We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1% SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 Mr. The 20,000 Mr protein band represents ~63% of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 Mr flagellar root protein into two components of nearly identical Mr, but of differing isoelectric points (i.e., pl's of 4.9 and 4.8), which we have designated 20,O00-Mr-c~ and 20,000-Mr-/3, respectively. Densitometric scans of twodimensional gels of cell extracts indicate that the 20,000-Mr-o~ and -/~ polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000-Mr-/3 component is phosphorylated and the 20,000-Mr-~ component contains no detectable label. These results suggest that the 20,000-Mr-~ component is converted to the more acidic 20,000-Mr-/3 form by phosphorylation. Both the 20,O00-Mr-a and -B flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-Mr protein binds to both the 20,000-Mr-~ and 20,000-Mr-/3 forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-lgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 Mr calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.Motility in eucaryotic cells in general appears to operate under calcium control through the regulatory action of a large family of calcium-modulated proteins (18,19,26). Calcium-based regulation of motility is best understood for troponin C and calmodulin in actomyosin contraction (sliding) in skeletal and smooth muscle, and in various nonmuscle actin-based cell movements (11,13,17,47,50). Evidence suggesting that calcium and calmodulin are involved in ciliary microtubulebased movement, i.e., sliding, (15,22,30) and in regulation of the mitotic apparatus (48, 49) is acc...

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Although trastuzumab (Herceptin) is an important advance in the treatment of breast cancer, a significant proportion of patients do not respond to trastuzumab either alone or in combination with chemotherapy. In this study, we observe that epidermal growth factor receptor (EGFR) and HER3 expression is substantially increased after long-term trastuzumab exposure of HER2-positive breast carcinoma-derived cell lines that show primary resistance to trastuzumab. Furthermore, long-term trastuzumab exposure of trastuzumab-resistant cell lines induces de novo sensitivity to the EGFR-targeted agents gefitinib or cetuximab in two of three cell lines accompanied by increased EGFR expression. Together, these results indicate that primary trastuzumab resistance is not synonymous with lack of responsiveness to trastuzumab and, importantly, suggest that trastuzumab priming may sensitize trastuzumab-resistant tumors to other HER family-directed therapeutics.

Abstract. Monoclonal and polyclonal antibodies raised against algal centrin, a protein of algal striated flagellar roots, were used to characterize the occurrence and distribution of this protein in interphase and Cell Biol. 105:1799Biol. 105: -1805. Immunofluorescence studies show dramatic changes in distribution of the centrin-based system during mitosis that include a transient contraction at preprophase; division, separation, and re-extension during prophase; and a second transient contraction at the metaphase/anaphase boundary. These observations suggest a fundamental role for centrin in motile events during mitosis.