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Growth and shortening of microtubules in the course of their polymerization and depolymerization have previously been observed to occur at variable rates. To gain insight into the meaning of this prominent variability, we studied the way in which its magnitude depends on the growth rate of experimentally observed and computer-simulated microtubules. The dynamic properties of plus-ended microtubules nucleated by pieces of Chlamydomonas flagellar axonemes were observed in real time by video-enhanced differential interference contrast light microscopy at differing tubulin concentrations. By means of a Monte Carlo algorithm, populations of microtubules were simulated that had similar growth and dynamic properties to the experimentally observed microtubules. By comparison of the experimentally observed and computer-simulated populations of microtubules, we found that 1) individual microtubules displayed an intrinsic variability that did not change as the rate of growth for a population increased, and 2) the variability was approximately fivefold greater than predicted by a simple model of subunit addition and loss. The model used to simulate microtubule growth has no provision for incorporation of lattice defects of any type, nor sophisticated geometry of the growing end. Thus, these as well as uncontrolled experimental variables were eliminated as causes for the prominent variability.

Dimerization of neural-cadherins occurs via formation of calcium dependent strand-crossover structures, which leads to cell-cell adhesion in multicellular organisms. Strand-crossover dimer forms exclusively between the first N-terminal extracellular modules (EC1) of adhesive partners via swapping of their βA-sheets and docking of tryptophan-2 in the hydrophobic pocket. Prolines recurrently occur in proteins that form strand-crossover dimer and are believed to be the source of the strain in the monomer. N-cadherin has two proline residues in the βA-sheet. Our studies address two interesting questions; why is the dimerization in neural-cadherin calcium dependent, and do all three calcium-binding sites at the EC1-EC2 interface play a role in dimerization. To investigate these questions we mutated three important calcium-binding amino acids, D134, D136, and D103, and three amino acids in the βA-sheet W2, P5 and P6, in NCAD12, a construct containing EC1 and EC2. Spectroscopic and chromatographic experiments showed that the calcium-binding sites are occupied sequentially in the order of site3, then site2 and site1, and cooperativity between site2 and site1 is essential for dimerization. Studies on the P5A, P6A double mutant showed that the proline mutations increased the dimerization affinity by ∼ 20 fold and relieved the requirement for calcium in dimerization. Studies on W2A showed that the binding of calcium creates strain in the hydrophobic interaction between the hydrophobic pocket and W2 in the closed monomer, which is relieved upon formation of the strand-crossover dimer. In summary, our findings confirm that the hydrophobic interaction involving W2 is the source of calcium-dependent dimerization and the proline residues at βA-sheet act as a switch to control the dynamics of the equilibrium between monomer and dimer which is crucial for the plasticity of synapses.