefficient and Manders colocalization coefficient (M2, the fraction of mKate2

30

overlapping with Venus) were used for correlation analysis. Two-tailed students’ t-test was


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used for comparison as appropriate using Prism (GraphPad).

2 3 4 5 6 7 8 9 10 11 12 13



1

Supporting information summary

2

Figure S1. Schematic representations of the constructions of four other ER-mito tethering

3

pairs and the representative fluorescent images of the NIH 3T3 cells expressing the

4

tethering systems.

5

Figure S2. Representative electron microscopic image of HEK 293T cells expressing the

6

Magnet-based tethering system in dark.

7

Figure S3. The representative confocal fluorescent images of an individual NIH 3T3 cell

8

expressing iLID light-insensitive mutant (C450M).

9

Figure S4. Control experiments to observe endogenous colocalization of ER and

10

mitochondria in NIH 3T3 cells with and without the LIT system in dark.

11

Figure S5. Light-induced mitochondria and ER tethering in primary neurons.

12

Figure S6. Measurement between OMM and ER estimated by structure modeling of iLID,

13

SspB-SsrA and mKate2.

14

Figure S7. The amino acid sequences of Venus-iLID-ActA and SspB-mKate2-CB5

15

constructs.

16

Movie S1. Confocal time-lapse imaging of the ER (SspB-mKate2-CB5) in an individual NIH

17

3T3 cell expressing the LIT system.

18

Movie S2. Confocal time-lapse imaging of the mitochondria (Venus-iLID-ActA) in an

19

individual NIH 3T3 cell expressing LIT system.

20

Movie S3. Confocal time-lapse imaging of the ER (SspB-mKate2-CB5) and mitochondria

21

(Venus-iLID-ActA) in an individual NIH 3T3 cell.

22

Movie S4. Confocal time-lapse imaging of the ER (red, SspB-mKate2-CB5) and

23

mitochondria (green, C450M mutant type of Venus-iLID-ActA) in an individual NIH3T3 cell.

24

Movie S5. Confocal time-lapse imaging of the ER in an individual NIH 3T3 cell expressing

25

the LIT system with global stimulation.

26

Movie S6. Confocal time-lapse imaging of the ER in an individual NIH 3T3 cell expressing

27

the LIT system with local stimulation.

28 29


Page 14 of 26

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1

Author Information

2

Corresponding Author

3

*Email: [email protected]

4 5

Author Contributions

6

F.P. and M.Y. conceived the approach. F.S., F.K., Y.H. F.P. and M.Y. designed the

7

experiments. F.S., Y.H., S.K., E.S.P. and M.Y. performed the experiments. F.S. and M.Y.

8

analyzed the data. F.S., F.K. and M.Y. wrote the manuscript. F.P. and Y.H. edited the

9

manuscript.

10 11

Acknowledgements

12

We thank J.R.N. Quejada and R. A. Bekdash for proofreading this manuscript and giving

13

helpful suggestions, C. Henderson for facilitating this collaborative project, H. He for

14

research support and B. Witover, T. Swayne and L. E. Munteanu for help with imaging. This

15

work was supported by grants from China Scholarship Council (CSC) to F.S., Japan

16

Society for the Promotion of Science (JSPS) Postdoctoral Fellowships for Research Abroad

17

to F.K., Precursory Research for Embryonic Science and Technology (PRESTO)-JST

18

(JPMJPR16F7 to Y.H.) and ImPACT Program of Council for Science, Technology and

19

Innovation (Cabinet Office, Government of Japan) to M.Y. as well as a grant from NIH

20

(NS089456) and the Roger De Spoelberch Award to F.P..

21 22

Notes

23

The authors declare no competing financial interest.

24 25 26 27 28 15 ACS Paragon Plus Environment


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Page 20 of 26

Figure Legends

2 3

Figure 1. The LIT system for optogenetic control of ER and mitochondria contacts.

4

(a) Schematic representation of light-inducible tethering (LIT) system using light-induced

5

heterodimerization to tether the endoplasmic reticulum (ER) and mitochondria. OMM, outer

6

mitochondrial membrane. (b) Schematic representation of the iLID based LIT system.

7 8

Figure 2. The LIT system to tether ER and mitochondria.

9

(a) Representative confocal fluorescent images of an individual NIH 3T3 cell expressing the

10

LIT system in dark and with blue light. The corresponding line profiles (yellow, in upper left

11

image) are shown on the right hand side panels. R value represents the correlation

12

coefficient of the red and green fluorescent signals in the upper and lower panel,

13

respectively. (b) Value change of Manders colocalization coefficient (MCC) M2 (mKate2

14

overlapping to Venus) following blue light stimulation in the NIH 3T3 cells. We observed a

15

significant increase of M2 in cells expressing WT LIT system while random minor change of

16

M2 was observed in cells expressing the C450M mutant. (n = 15 for WT, n = 16 for C450M,

17

three independent experiments). (c) Representative confocal fluorescent images of an

18

axon

19

(SspB-mKate2-CB5-IRES-iLID-ActA) and mitochondrial-localizing BFP (mito-BFP). After

20

stimulating blue light, the mKate2 red fluorescent proteins fused to SspB became

21

colocalized with the mitochondria (indicated by yellow arrowheads). Plot intensity profiles

22

are drawn along the axon as reference. (d) Value change of Pearson’s correlation

23

coefficient (PCC) after blue light stimulation of segments of axons at 12 s. (n = 29 for LIT, n

24

= 19 for Ctrl, 2 independent experiments). *P

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