Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth1,2,3,4,5,6,7,8. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.
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Small molecule-nanobody conjugate induced proximity controls intracellular processes and modulates endogenous unligandable targets
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We are grateful to C. Tucker, T. Inoue, Z.-H. Sheng, G. Banker, R. Prekeris, P. Schätzle and M. Esteves da Silva for sharing reagents and to C. Wierenga and A. Akhmanova for discussions. This research is supported by the Dutch Technology Foundation STW and the Foundation for Fundamental Research on Matter (FOM), which are part of the Netherlands Organisation for Scientific Research (NWO). Additional support came from NWO (NWO-ALW-VICI to C.C.H and NWO-ALW-VIDI to L.C.K.) and the European Research Council (ERC starting grant to L.C.K.).
The authors declare no competing financial interests.
Extended data figures and tables
a, b, Assay and constructs. A fusion construct of PEX3, monomeric red fluorescent protein (mRFP) and LOVpep (PEX–LOV) targets peroxisomes. After blue-light illumination, a fusion of the N terminus of the dynein adaptor BICD2 and ePDZb1 (BICDN–PDZ) is recruited to peroxisomes. c, Peroxisome distribution in a COS-7 cell expressing PEX–LOV and BICDN–PDZ before and during light-induced recruitment of dynein (inverted contrast). Red lines indicate cell outline. Scale bar, 10 μm. See Supplementary Video 1. d, Black: time trace of R90% (radius of circle enclosing 90% of cellular fluorescence; see Methods) in cells expressing PEX–LOV and BICDN–PDZ (n = 5 cells). Red: correlation index (frame-to-frame differences in the peroxisome recordings; see Methods) of the same cells. Blue-light illumination is indicated in blue; mean ± s.e.m.
Extended Data Figure 2 Light-induced organelle redistribution is organelle-specific and does not affect the cytoskeleton.
a, Images of fixed cells expressing PEX–LOV and KIF–PDZ, showing the distribution of peroxisomes and mitochondria (anti-cytochrome-c), or peroxisomes and the endoplasmic reticulum (anti-protein disulfide isomerase (PDI)) in the absence (left) or presence (right) of blue light. b, Images of fixed cells expressing PEX–LOV and KIF–PDZ, showing the distribution of peroxisomes and phalloidin, α-tubulin or EB1 staining in the absence or presence of blue light. c, Images of fixed cells expressing FKBP–RAB11, FRB–LOV and KIF–PDZ, showing the distribution of RAB11 recycling endosomes together with lysosomes (anti-Lamp1) or early endosomes (anti-EEA1) in the absence or presence of blue light. Red lines indicate cell outline. Scale bars, 10 μm.
Extended Data Figure 3 After light induced organelle displacement, peroxisomes remain at their newly obtained position whereas the distribution of recycling endosomes quickly reverses back to normal.
a, Peroxisome distribution before and after exposure to blue light in cells expressing PEX–LOV and KIF–PDZ. Blue-light illumination was terminated at t0:00. b, Distribution of RAB11 recycling endosomes before and after exposure to blue light in cells expressing FKBP–RAB11, FRB–LOV and KIF–PDZ. Blue light was turned off at t0:00. Red lines indicate cell outline. Scale bars, 10 μm. See Supplementary Video 4.
a, b, Assay and constructs. A fusion construct of Cry2PHR, tagRFPt and RAB11, (CRY–RAB11) targets RAB11 recycling endosomes. After blue-light illumination, a fusion of truncated KIF1A, GFP and CIBN (KIF–CIBN) or a fusion of truncated BICDN, GFP and CIBN (BICDN–CIBN) is recruited to RAB11 recycling endosomes. c, RAB11 vesicle distribution before and after light-induced recruitment of KIF1A (inverted contrast). Red lines indicate cell outline. Scale bar, 10 μm. d, Overlay of sequential binarized images from the recording in c, colour-coded by time as indicated. Orange marks the initial distribution of RAB11 vesicles, whereas green marks regions targeted after exposure to blue light. e, Time trace of the R60% and R90% (black) and the correlation index (red) of the cell shown in c and d. Blue box marks blue-light illumination. f, RAB11 distribution in a cell expressing CRY–RAB11 and BICDN–CIBN before and after blue-light illumination (inverted contrast). Red lines indicate cell outline. Scale bar, 10 μm. g, Time trace of the R60% and R90% (black) and correlation index for the cell shown in f. h, Irradiance response curve for cells transfected with CRY–PEX and KIF–CIBN (red), or PEX–LOV plus KIF–PDZ (black). To exclude activation failure due to poorly expressed motors, the number of cells reacting at each concentration was divided by the number of cells responding to subsequent high irradiance (∼1.3 W cm−2). Three biological