Abstract

Dr. Linck's long term objective is to elucidate the function of microtubules. This proposal concerns a new class of microtubule-associated proteins, called tektins. Three tektins (A-53-kd, B-51-kd and C-47-kd) form filaments in the walls of ciliary and flagellar microtubules. Tektin filaments are associated with a highly stable segment of the microtubule wall, the protofilament (pf)-ribbon. Pf-ribbons are composed of tubulin, tektins, and other polypeptides. This proposal combines the techniques of biochemistry, structural biology, and molecular genetics to elucidate the function of tektins in microtubules from sea urchin sperm and Chlamydomonas reinhardtii.
Aim 1 is to produce a molecular model of pf-ribbons. Tektin polymers will be reconstituted from purified subunits and analyzed biochemically and by EM to determine whether tektins A, B, and C form homopolymers or heteropolymers. Chemical crosslinking procedures will be used to map associations with neighboring polypeptides. EM, cryo-EM and STEM will be used to study the molecular arrangements of the proteins forming pf- ribbons.
Aim 2 is to identify and characterize tektin genes and proteins in Chlamydomonas. Cloned genomic and/or cDNA sequences will be determined and analyzed for their gene structure, predicted protein structure, and potential tubulin binding domains.Tektin proteins will be purified and characterized in order to confirm the DNA sequences and to construct oligonucleotides for PCR screening. Gene specific probes will be used to determine the number of tektin genes and their location on the genetic map. If tektin genes map to known loci for flagellar defects, the mutant proteins will be characterized to identify the defects.
Aim 3 is to investigate the function of tektins in Chlamydomonas using molecular/genetic and cell biological approaches. The in vivo function(s) of tektins will be studied by regulating the expression of native or mutant tektins in wild-type and mutant backgrounds. The assembly of epitope-tagged tektins will be followed in regenerating flagella. Finally, anti-tektin antibodies will be used to precipitate possible oligomeric tektin-tubulin intermediates and/or phosphorylated tektins.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM035648-10
Application #
2177980
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1985-02-01
Project End
1998-01-31
Budget Start
1994-02-01
Budget End
1995-01-31
Support Year
10
Fiscal Year
1994
Total Cost
Indirect Cost

  Institution

Name
University of Minnesota Twin Cities
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455

  Publications

Linck, Richard W; Chemes, Hector; Albertini, David F (2016) The axoneme: the propulsive engine of spermatozoa and cilia and associated ciliopathies leading to infertility. J Assist Reprod Genet 33:141-56
Setter, Peter W; Malvey-Dorn, Erika; Steffen, Walter et al. (2006) Tektin interactions and a model for molecular functions. Exp Cell Res 312:2880-96
Ikeda, Kazuho; Brown, Jennifer A; Yagi, Toshiki et al. (2003) Rib72, a conserved protein associated with the ribbon compartment of flagellar A-microtubules and potentially involved in the linkage between outer doublet microtubules. J Biol Chem 278:7725-34
Linck, Richard W; Norrander, Jan M (2003) Protofilament ribbon compartments of ciliary and flagellar microtubules. Protist 154:299-311
Norrander, J M; deCathelineau, A M; Brown, J A et al. (2000) The Rib43a protein is associated with forming the specialized protofilament ribbons of flagellar microtubules in Chlamydomonas. Mol Biol Cell 11:201-15
Hinchcliffe, E H; Linck, R W (1998) Two proteins isolated from sea urchin sperm flagella: structural components common to the stable microtubules of axonemes and centrioles. J Cell Sci 111 ( Pt 5):585-95
Norrander, J; Larsson, M; Stahl, S et al. (1998) Expression of ciliary tektins in brain and sensory development. J Neurosci 18:8912-8
Norrander, J M; Perrone, C A; Amos, L A et al. (1996) Structural comparison of tektins and evidence for their determination of complex spacings in flagellar microtubules. J Mol Biol 257:385-97
Norrander, J M; Linck, R W; Stephens, R E (1995) Transcriptional control of tektin A mRNA correlates with cilia development and length determination during sea urchin embryogenesis. Development 121:1615-23
Nojima, D; Linck, R W; Egelman, E H (1995) At least one of the protofilaments in flagellar microtubules is not composed of tubulin. Curr Biol 5:158-67

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