Serum-induced neurite retraction in CAD cells--involvement of an ATP-actin retractile system and the lack of microtubule-associated proteins.

Cultured catecholamine-differentiated cells [which lack the microtubule-associated proteins (MAPs): MAP1B, MAP2, Tau, STOP, and Doublecortin] proliferate in the presence of fetal bovine serum, and, in its absence, cease dividing and generate processes similar to the neurites of normal neurons. The reintroduction of serum induces neurite retraction, and proliferation resumes. The neurite retraction process in catecholamine-differentiated cells was partially characterized in this study. Microtubules in the cells were found to be in a highly dynamic state, and tubulin in the microtubules consisted primarily of the tyrosinated and deacetylated isotypes. Increased levels of acetylated or Δ2-tubulin (which are normally absent) did not prevent serum-induced neurite retraction. Treatment of differentiated cells with lysophosphatidic acid or adenosine deaminase induced neurite retraction. Inhibition of Rho-associated protein kinase, ATP depletion and microfilament disruption each (individually) blocked serum-induced neurite retraction, suggesting that an ATP-dependent actomyosin system underlies the mechanism of neurite retraction. Nocodazole treatment induced neurite retraction, but this effect was blocked by pretreatment with the microtubule-stabilizing drug paclitaxel (Taxol). Paclitaxel did not prevent serum-induced or lysophosphatidic acid-induced retraction, suggesting that integrity of microtubules (despite their dynamic state) is necessary to maintain neurite elongation, and that paclitaxel-induced stabilization alone is not sufficient to resist the retraction force induced by serum. Transfection with green fluorescent protein-Tau conferred resistance to retraction caused by serum. We hypothesize that, in normal neurons (cultured or in vivo), MAPs are necessary not only to stabilize microtubules, but also to establish interactions with other cytoskeletal or membrane components to form a stable structure capable of resisting the retraction force.

Quantification of acetylated tubulin.

The acetylation/deacetylation of Lys40 of the α-subunit is an important posttranslational modification undergone by tubulin during the life of a cell. Many previous studies have addressed the physiological role of this acetylation process using various approaches based on changes of acetylated tubulin (AcTubulin) content. In most of these studies, however, the actual amounts of AcTubulin were not known and it was difficult to draw conclusions. We present here a simple method to estimate the percentage of AcTubulin relative to total tubulin. The method is based on acetylation of the tubulin sample with acetic anhydride, Western blotting stained by antiAcTubulin antibody, and comparison of the optical density of the AcTubulin band with that of a corresponding sample that was not chemically acetylated.

A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype.

Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations that are enriched in AcTubulin (acetylated tubulin) and (for comparison) preparations lacking AcTubulin. Assembly-disassembly cycling of microtubules gives tubulin preparations that contain little or no AcTubulin. In the present study we demonstrated that this result is owing to the presence of high deacetylating activity in the extracts. This deacetylating activity in rat brain homogenates was inhibited by TSA (Trichostatin A) and tubacin, but not by nicotinamide, indicating that HDAC6 (histone deacetylase 6) is involved. TSA showed no effect on microtubule polymerization or depolymerization. We utilized these properties of TSA to prevent deacetylation during the assembly-disassembly procedure. The effective inhibitory concentration of TSA was 3 µM in the homogenate and 1 µM in the subsequent cycling steps. By comparison with immunopurified AcTubulin, we estimated that ~64% of the tubulin molecules in the three cycled preparations were acetylated. The protein profiles of these tubulin preparations, as assessed by SDS/PAGE and Coomassie Blue staining, were identical to that of a preparation completely lacking AcTubulin obtained by assembly-disassembly cycles in the absence of TSA. The tyrosination state and in vitro assembly-disassembly kinetics were the same regardless of the degree of acetylation.

Acetylated tubulin associates with the fifth cytoplasmic domain of Na(+)/K(+)-ATPase: possible anchorage site of microtubules to the plasma membrane.

We showed previously that NKA (Na(+)/K(+)-ATPase) interacts with acetylated tubulin resulting in inhibition of its catalytic activity. In the present work we determined that membrane-acetylated tubulin, in the presence of detergent, behaves as an entity of discrete molecular mass (320-400 kDa) during molecular exclusion chromatography. We also found that microtubules assembled in vitro are able to bind to NKA when incubated with a detergent-solubilized membrane preparation, and that isolated native microtubules have associated NKA. Furthermore, we determined that CD5 (cytoplasmic domain 5 of NKA) is capable of interacting with acetylated tubulin. Taken together, our results are consistent with the idea that NKA may act as a microtubule-plasma membrane anchorage site through an interaction between acetylated tubulin and CD5.