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First published online 13 September 2005
doi: 10.1242/jcs.02550

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Direct involvement of the isotype-specific C-terminus of ß tubulin in ciliary beating

Julia Vent^1,*, Todd A. Wyatt², D. David Smith¹, Asok Banerjee³, Richard F. Ludueña³, Joseph H. Sisson² and Richard Hallworth^1,

¹ Department of Biomedical Sciences, Creighton University, Omaha, Nebraska 68178, USA
² Department of Internal Medicine, Section of Pulmonary, Critical Care and Sleep Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA
³ Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

View larger version (39K): [in a new window]   Fig. 1. (A) Effect of isotype-specific antibodies against ß tubulin on CBF. Final antibody concentrations 0.33 µM, error bars indicate 1 s.e.m. Filled black squares, control; open pink circle, anti ßI tubulin; open upward blue triangle, anti ßII tubulin; open downward navy triangle, anti ßIII tubulin; open red diamond, anti ßIV tubulin; open purple triangle, anti ßV tubulin. The antibodies depicted in shades of red blocked ciliary beating, the antibodies in blue had no effect on CBF. (B) Concentration dependence of anti ßI tubulin; error bars indicate 1 s.e.m. Antibody concentrations: blue triangle, 0.33 µM; green triangle, 0.033 µM; red circle, 3.3 nM. Similar concentration curves were obtained for anti ßIV tubulin and anti ßV tubulin. (C) Effect of non-isotype-specific, non-C-terminal antibodies against and ß tubulin (DM1A, red circle and TUB 2.1, green triangle), error bars indicate 1 s.e.m.; antibody concentrations 0.33 µM. (D) Effect of anti tubulin C-(red circle) and N-terminal (green triangle), non-isotype-specific antibodies, error bars indicate 1 s.e.m.; antibody concentrations 0.33 µM.

View larger version (41K): [in a new window]   Fig. 2. Comparison of the effect of axonemal motif (red triangles) and C-terminal tail peptides (green circles) on CBF and controls (dotted line, black solid squares); error bars indicate 1 s.e.m.; peptide concentrations: 0.33 µM. For sequences, see Table 1. (A) sequences derived from ßI tubulin (433EEDFGEE and 438EEAEEEA). (B) sequences derived from ßII tubulin (433QGEFEEE and 440EGEDEA). (C) sequences derived from ßIII tubulin (433GEMYEDD and 443SESQGPK). (D) sequences derived from ßIV tubulin (433EGEFEEE and 439EAEEEVA). (E) sequences derived from ßV tubulin (433EEAFEDE and 441EEEINE). (F) Concentration-dependent effect of the axonemal motif peptide (EGEFEEE), concentrations: 0.33 µM (blue squares), 0.033 µM (green circles) and 3.3 nM (red triangles).

View larger version (21K): [in a new window]   Fig. 3. (A) Alanine scan of the axonemal motif peptide: when the central F is replaced by an A, there is no more effect of the peptide on CBF, red circles, 433EGEFEEE, green triangles, 433EGEAEEE. Peptide concentrations: 0.33 µM. Error bars indicate 1 s.e.m. (B) The effect of tubulin axonemal motif-like peptides on CBF, red circles, 441EGEGEEE, green triangles, 441EDEDEGE. Peptide concentrations: 0.33 µM. Error bars indicate 1 s.e.m.

View larger version (20K): [in a new window]   Fig. 4. (A) Example of a labeling of isolated bovine tracheal cilia by the isotype-specific antibody against ßIV tubulin. Scale bar 10 µm. (B) Immunoblotting of isolated bovine tracheal cilia by antibodies against and ß tubulin.