Research ArticleCELL BIOLOGY

A reconstituted mammalian APC-kinesin complex selectively transports defined packages of axonal mRNAs

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Science Advances  13 Mar 2020:
Vol. 6, no. 11, eaaz1588
DOI: 10.1126/sciadv.aaz1588
  • Fig. 1 APC and kinesin-2 are necessary and sufficient to transport β2Btubulin-mRNA.

    (A and B) Full-length APC binds KAP3 and β2Btubulin-RNA (β2Btubwtmin) with different affinities. Interactions of APC-GFP with β2Btubwtmin (A) and KAP3 (B) were measured by MST in solution. Inset in (B) shows a summary of affinities measured between RNA transport complex components. Error bars, SD. (C) KIF3ABK transports RNPs containing APC-TMR and Alexa647-β2Btubwt. Top: Time sequence from a TIRF-M assay containing 750 pM KIF3ABK, 80 pM APC-TMR, and 2 nM Alexa647-β2Btubwt. Bottom: A kymograph showing multiple run events of APC-TMR and Alexa647-β2Btubwt complexes, indicated by white arrowheads. (D) APC is essential for β2Btubwt transport. The kymographs show Alexa647-β2Btubwt signals from TIRF-M experiments with (left) or without (right) APC. (E) Quantification of processive β2Btubwt run events in the presence and absence of APC. N, number of independent experiments; n, total number of events. Error bars, SEM. Statistical significance was evaluated with an unpaired, two-tailed t test. ***P < 0.001. (F) APC recruits β2Btubwt to the microtubule lattice in the absence of KIF3ABK. Top: Time sequence from a TIRF-M assay containing 40 pM APC-TMR and 2 nM Alexa647-β2Btubwt. Bottom: Kymographs showing APC-RNA codiffusion events (white arrowheads). (G) MSD plots of APC-TMR and Alexa647-β2Btubwt from the experiments shown in (C) and (F). Error bars, SEM. Inset, the dwell times of APC-TMR and Alexa647-β2Btubwt in the absence of KIF3ABK on the microtubule lattice. Statistical significance was evaluated with a Mann-Whitney test on the raw data. (H and I) APC-β2Btubwt complex lattice diffusion is not biased. Velocity autocorrelations of transported and lattice-diffusing APC-TMR (H) and Alexa647-β2Btubwt (I) are shown. Error bars, SD.

  • Fig. 2 Function of the individual mRNA transport complex components.

    (A) KAP3 is required for processive RNA transport. Kymographs show Alexa647-β2Btubwt signals from TIRF-M experiments containing 750 pM KIF3AB, 150 pM APC, 2 nM Alexa647-β2Btubwt, and 500 pM KAP3 (left) or no KAP3 (right). (B) Quantification of processive Alexa647-β2Btubwt run events in the presence and absence of KAP3. Error bars, SEM. (C) MSD plots of Alexa647-β2Btubwt motility (displacement >1.1 μm to include nonprocessive events in experiments lacking KAP3) from experiments shown in (A). Error bars, SEM. (D) Mean instantaneous velocity distribution of processive (displacement >4 μm) Alexa647-β2Btubwt complexes. Gray line: A gauss fit to velocity distribution. (E) APC and β2Btubwt activate KIF3ABK. The kymographs show Alexa647-KIF3ABK (750 pM) signals from TIRF-M experiments containing either only KIF3ABK (left) or in addition 80 pM APC (middle) or 80 pM APC and 2 nM TMR-β2Btubwt (right). (F) Quantification of processive Alexa647-KIF3ABK run events in the presence and absence of APC or APC-β2Btubwt RNPs. Error bars, SEM. In (B) and (F), statistical significance was evaluated with an unpaired, two-tailed t test. **P < 0.01, *P < 0.05. N, number of independent experiments; n, total number of events.

  • Fig. 3 Fluorescence quantification of motile complexes reveals the mRNA transport complex architecture.

