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. 2020 Apr 7;10(4):563.
doi: 10.3390/biom10040563.

Bioenergetics of the Dictyostelium Kinesin-8 Motor Isoform

Michael P Koonce  1 , Irina Tikhonenko  1
Affiliations

Bioenergetics of the Dictyostelium Kinesin-8 Motor Isoform

Michael P Koonce et al. Biomolecules. .

Abstract

The functional organization of microtubules in eukaryotic cells requires a combination of their inherent dynamic properties, interactions with motor machineries, and interactions with accessory proteins to affect growth, shrinkage, stability, and architecture. In most organisms, the Kinesin-8 family of motors play an integral role in these organizations, well known for their mitotic activities in microtubule (MT) length control and kinetochore interactions. In Dictyostelium discoideum, the function of Kinesin-8 remains elusive. We present here some biochemical properties and localization data that indicate that this motor (DdKif10) shares some motility properties with other Kinesin-8s but also illustrates differences in microtubule localization and depolymerase action that highlight functional diversity.

Keywords: Dictyostelium; cell motility; kinesin; microtubules.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
A schematic of the DdKif10 kinesin motor constructs. The position of the motor domain is indicated in light blue, and the dark blue boxes mark prominent coiled-coil motifs in the coding sequence. The top row represents the full-length, 1238aa construct. A detailed representation of the domain organization of DdKif10 can be found in reference [17]. Despite multiple attempts, we were only able to detect the expression of the middle-length construct in D. discoideum cells.
Figure 2
Figure 2
Motor mechanics. Panel (A) shows microtubule (MT) pellet and ATP extract lanes following the binding and release of the DdKif10725 polypeptide (Coomassie-stained gel, arrowhead denotes position of the Kif10 fusion protein). Panel (B) shows a histogram of MT gliding activity induced by the DdKif10725 fragment, with an average rate of 0.17 μm/s. Panel (C) shows the MT affinity of the motor fragment, plotting % pelleted vs. MT concentration. Panel (D) illustrates motor catalytic activity, plotting the ATPase rate vs. MT concentration. The data in both panels (C) and (D) represent averages from three independent measurements, and error bars indicate standard deviations.
Figure 3
Figure 3
The cellular distribution of DdKif10725. The left column (green) shows the maximum intensity projections of the DdKif10 distribution in two interphase (A,D) and two mitotic cells (G,J). Antibody-labelled MTs (red) and Hoechst-stained nuclei (blue) are shown in the middle column (panels B,E,H,K), and the two frames are merged in the right column (panels C,F,I,L). Scale bar = 5 μm.
Figure 4
Figure 4
Complementary hairpin analysis. The top row shows MT arrangements (green) in three DdKif8 null cells (two interphase, one mitotic) in the presence of a constitutively expressed DdKif10 hairpin construct. The bottom three rows show the same arrangement, but with the null/hairpin constructs reversed. In all cases, the MT arrays look no different than those seen in wild-type cells. Nuclei and chromosomes are shown in blue. Scale bar = 5 μm.
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