MTs in pancreatic beta cells undergo extensive sliding driven by kinesin KIF5B.

A subset of RNA-sequencing data from primary mouse beta 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. GAPDH, loading control. Relevant ROIs from a Western blotting is shown. A representative example out of 3 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 area of MTs displaced into the bleached area after 5 minutes 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 over 1 µm-thick stacks by spinning disk confocal microscopy. (D1-F1) The last frame prior to photobleaching. (D2-F2) The first frame after photobleaching. (D3-F3) A frame 5 minutes (300 seconds) 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. (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-slice spinning disk confocal vmicroscopy. Halo-tag signal is shown as inverted gray-scale image. Top panels show cell overviews (scale bars 5µm). Below, boxed insets (scale bars 2 µm) are enlarged to show dynamics of fiducial marks (color arrows) at 1 second intervals (1-5 seconds). 0-to 5-second 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. Scrambled shRNA control N=1,421 tracks across 6 cells, shRNA#1 N=852 tracks across 5 cells, shRNA#2 N=679 tracks across 5 cells. P One-way ANOVA, p<0.001 (K) Summarized quantification of motile fraction of fiducial marks along the MT lattice. Scrambled shRNA control N=2,066 tracks across 6 cells, shRNA#1 N=390 tracks across 5 cells, shRNA#2 N=307 tracks across 5 cells. P One-way ANOVA, p<0.001

Microtubule 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). Laser scanning confocal microscopy maximum intensity projection of 1µm at the ventral side of the cell. N=12. Scale bars: 5um. (D) Quantification of mean tubulin intensity within the outer 2µm peripheral area of a cell, in data represented in (A-C). Mean values, black bars. One-way ANOVA, p<0.0001. N=5-9 cells. (E) Histograms of MT directionality within 1um of cell boundary using perfected thresholds (see Supplemental figure 2-1 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-test were performed across each bin for all cells, and a 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=106,741 pixels across 9 cells, shRNA #1 N=71,243 across 7 cells, shRNA #2 N= 54,101 across 7 cells. (F-H) Representative examples of MT directionality analysis quantified in (E). (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 threshold to detect individual peripheral MTs (see Supplemental figure 2-1 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 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 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 (shown as area of displaced MTs). MT displacement is shown as area of MTs displaced into the bleached area after 5 minutes 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, non-significant). (C-G”) Frames from representative FRAP live-cell imaging sequences. mEmerald-tubulin-expressing MIN6 cells. 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 minutes (300 seconds) 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. (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 non-functional in MT sliding.

Effects of ATP-independent MT-binding domain of KIF5B on microtubule abundance and alignment at the beta-cell 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 1µm at the ventral side of the cell. Scale bars: 5um. (D) Quantification of mean tubulin intensity within the outer 2µm peripheral area of a cell, in data represented in (A-C). Mean values, black bars. One-way ANOVA, p<0.0001. N=5-15 cells. (E) Histograms of MT directionality within 1um of cell boundary (see Supplemental figure 2-1 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-test were performed across each bin for all cells, and a 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 over-expressions. 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 threshold to detect individual peripheral MTs (see Supplemental figure 2-1 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 Supplemental figure 2-1 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

Enhanced 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 1µm at the ventral side of the cell. Scale bars: 5um. (C) Quantification of mean tubulin intensity within the outer 2µm 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 1um of cell boundary (see Supplemental figure 2-1 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-test were performed across each bin for all cells, and a 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 over-expressions. 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 Supplemental figure 2-1 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

MT sliding in beta 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 2.8mM glucose before the assay. (B) A cell pretreated with 2.8mM glucose and stimulated with 20 mM glucose before the assay. (A1-B1) The first frame after photobleaching. (A2-B2) A frame 5 minutes (300 seconds) 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 2.8mM versus 20mM glucose (see representative data in A-B). MT displacement is shown as area of MTs displaced into the bleached area after 5 minutes of recovery. One-way ANOVA test was performed for statistical significance (p-value <0.0001). N=16-24 cells per set. (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 2.8mM glucose (D) and a cell stimulated by 20mM glucose (E) are shown. Single-slice spinning disk confocal microscopy. Halo-tag signal is shown as inverted gray-scale image. Top panels show cell overviews (scale bars 5µm). Below, boxed insets are enlarged to show dynamics of fiducial marks (color arrows) at 1 second intervals (1-5 seconds). 0-to 5-second 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. (F) Histogram of all 5-second displacement of fiducial marks in Low vs High glucose (G) Summarized quantification of stationary fiducial marks along MT lattice. Low glucose N=5,615 tracks across 11 cells, High glucose 5min N=2,259 tracks across 6 cells, High Glucose 20min N=3,059 tracks across 6 cells. P One-way ANOVA, p<0.001 (H) Summarized quantification of moving fiducial marks along the MT lattice. Low glucose N=2,595 tracks across 11 cells, High glucose 5min N=2,642 tracks across 6 cells, High Glucose 20min N=4,049 tracks across 6 cells. One-way ANOVA, p<0.001.

(A) Full western blot of anti-kif5b from Fig. 1B. (B) Histogram of displacements over 5-second intervals for all fiducial marks in scrambled shRNA control vs KIF5B shRNA #1 and KIF5B shRNA #2.

Workflow of 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.

Illustration of thresholding variations and their influence on the output analysis.

(A) A KIF5B-depleted cell as an example of contrast/threshold optimization for different MT densities and corresponding outputs. (A1) An 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 1um of cell boundary using standard thresholds (see Supplemental figure 3/1 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-test were performed across each bin for all cells, and a 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.

DN effect of KIF5B tail domain on MT abundance at the beta-cell cell periphery requires ATP-independent MT-binding domain.

(A-B) MT organization in MIN6 cells expressing (A) KIFDNwt, (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 1µm at the ventral side of the cell. Scale bars: 5um. (E) Quantification of mean tubulin intensity within the outer 2µm 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.

Influence of thresholding variations on the output analysis of MT directionality in Figure 4 F-H. (A) Histograms of MT directionality within 1um of cell boundary using standard thresholds (see Supplemental figure 2-1 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 4 F-H). Unpaired t-test were performed across each bin for all cells, and a 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 over-expression. 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.

Influence of thresholding variations on the output analysis of MT directionality in Figure 5 D-E. (A) Histograms of MT directionality within 1um of cell boundary using standard thresholds (see Supplemental figure 2-1 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 5 D-E). Unpaired t-test were performed across each bin for all cells, and a 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= 94,841pixels across 9 cells, kinesore N= 29,796 pixels across 9 cells.