Single-motor and multi-motor motility properties of kinesin-6 family members

  1. Andrew Poulos
  2. Breane G. Budaitis
  3. Kristen J. Verhey

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Abstract

Kinesin motor proteins are responsible for orchestrating a variety of microtubule-based processes including intracellular transport, cell division, cytoskeletal organization, and cilium function. Members of the kinesin-6 family play critical roles in anaphase and cytokinesis during cell division as well as in cargo transport and microtubule organization during interphase, however little is known about their motility properties. We find that truncated versions of MKLP1 (HsKIF23), MKLP2 (HsKIF20A), and HsKIF20B largely interact statically with microtubules as single molecules but can also undergo slow, processive motility, most prominently for MKLP2. In multi-motor assays, all kinesin-6 proteins were able to drive microtubule gliding and MKLP1 and KIF20B were also able to drive robust transport of both peroxisomes, a low-load cargo, and Golgi, a high-load cargo, in cells. In contrast, MKLP2 showed minimal transport of peroxisomes and was unable to drive Golgi dispersion. These results indicate that the three mammalian kinesin-6 motor proteins can undergo processive motility but differ in their ability to generate forces needed to drive cargo transport and microtubule organization in cells.

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  1. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #4

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, the authors use both cellular and single molecule assays to compare the motility properties of three human kinesin-6 proteins: MKLP1, MKLP2, and KIF20B. The presented data indicate that the three motors are primarily non-processive as single molecules, but are capable of driving plus-end directed motility in ensembles. The ability of kinesin-6 motors to move organelles in cells was also tested using an FRB-FKBP induced dimerization assay. MKLP1 and KIF20B were more capable of driving the dispersion of both peroxisomes and golgi than MKLP2. These data are interpreted to indicate that MKLP2 ensembles exhibit less force than MKLP1 or KIF20B.

    Major Comments:

    1. The single molecule results from analyses of MKLP2 presented in this study contrast significantly with those presented in Adriaans et al. 2020. This previous study presented evidence that full-length MKLP2 moves processively (1.1 um run-length at 150 nm/s) as single molecules, and that this processivity is enhanced by binding to the chromosome passenger complex. The current study addresses these differences to some extent by indicating that a fraction of MKLP2 motors displayed slow, processive movement. However, the kymographs of MKLP2 in the two studies still look quite different (e.g. frequency of processive movement, pausing, velocity), and further explanation would be useful for understanding the apparent conflict in conclusions regarding MKLP2 motility. Does the use of a truncated MKLP2 construct in the current study change the behavior of the protein in the motility assay?
    2. The organelle dispersion assays shown in Figures 4 and 5 rely on co-transfection of motor-FRB and targeting-FKBP constructs. The extent of dispersion could be affected by expression levels of either construct in a particular cell. Controls indicating that similar expression levels were compared across experimental groups should be included.
    3. The authors speculate about the contributions of kinesin-6 extensions in the neck-linker and presence or absence of the N-latch residue to the motility properties observed. However, these predictions are not tested experimentally.

    Minor Comments:

    1. In the legend for Figure 2B- kymographs are of fluorescent microtubules?

    Significance

    The presented work provides an assessment of human kinesin-6 motors in a number of different motility assays. These motors play key roles during cell division and cytokinesis, and the multifaceted investigation of kinesin-6 motility presented in this manuscript complements previous studies that examined the same motors in one type of assay or assessed the activity of kinesin-6 motors from other organisms. The work, therefore, provides a framework for future structure, function studies that will be of interest to the molecular motor, cell division, and cytoskeleton fields.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript, "Differences in single-motor and multi-motor motility properties across the kinesin-6 family," by Poulos, et al, is a comprehensive study of the motility properties of the kinesin-6 motor proteins.

    The work has a high number of molecules, filaments, and cells used to have statistical significance and reliability of the results.

    Positive aspects

    1. Kinesin-6 family is an important class of motors that needed to be investigated in a systematic manner.
    2. The work was performed using highly reproducible assays that revealed novel information about this family of motor proteins.
    3. The data was presented in a clear and cogent manner, making the paper highly accessible to non-experts.

    Negative aspects

    My only suggestion is that the figures be discussed in order to make it a bit easier for the reader to follow. This is a minor suggestion.

