Glucose-stimulated KIF5B-driven microtubule sliding organizes microtubule networks in mouse pancreatic β cells
- Department of Cell and Developmental Biology, Vanderbilt University, United States
- Center for Stem Cell Biology, Vanderbilt University, United States
- Program of Developmental Biology, Vanderbilt University, United States
Figures
Figure 1 with 3 supplements
Microtubules (MTs) in pancreatic β cells undergo extensive sliding driven by kinesin KIF5B.
(A) A subset of RNA-sequencing data from primary mouse β cells showing highly expressed kinesins as indicated by mRNA counts. KIF5B (most-right bar, red data points) is the most abundant kinesin motor in this cell type. N=3. Note that this is a subset of the RNA-sequencing sets published in Sanavia et al., 2021. (B) Efficient depletion of KIF5B in MIN6 cells using two alternative shRNA sequences, as compared to a scrambled shRNA control. Based on immunofluorescent staining of KIF5B as in Figure 1—figure supplement 1 (A–C). Fold decrease of fluorescence signal per cell normalized to cells w/o shRNA expression in the same field of view. N=25–32 cells from 4 repeats. (C) Quantification of MT sliding FRAP assay in cells treated with scrambled control or one of the two KIF5B-specific shRNAs (see representative data in D–F). MT displacement is shown as the area of MTs displaced into the bleached area after 5 min of recovery. One-way ANOVA test was performed for statistical significance (p-value<0.0001). N=9–20 cells per set. (D–F) Frames from representative FRAP live-cell imaging sequences. mEmerald-tubulin-expressing MIN6 cells. Inverted grayscale images of maximum intensity projections of spinning disk confocal microscopy stacks over a 1-µm-thick ventral cell layer. Scale bars, 5 µm. (D1–F1) Overview of the whole cell prior to photobleaching. (D2–F3) Enlarged areas from (D1–F1) immediately after photobleaching (D2–F2) and 5 min (300 s) after photobleaching (D3–F3). Light-blue dotted lines indicate the edges of the photobleached areas. Red arrows indicate MTs displaced into the bleached area. Scale bars, 5 µm (D–F, Figure 1—video 1 ‘MT sliding FRAP’). (G–I) MIN6 cells featuring fiducial marks at MTs due to co-expression of SunTag-KIF5B-560Rigor construct and Halo-SunTag ligand. Representative examples for scrambled control shRNA-treated cell (G), KIF5B shRNA #1-treated cell (H), and KIF5B shRNA #2-treated cell (I) are shown. Single slices by spinning disk confocal microscopy. Halo-tag signal is shown as an inverted grayscale image. Top panels show cell overviews (scale bars, 5 µm). Below, boxed insets (scale bars, 2 µm) are enlarged to show dynamics of fiducial marks (color arrows) at 1 s intervals (1–5 s). 0–5 s tracks of fiducial mark movement are shown in the bottom panel, each track color-coded corresponding to the arrows in the image sequences. (J) Summarized quantification of stationary fraction of fiducial marks along MT lattice (5 s displacements below 0.15 µm). Scrambled shRNA control N=1421 tracks across 6 cells, shRNA#1 N=852 tracks across 5 cells, shRNA#2 N=2182 tracks across 7 cells. One-way ANOVA, p<0.001. (K) Summarized quantification of motile fraction of fiducial marks along the MT lattice (5 s displacements above 0.3 µm). Scrambled shRNA control N=2066 tracks across 6 cells, shRNA#1 N=390 tracks across 5 cells, shRNA#2 N=412 tracks across 7 cells. One-way ANOVA, p<0.001 (G–I, Figure 1—video 2 ‘MT sliding SunTag’).
-
Figure 1—source data 1
SunTag marks displacement 5 s intervals across each cell.
- https://cdn.elifesciences.org/articles/89596/elife-89596-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
KIF5B depletion controls.
(A–C) shRNA-based depletion of KIF5B in MIN6 cells. Cells expressing shRNA are detected by GFP expression (cyan in A1–C1, red arrows in A2–C2). Immunostained KIF5B signal (magenta in A1–C1 and grayscale in A2–C2) is significantly reduced by KIF5B shRNAs (B, C) and compared to scrambled control (A). See quantification in main Figure 1B. (D) Histogram of displacements over 5 s intervals for all fiducial marks in scrambled shRNA control versus KIF5B shRNA #1 and KIF5B shRNA #2.
