Sulfo-SMCC Prevents Annealing of Taxol-Stabilized Microtubules In Vitro

Microtubule structure and functions have been widely studied in vitro and in cells. Research has shown that cysteines on tubulin play a crucial role in the polymerization of microtubules. Here, we show that blocking sulfhydryl groups of cysteines in taxol-stabilized polymerized microtubules with a commonly used chemical crosslinker prevents temporal end-to-end annealing of microtubules in vitro. This can dramatically affect the length distribution of the microtubules. The crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfo-SMCC, consists of a maleimide and an N-hydroxysuccinimide ester group to bind to sulfhydryl groups and primary amines, respectively. Interestingly, addition of a maleimide dye alone does not show the same interference with annealing in stabilized microtubules. This study shows that the sulfhydryl groups of cysteines of tubulin that are vital for the polymerization are also important for the subsequent annealing of microtubules.Comment: 3 figure

Chemically treated and untreated MTs.

<p>The table shows the total numbers of microtubules (from one set of experiments), average calculated mean lengths (<i>L</i>_<i>mean</i>) and average mean lengths from the fitting curve (<i>L</i>_{<i>meanfit</i>}) for untreated MTs, and MTs treated with sulfo-SMCC, maleimide dye, and NHS ester dye for 24 h.</p

Lengths of untreated and sulfo-SMCC treated MTs at different time points.

<p>The table shows the total number of microtubules (from three independent sets of experiments), average calculated mean length (<i>L</i>_<i>mean</i>) and average mean length from the fitting curve (<i>L</i>_{<i>meanfit</i>}) for sulfo-SMCC treated and untreated MTs, measured after 0 h, 6 h, and 24 h of incubation.</p

Length distributions of chemically treated and untreated MTs after 24 h of incubation.

<p>Length distributions of 2 mg/ml (tubulin) solution of untreated MTs, MTs treated with 250 <i>μ</i>M sulfo-SMCC, 250 <i>μ</i>M maleimide dye and 250 <i>μ</i>M NHS ester dye are shown. Lengths of MTs are distributed exponentially in all cases; single-exponential fits shown as dashed lines. Treatment with sulfo-SMCC resulted in drastically shorter MTs in comparison to untreated MTs (mean length = 2 <i>μ</i>m and 17 <i>μ</i>m, respectively). Maleimide dye treated MTs, however, showed a less drastic effect with an intermediate mean length of 9.2 <i>μ</i>m. Addition of NHS ester dye to MTs as a control did not affect the lengths of MTs.</p

Dual-colored microtubule experiments.

<p>The table shows the total number of microtubules (from three independent sets of experiments), average calculated mean length (<i>L</i>_<i>mean</i>) and average mean length from the fitting curve (<i>L</i>_{<i>meanfit</i>}) for different time points of sulfo-SMCC treated and untreated MTs.</p

Length distribution of sulfo-SMCC treated and untreated MTs after different incubation times.

<p>Length distributions of 2 mg/ml (tubulin) solution of (A) untreated and (B) sulfo-SMCC treated (250 <i>μ</i>M) MTs after 0 h, 6 h, and 24 h are shown. Untreated MTs show a significant increase in their mean lengths after 6 h (p < 0.001) and 24 h (p < 0.001) of incubation, in comparison with those measured at 0 h. Sulfo-SMCC treated MTs show a constant mean length, independent of incubation time. Lengths of all samples are distributed exponentially; exponential fits with the normalized probability function <i>a</i>² <i>Lexp</i>(−<i>aL</i>) are shown as dashed lines. This functional form takes undersampling of short MTs due to the resolution limit into account.</p