    Significance

    Employing single molecule motility assays, gliding assays, and cellular transport assays, this study elucidates the physical abilities of this elusive and essential family of kinesin motors. Excitingly, it was shown that the kinesin 6 motors can move infrequently as single motors and more frequently as multi-motors. In cells, they can also transport cargos except MKLP2. These results clearly demonstrate that kinesin-6 motors have motility and can even move large objects under high load in multi-motor configurations. The work is well-articulated and should be accessible to a wide audience.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Poulos and colleagues perform experiments aimed at understanding the in vitro behavior of kinesin-6 family motor proteins. The results, which are well supported by the experiments, show that the truncated motors show mostly diffusive behavior in single molecule assays. Surface bound motor domains drive microtubule sliding at low velocity. Assays in which motors were coupled to organelles, using rapamycin induced dimerization of the FRB-tagged motor construct and either Golgi or lysosome proteins with a FKBP domain, further revealed that the MKLP1 and Kif20B could drive transport of both peroxisomes and Golgi, although MKLP2 could not transport the high load cargo (Golgi) and showed limited transport of peroxisomes.

    Comments.

    These experiments are important to show the properties of the kinesin-6 family motor domain; however, the general lack of robust single molecule processive motility supports the idea that in vivo, these motors contribute to cellular processes as part of complexes with other proteins (Central spindlin (MKLP1), Chromosome passenger complex (MKLP2)) which enhance motility. In fact others have shown that motorclustering is important for plus end accumulation of MKLP1 (using C.elegans proteins). More recently Adriaans etal showed that for MKLP2 that MKLP2 is a processive plus end directed motor, using purified homodimeres of full length protein. Importantly, addition of a recombinant CPC further increased processivity. What remains unclear is why imaging of MKLP2 in cells shows predominantly diffuse behavior with only a fraction of events showing directed motility. The authors might discuss this concept in more detail - how motility is impacted by binding partners and/or regions outside of the motor domain for some kinesin families. Alternatively, they could demonstrate the changes in motility by extending the study with longer constructs and additional components.

    The authors used truncated proteins for their assay, but also tested longer constructs. They state that the behavior was similar in single molecule assays, so they focus on the truncated motors. However, figure S2 looks like there are move processive motility events for MKLP1 and MKLP2, which is more in line with some other results (i.e. Adriaans et al, for MLP2). Can the authors comment on this?

    The authors perform assays to study organelle motility. What is already known about kinesin-6 motor contribution to this process? For MKLP1 and 2, the best studied role is anaphase and cytokinesis.

    Significance

    Overall, the work is well done; however, the main result is that motor domains of kinesin-6 show mostly diffuse motility. This strongly suggests that other binding partners or other parts of the motor are needed for processive motion. The mechanism responsible for mixture of directed and diffuse motion observed in cells remains unclear. The advance from these studies is not major, for the current manuscript. The authors could submit to a journal that supports publication of work that is well-executed, and an important part of the larger picture, but that is not a major advance. Alternatively, they could continue to address the additional cellular machanisms that may contribute to regulation of processive motion in dividing cells.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    This manuscript reports motility characteristics and load-bearing properties of three human kinesin-6 family proteins that function during late telophase/cytokinesis of mitosis. The authors report single molecule and multiple motor motility assays, and vesicle dispersion assays for the three motors. Because the kinesin motors are important for normal division, their motility characteristics are of interest to workers in the mitosis field. However, data presentation in this manuscript could be greatly improved, along with interpretations of functional differences based on kinesin-6 motility properties.

    Major points are the following:

    1. Quantitation and presentation of the data throughout the manuscript should be improved.

    The criteria used for identifying fluorescent spots as single motors are not given. This is typically based on photobleaching experiments and fluorescence intensity measurements - the authors should show these data to validate that the motility reported is due to single motors.

    A table should be included that shows the single molecule motility parameters that were analyzed and compared for the three motors, rather than just the dwell times for the assays shown in Fig. 1. Other motility characteristics should include run lengths, binding rates, detachment rates, and velocity. The percentage of time that the single motors move directionally, diffuse, or remain stationary should also be given.
    The authors refer to imaging rates (1 frame/50ms, p. 5), but do not state the total time of the assays, making the statements uninterpretable, as it is not clear what would be expected without knowledge of the total assay time. The authors also state that a slower imaging rate (1 frame/2 sec) was used to detect slow processive motility, but the logic underlying this statement is not clear, as a longer assay time should reveal the slow processive movement irrespective of the imaging rate. These statements should be clarified.
    The authors give the data for the dwell times in single motor assays and velocities in multiple motor assays as the mean + SEM, but the SD rather than SEM should be reported for these assays, given that the data are for individual single motors or individual gliding microtubules. The authors state the number of replicate experiments for the assays, but they should also state the number of data points that were obtained for each replicate. Further, they should evaluate the significance of differences in their data by giving P values obtained using appropriate statistical tests and indicate whether the differences among the motors are significant.
    The percentages of processive events (p. 5) are most likely dependent on the amount of inactive or denatured protein in a given preparation, rather than a motility property of the motor protein - this could be determined by analysis of whether the percentages differ from preparation to preparation of each motor and whether the mean+SD of the preparations of a given motor differs from the other motors. The statements by the authors on p. 8 that "the majority of proteins do not undergo unidirectional processive motility as single molecules but rather diffuse along the surface of the microtubule for several seconds" and "It is presently unclear why only a subset of kinesin-6 molecules are capable of directional motility (Figure 1 ..." are not meaningful, as they do not take into account the percentages of the kinesin-6 proteins that are inactivated or denatured during protein preparation.
    Again, given that inactive motors are produced during preparation of the proteins, it is not clear what the frequency of processive motility events means. If the authors think that the frequency of processive motility events is informative and a characteristic of each motor, they should present controls showing frequencies of processive motility events for specific well characterized motors. For example, does a control of kinesin-1 show 100% or only 95% processive motility events?
    For the multiple motor gliding assays, velocities are shown in Fig. 2 without controls demonstrating the dependence of the velocities on motor concentration in the assays - the gliding assays require dilution experiments to show that the velocities are within the linear range of motor concentration and do not fall within the range of higher concentrations in which motor gliding velocity is inhibited or lower motor concentrations in which the density of motors on the surface is too low to support processive movement. These control experiments of motor concentration vs velocity for the gliding assays should be shown for each of the three motors that was assayed. The authors should state whether the gliding velocities that were determined correspond to the Vmax for each of the motors that was assayed.