Figure 1—video 1
Microtubules (MT) sliding FRAP.
KIF5B-mediated MT sliding visualization via FRAP. Cells treated with scrambled control and two shRNAs against KIF5B are shown. Time, minutes:seconds.
Figure 1—video 2
Microtubule (MT) sliding SunTag.
KIF5B-mediated MT sliding visualization via SunTag-KIF5B-560Rigor. Cells treated with scrambled control and two shRNAs against KIF5B are shown. Sliding maximum intensity projection (15 time frames projected in each video frame). Time, seconds.
Figure 2 with 4 supplements
Microtubule (MT) abundance and alignment at the cell periphery depend on KIF5B.
(A–C) MT organization in MIN6 cells expressing scrambled control shRNA (A), KIF5B-targeting shRNA #1 (B), or KIF5B-targeting shRNA #2 (C). Top, immunofluorescence staining for tubulin (grayscale, inverted). Bottom, immunofluorescence staining for KIF5B (cyan). Maximum intensity projection of 1 µm at the ventral side of the cell. N=12. Scale bars, 5 µm. (D) Quantification of mean tubulin intensity within the outer 2 µm peripheral area of a cell, in data represented in (A–C). Mean values, black bars. One-way ANOVA, p<0.0001. N=7–9 cells. (E) Histograms of MT directionality within 1 µm of cell boundary using perfected thresholds (see Figure 2—figure supplements 3 and 4 for the analysis workflow and variants) in cells treated with scrambled control versus KIF5B-targeting shRNA. Data are shown for the summarized detectable tubulin-positive pixels in the analyzed single confocal slices of shRNA-treated cell population immunostained for tubulin, as represented in (F–H). Unpaired t-tests were performed across each bin for all cells, and a Kolmogorov-Smirnov (K-S) test was performed on the overall distribution. The share of MTs parallel to the edge (bin 0–10) is significantly higher in control as compared to KIF5B depletions. Pixel numbers in the analysis: SCR N=106,780 pixels across 9 cells, shRNA #1 N=71,243 across 7 cells, shRNA #2 N=60,087 across 7 cells. (F–H) Representative examples of MT directionality analysis in single confocal slices of shRNA-treated cells immunostained for tubulin, as quantified in (E). Single laser scanning confocal microscopy slices. (F) Scrambled control shRNA-treated cell. (G) KIF5B shRNA#1-treated cell. (H) KIF5B shRNA#1-treated cell. Overviews of cellular MT networks are shown as a threshold to detect individual peripheral MTs (see Figure 2—figure supplement 3, panel A5). (F1–H2) Directionality analysis outputs of regions from yellow boxes in (F–H) are shown color-coded for the angles between MTs and the nearest cell border. (I) Color code for (F1–H2): MTs parallel to the cell edge, blue; MTs perpendicular to the cell edge, red.
-
Figure 2—source data 1
Microtubule (MT) directionality source data: KIF5B depletion and KIF5B KO.
- https://cdn.elifesciences.org/articles/89596/elife-89596-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Microtubule (MT) abundance and alignment at the cell periphery depend on KIF5B in primary β cells in mouse islets.
