Kinesin's neck-linker determines its ability to navigate obstacles on the microtubule surface

Abstract

The neck-linker is a structurally conserved region among most members of the kinesin superfamily of molecular motor proteins that is critical for kinesin's processive transport of intracellular cargo along the microtubule surface. Variation in the neck-linker length has been shown to directly modulate processivity in different kinesin families; for example, kinesin-1, with a shorter neck-linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo by the coupling of most cargo to multiple motors, longer and more flexible neck-linkers may allow different kinesins to navigate more efficiently around the many obstacles, including microtubule-associated proteins (MAPs), that are found on the microtubule surface within cells. We hypothesize that, due to its longer neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than kinesin-1. We used total internal reflection fluorescence microscopy to observe single-molecule motility from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence or presence of two Tau isoforms, 3RS-Tau and 4RL-Tau, both of which are MAPs that are known to differentially affect kinesin-1 motility. Our results demonstrate that unlike kinesin-1, kinesin-2 is insensitive to the presence of either Tau isoform, and appears to have the ability to switch protofilaments while stepping along the microtubule when challenged by an obstacle, such as Tau. Thus, although kinesin-1 may be more processive, the longer neck-linker length of kinesin-2 allows it to be better optimized to navigate the complex microtubule landscape. These results provide new insight, to our knowledge, into how kinesin-1 and kinesin-2 may work together for the efficient delivery of cargo in cells.

Figure 1

Experimental reagents. (A) Schematic of kinesin constructs illustrating the N-terminal globular motor domains, C-terminal coiled-coil stalk, and the random-coil neck-linker connecting the two motor domains. The C-terminal end of Drosophila kinesin-1 was truncated at 559 and fused with an eGFP. The kinesin-2 construct contained two mouse kif3A motor domains and their neck-linkers fused with the coiled-coil stalk of the kinesin-1 construct, which has been shown to be functionally equivalent to the wild-type kif3A/B heterodimer (9). (B) Primary amino acid sequence of the neck-linker regions of all four kinesin constructs used in the experiments. Kinesin-1’s 14 amino acid neck-linker was lengthened to 17 (kinesin-1_+KAL), and kinesin-2’s 17 amino acid neck-linker was shortened to 14 (kinesin-2_{PA_ΔDAL}) as described by Shastry and Hancock (10). (C) Linear schematic of 3RS- and 4RL-Tau isoforms containing an acidic N-terminal region, a central proline-rich region (P1 and P2), and a microtubule-binding region with three or four microtubule-binding repeats (R1–R4). 4RL-Tau contains two additional N-terminal acidic inserts (I1 and I2) and one additional C-terminal microtubule-binding repeat (R2). Tau isoforms were labeled with Alexa 546 at a single cysteine residue in R3.

Figure 2

Characteristic run length comparison between kinesin-1 and kinesin-2 on paclitaxel microtubules. (A–C) Cumulative frequency plots of kinesin-1 in the absence or presence of 3RS- or 4RL-Tau. (D and E) Cumulative frequency plots of kinesin-2 in the absence or presence of 3RS- or 4RL-Tau. Black dots represent the raw run-length data and the gray curve is the observed cumulative frequency. The expected characteristic run length, derived from the microtubule length distribution, is shown within each graph. The error represents the 99% confidence interval and a p-value of less than 0.01 was considered significant. ^∗Represents a statistically significant difference from the characteristic run length observed in the absence of Tau.

Figure 3

Characteristic run length comparison between kinesin-1 and kinesin-2 on GMPCPP microtubules. (A–C) Cumulative frequency plots of kinesin-1 in the absence or presence of 3RS- or 4RL-Tau. (D and E) Cumulative frequency plots of kinesin-2 in the absence or presence of 3RS- or 4RL-Tau. Black dots represent the raw run-length data and the gray curve is the observed cumulative frequency. The expected characteristic run length, derived from the microtubule length distribution, is shown within each graph. The error represents the 99% confidence interval, and a p-value of less than 0.01 was considered significant.

Figure 4

Representative kymograph images of kinesin-2 pausing events observed during processive movement along paclitaxel microtubules. (A) Uninterrupted processive movement (nonpause event) in the absence of Tau. (B) Pause-termination event in the presence of 3RS-Tau. (C) Pause-step event in the presence of 3RS-Tau. Scale bars represent 2 μm. Animations in panels A–C are not drawn to scale and are for visual effect. (D) Kinesin-1 and kinesin-2’s percentage of pause-step events in the absence and presence of 3RS-Tau. Kinesin-2, in the presence of 3RS-Tau, is more likely to step after a pause relative to kinesin-1. Error bars represent SE. ^∗Represents significant difference between the absence and presence of 3RS-Tau, p = 0.05.

Figure 5

Characteristic run length comparison between kinesin-1_+KAL and kinesin-2_{pa_ΔDAL} on paclitaxel microtubules. (A–C) Cumulative frequency plots of kinesin-1_+KAL in the absence or presence of 3RS- or 4RL-Tau. (D and E) Cumulative frequency plots of kinesin-2_{pa_ΔDAL} in the absence or presence of 3RS- or 4RL Tau. Black dots represent the raw run length data and the gray curve is the observed cumulative frequency. The expected characteristic run length, derived from the microtubule length distribution, is shown within each graph. The error represents the 99% confidence interval and a p-value of less than 0.01 was considered significant. ^∗Represents a statistically significant difference from the characteristic run length observed in the absence of Tau.

Figure 6

Probability of kinesin sidestepping as a function of neck-linker contour length. (A) Force-extension curves of kinesin-1 and kinesin-2 (kif3A/A) neck-linker regions determined assuming a worm-like chain model (41–43). Kinesin-2’s longer neck-linker allows for a longer reach at the same force and thus an increased probability of stepping to an off-protofilament binding site. (B) Probability of kinesin stepping to nearby binding sites. Black dots represent binding sites for a 13-protofilament microtubule. Kinesin-1 only steps along a single protofilament, whereas kinesin-2 is predicted to sidestep left to the adjacent protofilament 2.1% of the time. The animation is not drawn to scale and is for visual effect; see Supporting Material for further details about the modeling.