Optogenetic control of molecular motors and organelle distributions in cells

Abstract

Intracellular transport and distribution of organelles play important roles in diverse cellular functions, including cell polarization, intracellular signaling, cell survival, and apoptosis. Here, we report an optogenetic strategy to control the transport and distribution of organelles by light. This is achieved by optically recruiting molecular motors onto organelles through the heterodimerization of Arabidopsis thaliana cryptochrome 2 (CRY2) and its interacting partner CIB1. CRY2 and CIB1 dimerize within subseconds upon exposure to blue light, which requires no exogenous ligands and low intensity of light. We demonstrate that mitochondria, peroxisomes, and lysosomes can be driven toward the cell periphery upon light-induced recruitment of kinesin, or toward the cell nucleus upon recruitment of dynein. Light-induced motor recruitment and organelle movements are repeatable, reversible, and can be achieved at subcellular regions. This light-controlled organelle redistribution provides a new strategy for studying the causal roles of organelle transport and distribution in cellular functions in living cells.

Figure 1. Schematic of Light-Controlled Motor Recruitment and Organelle Transport in Cells

(A) CRY2 is anchored to organelles via specific organelle targeting transmembrane domain. CIB1 is linked to truncated kinesin KIF5A. Upon blue light exposure, CIB1-CRY2 binding recruits kinesin motors to organelles, which drives organelles towards the plus end of microtubule. (B) CIB1 domain is linked to BICDN, a dynein/dynactin adaptor protein. When illuminated with blue light, CIB1 binds with CRY2 and thus recruits dynein to organelles. Consequently, organelles are propelled towards the minus end of microtubule.

Figure 2. Light-Induced Re-Distribution of Mitochondria in COS-7 Cells by Recruiting Molecular Motors

(A–D) The COS-7 cell was transfected with CRY2-mCherry-MiroTM and GFP-BICDN-CIB1. (A) Mitochondria were visualized in the red channel by CRY2-mCherry-MiroTM. Before blue light illumination, some mitochondria were positioned close to the nucleus while others spread throughout the cell. After blue light illumination, most mitochondria moved towards cell nucleus as indicated by the yellow circle. (N=20) (B) The green channel showed GFP-BICDN-CIB1 diffusing in the cytosol. (C) The percentage of mitochondria inside the yellow circle in (A) increased from 39% to 89% after intermittent light exposure for 60 minutes. (D) The kymograph extracted from the area indicated by yellow rectangle in (A) showed the clear trend of mitochondria moving toward the nucleus. (E–H) The COS-7 cell was transfected with CRY2-mCherry-MiroTM and KIF5A-GFP-CIB1. (E) After blue light illumination, many mitochondria moved away from the perinuclear region (yellow circle) and some clustered at cell edge as indicated by the arrows. (N=73) (F) KIF5A-GFP-CIB1 in the same cell distributed along microtubules. (G) The percentage of mitochondria at the perinuclear region (inside the yellow circle) decreased from 52% to 36% after intermittent light exposure for 30 minutes. (H) The kymograph extracted from the area indicated by yellow rectangle in (E) showed that over time, mitochondria were actively moved towards the cell periphery. Scale bars, 10µm. See also Figure S2, Movies S1 and S2.

Figure 3. The Distributions of Peroxisomes and Lysosomes in Cells Can Be Modulated by Light-Induced Recruitment of Motors

(A) In the cell co-transfected with GFP-BICDN-CIB1 and PEX-mCherry-CRY2, blue light illumination caused peroxisomes to become highly clustered around the cell nucleus indicated by the circle. (B) Kymographs generated along the yellow rectangular region in (A) shows the trajectories of peroxisomes moving toward the nucleus. (C) In the cell transfected with KIF5A-GFP-CIB1 and PEX-mCherry-CRY2, blue light illumination caused peroxisomes to move away from the cell nucleus. The circles marked the cell nuclei. (D) Kymographs generated along the yellow rectangular region in (C) shows peroxisomes moving away from the nucleus. (E) In the cell transfected with GFP-BICDN-CIB1 and LAMP-mCherry-CRY2, blue light illumination induced lysosomes clustering around the nucleus marked by the yellow circle. (F) Kymographs generated along the yellow rectangular region in (E) shows lysosomes moving toward the nucleus. (G) In the cell transfected with KIF5A-GFP-CIB1 and LAMP-mCherry-CRY2, lysosomes moved towards cell periphery with blue light exposure. The cell nucleus was indicated by the eclipse. (H) Kymographs generated along the yellow rectangular region in (G) shows lysosomes moving toward the cell periphery. Scale bars, 10µm. See also Figure S5.