(A–B) MT organization in β cells within intact islets isolated from a wild-type (wt) (A) or KIF5B KO mice. Immunofluorescence staining for tubulin (grayscale, inverted). Maximum intensity projections of the laser scanning confocal microscopy stacks through the cell. N=10–11. Scale bars, 5 µm. (C) Quantification of mean tubulin intensity within the outer 2 µm peripheral area of a cell, in data represented in (A–B). Mean values, black bars. One-way ANOVA, p<0.0001. N=10–11 cells. (D–E) Representative examples of MT directionality analysis in single confocal slices of β cells from mouse islets immunostained for tubulin, as quantified in (F). Single laser scanning confocal microscopy slices. (D) Cells from wt mouse islets. (E) Cells from KIF5B KO mouse islets. Overviews of cellular MT networks are shown as threshold to detect individual peripheral MTs (see Figure 2—figure supplement 3, panel A5). (D1–E2) Directionality analysis outputs of regions from yellow boxes in (D–E) are shown color-coded for the angles between MTs and the nearest cell border. (F) Color code for (D1–E2): MTs parallel to the cell edge, blue; MTs perpendicular to the cell edge, red. (G) Histograms of MT directionality within 1 µm of cell boundary using perfected thresholds (see Figure 2—figure supplements 3 and 4 for the analysis workflow and variants) in β cells from wt versus KIF5B KO mice. Data are shown for the summarized detectable tubulin-positive pixels in the analyzed single confocal slices of shRNA-treated cell population immunostained for tubulin, as represented in (D–E). Unpaired t-tests were performed across each bin for all cells, and a Kolmogorov-Smirnov (K-S) test was performed on the overall distribution. The share of MTs parallel to the edge (bin 0–10) is significantly higher in control as compared to KIF5B depletions. Pixel numbers in the analysis: wt N=180,709 pixels across 10 cells, KIF5B KO N=103,342 across 12 cells.
Figure 2—figure supplement 2
Overexpression of truncated KIF5B motor lacking cargo binding does not affect microtubule (MT) density at β-cell periphery.
(A–D) MT organization in MIN6 cells expressing scrambled control shRNA (A–B) or KIF5B-targeting shRNA #2 (C–D). BFP-tagged kinesin-1 motor construct is ectopically expressed in (B, D). Top, immunofluorescence staining for tubulin (grayscale, inverted). Bottom, BFP immunofluorescence for motor expression (cyan). Maximum intensity projections of the laser scanning confocal microscopy stacks through the whole cell. N=8. Scale bars, 5 µm.
Figure 2—figure supplement 3
Workflow of microtubule (MT) directionality analysis.
(A) Representation of the analysis workflow using a control DMSO-treated cell immunostained for tubulin as an example. (A1) An image of the original inverted grayscale confocal slice. (A2) A deconvolved image. (A3) Mask of the cell boundary. (A4) An image within the mask after application of standard % threshold. (A5) An image within the mask after application of a threshold optimizing detection of peripheral MTs for a particular cell. (A3–A5) are derivatives of (A2). (A6) A schematic illustrating map of distances from the nearest cell (mask) border per pixel (not to scale). (A7) A schematic illustrating map of angles per pixel (not to scale). A6 and A7 can be produced from A4 or A5. (A8) Color-coded output map of MT directionalities. A8 is a derivative of A7.
Figure 2—figure supplement 4
Illustration of thresholding variations and their influence on the output analysis.
(A) KIF5B-depleted cell as an example of contrast/threshold optimization for different microtubule (MT) densities and corresponding outputs. Upper row, contrast is adjusted for distinction of single MTs at the cell periphery. Lower row, contrast is adjusted for distinction of single MTs at the cell center. (A1) A deconvolved inverted grayscale confocal slice image contrasted to highlight peripheral MTs. (A2) The same deconvolved inverted grayscale confocal slice image contrasted to highlight central MTs. (A3) The image from A1 after application of a threshold optimized to highlight peripheral MTs. (A4) The image from A1 after application of a standard % threshold. (A5) Analysis outcome from (A3). (A6) Analysis outcome from (A4). (B) Histograms of MT directionality within 1 µm of cell boundary using standard thresholds (see Figure 2—figure supplement 3 for the analysis workflow) in cells treated with scrambled control versus KIF5B-targeting shRNA. Data are shown for the summarized detectable tubulin-positive pixels in the analyzed shRNA-treated cell population, as represented in (F–H). Unpaired t-tests were performed across each bin for all cells, and a Kolmogorov-Smirnov (K-S) test was performed on the overall distribution. The share of MTs parallel to the edge (bin 0–10) is significantly higher in control as compared to KIF5B depletions. Pixel numbers in the analysis: SCR N=61,437 pixels across 9 cells, shRNA#1 N=15,215 pixels across 7 cells, shRNA#2 N=21,125 pixels across 7 cells. (C) Histograms of MT directionality presented in this figure panel B with depicted outliers. (D) Histograms of MT directionality presented in Figure 2 panel E with depicted outliers. (E) Histograms of MT directionality presented in Figure 2—figure supplement 1 panel G with depicted outliers.
