Interaction Of Microtubule-associated Proteins Research Articles

755. Cytoplasmic Dynein-Dependent Adeno-Associated Virus Interaction with Microtubules

The importance of adeno-associated virus (AAV)-based gene therapy vectors as therapeutic vehicles is derived in part from efficient cell membrane penetration and translocation of the adeno-associated virus capsid to the nucleus where its genetic cargo is delivered. Other viruses have enhanced intracellular trafficking to the nucleus through interaction with the microtubule cytoskeleton with subsequent energy-dependent translocation in a retrograde manner towards the nucleus. The retrograde molecular motor, cytoplasmic dynein, participates in the microtubule-based nuclear-directed translocation of numerous viruses. Based on the hypothesis that the cytoplasmic dynein motor complex mediates a biochemical linkage of the AAV capsid with microtubules, a microtubule binding assay was developed in which binding of fluorophore-conjugated AAV capsids to microtubules was evaluated by mixing fluorophore-conjugated AAV capsid with polymerized microtubules, centrifuging to pellet the microtubules, and measuring the presence of the AAV-associated fluorescence in either the microtubule-free supernatant or microtubule-containing pellet. SDS-polyacrylamide gel electrophoresis of supernatant and pellet fractions showed that fluorophore-labeled AAV capsid was located predominantly in the supernatant when AAV was incubated with taxol-stabilized microtubules. However, when purified bovine microtubule-associated proteins were combined with microtubules, the majority of AAV was found in the pellet after centrifugation (69 ± 4 % vs 23 ± 4% in the absence of microtubule-associated proteins). The interaction of microtubule-associated proteins and AAV with microtubules did not occur in the presence of 500 mM NaCl. Using freshly isolated A549 lung epithelial cell lysate, the direct involvement of cytoplasmic dynein was evaluated. A549 cell lysate was depleted of endogenous cytoplasmic dynein by immunoprecipitation with a monoclonal antibody against the 74.1 kDa intermediate chain of cytoplasmic dynein. Microtubule-associated proteins isolated from whole A549 cell lysate versus cytoplasmic dynein-depleted A549 cell lysate were compared for the ability to support AAV-microtubule interaction. Whereas AAV capsid pelleted with microtubules when incubated with microtubule-associated proteins from whole A549 cell lysate, AAV capsid was found predominantly in the supernatant when cytoplasmic dynein-depleted cell lysate was used. No effect was observed when an irrelevant primary antibody was used for immunprecipitation. To confirm the involvement of cytoplasmic dynein in AAV-microtubule interaction, the nucleotide-sensitive binding property of cytoplasmic dynein was utilized. Under conditions that are known to disrupt cytoplasmic dynein interaction with microtubules (10 mM ATP), the efficiency of the AAV-microtubule interaction was reduced. However, the presence of an identical concentration of AMP-PNP, a non-hydrolyzable analog of ATP, had no effect on AAV-microtubule interactions. The results of this study suggest that the AAV capsid interacts with microtubules in a cytoplasmic dynein-dependent manner, providing a potential mechanism for the efficient translocation of the AAV capsid to the nucleus during infection.

Interaction of microtubule-associated proteins with microtubules: yeast lysyl- and valyl-tRNA synthetases and .tau. 218-235 synthetic peptide as model systems

The respective contributions of electrostatic interaction and specific sequence recognition in the binding of microtubule-associated proteins (MAPs) to microtubules have been studied, using as models yeast valyl- and lysyl-tRNA synthetases (VRS, KRS) that carry an exposed basic N-terminal domain, and a synthetic peptide reproducing the sequence 218-235 on tau protein, known to be part of the microtubule-binding site of MAPs. VRS and KRS bind to microtubules with a KD in the 10(-6) M range, and tau 218-235 binds with a KD in the 10(-4) M range. Binding of KRS and tau 218-235 is accompanied by stabilization and bundling of microtubules, without the intervention of an extraneous bundling protein. tau 218-235 binds to microtubules with a stoichiometry of 2 mol/mol of assembled tubulin dimer in agreement with the proposed binding sequences alpha[430-441] and beta[422-434]. Binding stoichiometries of 2/alpha beta S tubulin and 1/alpha S beta S tubulin were observed following partial or complete removal of the tubulin C-terminal regions by subtilisin, which localizes the site of subtilisin cleavage upstream residue alpha-441 and downstream residue beta-434. Quantitative measurements show that binding of MAPs, KRS, VRS, and tau 218-235 is weakened but not abolished following subtilisin digestion of the C-terminus of tubulin, indicating that the binding site of MAPs is not restricted to the extreme C-terminus of tubulin.

