Biochemistry of Metabolism: Cell Biology


Contents of this page:
a,b-Tubulin heterodimer & microtubule structure
Centriole & centrosome
Dynamic instability of microtubules
Microtubule associated proteins, drugs & toxins

Note: Page numbers for this topic refer to the textbook Molecular Biology of the Cell by Alberts et al. (A).

An a,b-tubulin heterodimer is the basic structural unit of microtubules. The heterodimer does not come apart, once formed. The a and b tubulins, which are each about 55 kDa MW, are homologous but not identical. Each has a nucleotide binding site. See Fig. 16.6 p. 915 of A.

A microtubule is a hollow cylinder about 24 nm in diameter. Along the microtubule axis, tubulin heterodimers are joined end-to-end to form protofilaments, with alternating a & b subunits. Staggered assembly of 13 protofilaments yields a helical arrangement of tubulin heterodimers in the cylinder wall.

A website depicts a 3-D reconstruction of the structure of an intact microtubule, based on cryo-electron microscopy and image processing (by the Visualization Group at Lawrence Berkeley National Laboratory, K. Downing's research group).

Electron microscopy of microtubules decorated with motor protein heads indicate a "3 start helix" in which each turn of the helix spans 3 tubulin monomers (e.g., a, b, a). This results in the microtubule wall having a "seam" where, instead of the predominant aa and bb lateral contacts, a subunits are laterally adjacent to b subunits. 

During in vitro microtubule assembly, tubulin heterodimers join end-to-end to form protofilaments. These associate laterally to form sheets, and eventually microtubules.

In vitro, heterodimers can add or dissociate at either end of a microtubule, but there is greater tendency for subunits to add at the plus end, where b-tubulin is exposed. As with actin filaments, microtubules can undergo treadmilling, with addition of tubulin heterodimers at the plus end and dissociation of tubulin heterodimers at the minus end.

GTP must be bound to both a and b subunits for a tubulin heterodimer to associate with other heterodimers to form a protofilament or microtubule. Subunit addition brings b-tubulin that was exposed at the plus end into contact with a-tubulin. This promotes hydrolysis of GTP bound to the now interior b-tubulin. Pi dissociates, but b-tubulin within a microtubule cannot exchange its bound GDP for GTP. The GTP on a-tubulin does not hydrolyze.

The minus end of a-tubulin may contribute an essential residue to the catalytic site of b-tubulin. Thus the minus end of an a subunit may serve as GAP (GTPase activating protein) for b-tubulin of the adjacent dimer in a protofilament. 

A homologous bacterial protein FtsZ is considered the ancestor of tubulin. FtsZ, which has a role in bacterial cytokinesis, also assembles into protofilaments, and the FtsZ protofilaments can associate to form sheets or tubules.

Protofilament structure has been determined at atomic resolution using cryo-EM (electron diffraction) analysis of 2-D crystals induced by treating tubulin with zinc ions in the presence of a derivative of the drug taxol. These "zinc sheets" consist of parallel arrays of protofilaments.

Each nucleotide in the tubulin protofilament is at an a-b interface. The inability of GTP to dissociate from the a-subunit is consistent with occlusion by a loop from the b subunit. A similar occlusion would account for the inability of b-tubulin within a protofilament to exchange bound GDP for GTP.

The nucleotide binds adjacent to a b-sheet (in magenta at right). The nucleotide binding site of tubulins is structurally similar to the nucleotide binding site of GTP-binding proteins of the Ras superfamily. Nucleotide binding sites of the motor proteins myosin and kinesin (to be discussed later) are also structurally similar to that of Ras. 

The nucleotide-binding domain of tubulins includes a highly conserved sequence GGGTG(T/S)G. This sequence, shown in black at right, is part of a loop and helix that extends from one of the b-strands (colored magenta) and passes near the nucleotide phosphates. 

Explore at right the structure of the a,b-tubulin heterodimer.

Also view an animation depicting assembly of microtubules.


of microtubule assembly 
In doublet and triplet microtubules the wall of one microtubule partly consists of the wall of an attached microtubule. 

The cartoon at right represents singlet, doublet, and triplet microtubules in cross section.

The A tubule of a doublet or triplet microtubule is a complete cylinder, made of 13 protofilaments. "Piggyback" B or C tubules are made of less than 13 protofilaments, usually 10. Micrographs in A p. 966, 968.

Centrioles are cylindrical structures, usually in pairs oriented at right angles to one another. The wall of each centriole cylinder is made of nine interconnected triplet microtubules, arranged as a pinwheel. The interior of each centriole appears empty, except for a "cartwheel" structure at one end. Electron micrographs show appendages that protrude from the outer surface at one end of a mature centriole, and fibrous structures connecting the two centriole cylinders. Diagrams in A p. 968 & 1031.

Centriolar microtubules are relatively stable. The a,b tubulin heterodimers present in centriolar triplet microtubules are modified by polyglutamylation. Additional tubulins, designated d, e, z & h, as well as other proteins, are either present in centrioles or required for their formation. There is some variability in composition among different organisms.

Basal bodies of cilia and flagella are also centrioles. See electron micrograph in an article by J. Beisson & M. Wright (requires subscription to Current Opinion in Cell Biology).

During centriole duplication prior to mitosis (G1 - S phase):
  • The 2 centriole cylinders separate.
  • A daughter centriole grows from a short disk-like structure at right angles to each parent centriole.
  • A line through each daughter centriole bisects the parent.

