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BMC Res Notes
2009 Jun 15;2:106. doi: 10.1186/1756-0500-2-106.
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Localization and function of Kinesin-5-like proteins during assembly and maintenance of mitotic spindles in Silvetia compressa.
Peters NT
,
Miller AC
,
Kropf DL
.
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BACKGROUND: Kinesin-5 (Eg-5) motor proteins are essential for maintenance of spindle bipolarity in animals. The roles of Kinesin-5 proteins in other systems, such as Arabidopsis, Dictyostelium, and sea urchin are more varied. We are studying Kinesin-5-like proteins during early development in the brown alga Silvetia compressa. Previously, this motor was shown to be needed to assemble a bipolar spindle, similar to animals. This report builds on those findings by investigating the localization of the motor and probing its function in spindle maintenance.
FINDINGS: Anti-Eg5 antibodies were used to investigate localization of Kinesin-5-like proteins in brown algal zygotes. In interphase zygotes, localization was predominantly within the nucleus. As zygotes entered mitosis, these motor proteins strongly associated with spindle poles and, to a lesser degree, with the polar microtubule arrays and the spindle midzone. In order to address whether Kinesin-5-like proteins are required to maintain spindle bipolarity, we applied monastrol to synchronized zygotes containing bipolar spindles. Monastrol is a cell-permeable chemical inhibitor of the Kinesin-5 class of molecular motors. We found that inhibition of motor function in pre-formed spindles induced the formation of multipolar spindles and short bipolar spindles.
CONCLUSION: Based upon these localization and inhibitor studies, we conclude that Kinesin-5-like motors in brown algae are more similar to the motors of animals than those of plants or protists. However, Kinesin-5-like proteins in S. compressa serve novel roles in spindle formation and maintenance not observed in animals.
Figure 1. Localization of Kinesin-5-like proteins (A-H) and morphology of short bipolar and multipolar spindles (I, J). Microtubules are in green in all images. Kinesin-5-like proteins are in red in A-C, E, F, H. Condensed chromatin is in blue in I and J. (A) 17-h old zygote in interphase. (B) Thallus cell of a 2-day old embryo entering prophase. (C-H) Two metaphase spindles are shown; anti-Kinesin-5 signal (C, F), microtubules (D, G), and merged images (E, H). In F and H, anti-Kinesin-5 signal was collected with increased amplifier gain and adjusted for contrast and brightness to enhance visualization of the weak signal; microtubule signal was not enhanced. Arrows indicate Kinesin-5-like motors colocalizing with microtubules in B and E, at the spindles poles and midzone in C and F, respectively. (I, J) Effect of monastrol treatment on metaphase spindles. Condensed chromatin is associated with short bipolar spindles in I and multipolar spindles in J following treatment with monastrol. Secondary antibodies commonly stick to zygotes' extracellular adhesive and is seen in A. Scale bars equal 10 μm.
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