A novel strategy to visualize vesicle-bound kinesins reveals the diversity of kinesin-mediated transport

Abstract

In mammals, 15 to 20 kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each kinesin or the functional significance of such diversity. To characterize the transport mediated by different kinesins, we developed a novel strategy to visualize vesicle-bound kinesins in living cells. We applied this method to cultured neurons and systematically determined the localization and transport parameters of vesicles labeled by different members of the Kinesin-1, -2, and -3 families. We observed vesicle labeling with nearly all kinesins. Only six kinesins bound vesicles that undergo long-range transport in neurons. Of these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased (KIF1A and KIF1Bβ), and one transported only in dendrites (KIF13A). Overall, the trafficking of vesicle-bound kinesins to axons or dendrites did not correspond to their motor domain preference, suggesting that on-vesicle regulation is crucial for kinesin targeting. Surprisingly, several kinesins were associated with populations of somatodendritic vesicles that underwent little long-range transport. This assay should be broadly applicable for investigating kinesin function in many cell types.

Keywords: Kinesin; membrane trafficking; motor protein; neuron; polarity; transport; vesicle.

FIGURE 1

Strategies to enhance vesicle labeling with fluorescent kinesins. Representative images of seven DIV hippocampal neurons expressing full-length GFP-KIF5B (A), HaloTag-KIF5B tail visualized with JF549 (B), full-length GFP-KIF13B (C) and GFP-KIF13B tail (D) for 6 to 8 hours at comparable levels. In (A) and (B), KLC1a was co-expressed. Yellow lines indicate the axons and dendrites from which high magnification views and kymographs were generated. For clarity, transport events from kymographs were redrawn as solid black lines. Kymograph lines with a positive slope indicate anterograde transport and those with a negative slope indicate retrograde transport. This convention is followed in all figures. Scale bars: low magnification = 20 μm; high magnification = 5 μm

FIGURE 2

Kinesin-1-labeled vesicles move bidirectionally in dendrites, but are biased to the axon. Representative images of seven DIV hippocampal neurons expressing HaloTag-KIF5B tail (A) or HaloTag-KIF5C tail (B), each co-expressed with KLC1a and visualized with JF549. Yellow lines indicate the axons and dendrites from which high magnification views and kymographs were generated. Kymographs show that both KIF5B- and KIF5C-labeled vesicles underwent short, bidirectional movement in dendrites and mostly long-range anterograde movements in axons. Scale bars: low magnification = 20 μm; high magnification = 5 μm. Graphs show quantification of the number of transport events per 100 μm (C), the average velocity of moving vesicles (D) and their average run length (E). KIF5B: 107 events in 17 cells. KIF5C: 386 events in 17 cells. Error bars show the SEM

FIGURE 3

Kinesin-2-labeled vesicles are largely restricted to the somatodendritic domain and undergo minimal long-ranged transport. Representative images of seven DIV hippocampal neurons expressing GFP-KIF3B (A) or GFP-KIF3C (B) tail together with KIF3A tail. Yellow lines indicate the axons and dendrites from which high magnification views and kymographs were generated. Kymographs show that these vesicles did not undergo long-range transport events. Scale bars: low magnification = 20 μm; high magnification = 5 μm

FIGURE 4

Kinesin-3-labeled vesicles exhibit a diverse range of transport parameters. Representative images of seven DIV hippocampal neurons expressing GFP-KIF1A tail (A), GFP-KIF1Bβ tail (B), GFP-KIF13A tail (C) and GFP-KIF13B tail (D). Yellow lines indicate the axons and dendrites from which high magnification views and kymographs were generated. Kymographs show that KIF1A- and KIF1Bβ-labeled vesicles undergo short, bidirectional movement in dendrites and axons. Vesicles labeled by KIF13A are restricted to dendrites. Vesicles labeled by KIF13B move bidirectionally in dendrites and have a strong preference for anterograde movement in the axon. Scale bars: low magnification = 20 μm; high magnification = 5 μm. Graphs show quantification of the number of transport events per 100 μm (C), the average velocity of moving vesicles (D) and their average run length (E). KIF1A: 430 events in 22 cells. KIF1Bβ: 683 events in 18 cells. KIF13A: 181 events in 290 cells. KIF13B: 649 events in 15 cells. Error bars show the SEM

FIGURE 5

Some Kinesin-3 family members label vesicles that do not undergo long-range transport. Representative images of seven DIV hippocampal neurons expressing GFP-KIF1Bα tail (A), GFP-KIF1C tail (B) and GFP-KIF16B tail (C). Yellow lines indicate the axons and dendrites from which kymographs were generated. Kymographs show that vesicles labeled by these kinesins underwent few long-range transport events. Low magnification scale bar: 20 μm; high magnification scale bar: 5 μm

FIGURE 6

Two-color live-cell imaging can identify the cargos moved by different kinesins. (A) Representative high magnification images and kymographs of cells expressing the indicated kinesin tails and cargoes. Yellow lines indicate transport events in both channels. HaloTags were visualized with JF549. Cells with KIF5B tails were cotransfected with KLC1. (B) A table indicating the percentage of kinesin-labeled vesicles that co-transported with various cargo markers. Anterograde axonal transport and bidirectional dendritic transport were analyzed separately

FIGURE 7

Quantitative analysis of kinesin-labeled vesicles that undergo long-ranged transport. (A) The directionality of axonal transport events of vesicles labeled with different Kinesin-1 and Kinesin-3 family members. (B) The directionality index of dendritic transport events of vesicles labeled with different kinesins. (C) The polarity index of vesicles labeled with these same kinesins. Indices were derived from kymograph analysis of seven to nine DIV hippocampal neurons expressing the designated kinesin tail labels. No axonal directional index was generated for vesicles labeled by KIF13A, as those vesicles were restricted to dendrites