replicates. Cells per intensity (for increasing intensities): 28, 21, 22, 20, 24, 22 and 20 for CRY, 30, 28, 33, 31, 28, 33, 33, 32 and 26 for LOV. Error bars depict s.e.m.; three biological replicates. Solid line shows fit to , with R the response, I the illumination intensity, I0 the intensity at which the response is 50%, and n the Hill coefficient. For CRY–PEX and PEX–LOV, I0 is 0.05 and 0.12 W cm−2, respectively. i, j, Assay and constructs. A fusion construct of FKBP, tagRFPt and RAB11 (FKBP–RAB11) targets RAB11 recycling endosomes. Rapalog addition couples FKBP to FRB, leading to recruitment of the FRB, tagBFP and LOVpep fusion protein (FRB–LOV). After blue-light illumination a fusion of truncated myosin-Vb, GFP and ePDZb1 (MYO–PDZ) is recruited to RAB11 vesicles. k, RAB11 distribution in a cell expressing FKBP–RAB11, FRB–LOV and MYO–PDZ before sequential blue-light illumination of the regions marked with numbered boxes (inverted contrast). Scale bar, 10 μm. See Supplementary Video 5. l, Time traces of the correlation index in the areas shown in k. Blue box marks whole-cell exposure to blue light, whereas colored boxes indicate local illumination. m, Example trajectories of two RAB11 recycling endosomes before, during and after recruitment of myosin-Vb, as indicated. Data was acquired with 1-s intervals. For each period 40 s are shown. n, Frame-to-frame displacements of RAB11 recycling endosomes before, during and after light-induced recruitment of myosin-Vb (5 s interval). Thick lines show the average of five tracks in shades of grey. o, FKBP–RAB11 distribution (inverted contrast) in a dendrite and dendritic spines before, during and after blue-light illumination. Images are maximum projections spanning 60 s. Red lines indicate cell outline, arrowheads mark spines targeted with recycling endosomes during blue-light illumination. Scale bar, 2 μm. p, Percentage of recycling endosome spine entry events per dendrite before, during and after illumination in bins of 100 s. Blue box indicates blue-light illuminated interval, n = 16 dendrites in three independent experiments. Red bar denotes mean ± s.e.m., ***P < 0.0001, one-way ANOVA, Bonferroni’s post-hoc test. q, Histogram of fraction of all (n = 237) recycling endosome spine entries in bins of 20 s. Blue box indicates blue-light-illuminated interval.
Extended Data Figure 5 Rapalog in the nanomolar range is sufficient to recruit FRB–LOV to FKBP–RAB11 and does not affect the number of spines or growth cones in hippocampal neurons.
a, Response curve of RAB11 recycling endosome relocalization in cells expressing FKBP–RAB11, FRB–LOV and KIF–PDZ exposed to blue light in relation to rapalog concentration. To exclude activation failure due to poorly expressed motors, the number of cells reacting at each concentration was divided by the number of cells responding to subsequent high rapalog concentration (1 μM). Solid line shows fit to , with R the response, c the rapalog concentration, c0 the concentration at which the response is 50%, n the Hill coefficient, and Rmin the response at 0 mM rapalog. Rmin is 22% and c0 is 15 nM. n = 30 (0.1 nM), 37 (1 nM), 30 (10 nM), 28 (100 nM) and 28 (500 nM) responsive cells from three independent experiments. Error bars depict s.e.m. b, Hippocampal neurons transfected with membrane–GFP incubated for 2.5 h in the presence or absence of 100 nM rapalog, co-stained with the post-synaptic marker Homer. c, Quantification of the number of Homer puncta per 100 μm dendrite length in the presence or absence of 100 nM rapalog (n = 13 neurons per condition). Error bars depict s.e.m. d, Hippocampal neurons transfected with GFP incubated for 2.5 h in the presence or absence of 100 nM rapalog, co-stained with phalloidin. e, Quantification of the number of growth cones per 50,000 μm2 in the presence or absence of 100 nM rapalog, co-stained with phalloidin. n = 19. Scale bars, 5 μm. Error bars depict s.e.m.
Extended Data Figure 6 RAB11 fusion constructs are recognized by the RAB11 antibody, partially co-localize with transferrin receptors and interact with RAB11FIP1.
a, Images of untransfected cells or cells transfected with CRY–RAB11, FKBP–RAB11 or tagRFPt–RAB11, co-stained with anti-RAB11 antibody (inverted contrast). Red lines indicate cell outline. Scale bar, 2.5 μm. b, Images of cells transfected with TfR-GFP only, or co-transfected with CRY–RAB11, FKBP–RAB11 or tagRFPt–RAB11 (inverted contrast). Red lines indicate cell outline. Scale bar, 2.5 μm. c–e, GFP pull-down assays with lysates of HEK cells expressing GFP or GFP–RAB11FIP1 together with tagRFPt–RAB11 (c), FKBP–RAB11 or FKBP–tagRFPt–RAB6 (d) or CRY–RAB11 (e) were analysed by western blotting using antibodies against tagRFPt and GFP.
