Microtubule Assembly from Single Flared Protofilaments-Forget the Cozy Corner?

Abstract

A paradigm shift for models of MT assembly is suggested by a recent cryo-electron microscopy study of microtubules (MTs). Previous assembly models have been based on the two-dimensional lattice of the MT wall, where incoming subunits can add with longitudinal and lateral bonds. The new study of McIntosh et al. concludes that the growing ends of MTs separate into flared single protofilaments. This means that incoming subunits must add onto the end of single protofilaments, forming only a longitudinal bond. How can growth of single-stranded protofilaments exhibit cooperative assembly with a critical concentration? An answer is suggested by FtsZ, the bacterial tubulin homolog, which assembles into single-stranded protofilaments. Cooperative assembly of FtsZ is thought to be based on conformational changes that switch the longitudinal bond from low to high affinity when the subunit is incorporated in a protofilament. This novel mechanism may also apply to tubulin assembly and could be the primary mechanism for assembly onto single flared protofilaments.

Figure 1

The classic cozy corner. Shaded subunits and arrows show the three ways subunits can add to the lattice: forming a single lateral bond with affinity Ka, a single longitudinal bond with affinity Kb, or both a lateral and longitudinal bond together with affinity Kc. With even a weak contribution from Ka, Kc can be orders of magnitude higher than Kb, as explained in detail in (3). The diagram is reprinted from (2) with permission.

Figure 2

A model of a MT showing a blunt tip with a cozy corner and a tapered tip where projecting sheets of PFs provide sites for adding a subunit with a single longitudinal bond (leading binding site) and variations on a cozy corner. Reprinted from (6) with permission. To see this figure in color, go online.

Figure 3

Models of flared PFs at the plus end of growing cytoplasmic MTs, reconstructed from cryoEM tomograms. The top row is from Chlamydomonas cells, the bottom row from PTK2 cells. Each green line is a single PF. Reprinted from (1) with permission. To see this figure in color, go online.

Figure 4

A diagram of the high- and low-affinity conformations. For monomeric subunits, the high-affinity conformation is highly disfavored, and the top and bottom surfaces form a poorly matched interface. In the high-affinity conformation, the top and bottom surfaces are rearranged so that they form a large and snugly fitting interface. The extra bond energy of this interface is sufficient to compensate for the free energy needed to switch to high affinity, so polymerization favors the switch to high affinity. The right-hand PF shows the process of elongation, in which a subunit is added in the low-affinity conformation and switches to high affinity. The high-affinity conformation involves internal rotations of the subdomains and movement of helix H7. These are not shown because the important switch is in the top and bottom interface surfaces. For simplicity, the diagram shows monomeric subunits, applicable to FtsZ. For dimeric tubulin, the conformational change would be transmitted across both α and β subunits, consistent with crystallography. To see this figure in color, go online.