[Show abstract][Hide abstract] ABSTRACT: gamma-Tubulin is assumed to participate in microtubule nucleation in acentrosomal plant cells, but the underlying molecular mechanisms are still unknown. Here, we show that gamma-tubulin is present in protein complexes of various sizes and different subcellular locations in Arabidopsis and fava bean. Immunoprecipitation experiments revealed an association of gamma-tubulin with alphabeta-tubulin dimers. gamma-Tubulin cosedimented with microtubules polymerized in vitro and localized along their whole length. Large gamma-tubulin complexes resistant to salt treatment were found to be associated with a high-speed microsomal fraction. Blue native electrophoresis of detergent-solubilized microsomes showed that the molecular mass of the complexes was >1 MD. Large gamma-tubulin complexes were active in microtubule nucleation, but nucleation activity was not observed for the smaller complexes. Punctate gamma-tubulin staining was associated with microtubule arrays, accumulated with short kinetochore microtubules interacting in polar regions with membranes, and localized in the vicinity of nuclei and in the area of cell plate formation. Our results indicate that the association of gamma-tubulin complexes with dynamic membranes might ensure the flexibility of noncentrosomal microtubule nucleation. Moreover, the presence of other molecular forms of gamma-tubulin suggests additional roles for this protein species in microtubule organization.
[Show abstract][Hide abstract] ABSTRACT: A-type cyclin-dependent kinases (CDKs), also known as cdc2, are central to the orderly progression of the cell cycle. We made a functional Green Fluorescent Protein (GFP) fusion with CDK-A (Cdc2-GFP) and followed its subcellular localization during the cell cycle in tobacco cells. During interphase, the Cdc2-GFP fusion protein was found in both the cytoplasm and the nucleus, where it was highly resistant to extraction. In premitotic cells, a bright and narrow equatorial band appeared on the cell surface, resembling the late preprophase band, which disintegrated within 10 min as followed by time-lapse images. Cdc2-GFP was not found on prophase spindles but left the chromatin soon after this stage and associated progressively with the metaphase spindle in a microtubule-dependent manner. Arresting cells in mitosis through the stabilization of microtubules by taxol further enhanced the spindle-localized pool of Cdc2-GFP. Toward the end of mitosis, Cdc2-GFP was found at the midzone of the anaphase spindle and phragmoplast; eventually, it became focused at the midline of these microtubule structures. In detergent-extracted cells, the Cdc2-GFP remained associated with mitotic structures. Retention on spindles was prevented by pretreatment with the CDK-specific inhibitor roscovitine and was enhanced by the protein phosphatase inhibitor okadaic acid. Furthermore, we demonstrate that both the endogenous CDK-A and Cdc2-GFP were cosedimented with taxol-stabilized plant microtubules from cell extracts and that Cdc2 activity was detected together with a fraction of polymerized tubulin. These data provide evidence that the A-type CDKs associate physically with mitotic structures in a microtubule-dependent manner and may be involved in regulating the behavior of specific microtubule arrays throughout mitosis.