Fig 2: Reporter mRNAs containing the Net1 3′UTR require the Kif1c motor for long, linear microtubule-dependent displacements. (A) Images are snapshots of live NIH/3T3 cells taken 30 min after plating. The cells stably expressed the β24bs/Net1 mRNA reporter and MCP-GFP (Green) and were treated with the indicated siRNAs. The green spots correspond to single mRNAs detected with the MCP-GFP. High speed imaging was performed over 1 min to track individual RNA movements. See Supplemental Movies S5–S8 for time lapse imaging. Scale bars are 5 microns. (B) Graphs plot the displacements of individual RNA tracks (x-axis) over the linearity of their forward progression (y-axis) (defined as the mean straight line speed divided by the mean speed), using the movies of cells as shown in A. Red lines indicate the thresholds used to filter tracks of molecules undergoing directed movement (based on Fig. 3). (C) Graph depicts the percentage of directed tracks per cell following treatment with the indicated siRNAs (see panel B). N = 11–14 cells. Stars represent P-values: (***) P < 0.001, ns: nonsignificant, estimated using one-way analysis of variance with Dunnett's multiple comparisons test. Error bars: standard error of the mean.

Fig 3: The KIF1C motor transports mRNAs containing the Net1 3′UTR to cell protrusions. (A) Images are micrographs of fixed NIH/3T3 cells expressing the β24bs/Net1 reporter mRNA, MCP-GFP (green), KIF1C-STx24 protein, and scFv-mScarletI (red). Single molecules of the β 24bs/Net1 reporter mRNA are visible in green, while single molecules of KIF1C-STx24 protein are red. The numbered white boxes are magnified in B. Blue: DNA stained with DAPI. Scale bar is 5 microns. (B) Insets represent magnifications of the boxed areas from the image shown in A. (Left) MCP-GFP signals labeling β24bs/Net1 mRNAs; (middle) scFv-mScarletI labeling KIF1C-STx24 protein; (right) merge with mRNAs in green and KIF1C-STx24 in red. Black and white arrowheads indicate colocalization of single molecules of β24bs/Net1 mRNAs and KIF1C-STx24. Scale bar is 5 microns. (C) Snapshot of a live NIH/3T3 cell expressing β24bs/Net1 mRNA, MCP-GFP (green), KIF1C-STx24 protein, and scFv-mScarletI (red). Snapshot is extracted from Supplemental Movie S9. The white arrowhead indicates a cotransport event of a single molecule of β24bs/Net1 mRNA (green) with a KIF1C-STx24 protein (red). The boxed area is magnified in panels D and E. Scale bar is 5 microns. (D) Magnification of the boxed area in panel C, highlighting a cotransport event. (Top) KIF1C-STx24; (middle) β24bs/Net1 mRNA; (bottom) merged panel with the β24bs/Net1 mRNA in green and KIF1C-STx24 in red. Scale bar is 1 micron. (E) Kymograph from Supplemental Movie S9, showing the trajectory of a single molecule of KIF1C-STx24 (top panel), a single molecule of β24bs/Net1 mRNA (middle panel), and the merge (bottom panel). The cellular area shown corresponds to panel D. (F) The graph depicts the distance traveled by cotransported molecules of KIF1C-STx24 and β24bs/Net1 mRNA. Each data point is a track (40 tracks in total), and the mean and 95% confidence intervals are shown by horizontal lines. Source data are provided in Supplemental Table S4. (G) Boxplot depicting the mean speed of cotransported molecules of KIF1C-STx24 and β24bs/Net1 mRNA in NIH/3T3 cells. Speed is microns/second. The vertical bars display the first and last quartile, the box corresponds to the second and third quartiles, and the horizontal line to the mean (40 tracks in total). Source data are provided in Supplemental Table S4.

