Polymerization kinetics of tubulin from mung seedlings modeled as a competition between nucleation and GTP‐hydrolysis rates

Abstract

Microtubules (MTs) form physiologically important cytoskeletal structures that are assembled by tubulin polymerization in nucleation‐ and guanosine triphosphate (GTP)‐dependent manner. GTP hydrolysis competes with the addition of monomers, to determine the GTP‐cap size, and the onset of shrinkage, which alternates with growth. Multiple theoretical models of MT polymerization dynamics have been reconciled to the kinetics of animal brain tubulins, but more recently, rapid kinetics seen in Arabidopsis tubulin polymerization suggest the need to sample a wider diversity in tubulin polymerization kinetics and reconcile it to theory. Here, we isolated tubulin from seedlings of Vigna sp. (mung bean), compared polymerization kinetics to animal brain tubulin, and used a computational model to understand the differences. We find that activity‐isolated mung tubulin polymerizes in a nucleation‐dependent manner, based on turbidimetry, qualitatively similar to brain tubulin, but with a 10‐fold smaller critical concentration. GTP‐dependent polymerization kinetics also appear to be transient, indicative of high rates of GTP hydrolysis. Computational modeling of tubulin nucleation and vectorial GTP hydrolysis to examine the effects of high nucleation and GTP‐hydrolysis rates predicts a dominance of the latter in determining MT lengths and numbers. Microscopy of mung tubulin filaments stabilized by GMPCPP or taxol results in few and short MTs, compared to the many long MTs arising from goat tubulin, qualitatively matching the model predictions. We find GTP‐hydrolysis outcompetes nucleation rates in determining MT lengths and numbers.

Mechanistic insights into GTP-dependence and kinetic polarity of FtsZ filament assembly

“…A similar effect is observed upon comparing SmFtsZ WT and EcFtsZ, where SmFtsZ WT filaments are shorter despite the higher GTPase activity in EcFtsZ. This might be attributed to the higher rate of addition to the filament when the CC is lower (nucleation rate = 1/CC), while the GTP binding affinity and rate of GTP hydrolysis at the active site remain the same (38). In the in vivo study, when EcFtsZ-Met225 is mutated to phenylalanine, we observe filamentous morphology of cells indicative of aberrant cell division, due to perturbation of FtsZ assembly.…”

Microtubule polymerization tuned by macromolecular crowdant size and density

“…We quantify the observations by fitting a nucleation dependent polymerization (NDP) kinetic model (Equation 1) to the absorbance kinetic data, that has been previously used to characterize MT polymerization kinetics by turbidimetry 23,26 . The model is used to estimate three parameters: the normalized steady state absorbance [A max - A 0 ] (where A max : steady state absorbance, A 0 : initial value), the polymerization rate r and half maximal time of polymerization t 1 / 2 .…”

“…Microtubule polymerization is nucleation-dependent and involves two steps, a rate limited MT nucleation 3 and elongation, together referred to as nucleation depen-dent polymerization, NDP 23,26,29,30 . We proceeded to test the effect of crowdants on both nucleation and elongation by polymerizing 30 µ M tubulin in the presence of 1 mM GTP and increasing concentrations of crowdants without the addition of seeds, as schematically depicted (Figure 4A).…”

Natural Taxanes: From Plant Composition to Human Pharmacology and Toxicity

“…Protofilaments arise in different numbers in different cells. Mostly thirteen protofilaments form through lateral α-α and β-β interactions in the cylindrical hollow structures—microtubules [ 68 , 69 , 70 ]. Monomer α-tubulin permanently binds guanosine-5’-triphosphate (GTP) at the dimer interface.…”

“…The critical concentration of tubulin may vary between species. For example, mung tubulin is up to 10 times lower than for brain tubulin [ 70 ]. The end of microtubules, where the polymerisation and depolymerisation of microtubules is more dynamic, is called plus (+), the other end is then minus (-).…”