The Arabidopsis thaliana Kinesin-5 AtKRP125b Is a Processive, Microtubule-Sliding Motor Protein with Putative Plant-Specific Functions.

Tobias Strauß,Wilhelm J. Walter,Saskia Schattner,Stefan Hoth

doi:10.3390/ijms222111361

Tobias Strauß, Wilhelm J. Walter + Show 2 more

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https://doi.org/10.3390/ijms222111361

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Abstract

The formation and maintenance of the mitotic spindle during cell division requires several microtubule-interacting motor proteins. Members of the kinesin-5 family play an essential role in the bipolar organization of the spindle. These highly conserved, homotetrameric proteins cross-link anti-parallel microtubules and slide them apart to elongate the spindle during the equal separation of chromosomes. Whereas vertebrate kinesin-5 proteins are well studied, knowledge about the biochemical properties and the function of plant kinesin-5 proteins is still limited. Here, we characterized the properties of AtKRP125b, one of four kinesin-5 proteins in Arabidopsis thaliana. In in vitro motility assays, AtKRP125b displayed the archetypal characteristics of a kinesin-5 protein, a low velocity of about 20 nm·s−1, and a plus end-directed, processive movement. Moreover, AtKRP125b was able to cross-link microtubules and to slide them apart, as required for developing and maintaining the mitotic spindle. In line with such a function, GFP-AtKRP125b fusion proteins were predominantly detected in the nucleus when expressed in Arabidopsis thaliana leaf protoplasts or Nicotiana benthamiana epidermis cells and analyzed by confocal microscopy. However, we also detected GFP signals in the cytoplasm, suggesting additional functions. By generating and analyzing AtKRP125b promoter-reporter lines, we showed that the AtKRP125b promoter was active in the vascular tissue of roots, lateral roots, cotyledons, and true leaves. Remarkably, we could not detect promoter activity in meristematic tissues. Taken together, our biochemical data support a role of AtKRP125b in mitosis, but it may also have additional functions outside the nucleus and during interphase.

Highlights

Transport processes and restructuring are an essential part of the cycle of a living cell and are largely mediated by motor proteins in almost all eukaryotic organisms
We examined the AtKRP125b promoter activity in transgenic promoter-GUS Arabidopsis lines and the intracellular localization of GFP-AtKRP125b after expression in transformed Arabidopsis and Nicotiana benthamiana cells
In an ATP-rich environment, fluorescently labelled microtubules were able to bind to the motor domains of AtKRP125b and were transported by them along the glass surface (Figure 1a–c, Supplementary Movie S1)

Summary

Introduction

Transport processes and restructuring are an essential part of the cycle of a living cell and are largely mediated by motor proteins in almost all eukaryotic organisms. Kinesins move along microtubules and are, often involved in processes such as organelle transport, mitotic spindle assembly and maintenance, and chromosome movement. Microtubules serve as a transport structure for the navigation of kinesins. The dynamics of microtubules themselves can be influenced by some kinesins by either stabilizing or depolymerizing them in a controlled manner [1,2]. The highly conserved motor domain forms the catalytic center of these proteins and is capable of hydrolyzing. The ATP-turnover allows a precisely timed binding and unbinding of the motor heads to the microtubule resulting in force transmission [3,4]

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