Figure 2.
RAB6A, ARHGEF10, and RAB8A co-localize with LAMP1Δ-RUSH post-Golgi carriers. (A) Heterologous expression of HALO-RAB6A in WT HeLa cells shows RAB6A present at the Golgi and in tubular structures that bud off and travel toward the plasma membrane (single micrograph from a Lattice-SIM time series), as evidenced by blue arrowheads. From Video 5. Scale bar: 10 μm; insert: 2 μm. (B) Lattice-SIM imaging showing HALO-RAB6A (magenta) co-localizing with LAMP1Δ-RUSH tubules (green) leaving the Golgi and moving toward the plasma membrane (∼35 min after biotin addition). Orange arrowheads indicate co-localizing structures. From Video 6. Scale bar: 10 μm; insert: 2 μm. (C) Percent of LAMP1Δ-RUSH carriers positive for HALO-RAB6A, untagged HALO as a control, three biological repeats (total carriers quantified = 359), statistical analysis = two-tailed t test. (D) Lattice-SIM imaging showing co-localization of HALO-ARHGEF10 (magenta) with LAMP1Δ-RUSH carriers (green) traveling toward the plasma membrane (∼35 min after biotin addition). Orange arrowheads indicate co-localizing structures. From Video 7. Scale bar: 10 μm; insert: 2 μm. (E) Percent of LAMP1Δ-RUSH carriers positive for HALO-ARHGEF10, untagged HALO as a control, three biological repeats (total carriers quantified = 389), statistical analysis = two-tailed t test. (F) Plasma membrane TIRF plane showing HALO-RAB8A (magenta) co-localizing with LAMP1Δ-RUSH carriers (green) near their fusion site at the plasma membrane. The color white denotes co-localizing structures. From Video 8. Scale bar: 10 μm; insert: 2 μm. (G) Percent of LAMP1Δ-RUSH carriers positive for HALO-RAB8A, untagged HALO as a control, three biological repeats (total carriers quantified within 3 μm of plasma membrane edge = 285), statistical analysis = two-tailed t test. (H) Transient RAB6A KO dramatically reduces LAMP1Δ-RUSH at the plasma membrane in a quantitative cell-surface assay carried out 35 min after biotin addition. Note that expression of HALO-RAB6A WT (rescue), and constitutively active RAB6A (QL), is able to restore plasma membrane expression but not the constitutively inactive form (TN). (I) Widefield imaging of LAMP1Δ-RUSH WT and RAB6A transient KO, 1 h after biotin addition. Image shows the LAMP1Δ reporter unable to leave the Golgi in the RAB6A KO cells. Scale bar: 10 μm. (J) Widefield imaging of LAMP1Δ-RUSH reporter (green) in RAB6A KO cells expressing HALO-RAB6A (*rescue—magenta). Upon RAB6A heterologous expression, the reporter is at the plasma membrane 1 h after biotin addition. Scale bar: 10 μm. Nucleus stain = DAPI. Error bar = SD of at least three independent experimental repeats. Student’s t test was performed on data in C, E, and G, and Tukey’s multiple comparisons test (HSD, FWER = 0.05) was performed on data in H. **P ≤ 0.01; ***P ≤ 0.001.

RAB6A, ARHGEF10, and RAB8A co-localize with LAMP1Δ-RUSH post-Golgi carriers. (A) Heterologous expression of HALO-RAB6A in WT HeLa cells shows RAB6A present at the Golgi and in tubular structures that bud off and travel toward the plasma membrane (single micrograph from a Lattice-SIM time series), as evidenced by blue arrowheads. From Video 5. Scale bar: 10 μm; insert: 2 μm. (B) Lattice-SIM imaging showing HALO-RAB6A (magenta) co-localizing with LAMP1Δ-RUSH tubules (green) leaving the Golgi and moving toward the plasma membrane (∼35 min after biotin addition). Orange arrowheads indicate co-localizing structures. From Video 6. Scale bar: 10 μm; insert: 2 μm. (C) Percent of LAMP1Δ-RUSH carriers positive for HALO-RAB6A, untagged HALO as a control, three biological repeats (total carriers quantified = 359), statistical analysis = two-tailed t test. (D) Lattice-SIM imaging showing co-localization of HALO-ARHGEF10 (magenta) with LAMP1Δ-RUSH carriers (green) traveling toward the plasma membrane (∼35 min after biotin addition). Orange arrowheads indicate co-localizing structures. From Video 7. Scale bar: 10 μm; insert: 2 μm. (E) Percent of LAMP1Δ-RUSH carriers positive for HALO-ARHGEF10, untagged HALO as a control, three biological repeats (total carriers quantified = 389), statistical analysis = two-tailed t test. (F) Plasma membrane TIRF plane showing HALO-RAB8A (magenta) co-localizing with LAMP1Δ-RUSH carriers (green) near their fusion site at the plasma membrane. The color white denotes co-localizing structures. From Video 8. Scale bar: 10 μm; insert: 2 μm. (G) Percent of LAMP1Δ-RUSH carriers positive for HALO-RAB8A, untagged HALO as a control, three biological repeats (total carriers quantified within 3 μm of plasma membrane edge = 285), statistical analysis = two-tailed t test. (H) Transient RAB6A KO dramatically reduces LAMP1Δ-RUSH at the plasma membrane in a quantitative cell-surface assay carried out 35 min after biotin addition. Note that expression of HALO-RAB6A WT (rescue), and constitutively active RAB6A (QL), is able to restore plasma membrane expression but not the constitutively inactive form (TN). (I) Widefield imaging of LAMP1Δ-RUSH WT and RAB6A transient KO, 1 h after biotin addition. Image shows the LAMP1Δ reporter unable to leave the Golgi in the RAB6A KO cells. Scale bar: 10 μm. (J) Widefield imaging of LAMP1Δ-RUSH reporter (green) in RAB6A KO cells expressing HALO-RAB6A (*rescue—magenta). Upon RAB6A heterologous expression, the reporter is at the plasma membrane 1 h after biotin addition. Scale bar: 10 μm. Nucleus stain = DAPI. Error bar = SD of at least three independent experimental repeats. Student’s t test was performed on data in C, E, and G, and Tukey’s multiple comparisons test (HSD, FWER = 0.05) was performed on data in H. **P ≤ 0.01; ***P ≤ 0.001.

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