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Single molecule imaging reveals differences in microtubule track selection between Kinesin motors.

Cai D, McEwen DP, Martens JR, Meyhofer E, Verhey KJ - PLoS Biol. (2009)

Bottom Line: In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules.Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip.These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
Cells generate diverse microtubule populations by polymerization of a common alpha/beta-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations.

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Related in: MedlinePlus

Single molecule imaging reveals preferential motility of Kinesin-1 on a subset of microtubules.(A) Schematic illustration of the domain structure of the KHC subunit of Kinesin-1 (left) and the constitutively active, truncated version KHC(1-560)-3xmCit (right). (B) Phase contrast image of a COS cell expressing KHC(1-560)-3xmCit. Scale bar, 10 µm. TIRF microscopy (C) was performed on a small region in the periphery of the cell (e.g., boxed area in B). From the image series, a SD Map (D) was created that provides a visual readout of all of the Kinesin-1 motility events. Scale bar, 3 µm. (E) TIRF image of a COS cell expressing mCherry-tubulin. Scale bar, 3 µm. (F) Two-color TIRF imaging. (1) COS cells coexpressing (2) KHC(1-560)-3xmCit and mCherry-tubulin were (3) imaged in the TIRF microscope. A SD Map of KHC(1-560)-3xmCit motility (KHC SD Map) and an average map of mCherry-tubulin (mChe-tub AVG Map) were created. The two images were (4) aligned and merged (Merged). Scale bar, 4 µm. (G) Retrospective immunofluorescence. (1) COS cells expressing (2) KHC(1-560)-3xmCit were (3) imaged by TIRF microscopy. The cells were (4) fixed immediately, stained with antibodies to total tubulin, and then (5) the same cell was imaged again in the TIRF microscope. A SD Map of the KHC(1-560)-3xmCit motility events was (6) aligned with the total tubulin image to create a merged image (Merged). Scale bar, 4 µm. Yellow lines in (D–G) indicate the edge of the cell.
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pbio-1000216-g001: Single molecule imaging reveals preferential motility of Kinesin-1 on a subset of microtubules.(A) Schematic illustration of the domain structure of the KHC subunit of Kinesin-1 (left) and the constitutively active, truncated version KHC(1-560)-3xmCit (right). (B) Phase contrast image of a COS cell expressing KHC(1-560)-3xmCit. Scale bar, 10 µm. TIRF microscopy (C) was performed on a small region in the periphery of the cell (e.g., boxed area in B). From the image series, a SD Map (D) was created that provides a visual readout of all of the Kinesin-1 motility events. Scale bar, 3 µm. (E) TIRF image of a COS cell expressing mCherry-tubulin. Scale bar, 3 µm. (F) Two-color TIRF imaging. (1) COS cells coexpressing (2) KHC(1-560)-3xmCit and mCherry-tubulin were (3) imaged in the TIRF microscope. A SD Map of KHC(1-560)-3xmCit motility (KHC SD Map) and an average map of mCherry-tubulin (mChe-tub AVG Map) were created. The two images were (4) aligned and merged (Merged). Scale bar, 4 µm. (G) Retrospective immunofluorescence. (1) COS cells expressing (2) KHC(1-560)-3xmCit were (3) imaged by TIRF microscopy. The cells were (4) fixed immediately, stained with antibodies to total tubulin, and then (5) the same cell was imaged again in the TIRF microscope. A SD Map of the KHC(1-560)-3xmCit motility events was (6) aligned with the total tubulin image to create a merged image (Merged). Scale bar, 4 µm. Yellow lines in (D–G) indicate the edge of the cell.

Mentions: Constitutively active kinesin motors can be generated by truncations that remove autoinhibitory and cargo-binding regions of the polypeptide. For this work, we generated KHC(1-560) (Figure 1A), a dimeric motor that has been well characterized in vitro and in vivo [16],[18],[19]. KHC(1-560) motors were tagged with three tandem copies of monomeric Citrine (mCit), a variant of enhanced yellow fluorescent protein (FP) (Figure 1A), and expressed in COS cells (Figure 1B). Single Kinesin-1 motors were tracked in live cells using a modified TIRF microscope (Figure 1C) in which the angle of illumination was varied to enable deeper imaging as described [17]. KHC(1-560)-3xmCit motors were observed to undergo both free diffusion and linear movement (Video S1). Linear motility occurred with an average speed of 0.83±0.08 µm/sec and average run length of 0.91±0.23 µm in live cells (Table 1, n = 372 events), consistent with previous work [16],[17].


