rab11 mcherry Search Results


rab11 mcherry  (Addgene inc)
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Addgene inc rab11 mcherry
Rab11 Mcherry, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rab11+mcherry/pCS2-CIB1-mCherry-Rab11+(Plasmid+%23140573)/pm32674302-94-8-11
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kif1a(1–365)-vvdfast-mvenus-sspb(micro)_p2a_ilid-mcherry-rab11  (Polysciences inc)
90
Polysciences inc kif1a(1–365)-vvdfast-mvenus-sspb(micro)_p2a_ilid-mcherry-rab11
Selecting optogenetic modules to reposition organelles. (A) Assay and constructs. Recycling endosomes (red) were tagged with fluorescently labeled <t>RAB11</t> fused to iLID. Blue light illumination induced conformational changes to expose Jα helix for transient binding to <t>KIF1A(1–383)-GFP-SSPB,</t> promoting transport to the cellular periphery. (B–D) Fixed-cell imaging (B) and quantification (C and D) of iLID-mCherry-RAB11 in COS-7 cells expressing KIF1A(1–383) fused to GFP-SSPB(milli), GFP-SSPB(micro), or GFP-SSPB(nano) in the dark or after 10 min of illumination with 20 µW cm −2 . Quantification in C shows the normalized iLID-mCherry-RAB11 intensity in the cellular periphery, defined as the 25% of cellular area that is most distal from the perinuclear region (gray in A). Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of control cells. Numbers show the percentage of responsive cells for each dataset (peripheral enrichment greater than mean + two times the SD of control). Data from at least three experiments. (D) Graph showing normalized cellular expression levels of KIF1A(1–383)-GFP-SSPB(micro) and iLID-mCherry-RAB11 per cell in the dark, color coded for peripheral RAB11 intensity to indicate cells that were not activated (gray) or showed dark-state activation (yellow, magenta, and blue). Scale bars are 10 µm.
Kif1a(1–365) Vvdfast Mvenus Sspb(micro) P2a Ilid Mcherry Rab11, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rab11+mcherry/ilid+mcherry+rab11/pmc07147098-255-6-11
Average 90 stars, based on 1 article reviews
kif1a(1–365)-vvdfast-mvenus-sspb(micro)_p2a_ilid-mcherry-rab11 - by Bioz Stars, 2026-10
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Image Search Results


Selecting optogenetic modules to reposition organelles. (A) Assay and constructs. Recycling endosomes (red) were tagged with fluorescently labeled RAB11 fused to iLID. Blue light illumination induced conformational changes to expose Jα helix for transient binding to KIF1A(1–383)-GFP-SSPB, promoting transport to the cellular periphery. (B–D) Fixed-cell imaging (B) and quantification (C and D) of iLID-mCherry-RAB11 in COS-7 cells expressing KIF1A(1–383) fused to GFP-SSPB(milli), GFP-SSPB(micro), or GFP-SSPB(nano) in the dark or after 10 min of illumination with 20 µW cm −2 . Quantification in C shows the normalized iLID-mCherry-RAB11 intensity in the cellular periphery, defined as the 25% of cellular area that is most distal from the perinuclear region (gray in A). Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of control cells. Numbers show the percentage of responsive cells for each dataset (peripheral enrichment greater than mean + two times the SD of control). Data from at least three experiments. (D) Graph showing normalized cellular expression levels of KIF1A(1–383)-GFP-SSPB(micro) and iLID-mCherry-RAB11 per cell in the dark, color coded for peripheral RAB11 intensity to indicate cells that were not activated (gray) or showed dark-state activation (yellow, magenta, and blue). Scale bars are 10 µm.

Journal: The Journal of Cell Biology

Article Title: An optimized toolbox for the optogenetic control of intracellular transport