    Again, the velocities given on p. 6 should include the SD and evaluation of the significance of the differences among the motors by obtaining P values.

    Proteins for motility assays: Western blots of the purified proteins should be shown as a supplemental figure.

    How are the motility characteristics of the three motors related to their spindle functions? This is the central point of the manuscript but is not clearly stated.

    1. Functional assays should be relevant to motor function.

    Given that the kinesin-6 motors under study are mitotic spindle motors that do not normally transport vesicles, it is not clear why the authors chose to show load dependence using peroxisome and Golgi dispersion assays, rather than assays of spindle function. The authors interpret peroxisomes and Golgi to differ in dispersion load, but this appears to be based on interpretations from assays of highly processive motors, kinesin-1 and myosin V, that function in vesicle trafficking, rather than quantitative data from appropriate controls showing that peroxisomes and Golgi can be dispersed by spindle motors that bear different loads. The problems inherent in the use of these assays for spindle motors are evidenced by the authors' observations on p. 6 that MKLP1- mNG-FRB and KIF20-mNG-FRB in midbodies could not be localized to peroxisomes by rapamycin. There are no data presented showing the dependence of dispersion on protein expression/presence in the cytoplasm, making the dispersion assays difficult to interpret.

    The kinesin-6 motor functional tests would be more relevant if they involved mitotic spindle assays, rather than peroxisome or Golgi dispersion assays. It is not clear how the loads involved in peroxisome or Golgi dispersion are related to kinesin motor function in the spindle. What are the implications of low- vs high-load motors in the spindle? How do the authors envision that motor loads in spindles relate to loads borne by vesicle transport motors?

    Minor points needed for clarity and reproducibility of the data:

    Methods

    Plasmids
    "MKLP1(1-711) lacks the insert present in KIF23 isoform 1" - the insert present in KIF23 isoform 1 but missing in MKLP1 (1-711) should be depicted/pointed out in Fig. S1 and information provided as to its predicted or actual structure.

    "KIF20B contained the protein sequence conflict E713K and natural variations N716I and H749L "- the sites of these changes should be indicated in Fig. S1 and information provided as to their effects on predicted or actual structure.
    Protein purification: "MKLP1(1-711)-3xFLAG-Avi was cloned by stitching four oligonucleotide primer sequences together into a digested MKLP1(1-711)-Avitag plasmid" - please explain what this means: what do the four oligonucleotide primer sequences correspond to? if they are the 3xFLAG-Avi tags, why were four sequences stitched together instead of three?
    The figures showing the kymographs should include labeled X and Y axes, rather than scale bars.

    The significance of the statement that "All motors displayed similar behaviors when tagged with Halo and Flag tags" is not clear, as the Halo and Flag tags were also C-terminal tags, like the 3xmCit tag.

    The figures (Fig. 3-5) that contain grey-scale cell depictions would be more readily interpretable by others if they were labeled with the authors' classification of the dispersion phenotype.

    Significance

    This manuscript reports motility characteristics and load-bearing properties of three human kinesin-6 family proteins that function during late telophase/cytokinesis of mitosis. The authors report single molecule and multiple motor motility assays, and vesicle dispersion assays for the three motors. Because the kinesin motors are important for normal division, their motility characteristics are of interest to workers in the mitosis field. However, data presentation in this manuscript could be greatly improved, along with interpretations of functional differences based on kinesin-6 motility properties.

    My expertise: motors, motor function in division, motility assays, microtubules

  5. Version published to 10.1242/bio.059533
  6. Version published to 10.1101/2022.07.13.499883 on bioRxiv