Figure 3 with 2 supplements
Microtubule (MT) sliding is facilitated through the ATP-independent MT-binding domain of kinesin-1.
(A) Schematic of kinesin-1 (KIF5) and the dominant-negative (KIFDN) and heterodimerization strategy. Top schematic shows full-length KIF5s, consisting of the motor domain, stalk coil-coil domain, and the tail. Three constructs utilized here include (1) The KIF5C motor domain tagged with a blue fluorescent protein (BFP) and the FKBP-rapamycin binding (FRB) for heterodimerization; (2) KIFDNwt construct with KIF5B tail domain tagged with the mCherry fluorescent protein and the FKBP for heterodimerization. (3) KIFDNmut construct is the same as (2) but features a set of point mutations (magenta) making the ATP-independent MT-binding domain unable to bind MT lattice. (B) Quantification of MT sliding in FRAP assay in cells subjected to DN construct expression and heterodimerization. MT displacement is shown as the area of MTs displaced into the bleached area after 5 min of recovery. See representative data (C–G). N=5–25 per condition. One-way ANOVA test was performed for statistical significance (p-value<0.0001; ns, nonsignificant). (C–G”) Frames from representative FRAP live-cell imaging sequences of MIN6 cells expressing mEmerald-tubulin. Inverted grayscale images of maximum intensity projections over 1-µm-thick stacks by spinning disk confocal microscopy. (C1–G1) The first frame after photobleaching. (C2–G2) A frame 5 min (300 s) after photobleaching. Light-blue dotted lines indicate the edges of the photobleached areas. Red arrows indicate MTs displaced into the bleached area. Scale bars, 5 µm. (C3–G3) Schematics of experimental manipulation: green represents MTs, blue represents endogenous KIF5B, magenta represents KIFDNwt, purple represents KIFDNmut, gray represents KIF5C motor, orange bracket represents heterodimerizing agent (rap, rapalog). Conditions: (C1–C3) Untreated control. Only endogenous KIF5B is present. (D1–D3) KIFDNwt overexpression. Endogenous KIF5B is unable to bind MTs. (E1–E3) KIFDNmut overexpression. It does not bind MTs and does not interfere with endogenous KIF5B. (C–E, Figure 3—video 1 ‘KFDN FRAP’) (F1–F3) KIFDNwt and KIF5C motor overexpression plus rapalog treatment. Heterodimerization creates a large pool of motors capable of MT sliding. (G–G”) KIFDNmut and KIF5C motor overexpression plus rapalog treatment. Heterodimerization creates a large pool of the motor nonfunctional in MT sliding (F–G, Figure 3—video 2 ‘KFDN + motor FRAP’).
Figure 3—video 1
KFDN FRAP.
dominant-negative microtubule (MT) sliding visualization via FRAP (tails). Cells overexpressing KIFDNwt versus KIFDNmut are shown. Time, minutes:seconds.
Figure 3—video 2
KFDN + motor FRAP.
dominant-negative microtubule (MT) sliding via FRAP (tails + motors). Cells expressing heterodimerized KIFDNwt with motor versus heterodimerized KIFDNmut with motor are shown. Time, minutes:seconds.
Figure 4 with 2 supplements
Effects of ATP-independent microtubule (MT)-binding domain of KIF5B on MT abundance and alignment at the β-cell periphery.
