Polarized cell growth, organelle motility, and cytoskeletal organization in conifer pollen tube tips are regulated by KCBP, the calmodulin-binding kinesin
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Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin 14 family, is a minus end directed C-terminal motor unique to plants and green algae. Its motor activity is negatively regulated by calcium/calmodulin binding, and its tail region contains a secondary microtubule-binding site. It has been identified but not functionally characterized in the conifer Picea abies. Conifer pollen tubes exhibit polarized growth as organelles move into the tip in an unusual fountain pattern directed by microfilaments but uniquely organized by microtubules. We demonstrate here that PaKCBP and calmodulin regulate elongation and motility. PaKCBP is a 140 kDa protein immunolocalized to the elongating tip, coincident with microtubules. This localization is lost when microtubules are disrupted with oryzalin, which also reorganizes microfilaments into bundles. Colocalization of PaKCBP along microtubules is enhanced when microfilaments are disrupted with latrunculin B, which also disrupts the fine network of microtubules throughout the tip while preserving thicker microtubule bundles. Calmodulin inhibition by W-12 perfusion reversibly slows pollen tube elongation, alters organelle motility, promotes microfilament bundling, and microtubule bundling coincident with increased PaKCBP localization. The constitutive activation of PaKCBP by microinjection of an antibody that displaces calcium/calmodulin and activates microtubule bundling repositions vacuoles in the tip before rapidly stopping organelle streaming and pollen tube elongation. We propose that PaKCBP is one of the target proteins in conifer pollen modulated by calmodulin inhibition leading to microtubule bundling, which alters microtubule and microfilament organization, repositions vacuoles and slows organelle motility and pollen tube elongation.
KeywordsCalmodulin Cytoskeleton KCBP Kinesin 14 Norway spruce Picea abies Pollen tube W-12
This work was supported by the College of Charleston through grants to M.D.L. from the Department of Biology, the Office of Research and Creative Activities, and the Faculty Research and Development Committee. E.Y.M. conducted part of this research as an undergraduate. We thank Robyn L. Overall at the University of Sydney for graciously providing bench space and lab support for M.D.L. Videos accompanying Figs. 1 and 4 are available at our lab website (http://lazzarom.people.cofc.edu) and as online resources at the journal website.
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