ABSTRACT
Microtubule doublets (MTDs), consisting of an incomplete B-microtubule at the surface of a complete A-microtubule, provide a structural scaffold mediating intraflagellar transport and ciliary beating. Despite the fundamental role of MTDs, the molecular mechanism governing their formation is unknown. We used a cell-free assay to demonstrate a crucial inhibitory role of the carboxyl-terminal (C-terminal) tail of tubulin in MTD assembly. Removal of the C-terminal tail of an assembled A-microtubule allowed for the nucleation of a B-microtubule on its surface. C-terminal tails of only one A-microtubule protofilament inhibited this side-to-surface tubulin interaction, which would be overcome in vivo with binding protein partners. The dynamics of B-microtubule nucleation and its distinctive isotropic elongation was elucidated by using live imaging. Thus, inherent interaction properties of tubulin provide a structural basis driving flagellar MTD assembly.
Subject(s)
Cilia/ultrastructure , Microtubules/ultrastructure , Tubulin/chemistry , Animals , Cattle , Cilia/chemistry , Computer Simulation , Cryoelectron Microscopy , Fluorescent Antibody Technique , Microtubules/chemistry , Models, Molecular , Protein Binding , Subtilisin , Swine , Tetrahymena thermophilaABSTRACT
How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically â¼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS-6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.
Subject(s)
Algal Proteins/metabolism , Cell Cycle Proteins/metabolism , Centrioles/metabolism , Chlamydomonas reinhardtii/metabolism , Organelles/metabolism , Algal Proteins/ultrastructure , Cell Cycle Proteins/ultrastructure , Centrioles/ultrastructure , Chlamydomonas reinhardtii/ultrastructure , Cryoelectron Microscopy/methods , Electron Microscope Tomography/methods , Models, Biological , Organelles/ultrastructureABSTRACT
FT infrared spectra of BrCN have been recorded in the region of the nu(2) band near 340 cm(-1), the nu(1) band near 580 cm(-1), and the 2nu(2) band near 690 cm(-1) with a resolution between 2.9 and 4.7 x 10(-3) cm(-1). The vibrational levels (01(1)0), (10(0)0), (02(0)0), (02(2)0), (11(1)0), and (20(0)0) have been analyzed employing cold bands, hot bands, and new millimeter-wave transitions. Band-by-band polynomial analyses and a combined fit of all data relevant to the 2v(1) + v(2) = 2 polyad levels have been performed. The latter fit considered l-resonance interactions between the (02(0)0), e and (02(2)0), e levels and Fermi resonance between the two Sigma states (10(0)0) and (02(0)0). Altogether about 1000 pieces of data up to J = 100 were fitted for each of the two isotopic species with rms of the residuals of 2-8 x 10(-4) cm(-1) for the infrared and 10-120 kHz for the pure rotational data. Copyright 2000 Academic Press.