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(a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, <t>CLIP170,</t> or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).
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(a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, <t>CLIP170,</t> or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).
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(a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, <t>CLIP170,</t> or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).
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mouse anti clip170  (Santa Cruz Biotechnology)
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(a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, <t>CLIP170,</t> or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).
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clip170  (Millipore)
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(a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, <t>CLIP170,</t> or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).
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(a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, <t>CLIP170,</t> or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).
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(a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, CLIP170, or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).

Journal: bioRxiv

Article Title: A single-component optogenetic toolkit for programmable control of microtubule

doi: 10.1101/2025.10.31.685931

Figure Lengend Snippet: (a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, CLIP170, or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).

Article Snippet: The plasmid templates for EB1 (#17234), CLIP170 (#54044), CAMSAP1 (# 59036),CAMSAP2 (#59037), KIF5A (#166954), and spastin (#134461) were purchased from Addgene.

Techniques: Binding Assay, Derivative Assay, Construct, Variant Assay, Activity Assay, Expressing, Fluorescence, Control, Labeling, Staining, Immunostaining, Live Cell Imaging