doi: 10.1083/jcb.201907149

Figure Lengend Snippet: Selecting optogenetic modules to reposition organelles. (A) Assay and constructs. Recycling endosomes (red) were tagged with fluorescently labeled RAB11 fused to iLID. Blue light illumination induced conformational changes to expose Jα helix for transient binding to KIF1A(1–383)-GFP-SSPB, promoting transport to the cellular periphery. (B–D) Fixed-cell imaging (B) and quantification (C and D) of iLID-mCherry-RAB11 in COS-7 cells expressing KIF1A(1–383) fused to GFP-SSPB(milli), GFP-SSPB(micro), or GFP-SSPB(nano) in the dark or after 10 min of illumination with 20 µW cm −2 . Quantification in C shows the normalized iLID-mCherry-RAB11 intensity in the cellular periphery, defined as the 25% of cellular area that is most distal from the perinuclear region (gray in A). Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of control cells. Numbers show the percentage of responsive cells for each dataset (peripheral enrichment greater than mean + two times the SD of control). Data from at least three experiments. (D) Graph showing normalized cellular expression levels of KIF1A(1–383)-GFP-SSPB(micro) and iLID-mCherry-RAB11 per cell in the dark, color coded for peripheral RAB11 intensity to indicate cells that were not activated (gray) or showed dark-state activation (yellow, magenta, and blue). Scale bars are 10 µm.

Article Snippet: U2OS cells were transfected with iLID-mCherry-RAB11, KIF1A(1–365)-VVDfast-mVenus-SSPB(micro)_P2A_iLID-mCherry-RAB11 or SSPB(micro)-mVenus-GCN4–ppKin14-VIb(861–1321)_P2A_iLID-mCherry-RAB11 using polyethylenimine (Polysciences).

Techniques: Construct, Labeling, Binding Assay, Imaging, Expressing, Activation Assay

Disrupting kinesin-3 dimerization prevents spatial segregation between motor and cargo . Related to . (A) Design of kinesin-3 constructs. Monomeric and constitutively dimerized versions were engineered by deletion of the neck coil (aa 366–383) of KIF1A(1–383)-GFP-SSPB or replacement of the neck coil with the leucine zipper GCN4. (B and C) Fixed-cell imaging (B) and quantification (C) of KIF1A-GFP-SSPB variants and mCherry-RAB11 in COS-7 cells. Quantification (C) shows the NPF of intensity. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of RAB11 distribution. Data from three independent experiments. Scale bars are 10 µm.

Journal: The Journal of Cell Biology

Article Title: An optimized toolbox for the optogenetic control of intracellular transport

doi: 10.1083/jcb.201907149

Figure Lengend Snippet: Disrupting kinesin-3 dimerization prevents spatial segregation between motor and cargo . Related to . (A) Design of kinesin-3 constructs. Monomeric and constitutively dimerized versions were engineered by deletion of the neck coil (aa 366–383) of KIF1A(1–383)-GFP-SSPB or replacement of the neck coil with the leucine zipper GCN4. (B and C) Fixed-cell imaging (B) and quantification (C) of KIF1A-GFP-SSPB variants and mCherry-RAB11 in COS-7 cells. Quantification (C) shows the NPF of intensity. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of RAB11 distribution. Data from three independent experiments. Scale bars are 10 µm.

Article Snippet: U2OS cells were transfected with iLID-mCherry-RAB11, KIF1A(1–365)-VVDfast-mVenus-SSPB(micro)_P2A_iLID-mCherry-RAB11 or SSPB(micro)-mVenus-GCN4–ppKin14-VIb(861–1321)_P2A_iLID-mCherry-RAB11 using polyethylenimine (Polysciences).

Techniques: Construct, Imaging

Engineering of opto-kinesin. (A) Design of opto-kinesin. A blue light–sensitive kinesin was engineered by replacing the neck coil (aa 366–383) of KIF1A(1–383)-GFP-SSPB with the blue light–sensitive homodimerization module VVD52C, generating KIF1A(1–365)-VVD52C-GFP-SSPB (opto-kinesin). (B) Schematic representation of the VVD52C and VVDfast modules. (C) Live-cell imaging of KIF1A(1–365)-VVD52C-mCherry-SSPB(micro) in U2OS cells before and after 10 min of illumination (see also ). Scale bar is 10 µm. (D–F) Quantification of experiments as in C depicting normalized peripheral intensity of the two opto-kinesins relative to preillumination state following continuous (D) or pulsed (E and F) illumination (470 nm; blue box). Graphs in D and E represent mean and SEM of at least four cells.

