Abstract
Kinesins are well known to power diverse long-range transport processes in virtually all eukaryotic cells. The ATP-dependent processive stepping as well as the regulation of kinesin' activity have, thus, been the focus of extensive studies over the past decades. It is widely accepted that kinesin motors can self-regulate their activity by suppressing the catalytic activity of the "heads." The distal random coil at the C terminus, termed "tail domain," is proposed to mediate this autoinhibition; however, a direct regulatory influence of the tail on the processive stepping of kinesin proved difficult to capture. Here, we simultaneously tracked the two distinct head domains in the kinesin-2 motor using dual-color super resolution microscopy (dcFIONA) and reveal for the first time their individual properties during processive stepping. We show that the autoinhibitory wild-type conformation selectively impacts one head in the heterodimer but not the other. Our results provide insights into the regulated kinesin stepping that had escaped experimental scrutiny so far.
Highlights
Maintenance of a eukaryotic cell is a daunting task of logistics
The motor is propelled by the energy provided by two alternating ATP hydrolysis cycles in the so-called “head” domains [7, 8]
To follow the two head domains independently, we introduced SNAP- and Halo-tags at the N termini of the KLP11/20 heterodimer
Summary
Maintenance of a eukaryotic cell is a daunting task of logistics. One key organizer of the eukaryotic cytoplasm is kinesin, a microtubuleassociated molecular motor that transports cargo in diverse settings throughout the cell [1, 2, 3, 4, 5, 6]. The capability to extract information simultaneously from both heads confirmed the previously suggested limping behavior as well as the inhibitory impact of the tail domain on the stepping of the kinesin motor. The dwell times for kinesin constructs that were labeled on one head only were shown to be distributed according to a convolution of two exponentials [7, 21].
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