The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

Bugiel, Michael; Böhl, Elisa; Schäffer, Erik

doi:10.1016/j.bpj.2015.03.022

Cited by 39 publications

(73 citation statements)

References 45 publications

(81 reference statements)

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“…Experiments were performed in flow cells that were constructed using silanized, hydrophobic glass cover slips and parafilm as described before (19). Full-length budding yeast Kip3 (Kip3-eGFP-His 6 ) was expressed and purified according to (16,26).…”

Section: Sample Preparation and Assaymentioning

confidence: 99%

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The kinesin-8 Kip3 depolymerizes microtubules with a collective force-dependent mechanism

Bugiel

Chugh²,

Jachowski

et al. 2019

Preprint

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Microtubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes in particular, during cell division. Motor proteins from the kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and length-dependent manner. However, it is still unclear how kinesin-8 depolymerizes microtubules. Here, we tracked the microtubule end-binding activity of yeast kinesin-8, Kip3, under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors' residence time at the microtubule end. On the microtubule lattice, Kip3 had a different binding behavior suggesting that the observations are distinct for the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism. Understanding the mechanics of kinesin-8's microtubule end activity will provide important insights into cell division with implications for cancer research. STATEMENT OF SIGNIFICANCEKinesin-8 motors are important for microtubule length regulation and overexpressed in different types of cancer. Yet, on the molecular level, it is unclear how these motors depolymerize microtubules. Using high-precision optical tweezers, we measured how single yeast kinesin-8 motors, Kip3, interacted with the microtubule end. Interestingly, we found that single Kip3 motors were still motile at the microtubule end. The force dependence of how long single motors were associated with the microtubule end enabled us to estimate what force motors must exert onto each other to achieve the collective microtubule depolymerization speed of many motors. Our data support a collective force-dependent depolymerization mechanism. A better understanding of Kip3's microtubule end activity has implications for cell division and associated diseases.

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Section: Sample Preparation and Assaymentioning

confidence: 99%

“…The bump-off model requires Kip3 to reach the MT end. While this feature can be well explained by Kip3's long run length and its suggested ability to bypass obstacles on the microtubule (17)(18)(19)(20), the activity of Kip3 at the MT end, i.e. the depolymerization mechanism itself, is poorly understood.…”

Section: Introductionmentioning

confidence: 99%

The kinesin-8 Kip3 depolymerizes microtubules with a collective force-dependent mechanism

Bugiel

Chugh²,

Jachowski

et al. 2019

Preprint

Self Cite

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“…The values for Kinesin-1 and Kip3 are consistent with previously reported values [31,36] and support the notion that the functionality of the coupled proteins is preserved. We expect that the anti-GFP-kinesin linkage can sustain forces of at least 10 pN for several seconds [34].…”

Section: Single-molecule Measurements With Kinesins Were Feasiblementioning

confidence: 99%

Versatile microsphere attachment of GFP-labeled motors and other tagged proteins with preserved functionality

et al. 2015

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Microspheres are often used as handles for protein purification or force spectroscopy. For example, optical tweezers apply forces on trapped particles to which motor proteins are attached. However, even though many attachment strategies exist, procedures are often limited to a particular biomolecule and prone to non-specific protein or surface attachment. Such interactions may lead to loss of protein functionality or microsphere clustering. Here, we describe a versatile coupling procedure for GFP-tagged proteins via a polyethylene glycol linker preserving the functionality of the coupled proteins. The procedure combines well-established protocols, is highly reproducible, reliable, and can be used for a large variety of proteins. The coupling is efficient and can be tuned to the desired microsphere-to-protein ratio. Moreover, microspheres hardly cluster or adhere to surfaces. Furthermore, the procedure can be adapted to different tags providing flexibility and a promising attachment strategy for any tagged protein.

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“…Despite their similarity, the origin of these rotations can be profoundly different. In processive MT motors it can result from side-steps (30,38,39), in processive myosins from a mismatch between the actin pitch and the step size (35) and in nonprocessive myosins from the distribution of "target zones" for motor attachment (40). When motors are attached to small beads their relative orientation could also play a role (34), which results in a helicity that varies from bead to bead.…”

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confidence: 99%

Working stroke of the kinesin-14, ncd, comprises two substeps of different direction

Nitzsche

Dudek

Hajdo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Single-molecule experiments have been used with great success to explore the mechanochemical cycles of processive motor proteins such as kinesin-1, but it has proven difficult to apply these approaches to nonprocessive motors. Therefore, the mechanochemical cycle of kinesin-14 (ncd) is still under debate. Here, we use the readout from the collective activity of multiple motors to derive information about the mechanochemical cycle of individual ncd motors. In gliding motility assays we performed 3D imaging based on fluorescence interference contrast microscopy combined with nanometer tracking to simultaneously study the translation and rotation of microtubules. Microtubules gliding on ncd-coated surfaces rotated around their longitudinal axes in an [ATP]-and [ADP]-dependent manner. Combined with a simple mechanical model, these observations suggest that the working stroke of ncd consists of an initial small movement of its stalk in a lateral direction when ADP is released and a second, main component of the working stroke, in a longitudinal direction upon ATP binding.kinesin | microtubule | gliding motility assay | fluorescence interference contrast microscopy | mathematical modeling S ingle-molecule techniques, in particular single-molecule fluorescence (1) and optical tweezers (2), have greatly advanced our understanding of motor proteins. Such experiments normally rely on the processivity of the motor, which makes it possible to observe multiple consecutive working cycles (i.e., steps). Although single-molecule mechanical measurements on nonprocessive motors can be performed with optical tweezers (3, 4), they require a sophisticated statistical analysis (5, 6) and, if the motor is to be studied under load, advanced feedback (7) or force-clamp mechanisms (8). The minus-end directed C-terminal kinesin-14, ncd, is such a nonprocessive motor. It has been shown to play an essential role in spindle focusing (9-11) and to slide and cross-link microtubules (12, 13).For the working stroke of a single ncd motor a lever arm model has been suggested (14, 15). However, as reviewed in ref. 16 the triggering event for the working stroke is still under debate. On the one hand, it has been suggested that the working stroke is connected to the ADP release (14, 17). On the other hand, it has been proposed to be coupled to the binding of ATP to the microtubule (MT)-ncd complex (4,15,18). In another study, a twostage displacement of the ncd stalk has been proposed where stalk displacement is initiated by binding of ncd to the MT and completed upon ATP binding (19). However, even though a variety of approaches including X-ray crystallography (15), EM (20-23), optical trapping (4, 24), FRET (19), and computational modeling (25) have been used to study the coupling between nucleotide states and mechanical states of ncd, the exact mechanism remains to be elucidated.Alternatively, nonprocessive motors can be studied in gliding motility assays (26) albeit with the disadvantage that single-motor behavior is difficult to access. On an ncd...

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The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

Cited by 39 publications

References 45 publications

The kinesin-8 Kip3 depolymerizes microtubules with a collective force-dependent mechanism

The kinesin-8 Kip3 depolymerizes microtubules with a collective force-dependent mechanism

Versatile microsphere attachment of GFP-labeled motors and other tagged proteins with preserved functionality

Working stroke of the kinesin-14, ncd, comprises two substeps of different direction

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