Abstract. We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37°C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 I.tM.Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 ~tM -t s -t for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s -t. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 lxM. The rate of rapid shortening was similar at the two ends (plus = 733 s-t; minus = 915 s-l), and did not vary with tubulin concentration.Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.T HE term "dynamic instability" describes microtubule assembly in which individual microtubules exhibit alternating phases of elongation and rapid shortening. Transitions between these phases are abrupt, stochastic, and infrequent in comparison to the rates of tubulin association and dissociation at the microtubule ends (17, 28). Substantial evidence has accumulated to indicate that dynamic instability is the basic mechanism of microtubule assembly in vitro (17, 28), and in both the mitotic spindle and the cyoplasmic microtubule complex (CMTC) l (10,11,34,35,37). Although microtubule dynamics within the cell may be regulated by various microtubule-associated proteins (MAPs) and other intracellular regulatory molecules, it is important 1. Abbreviations used in this paper: CMTC, cytoplasmic microtubule complex; DIC, differential interference contrast; MAP(s), microtubule-associated protein(s).first to understand the details of the inherent behavior of microtubules assembled from tubulin alone.Th...

A product encoded at the claret locus in Drosophila is needed for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. The predicted amino-acid sequence of the segregation protein was shown recently to be strikingly similar to Drosophila kinesin heavy chain. We have expressed the claret segregation protein in bacteria and have found that the bacterially expressed protein has motor activity in vitro with several novel features. The claret motor is slow (4 microns min-1), unlike either kinesin or dyneins. It has the directionality, the ability to generate torque and the sensitivity to inhibitors reported previously for dyneins. The finding of minus-end directed motor activity for a protein with sequence similarity to kinesin suggests that the dynein and kinesin motor domains are ancestrally related. The minus-end directed motor activity of the claret motor is consistent with a role for this protein in producing chromosome movement along spindle microtubules during prometaphase and/or anaphase.

SummaryBacteria can swim in liquid media by flagellar rotation and can move on surfaces via gliding or twitching motility. One type of gliding motility involves the extension, attachment and retraction of type IV pili (TFP), which pull the bacterium towards the site of attachment. TFP-dependent gliding motility has been seen in many Gram-negative bacteria but not in Grampositive bacteria. Recently, the genome sequences of three strains of Clostridium perfringens have been completed and we identified gene products involved in producing TFP in each strain. Here we show that C. perfringens produces TFP and moves with an unusual form of gliding motility involving groups of densely packed cells moving away from the edge of a colony in curvilinear flares. Mutations introduced into the pilT and pilC genes of C. perfringens abolished motility and surface localization of TFP. Genes encoding TFP are also found in the genomes of all nine Clostridium species sequenced thus far and we demonstrated that Clostridium beijerinckii can move via gliding motility. It has recently been proposed that the Clostridia are the oldest Eubacterial class and the ubiquity of TFP in this class suggests that a Clostridia-like ancestor possessed TFP, which evolved into the forms seen in many Gram-negative species.

Abstract. Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 p,M) was assembled at 37"C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution) . Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length . Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated NDIVIDUAI. microtubules reassembled from purified tubulin undergo alternating phases ofelongation and rapid AL shortening (19,26,49) . The transition (catastrophe) from elongation to rapid shortening, and the reverse transition (rescue), are abrupt, stochastic, and occur infrequently in comparison to the rates of subunit association and dissociation (49) . This behavior, termed dynamic instability (26), is also exhibited by the majority ofmicrotubules in the mitotic spindle and the interphase cytoplasmic microtubule complex (CMTC) ' (12-14, 33-36, 38, 39) and appears necessary for chromosome-spindle attachment, chromosome segregation, cellular morphogenesis, and establishment ofcell polarity (13,17,21,(29)(30)(31)(32) . Changes in frequencies ofcatastrophe R . A . Walker's current address is

The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of "stabilizing cap," possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.