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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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Preferential motility of Kinesin-1 does not occur on dynamic microtubules marked by EB3.(A) Two-color TIRF imaging of a COS cell coexpressing KHC(1-560)-3xmCit and EB3-mCherry. After imaging, a SD Map of KHC(1-560)-3xmCit motility events (KHC SD Map) and an average map of the EB3-mCherry fluorescence (EB3 AVG Map) were created and merged (Merged). Yellow line, edge of cell. Scale bar, 4 µm. In the boxed region, multiple KHC(1-560)-3xmCit motility events can be observed to occur adjacent to an EB3-mCherry-marked microtubule. (B) The KHC(1-560)-3xmCit motility events in the boxed region of (A) are depicted in the kymograph (left) and schematic representation (right). (C) Movement of EB3-mCherry in the boxed region of (A) is depicted in the kymograph (left) and schematic representation (right). Vertical scale bar, 0.5 s. Horizontal scale bar, 2 µm. (D) Schematic summary of the displacement over time of multiple KHC(1-560)-3xmCit motors and EB3-mCherry plus-ends in the cell in (A).
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pbio-1000216-g002: Preferential motility of Kinesin-1 does not occur on dynamic microtubules marked by EB3.(A) Two-color TIRF imaging of a COS cell coexpressing KHC(1-560)-3xmCit and EB3-mCherry. After imaging, a SD Map of KHC(1-560)-3xmCit motility events (KHC SD Map) and an average map of the EB3-mCherry fluorescence (EB3 AVG Map) were created and merged (Merged). Yellow line, edge of cell. Scale bar, 4 µm. In the boxed region, multiple KHC(1-560)-3xmCit motility events can be observed to occur adjacent to an EB3-mCherry-marked microtubule. (B) The KHC(1-560)-3xmCit motility events in the boxed region of (A) are depicted in the kymograph (left) and schematic representation (right). (C) Movement of EB3-mCherry in the boxed region of (A) is depicted in the kymograph (left) and schematic representation (right). Vertical scale bar, 0.5 s. Horizontal scale bar, 2 µm. (D) Schematic summary of the displacement over time of multiple KHC(1-560)-3xmCit motors and EB3-mCherry plus-ends in the cell in (A).

Mentions: We first tested whether Kinesin-1 motors move preferentially on dynamic microtubules. This population can be observed in live cells expressing FP-tagged plus-end tracking proteins (+TIPs) [21]. Two-color TIRF microscopy was used to analyze COS cells coexpressing KHC(1-560)-3xmCit with a mCherry-labeled version of the +TIP protein end binding (EB)3 [22]. Very few Kinesin-1 motility events could be observed on microtubules extending back from the EB3-mCherry-labeled plus ends (Video S3). A comparison of the SD Map of KHC(1-560)-3xmCit motility events with the average EB3-mCherry fluorescence (Figure 2A, representative of n = 12 cells in four experiments) demonstrates that the microtubule tracks utilized by KHC(1-560)-3xmCit motors are distinct from the dynamic microtubules marked by EB3-mCherry. In some cases, multiple Kinesin-1 motility events occurred on a microtubule track that appeared to lie directly adjacent to an EB3-marked dynamic microtubule (boxed region in Figure 2A, kymographs in Figure 2B, 2C). Kinesin-1 motility events overlapped with only 2.5%±1.1% of the EB3-mCherry-marked microtubules (Table 1). These results indicate that the preferential motility of Kinesin-1 motors does not occur on dynamic microtubules.


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)

Preferential motility of Kinesin-1 does not occur on dynamic microtubules marked by EB3.(A) Two-color TIRF imaging of a COS cell coexpressing KHC(1-560)-3xmCit and EB3-mCherry. After imaging, a SD Map of KHC(1-560)-3xmCit motility events (KHC SD Map) and an average map of the EB3-mCherry fluorescence (EB3 AVG Map) were created and merged (Merged). Yellow line, edge of cell. Scale bar, 4 µm. In the boxed region, multiple KHC(1-560)-3xmCit motility events can be observed to occur adjacent to an EB3-mCherry-marked microtubule. (B) The KHC(1-560)-3xmCit motility events in the boxed region of (A) are depicted in the kymograph (left) and schematic representation (right). (C) Movement of EB3-mCherry in the boxed region of (A) is depicted in the kymograph (left) and schematic representation (right). Vertical scale bar, 0.5 s. Horizontal scale bar, 2 µm. (D) Schematic summary of the displacement over time of multiple KHC(1-560)-3xmCit motors and EB3-mCherry plus-ends in the cell in (A).
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Related In: Results  -  Collection

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pbio-1000216-g002: Preferential motility of Kinesin-1 does not occur on dynamic microtubules marked by EB3.(A) Two-color TIRF imaging of a COS cell coexpressing KHC(1-560)-3xmCit and EB3-mCherry. After imaging, a SD Map of KHC(1-560)-3xmCit motility events (KHC SD Map) and an average map of the EB3-mCherry fluorescence (EB3 AVG Map) were created and merged (Merged). Yellow line, edge of cell. Scale bar, 4 µm. In the boxed region, multiple KHC(1-560)-3xmCit motility events can be observed to occur adjacent to an EB3-mCherry-marked microtubule. (B) The KHC(1-560)-3xmCit motility events in the boxed region of (A) are depicted in the kymograph (left) and schematic representation (right). (C) Movement of EB3-mCherry in the boxed region of (A) is depicted in the kymograph (left) and schematic representation (right). Vertical scale bar, 0.5 s. Horizontal scale bar, 2 µm. (D) Schematic summary of the displacement over time of multiple KHC(1-560)-3xmCit motors and EB3-mCherry plus-ends in the cell in (A).
Mentions: We first tested whether Kinesin-1 motors move preferentially on dynamic microtubules. This population can be observed in live cells expressing FP-tagged plus-end tracking proteins (+TIPs) [21]. Two-color TIRF microscopy was used to analyze COS cells coexpressing KHC(1-560)-3xmCit with a mCherry-labeled version of the +TIP protein end binding (EB)3 [22]. Very few Kinesin-1 motility events could be observed on microtubules extending back from the EB3-mCherry-labeled plus ends (Video S3). A comparison of the SD Map of KHC(1-560)-3xmCit motility events with the average EB3-mCherry fluorescence (Figure 2A, representative of n = 12 cells in four experiments) demonstrates that the microtubule tracks utilized by KHC(1-560)-3xmCit motors are distinct from the dynamic microtubules marked by EB3-mCherry. In some cases, multiple Kinesin-1 motility events occurred on a microtubule track that appeared to lie directly adjacent to an EB3-marked dynamic microtubule (boxed region in Figure 2A, kymographs in Figure 2B, 2C). Kinesin-1 motility events overlapped with only 2.5%±1.1% of the EB3-mCherry-marked microtubules (Table 1). These results indicate that the preferential motility of Kinesin-1 motors does not occur on dynamic microtubules.

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