Extended Data Figure 7 BICDN overexpression does not significantly inhibit dynein-based transport and the growth cone cytoskeleton is not affected by light-induced recruitment of BICDN to recycling endosomes.
a, b, Left: kymograph of dense-core vesicles motility in an axon expressing neuropeptide Y (NPY) fused to GFP (NPY–GFP) and empty vector (a) or BICDN–PDZ (b) (inverted contrast), representative of n = 5 and n = 10 axons, respectively. Right: corresponding binary image of traces used for further analysis of anterograde and retrograde movements. Scale bars, 5 μm and 10 s. c, Position of dense-core vesicles along an axon expressing NPY–GFP and BICDN–PDZ. Single coloured arrowheads point to the same vesicle, highlighting retrograde (red), anterograde (black) and non-moving (orange) vesicles. Scale bar, 5 μm. d, Quantification of the percentage of static, anterograde and retrograde moving vesicles from kymographs shown in a and b in axons with (n = 10) or without (n = 5) BICDN–PDZ overexpression. Graph shows mean ± s.e.m., *P > 0.05, one-way ANOVA and Bonferroni’s multiple comparison test. e, A fusion of FRB, tagBFP and LOVpep (FRB–LOV) and a fusion of GFP and ePDZb1 (PDZ) were expressed in neurons. After blue-light illumination, LOVpep undergoes a conformational change, allowing binding of PDZ to FRB–LOV. f, Actin dynamics in growth cones coexpressing mRFP–actin along with the constructs shown in e, in response to light-induced heterodimerization of FRB–LOV and PDZ, representative of n = 5 growth cones. The blue box indicates the interval of blue-light illumination. Scale bar, 5 μm. g, Imaging of growing microtubule (MT) plus ends using tdTomato-MACF18 shows the dynamics of microtubule plus-ends in growth cones before and during blue-light illumination in neurons co-expressing FKBP–RAB11, FRB–LOV and BICDN–PDZ. Red line indicates cell outline, arrowheads point at plus-ends. Scale bar, 5 μm. h, Kymograph of MACF18 comets of the growth cone shown in g and binarized traces used for analysis, representative of n = 4 growth cones. Blue box indicates blue-light illumination interval. Scale bars, 5 μm and 1 min. i, Area measurement of growth cone shown in g before and during blue-light illumination. j, Quantification of the number of MACF18 comets per minute in growth cones before and during blue-light illumination (n = 4 neurons). Graph shows mean ± s.e.m. Paired two-tailed t-test, n = 4 cells. k, Quantification of the growth length of MACF18 comets in growth cones before and during blue-light illumination (n = 4 neurons). Graph shows mean ± s.e.m. Paired two-tailed t-test, n = 4 cells. l, Distribution of fraction of MACF18 comets per growth length in bins of 0.5 μm (n = 214 traces). m, Quantification of the growth speed of MACF18 comets in growth cones before and during blue-light illumination (n = 4 neurons). Graph shows mean ± s.e.m. Paired two-tailed t-test, n = 4 cells. n, Distribution of fraction of MACF18 comets per growth speed in bins of 0.1 μm s−1 (n = 214 traces).
Extended Data Figure 8 Intensity rescaling and accurate growth cone area measurements based on RAB11 fluorescence.