(A–C) MT organization in MIN6 cells expressing (B) KIFDNwt and KIF5C motor heterodimerized via rapalog treatment, (C) KIFDNmut and KIF5C motor heterodimerized via rapalog treatment and compared to a control cell with no ectopic expressions (A). Top, immunofluorescence staining for tubulin (grayscale, inverted). Blue dotted line indicates the borders of a cell expressing constructs of interest. Bottom in A, F-actin (phalloidin, red) and DAPI (blue). Bottom in B and C, ectopically expressed mCherry-labeled KIFDN constructs (magenta) and BFP-labeled KIF5C motor (green). Laser scanning confocal microscopy maximum intensity projection of 1 µm at the ventral side of the cell. Scale bars, 5 um. (D) Quantification of mean tubulin intensity within the outer 2 µm peripheral area of a cell, in data represented in (A–C). Mean values, black bars. One-way ANOVA, p<0.0001. N=7–15 cells. (E) Histograms of MT directionality within 1 µm of cell boundary (see Figure 2—figure supplement 3 for the analysis workflow) in control cells compared to cells expressing heterodimerized KIFDN variants. Data are shown for the summarized detectable tubulin-positive pixels in the analyzed cell population, as represented in (F–H). Unpaired t-tests were performed across each bin for all cells, and a Kolmogorov-Smirnov (K-S) test was performed on the overall distribution. The share of MTs parallel to the edge (bin 0–10) is significantly higher in control as compared to the overexpressions. NT control N=138,810 pixels across 9 cells, KIFDNwt + motor N=48,285 pixels across 9 cells, KIFDNmut + motor N=40,832 pixels across 10 cells. (F–H) Representative examples of MT directionality analysis quantified in (E). (F) Control cell, no ectopic expressions. (G) Cell expressing KIFDNwt + motor. (H) Cell expressing KIFDNmut+ motor. Overviews of cellular MT networks are shown as a threshold to detect individual peripheral MTs (see Figure 2—figure supplement 3 panel A5). (F1–H2) Directionality analysis outputs of regions from yellow boxes in (F–H) are shown color-coded for the angles between MTs and the nearest cell border (see Figure 2—figure supplement 3 panel A8). (I) Color code for (F1–H2): MTs parallel to the cell edge, blue; MTs perpendicular to the cell edge, red.
-
Figure 4—source data 1
Microtubule (MT) directionality source data: KIFDN overexpression.
- https://cdn.elifesciences.org/articles/89596/elife-89596-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Dominant-negative (DN) effect of KIF5B tail domain on microtubule (MT) abundance at the β-cell periphery requires ATP-independent MT-binding domain.
(A–B) MT organization in MIN6 cells expressing (A) KIFDNwt and (B) KIFDNmut. Top, immunofluorescence staining for tubulin (grayscale, inverted). Blue dotted line indicates the borders of a cell expressing constructs of interest. Bottom, ectopically expressed mCherry-labeled KIFDN constructs (magenta). Laser scanning confocal microscopy maximum intensity projection of 1 µm at the ventral side of the cell. Scale bars, 5 um. (C) Quantification of mean tubulin intensity within the outer 2 µm peripheral area of a cell, in data represented in (A–B) as compared to untreated controls (see Figure 4A). Mean values, black bars. One-way ANOVA, p<0.0001. N=7–11 cells.
Figure 4—figure supplement 2
Influence of thresholding variations on the output analysis of microtubule (MT) directionality in Figure 4F–H.
(A) Histograms of MT directionality within 1 µm of cell boundary using standard thresholds (see Figure 2—figure supplement 3 for the analysis workflow) in cells expressing ectopic heterodimerized KIF5C motor. Data are shown for the summarized detectable tubulin-positive pixels in the analyzed shRNA-treated cell population, as represented in Figure 4F–H. Unpaired t-tests were performed across each bin for all cells, and a Kolmogorov-Smirnov (K-S) test was performed on the overall distribution. The share of MTs parallel to the edge (bin 0–10) is significantly higher in control as compared to heterodimerized KIF5B overexpression. Pixel numbers in the analysis: NT control N=120,959 pixels across 9 cells, KIFDNwt + motor N=26,110 pixels across 7 cells, KIFDNmut + motor N=41,091 pixels across 10 cells. (B) Histograms of MT directionality presented in this figure panel A with depicted outliers. (C) Histograms of MT directionality presented in main Figure 4 panel E with depicted outliers.
Figure 5 with 2 supplements
Enhanced microtubule (MT) sliding results in loss of peripheral MT alignment at the border.
