ATP-dependent formation and motility of aster-like structures with isolated calf brain microtubule proteins.

Abstract

Microtubule proteins isolated from calf brain will undergo gelation-contraction in the presence of ATP. We have now examined this process by video-enhanced contrast microscopy. After ATP addition to steady-state microtubules, slow (1-5 'Am/min), linear movements of particles and microtubules toward aggregation centers occur. Th~eresulting structures resemble mitotic spindle asters. During the time when gel contraction occurs, asters move (at 1-5 ,um/min) toward other nearby asters. This is accompanied by the apparent shortening of the microtubules running between the asters. This is the first example of isolated microtubules undergoing a process that has similarities to half-spindle shortening during anaphase A. Formation of aster-like structures without preformed microtubule organizing centers raises the possibility that a similar process may contribute to microtubule organization in vivo. (2,3,5), but the mechanism of action of ATP in these processes remains unknown.It has been found recently that microtubules isolated from calf brain by three cycles of assembly and disassembly will undergo "gelation-contraction" in the presence of ATP (6).We have now examined this process by computer-assisted, video-enhanced contrast microscopy (7-9). These observations reveal the formation and motility of structures with a striking resemblance to mitotic asters and spindles. The motility observed differs significantly from that reported previously by isolated microtubules but displays some similarities to half-spindle shortening during anaphase A. METHODSFor each experiment, microtubule proteins were isolated as described (6) except that the third-cycle microtubule protein was frozen by dripping the solution into liquid nitrogen. The protein was stored at liquid-nitrogen or dry-ice temperature until needed, at which time the frozen protein pellets were rapidly thawed and then polymerized at 370C in 1 mM GTP/1 mM EGTA/0.5 mM MgCl2/O.1 M4-morpholeneethanesulfonic acid (Mes), pH 6.6. The protein was Used either undiluted, at about 5 mg/ml, or was diluted 1:1 to 1:2 with buffer before ATP addition to make individual microtubules more visible. Incubation was carried out for 30 min to assemble microtubules to a steady-state array, at which time 2 mM ATP was added. Slides were immediately prepared by using a single drop of solution, and the coverslip was sealed with VALAP [a 1:1:1 (wt/wt) mixture of Vaseline, lanolin, and paraffin) or clear nail polish. The sample was maintained at approximately 370C either by placing the microscope in a heated room (when using a x 100 oil immersion objective) or by heating the stage with an air curtain incubator (when using a dry objective). The air curtain incubator was positioned to operate continuously while holding the slide at the desired temperature to eliminate artifacts caused by cyclic temperature changes.Observations were performed by using either phasecontrast, differential-interference-contrast (DIt), or polarization optics. Details and operation of the image-enhancement syste...

Nerve Terminal Remodeling Visualized in Living Mice by Repeated Examination of the Same Neuron

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