Journal: The Journal of Cell Biology

Article Title: An optimized toolbox for the optogenetic control of intracellular transport

doi: 10.1083/jcb.201907149

Figure Lengend Snippet: Engineering of opto-kinesin. (A) Design of opto-kinesin. A blue light–sensitive kinesin was engineered by replacing the neck coil (aa 366–383) of KIF1A(1–383)-GFP-SSPB with the blue light–sensitive homodimerization module VVD52C, generating KIF1A(1–365)-VVD52C-GFP-SSPB (opto-kinesin). (B) Schematic representation of the VVD52C and VVDfast modules. (C) Live-cell imaging of KIF1A(1–365)-VVD52C-mCherry-SSPB(micro) in U2OS cells before and after 10 min of illumination (see also ). Scale bar is 10 µm. (D–F) Quantification of experiments as in C depicting normalized peripheral intensity of the two opto-kinesins relative to preillumination state following continuous (D) or pulsed (E and F) illumination (470 nm; blue box). Graphs in D and E represent mean and SEM of at least four cells.

Article Snippet: U2OS cells were transfected with iLID-mCherry-RAB11, KIF1A(1–365)-VVDfast-mVenus-SSPB(micro)_P2A_iLID-mCherry-RAB11 or SSPB(micro)-mVenus-GCN4–ppKin14-VIb(861–1321)_P2A_iLID-mCherry-RAB11 using polyethylenimine (Polysciences).

Techniques: Live Cell Imaging

Dual-layer optogenetic control limits dark-state activation of transport. (A) Assay: blue light illumination induces both dimerization of the opto-kinesin KIF1A(1–365)-VVD-GFP-SSPB(micro) and binding of the KIF1A motor to iLID-mCherry-RAB11, together stimulating plus end–directed intracellular transport of recycling endosomes into the cellular periphery. (B–D) Fixed-cell imaging (B) of indicated constructs in COS-7 cells in the dark or after illumination with low-intensity (20 µW cm −2 , as in ) or high-intensity (100 µW cm −2 ) blue light. Quantification in C shows the NPF of RAB11 intensity. Each dot represents one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of control cells. Numbers show the percentage of responsive cells for each dataset (peripheral enrichment greater than mean + two times the SD of control). Data were from at least three experiments. Graph in D shows normalized cellular expression levels of indicated constructs per cell in the dark, color coded for peripheral RAB11 intensity to indicate cells that were nonresponsive (gray) or showed dark-state activation (yellow, magenta, and blue). Scale bars are 10 µm.

Journal: The Journal of Cell Biology

Article Title: An optimized toolbox for the optogenetic control of intracellular transport

doi: 10.1083/jcb.201907149

Figure Lengend Snippet: Dual-layer optogenetic control limits dark-state activation of transport. (A) Assay: blue light illumination induces both dimerization of the opto-kinesin KIF1A(1–365)-VVD-GFP-SSPB(micro) and binding of the KIF1A motor to iLID-mCherry-RAB11, together stimulating plus end–directed intracellular transport of recycling endosomes into the cellular periphery. (B–D) Fixed-cell imaging (B) of indicated constructs in COS-7 cells in the dark or after illumination with low-intensity (20 µW cm −2 , as in ) or high-intensity (100 µW cm −2 ) blue light. Quantification in C shows the NPF of RAB11 intensity. Each dot represents one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of control cells. Numbers show the percentage of responsive cells for each dataset (peripheral enrichment greater than mean + two times the SD of control). Data were from at least three experiments. Graph in D shows normalized cellular expression levels of indicated constructs per cell in the dark, color coded for peripheral RAB11 intensity to indicate cells that were nonresponsive (gray) or showed dark-state activation (yellow, magenta, and blue). Scale bars are 10 µm.

Article Snippet: U2OS cells were transfected with iLID-mCherry-RAB11, KIF1A(1–365)-VVDfast-mVenus-SSPB(micro)_P2A_iLID-mCherry-RAB11 or SSPB(micro)-mVenus-GCN4–ppKin14-VIb(861–1321)_P2A_iLID-mCherry-RAB11 using polyethylenimine (Polysciences).

