A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations

Abstract

Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin-binding domain-tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations.

Figure 1.

Linking Rab5 to dynein by inducible dimerization causes early endosomes to accumulate in the cell center. (A) As shown in the schematic, FRB-3myc-Rab5 and tdTM-BicD2⁵⁹⁴-FKBP were coexpressed along with GFP-Rab5, a vesicle marker. After adding the linker drug (AP21967), the FRB and FKBP domains were fused together, linking the FRB-3myc-Rab5 to dynein. The second diagram shows the predicted redistribution of GFP-Rab5 vesicles to the minus end of microtubules (gray). (B) Representative images showing the distribution of tdTM-BicD2⁵⁹⁴-FKBP and GFP-Rab5 in control cells and in cells treated with the linker drug. In control cells BicD2 was mostly soluble, with small aggregations throughout the cell. GFP-Rab5 vesicles were distributed through the cell. In treated cells, both GFP-Rab5 vesicles and BicD2 became concentrated in the center of the cell. The yellow lines outline the cell boundaries. Bar, 30 µm.

Figure 2.

Rab7 interacts primarily with late endosomes. (A) A schematic showing the components of the assay. GFP-BicD2⁵⁹⁴-FKBP and FRB-3myc-Rab7 were coexpressed. Vesicles were labeled by anti-EEA1 staining or by uptake of Tf555 or LysoTracker red. (B) Representative images showing the distribution of each vesicle population and the distribution of BicD2. In controls, EEA1- and Tf555-labeled vesicles were found throughout the cell, whereas LysoTracker red vesicles were more concentrated in the perinuclear region. BicD2 had a soluble distribution. In cells treated with the linker drug, the distribution of EEA1- and Tf555-labeled vesicles was largely unchanged. Vesicles labeled with LysoTracker red (LysoRed) were massively misdirected to the cell center. BicD2 also accumulated in the cell center. The yellow lines outline the cell boundaries. Bar, 30 µm.

Figure 3.

Different kinesins bind to different endosomal populations. (A) As shown in the schematic, GFP-BicD2⁵⁹⁴-FKBP was expressed together with a series of FRB-tagged kinesin tails. Vesicles were labeled by anti-EEA1 staining or uptake of Tf555 or LysoTracker red. (B) Representative cells showing the distribution of labeled vesicles. In controls, EEA1- and Tf555-labeled vesicles were found throughout the cell, whereas LysoTracker red vesicles were concentrated in the perinuclear region. In cells treated with the linker drug, EEA1- and Tf555-labeled vesicles were moved toward the cell center only in cells expressing FRB-3myc-KIF13A_tail or FRB-3myc-KIF13B_tail. Vesicles labeled with LysoTracker red moved to the cell center only in cells expressing FRB-3myc-KIF1A_tail or FRB-3myc-KIF1Bβ_tail. The control cell expressed FRB-3myc-KIF1A_tail and was not exposed to the linker drug. The yellow lines outline the cell boundaries. Bars: (EEA1) 25 µm; (Tf555 and LysoTracker red) 30 µm.

Figure 4.

Two different kinesins can bind the same vesicle. As shown in the schematic, vesicles were labeled by expressing GFP-KIF1A_tail, which was coexpressed with tdTM-BicD2⁵⁹⁴-FKBP and one of four different FRB-tagged kinesin tails. The remaining panels are representative images showing the distribution of vesicles labeled with GFP-KIF1A_tail. In control cells, GFP-KIF1A_tail labeled small vesicles that were concentrated in the perinuclear region. After treatment with the linker drug, GFP-KIF1A_tail vesicles accumulated in the cell center only in cells expressing FRB-3myc-KIF1A_tail or FRB-3myc-KIF1Bβ_tail. The control cell expressed FRB-3myc-KIF1A_tail but was not exposed to linker drug. The yellow lines outline the cell boundaries. Bar, 25 µm.

Figure 5.

Identifying the domain of KIF13B that binds to endosomes. (A) As shown in the schematic, different regions of KIF13B tail tagged with FRB were coexpressed with tdTM-BicD2⁵⁹⁴-FKBP and TfR-GFP. (B) In control cells, TfR-GFP was distributed throughout the cell. After treatment with the linker drug TfR-GFP, vesicles were moved to the cell center in cells expressing FRB-3myc-KIF13B^832–1,826 but not FRB-3myc-KIF13B^442–831. The yellow lines outline the cell boundaries. Bar, 30 µm.