Raymond E. Stephens, Ph.D.
Professor of Physiology and Biophysics, Emeritus
B.S. (1962), Geneva College
M.S.P.H. (1963), University of Pittsburgh Graduate School of Public Health
Ph.D. (1965), Dartmouth Medical School
Dynamics of stable microtubule assembly and turnover
The macromolecular assemblage of structural and enzymatic proteins in fully assembled, fully functional cilia can undergo rapid turnover at an incorporation rate approaching that of total organelle regeneration. Paradoxically, this is true of the tubulin within the stable, fixed-length outer doublet microtubules of the 9+2 axoneme, even when tubulin synthesis and normal polymerization are blocked. Whereas microtubule-associated proteins can exchange by simple displacement from the tubule surface, tubulin itself must either 1) incorporate at microtubule tips and leave near the base since length is constant or 2) exchange at the junctions or seams between and within doublet microtubules where tubulin dimer lattice mismatches exist. Furthermore, precursor tubulin is associated with the ciliary membrane compartment, possibly complexed with lipids, while most other 9+2 building blocks are conveyed in direct association with the axoneme by intraflagellar transport (IFT).
It is well established that mature cilia contain abundant chaperones and that microtubules can accommodate a variable number of protofilaments along their lengths, accompanied by consequent lattice defects. Based on this, we have proposed two closely related hypotheses for tubulin exchange: 1) Chaperones may promote tubulin incorporation at the distal tip and concurrent removal at or toward the base; 2) Lattice defects may permit direct incorporation and passive propagation of tubulin, “treadmilling” it from the tip of the cilium, where incorporation is known to occur, toward more proximal regions, where the steric excess of tubulin must exit. These two concepts are not mutually exclusive and may be cooperative (click to see animated PowerPoint).
A further curious observation is that tubulin incorporates first in association with the highly stable tektin filament that serves as a “molecular ruler” within the A-tubule of the doublet, specifying the axial periodicity of many of its associated components. Present in twice the amount per unit length as in doublet microtubules, tektins likely perform a similar function within the triplet microtubules of basal bodies. The tektin filament is a coiled-coil co-polymer of equimolar tektins A, B and C that, in turn, associates 1:1 with tightly bound tubulin. It may be located within or at the discontinuity or seam required by the A-tubule dimer lattice. In spite of the filament’s relative chemical stability and critical, integral structure, a subset of tektins within it can exchange as well.
1) seeking direct evidence for the postulated surface lattice incorporation-dislocation-propagation mechanism for tubulin turnover within stable microtubules.
2) investigating how co-polymeric tektin filaments are structurally integrated into axonemal doublet and basal body triplet microtubules (collaborator: Dr. Richard Linck, University of Minnesota).
3) determining the kinetics and mechanism of tektin exchange within these unusually stable, copolymeric filaments.
4) analyzing the association of 9+2 precursor tubulin with membrane lipids and integral membrane proteins during turnover and regeneration of cilia.
Stephens, R.E. (2008). Ciliogenesis, ciliary function, and selective isolation. ACS Chemical Biology 3: 84-86.
Linck, R.W., and Stephens, R.E. (2007). Functional protofilament numbering of ciliary, flagella, and centriolar microtubules. Cell Motil. Cytoskel. 64: 489-495.
Setter, P.W., Malvey-Dorn, E., Steffen, W., Stephens, R.E., and Linck, R.W. (2006). Tektin interactions and a model for molecular functions. Exp. Cell Res. 312: 2880-2896.
Stephens, R.E. (2001). Ciliary protein turnover continues in the presence of inhibitors of Golgi function: evidence for membrane protein pools and unconventional intracellular membrane dynamics. J. Exp. Zool. 289: 335-349.
Stephens, R.E. (2000). Preferential incorporation of tubulin into the junctional region of ciliary outer doublet microtubules: a model for treadmilling by lattice dislocation. Cell Motil. Cytolskel. 47: 130-140.
Stephens, R.E. (1999). Turnover of tubulin in ciliary outer doublet microtubules. Cell Struct. Funct. 24: 413-418.
Stephens, R.E., and Lemieux, N.A. (1999). Molecular chaperones in cilia and flagella: implications for protein turnover. Cell Motil. Cytoskel. 44: 274-283.
Stephens, R.E., and Lemieux, N.A. (1998). Tektins as structural determinants in basal bodies. Cell Motil. Cytoskel. 40: 379-393.
Stephens, R.E. (1997). Synthesis and turnover of embryonic sea urchin ciliary proteins during selective inhibition of tubulin synthesis and assembly. Molec. Biol. Cell 8: 2187-2198.
Stephens, R.E. (1996). Selective incorporation of architectural proteins into terminally-differentiated molluscan gill cilia. J. Exp. Zool. 274: 300-309.
Stephens, R.E. (1995). Ciliogenesis in sea urchin embryos – a subroutine in the program of development. BioEssays 17: 331-340.
Norrander, J.M., Linck, R.W, and Stephens, R.E. (1995). Transcriptional control of tektin A mRNA correlates with cilia development and length determination during sea urchin embryogenesis. Development 121: 1615-1623.
Stephens, R.E. (1994). Tubulin and tektin in sea urchin embryonic cilia: pathways of protein incorporation during turnover and regeneration. J. Cell Sci. 107: 683-692.
Department of Physiology and Biophysics L-715
Boston University School of Medicine
72 East Concord Street
Boston MA 02118-2526
Fax: (617) 638-4273