Taxol interferes with the interaction of microtubule-associated proteins with microtubules in cultured neurons

Treatment of neurons with taxol leads to the formation of microtubule bundles in which individual microtubules are much more closely spaced than in untreated neurons (Letourneau and Ressler, 1984). This suggests that taxol interferes with the mechanisms that regulate microtubule spacing in situ. I have determined whether treatment of neurons with taxol alters the composition of their microtubules, reasoning that such alterations may be related to the taxol-induced alterations in microtubule spacing. Cultures of sympathetic neurons were incubated with 35S-methionine and either taxol, podophyllotoxin, a potent microtubule-depolymerizing agent, or dimethyl sulfoxide (DMSO), the solvent for the drugs. The levels of labeled microtubule-associated proteins (MAPs) assembled into microtubules in the various cultures were then assayed biochemically. I focused on 4 MAPs: tau, chartins, MAP-2, and the MAP with a molecular mass of 210,000 Da (210 kDa). In untreated cultures, these MAPs are prominent components of microtubules. The levels of all MAPs, as well as tubulin, in microtubules were greatly reduced in cultures treated with podophyllotoxin, compared to controls. Taxol had varied effects on the interaction of MAPs with microtubules in situ. Microtubules formed in the presence of taxol contained normal or slightly elevated levels of tau and the 210 kDa MAP compared to microtubules in control cultures. In contrast, microtubules formed in the presence of taxol were almost completely devoid of chartin MAPs and MAP-2 compared to controls. These results show that taxol interferes with the interaction of some, but not all, MAPs with microtubules in situ. The altered MAP composition of microtubules in taxol-treated neurons may contribute to the abnormal spacing of microtubules seen in such neurons.

Interaction of microtubule-associated proteins with actin filaments. Studies using the fluorescence-photobleaching recovery technique.

The interaction of microtubule-associated proteins with actin filaments has been investigated by measuring the diffusion coefficient of either the filament or the microtubule-associated proteins. Experiments were performed using the technique of fluorescence photobleaching recovery with actin labeled with iodoacetamidotetramethyl rhodamine or microtubule-associated proteins labeled with iodoacetamidofluorescein. Actin filaments composed of pure rhodamine-labeled actin are not immobilized under a variety of conditions (Tait, J. F., and Frieden, C. (1982c) Biochemistry 21, 6046-6053). We find that addition of microtubule-associated proteins to rhodamine-labeled actin in a ratio as low as 1:1000 can cause immobilization, presumably cross-linking actin into a network of nondiffusible filaments. Immobilization occurs after polymerization is complete, suggesting either a length redistribution of actin filaments, a redistribution of the cross-links between filaments, or the slow addition of actin filaments to other filaments via the microtubule-associated protein. Experiments using fluorescein-labeled microtubule-associated proteins show that these proteins are bound to actin filaments as they are formed and that binding depended on actin concentration, indicating that there are a number of binding sites on the actin filaments. However, while the actin filaments become completely immobilized, the microtubule-associated proteins become only partially immobilized suggesting at least two different classes of binding affinities. The large peptide obtained from trypsin-treated fluorescein-labeled microtubule-associated proteins is not able to immobilize actin filaments since it does not bind to the filaments.

Phosphorylation of microtubule-associated proteins (MAPs) and pH of the medium control interaction between MAPs and actin filaments.

Interaction of microtubule-associated proteins (MAPs) with actin filaments at neutral pH is inhibited by phosphorylation of MAPs. Phosphorylated MAPs are less potent than unphosphorylated ones in increasing the low-shear viscosity of actin filaments in the neutral pH range. The ability of unphosphorylated MAPs to crosslink actin filaments falls off sharply above pH 7.5. Upon phosphorylation, the crosslinking ability of the MAPs peaks sharply between pH 6.2 and 6.3. Thus, the MAPs-actin interaction can be regulated by phosphorylation of MAPs and small changes in the physiological range of pH.