pgfp eb1  (Addgene inc)

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

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 illustrating the design and light-induced association of OptoTIP with EB1 to track MT plus-ends. (b) OptoTIP variants were constructed by fusing CRY2 with EB1-binding motifs derived from APC, DST, or STIM1. (c) Confocal images and quantification of OptoTIP-mediated MT plus-end tracking during repeated dark-light cycles, followed by nocodazole treatment (20 μM). Blue bars indicate periods of illumination (470 nm, 40 μW/mm 2 ). Data were fitted with a single exponential decay function (t 1/2, on = 11.1 ± 4.7 sec; t 1/2,off = 204 ± 44 sec). n = 10 cells from three independent biological replicates. Also see Supplementary Video 5 . (d) Confocal images showing photo-activatable colocalization of mCh-OptoTIP (red) with GFP-EB1 (green) at growing MT plus-ends following blue light exposure, demonstrating faithful tracking of EB1-marked comets. Also see Supplementary Video 6. (e) Confocal images showing precise spatiotemporal control of OptoTIP tracking by visualization of successively selected photo-stimulation regions. (f) Confocal images of L3 stage C.elegans expressing GFP::α-tubulin and mCh::OptoTIP, showing blue light-inducible OptoTIP tracking of growing MT plus-ends. Also see Supplementary Video 7. (g) Enlarged region (white boxed inset) from panel (f) showing reversible OptoTIP relocalization upon alternating dark-light cycles.. Also see Supplementary Video 8. (h) Schematic depicting the design of LOV2-based OptoTIP variants. To enable tracking of MT plus-ends with faster deactivation kinetics, the SxIP motif derived from dystonin (DST 5469-5485 ) was positioned downstream of the C-terminal Jα helix of LOV2, which was then fused either to a tetrameric DsRed (D series; D1-D6) or the light-inducible oligomerization module CRY2 (V20). (i) Quantification of comet-to-cytosol fluorescence intensity ratios for LOV2-based OptoTIP constructs. mCherry fluorescence at comets and adjacent cytosolic regions was measured before and after 30 s blue light exposure (470 nm, 40 μW/mm²). n = 17-25 cells from three independent biological replicates. (j) Confocal images of HeLa cells expressing mCherry-CRY2-LOV2-SxIP (V20, DST 5470-5485 ) showing light-dependent plus-end tracking with rapid deactivation kinetics during sequential dark-light-dark cycles. Also see Supplementary Video 9. (k) Quantitative analysis of comet-to-cytosol fluorescence intensity ratios for the mCherry-CRY2-LOV2-SxIP (V20) construct. The measured kinetics revealed efficient MT plus-end tracking with an activation half-life of 9.1 ± 4.1 s and deactivation half-life of 40.2 ± 6.6 s. Data were fitted with a single exponential decay function. n = 16 cells from three independent biological replicates.

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

Techniques: Construct, Binding Assay, Derivative Assay, Control, Expressing, Fluorescence, Activation Assay

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) Modeled 3D structure of the STIM1 EB1-binding motif ( 642 TRIP 645 ; shown as sticks) in complex with human EB1 (brown surface; PDB entry: 3GJO). (b) Sequence-logo representation of the tested EB1-binding motif derived from screening of 80 STIM1-TRIP mutants. The relative height of each residue reflects its MT plus-end tracking capability. OptoTIP-V17 construct was used in the assay (see Supplementary Fig. 3a ), which was generated by fusing CRY2 to a fragment of the STIM1 cytoplasmic domain (aa 630–685). This region contains the EB1-binding TRIP motif (aa 642–645) and the polybasid domain (PB, aa 666–685) in the C-tail that mediates phosphoinositide interactions with the inner leaflet of the plasma membrane (PM). Also see Supplementary Fig. 3 for representative images. ( c-d ) Heat-map representation of the extent of comet formation ( c ) or PM translocation ( d ) for all 80 OptoTIP-V17 variants. Each residue within the TRIP template (left column) was individually substituted with the 19 other amino acids indicated above the matrices. (e) Scatter plot summarizing the behaviors of 80 mCh-OptoTIP-V17 variants. Single-residue substitutions within the TRIP motif produced a bimodal distribution: mutants that lost plus-end tracking frequently accumulated at the plasma membrane, whereas those with enhanced plus-end tracking showed diminished membrane association, supporting a competitive interplay between EB1- and PM-targeting determinants within the STIM1 C-terminus. (f) Schematic summarizing four possible photo-inducible scenarios visualized in HeLa cells expressing mCh-OptoTIP-V17 variants: (i) MT plus-end tracking via EB1 binding; (ii) PM translocation via interactions with PM-resident phosphoinositides; (iii) diffuse cytosolic distribution; or (iv) aggregation due to CRY2 homo-oligomerization. Loss or weakening of EB1 binding typically results in one of the latter three outcomes.

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, Sequencing, Derivative Assay, Residue, Construct, Generated, Clinical Proteomics, Membrane, Translocation Assay, Produced, Expressing

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 illustration of the single-component OptoSAW constructs generated by directly fusing the minimal MT-severing domain of spastin (residues 228-616 carrying the K3/Q3 mutation, ³⁰⁹KKK³¹¹ → QQQ) to either OptoMT or OptoTIP. See Supplementary Fig. 8 for spastin engineering and characterization details. (b) Time-lapse confocal images monitoring light-inducible MT severing in single HeLa cells expressing mCh-OptoMT-SAW (red) and GFP-OligoMT (green), the latter serving as a MT marker. See Supplementary Video 12 . (c) Fluorescence intensity profiles of GFP (green) and mCherry (red) signals measured along the dashed line shown in panel b, demonstrating the progressive decline of filament continuity during blue light illumination. (d) Quantification of filament-associated GFP intensity from images shown in panel b. n = 32 cells from three independent biological replicates. (e) Time-lapse confocal images of HeLa cells co-expressing EB1-GFP (green) and mCherry-OptoTIP-SAW (red), showing light-induced disruption of +TIP comet dynamics. See Supplementary Video 13 . (f) Confocal images demonstrating spatially restricted MT severing. Patterned blue light illumination selectively induced MT disassembly within targeted regions. (g) Time-lapse imaging of HeLa cells co-expressing mCherry-OligoTIP (+TIP marker) and OptoTIP-SAW following blue light stimulation, revealing progressive disassembly of +TIP comets. (h) Quantitative analysis of the ratio of the comet-to-cytosolic mCherry fluorescence intensities in cells expressing OptoTIP-SAW under dark (black) and illuminated (blue) conditions, demonstrating efficient, light-dependent +TIP severing. n = 16 cells from three independent biological replicates.

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

Techniques: Construct, Generated, Mutagenesis, Expressing, Marker, Fluorescence, Disruption, Imaging