Techniques: Activation Assay, Binding Assay, Imaging, Construct, Expressing

Robust and multicycle peripheral organelle redistribution with opto-kinesin. (A–E) Live-cell imaging (A) and quantification (B–E) of U2OS cells expressing the indicated constructs before or after 10 min of illumination with blue light (see also and for top and bottom). (B–E) Graphs depicting peripheral enrichment (B) or perinuclear enrichment (C) of iLID-mCherry-RAB5 before and during illumination. Peripheral enrichment index was defined as (peripheral RAB5/total RAB5) − (nonperipheral RAB5/total RAB5), with the peripheral region defined as a 9.13-µm band along the cellular outline (see cartoon in B). Perinuclear enrichment index was similarly defined for the area around the nucleus and the perinuclear vesicle cloud (see cartoon in C). Plots in D and E depict per-cell change in peripheral (D) or perinuclear (E) enrichment index after 10 min of illumination. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean of control cells. Boxed numbers show the percentage of responsive cells for each dataset (peripheral or perinuclear enrichment greater than mean + two times the SD of control). Graphs represent mean and 95% confidence intervals of at least 15 cells from a total of at least three experiments. (F and G) Live-cell imaging (F) and quantification (G) of iLID-mCherry-RAB5 in U2OS cell expressing opto-kinesin KIF1A(1–365)-VVDfast before and after global illumination with blue light (see also ). Left and middle panels show maximum intensity projections of a 30-frame time-lapse during 30 s. Right panel shows manually tracked trajectories of individual early endosomes before (gray) and after illumination (cyan). (G) Quantification of change in displacement of early endosomes tracked in (F) over 30 s before (left, gray) and during illumination (right, cyan). Asterisks indicate significance (Student’s t test, paired). ∗∗∗, P = 0.0009. (H and I) Live-cell imaging (H) and quantification (I) of iLID-mCherry-RAB5 in U2OS cell expressing opto-kinesin KIF1A(1–365)-VVDfast during pulsed global illumination (see also ). Cell was exposed to four illumination cycles of 200 s of blue light, followed by 400 s of darkness. Bottom panels show enlarged region indicated by arrow in top panels. (I) Quantification of peripheral enrichment index of iLID-mCherry-RAB5 (magenta). Graph represents mean and 95% confidence intervals of six experiments. (J and K) Live-cell imaging (J) and quantification (K) of iLID-mCherry-RAB5 and opto-kinesin KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) in U2OS cells treated with nocodazole (10 µM) to depolymerize microtubules. Cells were illuminated continuously for 30 s with 100 µW cm −2 of blue light, followed by 150 s of darkness. Graph in K shows normalized intensity of opto-kinesin on individual endosomes. Blue boxes indicate 470-nm illumination. Scale bars are 10 µm or 5 µm (K).

Journal: The Journal of Cell Biology

Article Title: An optimized toolbox for the optogenetic control of intracellular transport

doi: 10.1083/jcb.201907149

Figure Lengend Snippet: Robust and multicycle peripheral organelle redistribution with opto-kinesin. (A–E) Live-cell imaging (A) and quantification (B–E) of U2OS cells expressing the indicated constructs before or after 10 min of illumination with blue light (see also and for top and bottom). (B–E) Graphs depicting peripheral enrichment (B) or perinuclear enrichment (C) of iLID-mCherry-RAB5 before and during illumination. Peripheral enrichment index was defined as (peripheral RAB5/total RAB5) − (nonperipheral RAB5/total RAB5), with the peripheral region defined as a 9.13-µm band along the cellular outline (see cartoon in B). Perinuclear enrichment index was similarly defined for the area around the nucleus and the perinuclear vesicle cloud (see cartoon in C). Plots in D and E depict per-cell change in peripheral (D) or perinuclear (E) enrichment index after 10 min of illumination. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean of control cells. Boxed numbers show the percentage of responsive cells for each dataset (peripheral or perinuclear enrichment greater than mean + two times the SD of control). Graphs represent mean and 95% confidence intervals of at least 15 cells from a total of at least three experiments. (F and G) Live-cell imaging (F) and quantification (G) of iLID-mCherry-RAB5 in U2OS cell expressing opto-kinesin KIF1A(1–365)-VVDfast before and after global illumination with blue light (see also ). Left and middle panels show maximum intensity projections of a 30-frame time-lapse during 30 s. Right panel shows manually tracked trajectories of individual early endosomes before (gray) and after illumination (cyan). (G) Quantification of change in displacement of early endosomes tracked in (F) over 30 s before (left, gray) and during illumination (right, cyan). Asterisks indicate significance (Student’s t test, paired). ∗∗∗, P = 0.0009. (H and I) Live-cell imaging (H) and quantification (I) of iLID-mCherry-RAB5 in U2OS cell expressing opto-kinesin KIF1A(1–365)-VVDfast during pulsed global illumination (see also ). Cell was exposed to four illumination cycles of 200 s of blue light, followed by 400 s of darkness. Bottom panels show enlarged region indicated by arrow in top panels. (I) Quantification of peripheral enrichment index of iLID-mCherry-RAB5 (magenta). Graph represents mean and 95% confidence intervals of six experiments. (J and K) Live-cell imaging (J) and quantification (K) of iLID-mCherry-RAB5 and opto-kinesin KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) in U2OS cells treated with nocodazole (10 µM) to depolymerize microtubules. Cells were illuminated continuously for 30 s with 100 µW cm −2 of blue light, followed by 150 s of darkness. Graph in K shows normalized intensity of opto-kinesin on individual endosomes. Blue boxes indicate 470-nm illumination. Scale bars are 10 µm or 5 µm (K).