(Biogenesis of ciliary basal bodies, which is somewhat different, will not be discussed here.)

The centrosome, a mass of protein also called the microtubule organizing center (MTOC) or pericentriolar material, surrounds centrioles in animal cells. Following duplication, the pericentriolar material initially is associated only with each parent centriole cylinder. 

Proteins present in the pericentriolar material or on the surface of centrioles include centrin, pericentrin, ninein, cenexin, CEP110, CEP250 (C-Nap1), g-tubulin, and others. Labeling studies have shown such proteins to be located at one end and along lateral margins of the centriolar cylinder, forming a mass the shape of a tube. See Fig. 2 in article by Ou et al. (requires subscription to Cell Motility & Cytoskeleton).

g-Tubulin, which is homologous to a and b tubulins, nucleates microtubule assembly within the centrosome. Several (12-14) copies of g-tubulin associate in a complex with other proteins called "grips" (gamma ring proteins). This g-tubulin ring complex is seen by electron microscopy to have an open ring-like structure resembling a lock washer, capped on one side.

Microtubules nucleated by the g-tubulin ring complex appear capped at one end, assumed from other data to be the minus end. Polymerization at the minus end of these microtubules is inhibited. Grip proteins of the cap may be involved in mediating binding to the centrosome.

Phosphorylation of a conserved tyrosine residue of g-tubulin has been shown to regulate microtubule nucleation in yeast cells.

At a website maintained by the Agard Lab, see micrographs and diagrams showing the structure of the g-tubulin ring complex and the capping of microtubules by this complex. See also A. p. 930.

During cell division, the duplicated centrosome helps to organize the mitotic spindle. See also A p. 1037.

During interphase, the centrosome (MTOC) is usually located near the nucleus. Microtubules grow out from the MTOC, forming a hub and spoke array, even during interphase. See A p. 930-932.

With minus ends of most microtubules anchored in the centrosome, microtubules grow and shrink mainly through addition and loss of tubulin heterodimers at their plus ends.

A sub-population of microtubules with free minus ends exists in some cells. These may arise by breakage or cleavage of microtubules.

Dynamic instability: Microtubules may grow steadily, and then shrink rapidly by loss of tubulin dimers at the plus end. The rapid disassembly is referred to as catastrophe. (The graph at right is representative, but is not a depiction of actual data.) See also A p. 918-920.

In vitro, the tendency to grow or shrink may be a function of tubulin concentration. As microtubules grow, tubulin dimers are depleted. Below a critical tubulin concentration, rapid shrinkage at the plus end has been attributed to loss of a "GTP cap." Hydrolysis of GTP by b-tubulin, as polymerization brings it into contact with a-tubulin, takes time. A rapidly growing microtubule may accumulate a few layers of tubulin-GTP at the plus end. 

A GTP cap stabilizes the plus end of a microtubule. If the concentration of tubulin heterodimers is low, dissociation of tubulin-GTP may expose tubulin-GDP at the plus end, causing that end to become unstable. Rapid shrinkage ensues. 

Fraying or curving of protofilaments is observed at the ends of rapidly disassembling microtubules. This may be due to a change in conformation when b-subunits at the plus end have bound GDP instead of GTP. Tubulin heterodimers with GDP bound to the b subunit form ring shaped assemblies in vitro. Straight protofilaments form only when both tubulins have bound GTP. Diagrams & micrographs in A. p. 919.

Dynamic instability of microtubules in vivo is regulated by interaction with other proteins. For example, during prophase of mitosis, microtubules grow out from the centrosome. If the plus end of a microtubule makes contact with a chromosome, it becomes stabilized. Otherwise rapid disassembly at the plus end ensues, and the tubulin dimers are available for growth of another microtubule.

A website of the Borisy lab has movies depicting microtubule instability. See, e.g., movie #1 on centrosomal control of microtubule dynamics.

MAPs (microtubule-associated proteins) are a diverse class of proteins that bind to microtubules. See A p. 935-936. Binding of MAPs may be regulated by phosphorylation, which causes some MAPs to detach from microtubules.

+TIPS or plus-end tracking proteins, are proteins that associate with the plus ends of microtubules. Many +TIPs are motor proteins. Some mediate interaction with the actin cytoskeleton in the cell cortex, adjacent to the plasma membrane. Some +TIPS regulate microtubule dynamics and stability at the plus end.

+TIPS that stabilize or promote growth of microtubules include the following, for example:

Catastrophe-promoting proteins (catastrophins) bind to plus ends of microtubules and promote dissociation of tubulin dimers. They may activate GTP hydrolysis or induce a curved protofilament conformation.
  • For example, MCAK, a member of the kinesin family of proteins, promotes dissociation of tubulin dimers at the kinetochore, as kinetochore-linked microtubules shorten during anaphase of mitosis.
    In vitro
    , MCAK can cause microtubules to shrink at both ends.
    See a movie showing shortening of fluorescent-labeled microtubules after addition of MCAK (video supplement #1 from article by Helenius et al.; requires a subscription to Nature).

Other proteins that promote microtubule disassembly include the following:

Some MAPs cross-link adjacent microtubules or link microtubules to membranes or to intermediate filaments. The length of intervening segments between microtubule-binding domains in particular MAPs may determine the spacing of microtubules in parallel arrays. Some examples:

Some toxins and drugs (all of which inhibit mitosis) affect polymerization or depolymerization of tubulin.

Copyright 1999-2006 by Joyce J. Diwan. All rights reserved.

Additional Material on Microtubules:
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