Figure 4. Characterization of Organelle Transport by Light-Induced Recruitment of Molecular Motors

(A) The peroxisome indicated by the yellow arrow moved along the microtubule and towards the cell periphery by light-induced recruitment of kinesins. The cell was triple-transfected with KIF5A-CIB1, TAU-YFP and PEX-mCherry-CRY2. Microtubules were labeled by TAU-YFP. Z projection of peroxisomes was obtained by projecting the maximum intensity of PEX-mCherry-CRY2 during 20 frames acquisition at 1frame/second and was merged with GFP image, which overlaps with the underlying microtubule. (B) Z projection of light-induced peroxisome trajectories spreads radially from the nucleus to cell periphery, resembling the distribution of microtubule network. The cell was triple-transfected with KIF5A-CIB1, TAU-YFP and PEX-mCherry-CRY2. Z projection of peroxisomes was obtained by recording the maximum intensity of PEX-mCherry-CRY2 during 100 frames acquisition at 1frame/second. (C) Kymographs of the indicated yellow lines (I) and (II) in (B) show that many peroxisomes moved towards cell periphery on the same microtubules and at a relatively constant speed. (D) Histogram of peroxisomes moving speeds for 177 peroxisomes in 4 cells shows an average speed of 0.55 µm/s. (E) Trajectories of light-induced peroxisome retrograde movement resemble the distribution of microtubules around the center where many microtubules merged. The cell was triple-transfected with BICDN-CIB1, TAU-YFP and PEX-mCherry-CRY2. (F) Kymographs of the indicated lines (III) and (IV) show that peroxisomes moved along microtubules towards cell nucleus. (G) Histogram of peroxisomes speeds was obtained by calculating the speeds for 119 peroxisomes in 4 cells shows an average speed of 0.57 µm/s. Scale bars, 5 µm (A), 10µm (B, E). See also Movie S3 and S4.

Figure 5. Light-Induced Organelle Redistribution Is Reversible and Is Dependent on Motor Expression Level

(A) CRY2 and CIB1 binding on mitochondria membrane is reversible and repeatible. The cell was transfected with CIB1-GFP-Miro1TM and CYR2-mCherry. Before blue light, CIB1-GFP-Miro1TM was targeted to mitochondria while CYR2PHR-mCherry was cytosolic. Upon blue light exposure, CRY2-mCherry bound to CIB1-GFP-Miro1TM and localized to mitocondria. (B) The same transfected cell shown in (A) was repeatedly subjected to four cycles of blue light and dark periods. For each cycle, CRY2-mCherry was recruited to mitochondria within seconds of blue light illumination, while it returned to its cytosolic distribution after incubating in dark for 10–15 minutes. Within each period of blue light illumination, the cell was stimulated with 200-ms exposure of blue light at 9.7 W/cm² per 1-second interval. (C) In the cell transfected with CRY2-mCherry-MiroTM and KIF5A-GFP-CIB1, mitochondria were driven towards the cell periphery by intermittent blue light exposure (one 200-ms pulse per 10 second) for 30 minutes. Then the cell was incubated without blue light stimulation and mitochondria were observed to gradually move back towards the cell nucleus. Quantification of the percentage of the mitochondria inside the yellow circle shows the percentage decreased from 61% to 40% after blue light stimulation and increased back to 63% after 3-hour dark incubation. (D) The average moving distances of mitochondria after 15-minute intermittent blue light illumination were calculated and plotted along with motor expression level. In 20 cells double-transfected with CRY2-mCherry-MiroTM and KIF5A-GFP-CIB1, the higher the KIF5A expression level was, the further the mitochondria moved towards cell periphery at the end of 15-minute period (left graph). However, in 17 cells transfected with CRY2P-mCherry-MiroTM and GFP-BICDN-CIB1, the expression level of BICDN had no obvious influence on how far mitochondria moved towards cell nucleus during 15-minute period (right graph). Scale bars, 10µm. See also Movie S5.

Figure 6. Subcellular Spatial Control of Light-Induced Mitochondria Movement

(A) The COS-7 cell was transfected with CRY2-mCherry and CIB1-GFP-MiroTM. Blue light illumination was restricted within a subcellular region of the cell (indicated by the blue circle). Upon blue light activation, CRY2-mCherry was recruited to mitochondria only in the targeted region, whereas it remained cytosolic in the rest of the cell. The local recruitment can be more clearly seen in the zoomed-in images shown in the lower panel. Later, the whole cell was illuminated with blue light and CRY2-mCherry got colocalized with mitochondria in the whole cell (the rightmost image). (B) In the cell co-expressing CRY2-mCherry-MiroTM and KIF5A-GFP-CIB1, only the marked region was illuminated with intermittent blue light for 1 minute at 5-second interval. Only in this region, mitochondria moved outwards to the cell periphery as indicated by the white arrow while in the remaining part of the cell, mitochondria distribution remained mostly unchanged (the middle image). Later, the whole cell was illuminated with intermittent blue light for 15 minutes and mitochondria moved out towards cell periphery throughout the cell as indicated by the white arrows. Scale bars, 10µm.