    (A) Bleaching of an Alexa647-KIF3A homodimer calibration sample. The cartoon on the top schematizes the experimental setup; 10 pM homodimeric SNAP-KIF3A labeled either with Alexa647 or TMR was imaged on paclitaxel-stabilized microtubules in the presence of AMPPNP to immobilize the kinesin. The central three images and kymograph at the bottom show an exemplary time course of Alexa647-KIF3A bleaching. (B) A bleaching trace obtained from a single Alexa647-KIF3A dimer indicating total intensities of one and two Alexa647 fluorophores. (C) The blue distributions show total intensities derived from single-particle tracking of immobilized KIF3A-Alexa647 dimers (dark blue, derived from the first 10; light blue, derived from the last 20 frames). Correspondingly, the light and dark blue lines indicate kernel density function (KDF) fits to the shown distributions. The dotted magenta line shows the least square fit of the sum of the monomer and multimer fractions (black and gray dotted lines), derived from iterative convolutions of the monomer distribution (light blue line). The inset bar graphs show the measured fraction of KIF3A mono- and multimers and the real fractions obtained after correcting for the labeling ratio of KIF3A (table S2). (D to F) Quantification of molecules in individual RNA transport complex components. Using single-particle tracking, the total intensities of KIF3ABK, APC, and β2Btubwt was measured in processive (displacement >4 μm) complexes. For (D) and (E), experiments contained 750 pM KIF3ABK, 80 pM APC-TMR, and 2 nM Alexa647-β2Btubwt. For (F), experiments contained 750 pM Alexa647-KIF3ABK, 80 pM APC, and 2 nM TMR-β2Btubwt. (G and H) Intensity distributions of microtubule lattice-diffusing RNPs from assays containing 40 pM APC-TMR and 2 nM Alexa647-β2Btubwt. (I) Number of molecules of RNA transport complex components derived from (D) to (F) after labeling ratio (table S2) correction. (J) Number of molecules in diffusive RNPs derived from (G) and (H) after labeling ratio correction. The schematized complexes depicted in (D) to (H) illustrate the experimental setup and the transport complex component analyzed (colored). For (D) to (H), at least three independent experiments were analyzed, providing at least 75 tracks containing >11,000 total intensities from detected particles. Light-red or light-yellow distributions show intensity distributions of Alexa647- or TMR-Kif3A from calibration experiments. Colored lines (red or yellow) indicate KDF. Dotted lines indicate monomer and multimer fractions grayscale according to legend in (J).

  • Fig. 4 APC selectively links β-actin and β2B-tubulin mRNAs to kinesin-2.

    (A) Kymographs showing Alexa647-β2Btubwt and TMR-β2Btubmut in experiments containing 750 pM KIF3ABK and 150 pM APC. Equimolar RNA concentrations of 500 pM were used. Right: Quantification of Alexa647-β2Btubwt and TMR-β2Btubmut microtubule binding events (diffusive and processive). (B) Same as in (A) but comparing TMR-βactinwt to Alexa647-βactinmut RNA using equimolar RNA concentrations of 3000 pM. Right: Quantification of TMR-βactinwt and Alexa647-βactinmut microtubule binding events (diffusive and processive). Error bars in (A) and (B), SEM. In (A) and (B), statistical significance was evaluated with an unpaired, two-tailed t test. ***P < 0.001, **P < 0.01.

  • Fig. 5 The affinity of APC to different mRNAs controls mRNA transport frequency.