Article Snippet: U2OS cells were transfected with iLID-mCherry-RAB11, KIF1A(1–365)-VVDfast-mVenus-SSPB(micro)_P2A_iLID-mCherry-RAB11 or SSPB(micro)-mVenus-GCN4–ppKin14-VIb(861–1321)_P2A_iLID-mCherry-RAB11 using polyethylenimine (Polysciences).

Techniques: Live Cell Imaging, Expressing, Construct

Dual color live-cell imaging of opto-kinesin and iLID mCherry-RAB5 . Related to . (A) Live-cell imaging of iLID-mCherry-RAB5 and opto-kinesin KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) in U2OS before and after 10 min of illumination (see also ). Scale bar is 10 µm. (B) Kymograph showing early endosome motility and induced colocalization with opto-kinesin as in A. (C) Example of induced early endosome motility (marked by arrowheads) as in A, before and immediately after illumination. Scale bar is 5 µm.

Journal: The Journal of Cell Biology

Article Title: An optimized toolbox for the optogenetic control of intracellular transport

doi: 10.1083/jcb.201907149

Figure Lengend Snippet: Dual color live-cell imaging of opto-kinesin and iLID mCherry-RAB5 . Related to . (A) Live-cell imaging of iLID-mCherry-RAB5 and opto-kinesin KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) in U2OS before and after 10 min of illumination (see also ). Scale bar is 10 µm. (B) Kymograph showing early endosome motility and induced colocalization with opto-kinesin as in A. (C) Example of induced early endosome motility (marked by arrowheads) as in A, before and immediately after illumination. Scale bar is 5 µm.

Article Snippet: U2OS cells were transfected with iLID-mCherry-RAB11, KIF1A(1–365)-VVDfast-mVenus-SSPB(micro)_P2A_iLID-mCherry-RAB11 or SSPB(micro)-mVenus-GCN4–ppKin14-VIb(861–1321)_P2A_iLID-mCherry-RAB11 using polyethylenimine (Polysciences).

Techniques: Live Cell Imaging

Minus end–directed organelle redistribution using moss-derived kinesin-14. (A) Fixed-cell imaging of recycling endosomes (middle) tagged with FKBP-mCherry-RAB11 and the dynein adaptor FRB-BICDN-HA (top) in COS-7 cells, with or without rapalog (100 nM, 1-h treatment). (B) Moss-derived kinesin-14 VIb constructs. Addition of GCN4 leucine zipper creates a dimer of dimers, effectively creating a tetrameric motor. (C) Fixed-cell imaging of kinesin-14 VIb constructs in COS-7 cells. (D) Similar to A, but with cells expressing dimeric FRB-GFP–ppKin14-VIb. (E) Assay: early endosomes were tagged with iLID-mCherry-RAB5. Blue light illumination induced unfolding of iLID and binding to SSPB-GFP–ppKin14-VIb or SSPB-GFP-GCN4–ppKin14-VIb, stimulating anterograde transport (gray). (F) Quantification of live-cell imaging of iLID-mCherry-RAB5 and opto-kinesin KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) in U2OS cells treated with nocodazole (10 µM) to depolymerize microtubules. Cells were illuminated continuously for 30 s with 100 µW cm −2 of blue light, followed by 150 s of darkness. Graph shows normalized intensity of opto-kinesin on individual endosomes. (G) Live-cell imaging and quantifications (H–K) of iLID-mCherry-RAB5 in U2OS cells expressing the indicated constructs before or after 10 min of illumination. Quantifications in H–K show peripheral enrichment index (H) or perinuclear enrichment index (I) of iLID-mCherry-RAB5. Plots in J and K show per-cell change in peripheral (J) or perinuclear (K) enrichment index after 10 min. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean of control cells. Boxed numbers show the percentage of responsive cells for each dataset (peripheral or perinuclear enrichment greater than mean + two times the SD of control). Graphs represent mean and 95% confidence intervals of at least 15 cells from a total of at least three experiments. Blue box indicates 470-nm illumination. Scale bars are 10 µm.