Single molecule imaging reveals differences in microtubule track selection between Kinesin motors.

Cai D, McEwen DP, Martens JR, Meyhofer E, Verhey KJ - PLoS Biol. (2009)

Single molecule imaging reveals preferential motility of Kinesin-1 on a subset of microtubules.(A) Schematic illustration of the domain structure of the KHC subunit of Kinesin-1 (left) and the constitutively active, truncated version KHC(1-560)-3xmCit (right). (B) Phase contrast image of a COS cell expressing KHC(1-560)-3xmCit. Scale bar, 10 µm. TIRF microscopy (C) was performed on a small region in the periphery of the cell (e.g., boxed area in B). From the image series, a SD Map (D) was created that provides a visual readout of all of the Kinesin-1 motility events. Scale bar, 3 µm. (E) TIRF image of a COS cell expressing mCherry-tubulin. Scale bar, 3 µm. (F) Two-color TIRF imaging. (1) COS cells coexpressing (2) KHC(1-560)-3xmCit and mCherry-tubulin were (3) imaged in the TIRF microscope. A SD Map of KHC(1-560)-3xmCit motility (KHC SD Map) and an average map of mCherry-tubulin (mChe-tub AVG Map) were created. The two images were (4) aligned and merged (Merged). Scale bar, 4 µm. (G) Retrospective immunofluorescence. (1) COS cells expressing (2) KHC(1-560)-3xmCit were (3) imaged by TIRF microscopy. The cells were (4) fixed immediately, stained with antibodies to total tubulin, and then (5) the same cell was imaged again in the TIRF microscope. A SD Map of the KHC(1-560)-3xmCit motility events was (6) aligned with the total tubulin image to create a merged image (Merged). Scale bar, 4 µm. Yellow lines in (D–G) indicate the edge of the cell.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2749942&req=5

pbio-1000216-g001: Single molecule imaging reveals preferential motility of Kinesin-1 on a subset of microtubules.(A) Schematic illustration of the domain structure of the KHC subunit of Kinesin-1 (left) and the constitutively active, truncated version KHC(1-560)-3xmCit (right). (B) Phase contrast image of a COS cell expressing KHC(1-560)-3xmCit. Scale bar, 10 µm. TIRF microscopy (C) was performed on a small region in the periphery of the cell (e.g., boxed area in B). From the image series, a SD Map (D) was created that provides a visual readout of all of the Kinesin-1 motility events. Scale bar, 3 µm. (E) TIRF image of a COS cell expressing mCherry-tubulin. Scale bar, 3 µm. (F) Two-color TIRF imaging. (1) COS cells coexpressing (2) KHC(1-560)-3xmCit and mCherry-tubulin were (3) imaged in the TIRF microscope. A SD Map of KHC(1-560)-3xmCit motility (KHC SD Map) and an average map of mCherry-tubulin (mChe-tub AVG Map) were created. The two images were (4) aligned and merged (Merged). Scale bar, 4 µm. (G) Retrospective immunofluorescence. (1) COS cells expressing (2) KHC(1-560)-3xmCit were (3) imaged by TIRF microscopy. The cells were (4) fixed immediately, stained with antibodies to total tubulin, and then (5) the same cell was imaged again in the TIRF microscope. A SD Map of the KHC(1-560)-3xmCit motility events was (6) aligned with the total tubulin image to create a merged image (Merged). Scale bar, 4 µm. Yellow lines in (D–G) indicate the edge of the cell.
Mentions: Constitutively active kinesin motors can be generated by truncations that remove autoinhibitory and cargo-binding regions of the polypeptide. For this work, we generated KHC(1-560) (Figure 1A), a dimeric motor that has been well characterized in vitro and in vivo [16],[18],[19]. KHC(1-560) motors were tagged with three tandem copies of monomeric Citrine (mCit), a variant of enhanced yellow fluorescent protein (FP) (Figure 1A), and expressed in COS cells (Figure 1B). Single Kinesin-1 motors were tracked in live cells using a modified TIRF microscope (Figure 1C) in which the angle of illumination was varied to enable deeper imaging as described [17]. KHC(1-560)-3xmCit motors were observed to undergo both free diffusion and linear movement (Video S1). Linear motility occurred with an average speed of 0.83±0.08 µm/sec and average run length of 0.91±0.23 µm in live cells (Table 1, n = 372 events), consistent with previous work [16],[17].

Bottom Line: In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules.Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip.These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
Cells generate diverse microtubule populations by polymerization of a common alpha/beta-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations.

Show MeSH
Related in: MedlinePlus