    (A) An overview TIRF-M image (left) and kymograph (right) showing Alexa647-β2Btubwt and TMR-βactinwt transport in the same experiments at 1:10 molar ratio (200 pM:2 nM). Arrows and arrowheads point to TMR-βactinwt and Alexa647-β2Btubwt RNPs, respectively. (B) A titration of Alexa647-β2Btubwt from 2000 to 40 pM leads to an increase in the relative amount of TMR-β2Btubmut transport per experiment. (C) Plot of processive run events of TMR-βactinwt (displacement >4 μm) per experiment in dependence of the free Alexa647-β2Btubwt concentration. Gray line: Linear regression fit. Yellow-shaded area: 95% confidence band. (D) Dwell time distribution of β2Btubwt and βactwt RNAs in experiments containing 750 pM KIF3ABK, 150 pM APC, 40 pM Alexa647-β2Btubwt, and 2000 pM TMR-βactwt. Colored solid lines: Monoexponential decay fits to dwell time distributions. (E) MST measurement of the affinity of βactinwt and β2Btubwt RNA to APC-GFP. Error bars, SD. Solid lines: One-site binding model fit to the measured normalized fluorescence. (F) Model of transport RNP assembly. Oligomerizing APC binds guanine-rich motifs located in mRNA 3′UTRs, microtubules and KAP3 with different domains. mRNAs with different G-motif variants compete for APC binding. APC-RNPs move by two different modes: (i) active transport by kinesin-2 and (ii) microtubule lattice diffusion. In (D), statistical significance was evaluated with a Mann-Whitney test on the raw data. N, number of independent experiments; n, total number of events.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/11/eaaz1588/DC1

    Fig. S1. Assay schematic, coomassie gels of used proteins, tracking data and bleaching controls.

    Fig. S2. Coomassie gels of used proteins, SEC-MALS data and minimal APC-fragment controls.

    Fig. S3. Coomassie gel of the used protein and tracking data of immobilized motor proteins.

    Table S1. Sequence of RNA fragments used.

    Table S2. Labeling ratios (in percentage) of proteins and RNAs used in this study.

    Movie S1. The kinesin-2 KIF3A/B/KAP3 and APC transport an axonal mRNA.

    Movie S2. Cotransport of APC–β2B-tubulin RNA complexes.

    Movie S3. APC binds and diffuses on the microtubule lattice in the absence of kinesin-2.

    Movie S4. APC–β2B-tubulin RNA complexes diffuse on the microtubule lattice.

    Movie S5. APC recruits and activates the heterotrimeric kinesin-2 KIF3A/B/KAP3.

    Movie S6. Single-particle tracking of transported β2B-tubulin RNA.

    Movie S7. Quadruple-color movie illustrating the selectivity of the reconstituted mRNA transport system.

    Movie S8. The APC-KIF3A/B/KAP3 mRNA transport system selectively transports β-actin RNA.

    Movie S9. β-actin and β2B-tubulin RNAs are transported in individual packages by the APC-KIF3A/B/KAP3 complex.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Assay schematic, coomassie gels of used proteins, tracking data and bleaching controls.
    • Fig. S2. Coomassie gels of used proteins, SEC-MALS data and minimal APC-fragment controls.
    • Fig. S3. Coomassie gel of the used protein and tracking data of immobilized motor proteins.
    • Table S1. Sequence of RNA fragments used.
    • Table S2. Labeling ratios (in percentage) of proteins and RNAs used in this study.
    • Legends for movies S1 to S9

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). The kinesin-2 KIF3A/B/KAP3 and APC transport an axonal mRNA.
    • Movie S2 (.avi format). Cotransport of APC–β2B-tubulin RNA complexes.
    • Movie S3 (.avi format). APC binds and diffuses on the microtubule lattice in the absence of kinesin-2.
    • Movie S4 (.avi format). APC–β2B-tubulin RNA complexes diffuse on the microtubule lattice.
    • Movie S5 (.avi format). APC recruits and activates the heterotrimeric kinesin-2 KIF3A/B/KAP3.
    • Movie S6 (.avi format). Single-particle tracking of transported β2B-tubulin RNA.
    • Movie S7 (.avi format). Quadruple-color movie illustrating the selectivity of the reconstituted mRNA transport system.
    • Movie S8 (.avi format). The APC-KIF3A/B/KAP3 mRNA transport system selectively transports β-actin RNA.
    • Movie S9 (.avi format). β-actin and β2B-tubulin RNAs are transported in individual packages by the APC-KIF3A/B/KAP3 complex.

    Files in this Data Supplement:

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