Binding of SFPQ to KIF5A/KLC1 by the Y-acidic motif is required for anterograde transport. (A) Alignment of the sequence within the coiled-coil domain of SFPQ containing the Y-acidic motif. Bottom: Schematic of the domains of SFPQ; red bracket indicates the region containing the Y-acidic motif. (B) ITC measurements of the reference KLC1 (TPR1-6) fragment with either the WT SFPQ peptide (ESEMEDAYHEHQANLLR) or the SFPQY527A peptide (ESEMEDAAHEHQANLLR). (C) HEK 293T cells transfected with HA-KIF5A, KLC1-Myc, and with either empty vector, full-length FLAG-tagged WT SFPQ, or SFPQY527A. HA was immunoprecipitated and blotted for FLAG, Myc, and HA. Input represents 0.5% of 0.5 mg of protein lysate used for SFPQ IP, and total elute from the bead was run on the gel. (D) Quantification of pull down in C relative to input; ****P < 0.0001 by unpaired two-tailed t test; n = 3; data represent mean ± SEM. (E) Kymograph of Halo-tagged WT SFPQ and SFPQY527A in axons of DRG neurons. Scale Bars: 2 µm and 15 s. (F) Average number of Halo-tagged WT SFPQ and SFPQY527A granules per micron of axon length. Data from WT SFPQ control (n = 31) and SFPQY527A (n = 34) axons analyzed across at least four independent microfluidic experiments. ***P = 0.0003 by unpaired two-tailed t test; data represent mean ± SEM. (G) Average percentage of time spent in anterograde transport for Halo-tagged WT SFPQ and SFPQY527A in axons of DRG sensory neurons. Data represents 258 granules for WT SFPQ control and 202 granules for SFPQY527A analyzed from at least 30 axons across at least four independent microfluidic experiments. ***P = 0.0004 by unpaired two-tailed t test; data represent mean ± SEM. GPQ, glycine proline glutamine-rich; NOPS, NONA/paraspeckle domain; RRM, RNA recognition motif.
Figure 4.

Binding of SFPQ to KIF5A/KLC1 by the Y-acidic motif is required for anterograde transport. (A) Alignment of the sequence within the coiled-coil domain of SFPQ containing the Y-acidic motif. Bottom: Schematic of the domains of SFPQ; red bracket indicates the region containing the Y-acidic motif. (B) ITC measurements of the reference KLC1 (TPR1-6) fragment with either the WT SFPQ peptide (ESEMEDAYHEHQANLLR) or the SFPQY527A peptide (ESEMEDAAHEHQANLLR). (C) HEK 293T cells transfected with HA-KIF5A, KLC1-Myc, and with either empty vector, full-length FLAG-tagged WT SFPQ, or SFPQY527A. HA was immunoprecipitated and blotted for FLAG, Myc, and HA. Input represents 0.5% of 0.5 mg of protein lysate used for SFPQ IP, and total elute from the bead was run on the gel. (D) Quantification of pull down in C relative to input; ****P < 0.0001 by unpaired two-tailed t test; n = 3; data represent mean ± SEM. (E) Kymograph of Halo-tagged WT SFPQ and SFPQY527A in axons of DRG neurons. Scale Bars: 2 µm and 15 s. (F) Average number of Halo-tagged WT SFPQ and SFPQY527A granules per micron of axon length. Data from WT SFPQ control (n = 31) and SFPQY527A (n = 34) axons analyzed across at least four independent microfluidic experiments. ***P = 0.0003 by unpaired two-tailed t test; data represent mean ± SEM. (G) Average percentage of time spent in anterograde transport for Halo-tagged WT SFPQ and SFPQY527A in axons of DRG sensory neurons. Data represents 258 granules for WT SFPQ control and 202 granules for SFPQY527A analyzed from at least 30 axons across at least four independent microfluidic experiments. ***P = 0.0004 by unpaired two-tailed t test; data represent mean ± SEM. GPQ, glycine proline glutamine-rich; NOPS, NONA/paraspeckle domain; RRM, RNA recognition motif.

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