Fig 4: Identification of mRNAs associated with the KIF1C motor. (A) The KIF1C kinesin colocalizes with its mRNA in protrusions of HeLa cells. Images are micrographs of a H9 FlipIn HeLa cell line stably expressing a KIF1C-GFP cDNA. (Top left) KIF1C mRNA detected by smFISH with probes against the endogenous mRNA; (top right) KIF1C-GFP protein; (bottom left) DNA stained with DAPI; (bottom right) merge of the two signals with the KIF1C-GFP protein in green and KIF1C mRNA in red. Orange arrow: a cell protrusion. Blue: DNA stained with DAPI. Scale bar: 10 microns. Insets represent zooms of the boxed areas in the merge panel. White and black arrowheads indicate the colocalization of KIF1C mRNA with KIF1C-GFP protein. (B) Transcripts associating with the KIF1C-GFP protein. The graph depicts the microarray signal intensity of RNAs detected in a KIF1C-GFP pull-down (x-axis), versus the control IP (y-axis). Each dot represents an mRNA. Red dot: KIF1C mRNA; blue dot: mRNAs enriched in the KIF1C-GFP IP. N = 2. (C) Colocalization of KIF1C-GFP with KIF1C, NET1, TRAK2, and RAB13 mRNAs. Images are micrographs of HeLa H9 Flip-In cells stably expressing a KIF1C-GFP cDNA, labeled by smiFISH with probes against the indicated mRNAs. (Top) Cy3 fluorescent signals corresponding to endogenous KIF1C, NET1, TRAK2, and RAB13 mRNAs. (Middle) KIF1C-GFP signal. (Lower) Merge with the Cy3 signal in red and GFP signal in green. Blue: DNA stained with DAPI. Scale bar: 10 microns. Arrowheads indicate accumulation of single mRNA molecules at cell protrusions. (D) Single-molecule colocalization of KIF1C-ST-x24 with RAB13 mRNAs. Images are micrographs of Hela cells stably expressing KIF1C-STx24 and scFv-GFP. Red: Cy3 fluorescent signals corresponding to RAB13 mRNAs labeled by smiFISH with probes against endogenous RAB13 mRNA. Green: GFP signal corresponding to single molecules of KIF1C protein. Blue: DNA stained with DAPI. Scale bar: 10 microns. (E) Insets represent zooms of the numbered areas from panel D. Legend as in D. Arrowheads indicate molecules of RAB13 mRNA and KIF1C-STx24 protein. Micrographs show cells representative of the population.

Fig 5: KIF1C associates with APC and is required for the localization of APC-dependent mRNAs to cytoplasmic protrusions. (A) Coimmunoprecipitation of KIF1C with APC. GFP or GFP-APC were immunoprecipitated from cells also expressing KIF1C-mCherry and analyzed by western blot to detect the indicated proteins. Results are representative of three independent experiments. (B) Colocalization of GFP-APC and KIF1C-mCherry at cytoplasmic protrusions (arrows). Images are representative of multiple cells observed in two independent experiments. Scale bar: 10 microns. (C) Depletion of Kif1c prevents mRNA accumulation in protrusions. Images are micrographs of NIH/3T3 cells labeled by smFISH with probes against Net1, Rab13, Ddr2, and Dynll2 mRNAs, following treatment with siRNAs against Kif1C (panels si-Kif1c) or a control sequence (panel si-Control). Scale bars: 10 microns. Green: outline of the cells; blue: outline of the nuclei; black: smFISH signals. Insets represent magnifications of the boxed areas. (D) Quantification of mRNA localization of cells described in C. Graphs represent the intracellular distribution of the indicated mRNAs as measured by PDI index, with and without treatment of cells with the indicated siRNAs. Red bars represent the mean and 95% confidence interval. Points indicate individual cells observed in two independent experiments. (E) Detection of Kif1C protein (upper panels) or Kif1C RNA levels (lower graph) from cells treated with the indicated siRNAs. Stars in D and E are P-values: (****) P < 0.0001, (***) P < 0.001, estimated by analysis of variance with Bonferroni's multiple comparisons test.