a, Mean intensity of growth cone FKBP–RAB11 fluorescence from neurons expressing BICDN–PDZ in the absence (black, n = 21) or presence of FRB–LOV (red, n = 25) normalized to the intensity before (t−2:30 min) (left axis) and rescaled relatively to the intensity of –LOV growth cones at t8 min (right axis). Blue box indicates blue-light illuminated interval. Graph shows mean ± s.e.m. b, Quantification of FKBP–RAB11 fluorescence intensity in the same neurons as shown in a after 8 min of blue-light illumination, normalized to the average fluorescence at t8 min in control neurons. Graph shows mean ± s.e.m., ***P < 0.0001, Mann–Whitney test. c, Area measurements of two representative growth cones from neurons expressing FKBP–RAB11, FRB–LOV, BICDN–PDZ and soluble GFP over time. Representative of five growth cones (shown in d and e). d, Normalized tagRFPt-RAB11 intensity of five growth cones as in c plotted against their normalized GFP intensity. Intensity values are averaged over the first five frames per growth cone. Pearson correlation coefficient (r) for each growth cone is indicated in top left corner. Same colour indicates measurements of the same growth cone. e, FKBP–RAB11-based area measurements plotted against GFP-based area measurements of the same growth cones as in d. Pearson correlation coefficient (r) for each growth cone is indicated in top left corner. Same colour indicates measurements of the same growth cone. f, Traces of growth cone area measurements based on FKBP–RAB11 signal in the absence (n = 25, red trace) and presence of FRB–LOV (n = 21, black trace) in growth cones before and during blue-light illumination (see Methods). Graph shows mean ± s.e.m. Blue box indicates blue light-exposed interval. g, Quantification of the area increase in the absence and presence of FRB–LOV in growth cones during blue-light illumination (−4 to 0 min).Values per growth cone are averaged over three frames. Graph shows mean ± s.e.m. P = 0.4145 (n.s., not significant), Mann–Whitney test. h, Cumulative histogram showing the fraction of growth cones with area shrinkage or growth (left or right of dashed line, respectively) before blue-light illumination (−4 to 0 min). Values per growth cone are averaged over three frames. i, Quantification of the area change of −FRB–LOV and +FRB–LOV growth cones during blue-light illumination (0 to 8 min). Values per growth cone are averaged over three frames. Graph shows mean ± s.e.m., *P = 0.0206, Mann–Whitney test. j, Cumulative histogram showing the fraction of growth cones with area shrinkage or growth (left or right of dashed line, respectively) during blue-light illumination (0–8 min). Values per growth cone are averaged over three frames. k, Scatter plot showing net growth during blue-light illumination and normalized fluorescence intensity after blue-light illumination per +FRB-LOV (red) or −FRB-LOV (black) growth cone.
This file contains Supplementary Text and an additional reference. (PDF 145 kb)
This video complements Extended Data Figure 1. Peroxisome motility in COS7 cells before and after recruitment of BICDN-PDZ using blue light. 1 minute between frames and total time is 44.5 minutes. 1200x sped up. (MOV 2172 kb)
This video complements Figure 1f-h. Peroxisome motility in COS7 cells during reversible recruitment of KIF-PDZ through repeated exposure to blue light. 5 seconds between frames and total time is 24 minutes and 10 seconds. 150x sped up. (MOV 3712 kb)
This video complements Figure 1i-l. Peroxisome motility in COS7 cells upon local recruitment of KIF-PDZ using blue light. White boxes indicate area and time of illuminations. 10 seconds between frames and total time is 13 minutes and 45 seconds. 200x sped up. (MOV 6205 kb)
This video complements Extended Data Figure 3b. Distribution of RAB11 vesicles in a COS7 cell before, during and after recruitment of KIF-PDZ using blue light. 10 seconds between frames and total time is 17 minutes and 5 seconds. 100x sped up. (MOV 1096 kb)
Anchoring recycling endosomes with spatiotemporal precision using light-induced myosin-Vb recruitment
This video complements Extended Data Figure 4k, l. Motility of RAB11 vesicles in COS7 cells after recruitment of MYO-PDZ using blue light. White boxes indicate area and time of blue-light illuminations. 10 seconds between frames and total time is 7 minutes and 20 seconds for global illumination and 10 minutes and 30 seconds for local illumination. 200x sped up. (MOV 2137 kb)
This video complements Figure 2g, h. Peroxisome motility upon blue light-induced recruitment of MYO-PEX in an illuminated and a control dendrite of the same hippocampal neuron. Solid lines indicate cell outline. 2 seconds between frames and total time is 6 minutes and 40 seconds. Scale bar is 5μm. 60x sped up. (MOV 3406 kb)
This video complements Figure 3a-f. Growth cones visualized by FKBP-RAB11 and soluble mCherry fluorescence in the presence (left) and absence of FRB-LOV (right) in neurons exposed to blue light. In the first experiment, growth cones co-express BICDN-PDZ as shown in Figure 3a; in the second experiment, growth cones co-express KIF-PDZ as shown in Figure 3d. 10 seconds between frames and total time is 2x12 minutes. Time is indicated relatively to blue-light illumination onset. Scale bar is 5μm.100x sped up. (MOV 8091 kb)
This video complements Figure 4c-g. Mitochondrial motility visualized with TOM-LOV in a neuron co-expressing KIF-PDZ before and during global and local blue-light illumination. Box indicates area of local illumination. 10 seconds between frames and total time is 25 minutes. Scale bar is 5μm. 200x sped up. (MOV 3291 kb)
This video complements Figure 4h-j. Mitochondrial motility visualized with TOM-LOV in a neuron co-expressing SNPH-PDZ before and during global blue-light illumination. 20 seconds between frames and total time is 43 minutes and 40 seconds. Scale bar is 5μm. 400x sped up. (MOV 2717 kb)
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van Bergeijk, P., Adrian, M., Hoogenraad, C. et al. Optogenetic control of organelle transport and positioning. Nature 518, 111–114 (2015). https://doi.org/10.1038/nature14128
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