Journal: The Journal of Cell Biology

Article Title: An optimized toolbox for the optogenetic control of intracellular transport

doi: 10.1083/jcb.201907149

Figure Lengend Snippet: Minus end–directed organelle redistribution using moss-derived kinesin-14. (A) Fixed-cell imaging of recycling endosomes (middle) tagged with FKBP-mCherry-RAB11 and the dynein adaptor FRB-BICDN-HA (top) in COS-7 cells, with or without rapalog (100 nM, 1-h treatment). (B) Moss-derived kinesin-14 VIb constructs. Addition of GCN4 leucine zipper creates a dimer of dimers, effectively creating a tetrameric motor. (C) Fixed-cell imaging of kinesin-14 VIb constructs in COS-7 cells. (D) Similar to A, but with cells expressing dimeric FRB-GFP–ppKin14-VIb. (E) Assay: early endosomes were tagged with iLID-mCherry-RAB5. Blue light illumination induced unfolding of iLID and binding to SSPB-GFP–ppKin14-VIb or SSPB-GFP-GCN4–ppKin14-VIb, stimulating anterograde transport (gray). (F) Quantification of live-cell imaging of iLID-mCherry-RAB5 and opto-kinesin KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) in U2OS cells treated with nocodazole (10 µM) to depolymerize microtubules. Cells were illuminated continuously for 30 s with 100 µW cm −2 of blue light, followed by 150 s of darkness. Graph shows normalized intensity of opto-kinesin on individual endosomes. (G) Live-cell imaging and quantifications (H–K) of iLID-mCherry-RAB5 in U2OS cells expressing the indicated constructs before or after 10 min of illumination. Quantifications in H–K show peripheral enrichment index (H) or perinuclear enrichment index (I) of iLID-mCherry-RAB5. Plots in J and K show per-cell change in peripheral (J) or perinuclear (K) enrichment index after 10 min. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean of control cells. Boxed numbers show the percentage of responsive cells for each dataset (peripheral or perinuclear enrichment greater than mean + two times the SD of control). Graphs represent mean and 95% confidence intervals of at least 15 cells from a total of at least three experiments. Blue box indicates 470-nm illumination. Scale bars are 10 µm.

Article Snippet: U2OS cells were transfected with iLID-mCherry-RAB11, KIF1A(1–365)-VVDfast-mVenus-SSPB(micro)_P2A_iLID-mCherry-RAB11 or SSPB(micro)-mVenus-GCN4–ppKin14-VIb(861–1321)_P2A_iLID-mCherry-RAB11 using polyethylenimine (Polysciences).

Techniques: Derivative Assay, Imaging, Construct, Expressing, Binding Assay, Live Cell Imaging

