There are two objectives of this proposal: (a) To study the macromolecular structure and assembly of ciliary and flagellar microtubules; and (b) to investigate the regulatory mechanisms that control motility. Microtubules are involved in vital cell processes including mitosis, cytoskeletal-changes, secretion, particle transport in nerve cells and ciliary and flagellar motility in the respiratory and reproductive tracts. Microtubule function is severely affected by malignancies and environmental toxins. Our understanding of microtubule function will benefit from studies on flagella, the best characterized of the microtubule systems for it is with flagella that the functional roles of microtubule proteins can best be determined. The research will proceed with the following aims: (1) Microtubules will be isolated without loss of their native structure and the arrangement of protein subunits ("tubulin") forming the microtubule wall will be determined by optical diffraction and computer analysis of electron microscope images and by X-ray diffraction. (2) Flagellar doublet microtubules can be fractionated into resistant ribbons of 3-4 protofilaments containing 5 "ribbon proteins" plus tubulin. These proteins will be characterized using biochemical procedures and tested for biological activity (e.g., enzymatic-, calcium-binding-, ability to promote microtubule assembly. (3) Evidence indicates that flagellar microtubules twist in the presence of calcium ions and that such twisting may be important in the regulation of flagellar motility. The molecular basis of this twisting will be investigated using electron microscopy (EM) and darkfield light microscopy to visualized individual microtubules. This twisting behavior will be correlated with the presence and function of ribbon proteins and other microtubule-associated proteins in fractionated (e.g., ribbons) and reconstituted models. (4) The molecular mechanisms that regulate microtubule motility will be studied further in the axostyle of certain protozoa. Polarization microscopy, high resolution EM techniques and biochemical procedures will be used to analyze the macromolecular structure of this contractile organelle and to establish the identity and locus of the force-generating and control proteins.
NIH ExPorter
NIH ExPorter