UV cutting of MAPs-bound microtubules.

Abstract

Microtubules exhibit dynamic instability in vivo and in vitro (with purified tubulin), stochastically switching between phases of persistent growth and shortening (reviewed in ref. 1). The growth phase is governed by the presence of a tubulin GTP cap at the end of the microtubule, and the structure we observe is tubulin protofilament sheet folded up into a blunt and straight microtubule. The shortening phase is initiated by the loss of the tubulin GTP cap, and we see the blossom structure of protofilaments, curved inside-out. The transitions back and forth between the growth and shortening phases include an intermediate phase in which the microtubule has lost its tubulin GTP cap but still, transiently, retains the straight, blunt structure chracteristic of the growth phase (2). Thus, for a microtubule to grow, new subunits must be added to the tubulin GTP cap; loss of the cap puts the microtubule in the intermediate phase. And for a microtubule to shorten, the protofilaments must first peel away from the central lattice and subsequently break off (3). If microtubules are polymerized with purified tubulin, cutting off the tubulin GTP cap, either mechanically or with UV, produces microtubules in the intermediate phase (2). For plus ends, the intermediate phase quickly switches to the shortening phase. For minus ends, the intermediate phase quickly switches back to the growth phase (2). Microtubule-associated-proteins (MAPS)-particularly neuronal MAPS such as MAP2 or Tauhave been shown to suppress microtubule dynamics (4). To test the hypothesis that MAPS promote switching from the intermediate to the growth phase, we used UV irradiation to cut the ends off MAPS-bound microtubules. Sea urchin axonemes were added to a coverslip and glass slide chamber. The axonemes adhered to the surface of the coverslip. Then partially purified (i.e., 2-cycles) porcine brain tubulin (1.3 mg/ml) was added to polymerize microtubules. Note that porcine brain tubulin, after 2 cycles of warm-cold purification, still retains the endogenous MAPS from the homogenate, particularly MAP2 and Tau (5). MAPS-bound microtubules were observed by video-enhanced differential interference contrast microscopy, and severed with a UV microbeam. The experimental apparatus has been described in detail (6). Using a 5-s exposure of a UV microbeam (a 200-W mercury arc lamp served as the source), we cleanly severed single microtubules extending from the axonemes. We severed 23 minus ends and 39 plus ends. All were stable after being severed and did not exhibit immediate shortening. Instead, they continued to grow (Fig. 1). The behavior of severed minus ends of MAPS-bound microtubules was similar to that of severed minus ends of purifiedtubulin microtubules: they quickly switched from the interme-