Effects of ionic salt on the MDCT-induced assembly of tubulin.

<p>A, Tubulin (10 µM) was incubated with 4 µM MDCT in PEM buffer in the absence (▪) and presence of 50 (▴), 75 (▾), 100 (♦) and 200 (o) mM NaCl for 5 min on ice. Subsequently, 1 mM GTP was added to the reaction mixtures and the assembly kinetics was monitored at 37°C. The assembly of tubulin (10 µM) without MDCT and NaCl (Δ) was also monitored. B, NaCl inhibited MDCT-induced assembly of tubulin. The data represent average ± SD of three independent sets.</p

MDCT enhanced tubulin assembly in a concentration dependent manner <i>in vitro</i>.

<p>A, Effects of MDCT on the assembly kinetics of tubulin was monitored by light scattering at 400(10 µM) was polymerized in the absence (•) and presence of 1 (▪), 2 (▴) and 4 µM (▾) MDCT or 4 µM BSA (♦) in PEM buffer containing 1 mM GTP. B, The effect of MDCT on tubulin polymerization was determined by sedimentation assay. One of the three experiments is shown. C, MDCT caused an increase in the number of microtubules per field of view. Electron micrographs of polymerized tubulin in the absence (a) and in presence of MDCT (b) are shown. Polymers formed in presence of MDCT showed microtubule like morphology (c). D, MDCT stabilized microtubules against dilution induced disassembly. Preassembled microtubules were diluted in warm PEM buffer without or with 1, 2 and 4 µM MDCT and incubated for 10 min at 37°C. Microtubules were collected by centrifugation and the amount of microtubules recovered was estimated from a Coomassie-stained 12% SDS-PAGE. One of the three sets is shown.</p

MDCT showed greater binding to microtubules as compared to MD and CT.

<p>A, preformed microtubules were incubated with MD, CT or MDCT for 5°C and microtubule bound protein fragments were separated by centrifugation. Polymeric (pellet, P) and soluble (supernatant, S) tubulin fractions were analyzed on 12% SDS-PAGE. B, Percent of each fragment bound to microtubules is plotted as mean ± SD of three independent sets.</p

MDCT bound to tubulin in HeLa cell extract.

<p>Tubulin was precipitated from the HeLa cell extract using MDCT bound with Ni-NTA agarose resin as bait. Anti α-tubulin and anti- MAP7D3 antibodies were used to detect tubulin and MDCT, respectively.</p

Characterization of MD, CT and MDCT.

<p>A, Purified MD, CT and MDCT were analyzed on 12% SDS-PAGE. The expected molecular weights of MD, CT and MDCT are 22, 18 and 37 kDa respectively. B, Far-UV CD spectra of MD, CT and MDCT are shown.</p

MDCT bound at the C-terminal tail of tubulin subunits in microtubules.

<p>A, MDCT shares binding site with tau on microtubules. Taxol stabilized microtubules were incubated with 4 µM tau for 10 min at 25°C and then, incubated without (2) or with 4 (3) or 8 µM (4) MDCT for an additional 10 min. Polymers were collected through centrifugation. A mixture of tau and MDCT without microtubules (5) was also treated the same way. Supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE. Only tubulin (6) was also run in the same gel to compare its position with tau. B, Taxol stabilized microtubules were first incubated with 4 µM MDCT for 10 min at 25°C and then, incubated without (2) or with 4 (3) and 8 µM (4) tau for another 10 min. The samples were analyzed as described previously. C, Taxol stabilized microtubules were treated with subtilisin to cleave C-terminal tail of β-tubulin alone (αβ_s) (lane 3) and C-terminal tail of both α and β tubulin (α_sβ_s) (lane 4) and analyzed on 7.5% SDS-polyacrylamide gel. Lanes 1 and 2 have protein marker and taxol stabilized microtubules without subtilisin treatment, respectively. D, MDCT showed lesser binding to subtilisin treated microtubules. Microtubules composed of αβ, αβ_s or α_sβ_s tubulin dimers were incubated with MDCT and centrifuged to pellet the microtubules. Pellets were analyzed on 7.5% polyacrylamide gel to check the amount of MDCT bound to αβ, αβ_s or α_sβ_s containing microtubules, lanes 2, 3 and 4, respectively. Lanes 1 and 5 respectively, have protein molecular weight marker and MDCT that pelleted down in the absence of microtubules. One of the three sets is shown.</p

Effects of MD, CT and MDCT on the assembly of purified tubulin.

<p>A, Tubulin (10 µM) was polymerized in the absence and presence of 4 µM MD, CT, MDCT or MD and CT in PEM buffer containing 1 mM GTP. Supernatant and pellet fractions were analyzed using 12% SDS-PAGE. B, Ratio of polymerized tubulin to soluble tubulin in the presence of different fragments is plotted as mean ± SD of three sets of experiments.</p

MDCT bound along the length of microtubules <i>in vitro</i>.

<p>A, tubulin (red) and MDCT (green) were detected using anti-α-tubulin and anti-MAP7D3 antibodies. Scale bar represents 2 µm. B, Determination of the dissociation constant for the interaction of MDCT and microtubules. Taxol stabilized microtubules were incubated with different concentrations (0.5 to 8 µM) of MDCT. Microtubule bound MDCT was estimated from the Coomassie blue stained 12% SDS-polyacrylamide gels. MDCT sedimented in the presence (a) and absence (b) of microtubules. A dissociation constant for the interaction of MDCT and microtubules was determined by fitting the data in a binding isotherm (c).</p

Construction and Optimization of TD domain Purification, and Initial <i>in vitro</i> Screening.

<p>(A) Representation of overlapping PCR primers for generation of novel His-tagged PCR products for cloning to protein expression vectors. (B) Western Blot of expressed pET-Transduction domain (TD) recombinant proteins purified under insoluble (denaturing) conditions (top blot) and soluble (bottom blot) conditions. Predicted proteins are indicated to right of each gel. (C) Oligodendrocyte cultures treated with TD2.2 recombinant protein showing TD2.2 localized to apparent cytosolic (white arrows) and nuclear (red arrows) compartments. Scale bars: 50 µM.</p

Kinetics of EGFP-TD2.2 Recombinant Protein Transduction to Mature Human Oligodendrocytes by Sectional Confocal Microscopy.

<p>A single field of view (single mid-sectional depth) of mCherry⁺ human oligodendrocytes, with hourly differential interference contract (DIC) and mCherry/EGFP images taken following application of 1 µM EGFP-TD2.2 recombinant protein. Arrows track transduction of extracellular EGFP-TD2.2 protein into an oligodendrocyte.</p