Application of the optimized optogenetic toolbox to populations of cells. (A) Constructs: use of a P2A self-cleaving peptide enables expression of iLID-mCherry-RAB11 with SSPB-Kin14-VIb or opto-kinesin-SSPB at equimolar levels from a single construct. (B) Immunoblot (IB) of whole-cell lysates from U2OS cells expressing the indicated proteins. Dotted lines indicate the excision of lanes. Exp. MW, expected molecular weight. (C–F) Fixed-cell imaging (C) and quantification (D–F) of iLID-mCherry-RAB11 and opto-kinesin KIF1A(1–365)-VVDfast-GFP-SSPB(micro) from a single plasmid in COS-7 cells in the dark or after illumination with blue light (100 µW cm −2 ). (D) Quantification showing the NPF of RAB11 intensity. (E) Graph showing normalized cellular expression levels of KIF1A(1–365)-VVDfast-GFP-SSPB(micro) and iLID-mCherry-RAB11 when expressed from a single plasmid (magenta) as in A or using two separate constructs for motor and cargo (gray). (F) Graph showing normalized cellular expression levels of KIF1A(1–365)-VVDfast-GFP-SSPB(micro) and the peripheral fraction of RAB11 per cell in the dark (gray) and after illumination (blue). (G and H) Fixed-cell imaging (G) and quantification (H) of iLID-mCherry-RAB11 and SSPB(micro) in COS-7 from a single plasmid in COS-7 cells in the dark or after illumination with blue light (100 µW cm −2 ). Graph in H shows the normalized perinuclear fraction of RAB11 intensity (mean RAB11 MTOC intensity/mean cellular RAB11 intensity). (I) Live-cell imaging of iLID-mCherry-RAB11 in HeLa Flp-in cells treated with doxycycline to induce the equimolar expression of SSPB-GCN4-mVenus–ppKin14-VIb and iLID-mCherry-RAB11 (top; see also ) or of KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) and iLID-mCherry-RAB11 (bottom; see also ) before or during illumination. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of control cells. Numbers show the percentage of responsive cells for each dataset. Data were from at least three experiments. Scale bars are 10 µm.

Journal: The Journal of Cell Biology

Article Title: An optimized toolbox for the optogenetic control of intracellular transport

doi: 10.1083/jcb.201907149

Figure Lengend Snippet: Application of the optimized optogenetic toolbox to populations of cells. (A) Constructs: use of a P2A self-cleaving peptide enables expression of iLID-mCherry-RAB11 with SSPB-Kin14-VIb or opto-kinesin-SSPB at equimolar levels from a single construct. (B) Immunoblot (IB) of whole-cell lysates from U2OS cells expressing the indicated proteins. Dotted lines indicate the excision of lanes. Exp. MW, expected molecular weight. (C–F) Fixed-cell imaging (C) and quantification (D–F) of iLID-mCherry-RAB11 and opto-kinesin KIF1A(1–365)-VVDfast-GFP-SSPB(micro) from a single plasmid in COS-7 cells in the dark or after illumination with blue light (100 µW cm −2 ). (D) Quantification showing the NPF of RAB11 intensity. (E) Graph showing normalized cellular expression levels of KIF1A(1–365)-VVDfast-GFP-SSPB(micro) and iLID-mCherry-RAB11 when expressed from a single plasmid (magenta) as in A or using two separate constructs for motor and cargo (gray). (F) Graph showing normalized cellular expression levels of KIF1A(1–365)-VVDfast-GFP-SSPB(micro) and the peripheral fraction of RAB11 per cell in the dark (gray) and after illumination (blue). (G and H) Fixed-cell imaging (G) and quantification (H) of iLID-mCherry-RAB11 and SSPB(micro) in COS-7 from a single plasmid in COS-7 cells in the dark or after illumination with blue light (100 µW cm −2 ). Graph in H shows the normalized perinuclear fraction of RAB11 intensity (mean RAB11 MTOC intensity/mean cellular RAB11 intensity). (I) Live-cell imaging of iLID-mCherry-RAB11 in HeLa Flp-in cells treated with doxycycline to induce the equimolar expression of SSPB-GCN4-mVenus–ppKin14-VIb and iLID-mCherry-RAB11 (top; see also ) or of KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) and iLID-mCherry-RAB11 (bottom; see also ) before or during illumination. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of control cells. Numbers show the percentage of responsive cells for each dataset. Data were from at least three experiments. Scale bars are 10 µm.

Article Snippet: U2OS cells were transfected with iLID-mCherry-RAB11, KIF1A(1–365)-VVDfast-mVenus-SSPB(micro)_P2A_iLID-mCherry-RAB11 or SSPB(micro)-mVenus-GCN4–ppKin14-VIb(861–1321)_P2A_iLID-mCherry-RAB11 using polyethylenimine (Polysciences).