(A–B) MT organization in MIN6 cells pretreated with DMSO and kinesore, respectively. Immunofluorescence staining for tubulin (grayscale, inverted). Laser scanning confocal microscopy maximum intensity projection of 1 µm at the ventral side of the cell. Scale bars, 5 um. (C) Quantification of mean tubulin intensity within the outer 2 µm peripheral area of a cell, in data represented in (A–B). Mean values, black bars. One-way ANOVA, p<0.0001. N=10 cells per condition. Histograms of MT directionality within 1 µm of cell boundary (see Figure 2—figure supplement 3 for the analysis workflow) in DMSO-treated control cells compared to kinesore-treated cells. Data are shown for the summarized detectable tubulin-positive pixels in the analyzed cell population, as represented in (D–E). Unpaired t-tests were performed across each bin for all cells, and a Kolmogorov-Smirnov (K-S) test was performed on the overall distribution. The share of MTs parallel to the edge (bin 0–10) is significantly higher in control as compared to the overexpressions. DMSO control N=136,840 pixels across 10 cells, kinesore-treated N=87,361 pixels across 9 cells. (D–E) Representative examples of MT directionality analysis quantified in (F). Directionality analysis outputs of regions from yellow boxes in (D–E) are shown color-coded for the angles between MTs and the nearest cell border (see Figure 2—figure supplement 3 panel A8). (G) Color code for (D1–E2): MTs parallel to the cell edge, blue; MTs perpendicular to the cell edge, red.
-
Figure 5—source data 1
Microtubule (MT) directionality source data: kinesore treatment.
- https://cdn.elifesciences.org/articles/89596/elife-89596-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Influence of thresholding variations on the output analysis of microtubule (MT) directionality in Figure 5D and E.
(A) Histograms of MT directionality within 1 µm of cell boundary using standard thresholds (see Figure 2—figure supplement 3 for the analysis workflow) in cells treated with DMSO or kinesore. Data are shown for the summarized detectable tubulin-positive pixels in the analyzed respective cell population, as represented in Figure 5D and E. Unpaired t-tests were performed across each bin for all cells, and a Kolmogorov-Smirnov (K-S) test was performed on the overall distribution. The share of MTs parallel to the edge (bin 0–10) is significantly higher in control as compared to kinesore treated. Pixel numbers in the analysis: DMSO control N=94,841 pixels across 9 cells, kinesore N=29,796 pixels across 9 cells. (B) Histograms of MT directionality presented in this figure panel A with depicted outliers. (C) Histograms of MT directionality presented in main Figure 5 panel F with depicted outliers.
Figure 5—figure supplement 2
Kinesore has no effect in microtubule (MT) network in KIF5B-depleted cells.
(A–F) Representative examples of MT organization in MIN6 cells expressing scrambled control shRNA (A, D), KIF5B-targeting shRNA #1 (B, E), or KIF5B-targeting shRNA #2 (C, F). A DMSO-treated control (A–C) and kinesore-treated cells (D–F) are shown. Immunofluorescence staining for tubulin (grayscale, inverted). Maximum intensity projections of the laser scanning confocal microscopy stacks through the whole cell. N=8 cells/condition. Scale bars, 5 µm.
Figure 6 with 2 supplements
Microtubule (MT) sliding in β cells is stimulated by glucose.
(A–B) Frames from representative FRAP live-cell imaging sequences of MT sliding response to glucose stimulation. mEmerald-tubulin-expressing MIN6 cells. Inverted grayscale images of maximum intensity projections over 1-µm-thick stacks by spinning disk confocal microscopy. (A) A cell pretreated with 2.8 mM glucose before the assay. (B) A cell pretreated with 2.8 mM glucose and stimulated with 20 mM glucose before the assay. (A1–B1) The first frame after photobleaching. (A2–B2) A frame 5 min (300 s) after photobleaching. Light-blue dotted lines indicate the edges of the photobleached areas. Red arrows indicate MTs displaced into the bleached area. Scale bars, 5 µm. (C) Quantification of MT sliding FRAP assay in cells in 2.8 mM versus 20 mM glucose (see representative data in A–B). MT displacement is shown as the area of MTs displaced into the bleached area after 5 min of recovery. One-way ANOVA test was performed for statistical significance (p-value<0.0001). N=16–24 cells per set (A–B, Figure 6—video 1 ‘FRAP low and high glucose’). (D–E) MIN6 cells featuring fiducial marks at MTs due to co-expression of SunTag-KIF5B-560Rigor construct and Halo-SunTag ligand. Representative examples for cells in 2.8 mM glucose (D) and a cell stimulated by 20 mM glucose (E) are shown. Single-slice spinning disk confocal microscopy. Halo-tag signal is shown as inverted grayscale image. Top panels show cell overviews (scale bars, 5 µm). Below, boxed insets are enlarged to show dynamics of fiducial marks (color arrows) at 1 s intervals (1–5 s). 0–5 s tracks of fiducial mark movement are shown in the bottom panel, each track color-coded corresponding to the arrows in the image sequences. N=6–11 cells (A–B Figure 6—video 2 ‘SunTag low and high glucose’). (F) Histogram of all 5 s displacement of fiducial marks in low versus high glucose. (G) Summarized quantification of stationary fiducial marks along MT lattice (5 s displacements below 0.15 µm). Low glucose N=5615 tracks across 11 cells, high glucose 5 min N=2259 tracks across 6 cells, high glucose 20 min N=3059 tracks across 6 cells. One-way ANOVA, p<0.001. (H) Summarized quantification of moving fiducial marks along the MT lattice (5 s displacements over 0.3 µm). Low glucose N=2595 tracks across 11 cells, high glucose 5 min N=2642 tracks across 6 cells, high glucose 20 min N=4049 tracks across 6 cells. One-way ANOVA, p<0.001.
