Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1997 Jul 14;138(1):105-17.
doi: 10.1083/jcb.138.1.105.

A metastable intermediate state of microtubule dynamic instability that differs significantly between plus and minus ends

Affiliations

A metastable intermediate state of microtubule dynamic instability that differs significantly between plus and minus ends

P T Tran et al. J Cell Biol. .

Abstract

The current two-state GTP cap model of microtubule dynamic instability proposes that a terminal crown of GTP-tubulin stabilizes the microtubule lattice and promotes elongation while loss of this GTP-tubulin cap converts the microtubule end to shortening. However, when this model was directly tested by using a UV microbeam to sever axoneme-nucleated microtubules and thereby remove the microtubule's GTP cap, severed plus ends rapidly shortened, but severed minus ends immediately resumed elongation (Walker, R.A., S. Inoué, and E.D. Salmon. 1989. J. Cell Biol. 108: 931-937). To determine if these previous results were dependent on the use of axonemes as seeds or were due to UV damage, or if they instead indicate an intermediate state in cap dynamics, we performed UV cutting of self-assembled microtubules and mechanical cutting of axoneme-nucleated microtubules. These independent methods yielded results consistent with the original work: a significant percentage of severed minus ends are stable after cutting. In additional experiments, we found that the stability of both severed plus and minus ends could be increased by increasing the free tubulin concentration, the solution GTP concentration, or by assembling microtubules with guanylyl-(alpha,beta)-methylene-diphosphonate (GMPCPP). Our results show that stability of severed ends, particularly minus ends, is not an artifact, but instead reveals the existence of a metastable kinetic intermediate state between the elongation and shortening states of dynamic instability. The kinetic properties of this intermediate state differ between plus and minus ends. We propose a three-state conformational cap model of dynamic instability, which has three structural states and four transition rate constants, and which uses the asymmetry of the tubulin heterodimer to explain many of the differences in dynamic instability at plus and minus ends.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Schematic diagram of the darkfield UV microbeam apparatus. Self-assembled microtubules were observed by video darkfield microscopy using an HBO 100-W mercury arc lamp, a darkfield condenser (Carl Zeiss, Inc.), a 100×/0.85 NA glycerin immersion objective (Carl Zeiss, Inc.), and a camera (model 66 SIT; Dage-MTI, Inc.). An HBO 100-W mercury arc lamp served as the UV source. A quartz collector lens directed the lamp output through an adjustable diaphragm (the slit) and onto a dichroic mirror, which reflected wavelengths <400 nm to the Ultrafluar objective. The slit image was then projected by the objective onto the specimen image plane. Design details for the integration of the UV microbeam and the darkfield microscope are provided in the text.
Figure 2
Figure 2
UV cutting of self-assembled microtubules. Microbeam irradiation of free microtubules produces two severed ends: typically one shortens, the other is stable. The microtubule shown extended out of the field of view for over 100 μm in either direction. The microbeam area is indicated by the circle. After irradiation, the severed end on the right is stable, and the severed end on the left shortened toward the left edge of the field through the observed time. Bar, 5 μm.
Figure 3
Figure 3
Schematic diagram of VE-DIC microscope setup for mechanical severing of single microtubules. Axoneme-nucleated microtubules were observed by VE-DIC microscopy using an HBO 100-W mercury arc lamp, a 100×/1.25 NA oil immersion objective (Carl Zeiss, Inc.), DIC condenser (Carl Zeiss, Inc.), DIC optics, and a video camera (model C2400 Newvicon; Hamamatsu Corp.) (Walker et al., 1990). The cutting apparatus consists of an aluminum Kiehart-Ellis chamber (Kiehart, 1981) and an Ellis piezoelectric cube attached to a joystick controller (Begg and Ellis, 1979).
Figure 4
Figure 4
(a) Diagram of the Kiehart-Ellis chamber preparation, microneedle shape and alignment, and the mechanical severing motion. (b) Video image of the field of view. Shown is the glass microneedle tip pressing down on the coverslip surface and axoneme-nucleated microtubules. Bar, 5 μm.
Figure 5
Figure 5
Glass microneedle severing of axoneme-nucleated microtubules. Sequence showing the behaviors typical of severed plus and minus ends. After severing, the plus end immediately and rapidly shortened back to the axoneme; the minus end continued growing without rapid shortening. Bar, 10 μm.
Figure 6
Figure 6
In the three-state model of dynamic instability, k c is the frequency of spontaneous catastrophe, k r is the frequency of spontaneous rescue, k EI is the frequency of transition between elongation (E) and the intermediate state (I), k IE is the frequency of transition from I to E, k IS is the frequency of transition from I to S, and k SI is the frequency of transition from S to I. Note that each of these transition rate constants corresponds to changes in the structure of the microtubule ends as described in the text.
Figure 7
Figure 7
The opposite orientation of tubulin dimers at plus and minus ends is probably responsible for the differences in dynamic instability as described in the text. The values of the frequencies of catastrophe, k c, and rescue, k r, are taken from Table II for 16 μM tubulin. The values for the transition rate constants at each end were calculated as described in the Appendix.

Similar articles

Cited by

References

    1. Begg DA, Ellis GW. Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers. J Cell Biol. 1979;82:528–541. - PMC - PubMed
    1. Bell CW, Fraser C, Sale WS, Tang W-JY, Gibbons IR. Preparation and purification of dynein. Methods Cell Biol. 1982;24:373–397. - PubMed
    1. Billger MA, Bhatacharjee G, Williams RC., Jr Dynamic instability of microtubules assembled from microtubule-associated-protein-free tubulin: neither variability of growth and shortening rates nor “rescue” requires microtubule-associated proteins. Biochemistry. 1996;35:13656–13663. - PubMed
    1. Caplow M. Microtubule dynamics. Curr Opin Cell Biol. 1992;4:58–65. - PubMed
    1. Caplow M, Shanks J. Induction of microtubule catastrophe by formation of tubulin-GDP and apotubulin subunits at microtubule ends. Biochemistry. 1995;34:15732–15741. - PubMed

Publication types