Techniques: Construct, Expressing, Western Blot, Molecular Weight, Imaging, Plasmid Preparation, Live Cell Imaging

Application of the optimized optogenetic toolbox to endogenously tagged cargo. (A and B) Fixed-cell imaging (A) and quantification (B) of recycling endosomes, endogenously tagged with iLID-mCherry-RAB11 and transiently expressed SSPB(nano)-GFP-GCN4–ppKin14-VIb in HeLa cells, in the dark or after 30 min of blue light. Insets show 2.5-fold magnifications of the MTOC region. (B) Quantification shows the normalized enrichment of RAB11 at the MTOC relative to nonilluminated cells. Dots represent one cell and bars indicate mean and 95% confidence intervals. Data were from three independent experiments. Scale bars are 10 µm. Asterisks indicate significance (Student’s t test, unpaired). ∗∗∗∗, P < 0.0001.

Journal: The Journal of Cell Biology

Article Title: An optimized toolbox for the optogenetic control of intracellular transport

doi: 10.1083/jcb.201907149

Figure Lengend Snippet: Application of the optimized optogenetic toolbox to endogenously tagged cargo. (A and B) Fixed-cell imaging (A) and quantification (B) of recycling endosomes, endogenously tagged with iLID-mCherry-RAB11 and transiently expressed SSPB(nano)-GFP-GCN4–ppKin14-VIb in HeLa cells, in the dark or after 30 min of blue light. Insets show 2.5-fold magnifications of the MTOC region. (B) Quantification shows the normalized enrichment of RAB11 at the MTOC relative to nonilluminated cells. Dots represent one cell and bars indicate mean and 95% confidence intervals. Data were from three independent experiments. Scale bars are 10 µm. Asterisks indicate significance (Student’s t test, unpaired). ∗∗∗∗, P < 0.0001.

Article Snippet: U2OS cells were transfected with iLID-mCherry-RAB11, KIF1A(1–365)-VVDfast-mVenus-SSPB(micro)_P2A_iLID-mCherry-RAB11 or SSPB(micro)-mVenus-GCN4–ppKin14-VIb(861–1321)_P2A_iLID-mCherry-RAB11 using polyethylenimine (Polysciences).

Techniques: Imaging

Spontaneous recovery from induced repositioning reveals transport dynamics of different organelles. (A) Assay. (B–J) Live-cell imaging (B, E, and H) and quantifications (C, D, F, G, I, and J) of LAMP1-mCherry-iLID (B–D), iLID-mCherry-RAB5 (E–G), or iLID-mCherry-RAB11 (H–J) in U2OS cells expressing opto-kinesin KIF1A(1–365)-VVDfast-GFP-SSPB(micro) or SSPB(micro)-GFP-GCN4-ppKin14-VIb before, during (middle), or 800 s after 200 s of illumination with blue light. Quantifications show change in peripheral organelle enrichment index (C, F, and I) or change in perinuclear enrichment index (D, G, and J). Graphs represent mean and SEM of at least three cells. Blue box indicates 470-nm illumination. Scale bars are 10 µm.

Journal: The Journal of Cell Biology

Article Title: An optimized toolbox for the optogenetic control of intracellular transport

doi: 10.1083/jcb.201907149

Figure Lengend Snippet: Spontaneous recovery from induced repositioning reveals transport dynamics of different organelles. (A) Assay. (B–J) Live-cell imaging (B, E, and H) and quantifications (C, D, F, G, I, and J) of LAMP1-mCherry-iLID (B–D), iLID-mCherry-RAB5 (E–G), or iLID-mCherry-RAB11 (H–J) in U2OS cells expressing opto-kinesin KIF1A(1–365)-VVDfast-GFP-SSPB(micro) or SSPB(micro)-GFP-GCN4-ppKin14-VIb before, during (middle), or 800 s after 200 s of illumination with blue light. Quantifications show change in peripheral organelle enrichment index (C, F, and I) or change in perinuclear enrichment index (D, G, and J). Graphs represent mean and SEM of at least three cells. Blue box indicates 470-nm illumination. Scale bars are 10 µm.

Article Snippet: U2OS cells were transfected with iLID-mCherry-RAB11, KIF1A(1–365)-VVDfast-mVenus-SSPB(micro)_P2A_iLID-mCherry-RAB11 or SSPB(micro)-mVenus-GCN4–ppKin14-VIb(861–1321)_P2A_iLID-mCherry-RAB11 using polyethylenimine (Polysciences).

Techniques: Live Cell Imaging, Expressing