-
Figure 6—source data 1
SunTag marks displacement 5 s intervals across each cell and population analysis referenced in panels G and H.
- https://cdn.elifesciences.org/articles/89596/elife-89596-fig6-data1-v1.xlsx
Figure 6—video 1
FRAP low and high glucose.
Glucose-dependent microtubule (MT) sliding visualization via FRAP. Cells in low versus high glucose are shown. Time, minutes:seconds.
Figure 6—video 2
SunTag low and high glucose.
Glucose-dependent microtubule (MT) sliding visualization via SunTag-KIF5B-560Rigor. Insets from cells in low versus high glucose are shown. Sliding maximum intensity projection (15 time frames projected in each video frame). Top, SunTag puncta highlighted. Bottom, SunTag puncta tracked. Time, seconds.
Figure 7
Schematic of the main results and predictions.
(A) Role of KIF5B-dependent microtubule (MT) sliding in MT density at the cell periphery. (B) Role of KIF5B-dependent MT sliding in the alignment of peripheral MTs. (C) Potential role of glucose-facilitated KIF5B-dependent MT sliding in forming temporal openings in the peripheral MT array by moving fragmented MTs aside or MT looping. (D) Potential role of glucose-facilitated KIF5B-dependent MT sliding in repair of the peripheral MT array, partially destabilized/fragmented downstream of glucose. Track MTs, green. Cargo MTs, blue. Kinesin-1, black. Direction of kinesin movement shown as a solid arrow. Subsequent steps of the process are shown as dashed double arrows.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (M. musculus) | MIN6 | Gift from Dr. Miyazaki, Osaka University Medical School, Japan | RRID:CVCL_0431 | Mouse Insulinoma Cell Line |
| Chemical compound, drug | Kinesore | Tocris Bioscience | Cat#: 6664 | Final concentration (50 µm) |
| Chemical compound, drug | A/C Heterodimerizing Drug | Takara Bio Inc | Cat#: 635056 | Final concentration (25 µm) |
| Antibody | Anti-KIF5B rabbit monoclonal antibody | Abcam | Cat#: Ab167429 | (1:500 dilution) |
| Antibody | Anti-alpha-Tubulin mouse monoclonal antibody (DM1a) | Sigma-Aldrich | Cat#: T9026 | (1:1000 dilution) |
| Antibody | Anti-alpha-Tubulin rabbit polyclonal antibody | Sigma-Aldrich | Cat#: T3526 | (1:1000 dilution) |
| Other | Janelia Fluor 585 (JF585) and 646 (JF646), HaloTag Ligands | Promega | CS315105, GA1120 | Final concentration (2 µl/mL) |
| Strain, strain background (M. musculus) | C57BL/6J | Jackson Laboratory | Strain #:000664 RRID:IMSR_JAX:000664 | Control mice (wt) |
| Strain, strain background (M. musculus) | Kif5bfl/−:RIP2-Cre | Cui et al., 2011 | Strain #:008637; RRID:IMSR_JAX:008637 | Kif5b KO mice; derived from Jackson Laboratory fl/fl strain (see identifiers) |
Additional files
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Glucose-stimulated KIF5B-driven microtubule sliding organizes microtubule networks in mouse pancreatic β cells
eLife 12:RP89596.
https://doi.org/10.7554/eLife.89596.4
