Electron tomography reveals a flared morphology on growing microtubule ends - PubMed (original) (raw)
. 2011 Mar 1;124(Pt 5):693-8.
doi: 10.1242/jcs.072967. Epub 2011 Feb 8.
Affiliations
- PMID: 21303925
- PMCID: PMC3039015
- DOI: 10.1242/jcs.072967
Electron tomography reveals a flared morphology on growing microtubule ends
Johanna L Höög et al. J Cell Sci. 2011.
Abstract
Microtubules (MTs) exhibit dynamic instability, alternating between phases of growth and shortening, mostly at their uncapped plus ends. Based on results from cryo-electron microscopy it was proposed that growing MTs display mainly curved sheets and blunt ends; during depolymerisation curled 'ramshorns' predominate. Observations of MTs in mitotic cells have suggested that the situation in vivo differs from that in vitro, but so far, a clear comparison between in vivo and in vitro results has not been possible because MT end structures could not be correlated directly with the dynamic state of that particular MT. Here we combine light microscopy and electron tomography (ET) to show that growing MT plus ends in the fission yeast Schizosaccharomyces pombe display predominantly a flared morphology. This indicates that MT polymerisation in vivo and in vitro can follow different paths.
Figures
Fig. 1.
Dynamics of regrowing MTs and MT ‘stubs’. (A) Live S. pombe cells expressing GFP–α-tubulin before and after MBC addition. (B) Kymograph of the bundle highlighted in C. An initial lag phase (yellow arrow), was followed by a period of polymerisation (green arrows) that was not faster than polymerisation measured in untreated bundles. This growth was followed by MT depolymerisation (red arrows). (C) MT bundle (GFP–α-tubulin) regrowth after MBC wash-out (time 0). (D) Fluorescence recovery after photobleaching experiment (FRAP) shows that MT stubs have little or no turn-over. Blue arrow and line indicate bleached stub; red arrowhead and line indicate control stub. Scale bars: 2 μm.
Fig. 2.
Most regrowing MT plus ends are flared. (A) We used cell length to select cells in early G2 for tomography reconstructions (mean ± s.d.). (B) Tomographic 3D models of MT bundle architecture 90 seconds after MBC wash-out. MTs (green filaments) have flared (turquoise balls), capped (red), curled (purple), sheeted (yellow) or ambiguous (white) ends. The SPB (centrosome equivalent) is visible in yellow, sitting on the nuclear envelope (pink). (C) Individual MT lengths were short during MBC treatment and remained shorter than in the wild type after release. (D) A gallery of MT end structures, percentage of the total population of ends (_n_=174) in MBC-released cells, and a cartoon illustrating how the flared ends (turquoise lines) were separated from the curled ends (purple lines). Ends are curled if the protofilament end meets a line positioned where the protofilaments leave the main MT axis (dashed line). (E) Plus ends were defined as the end opposite to a capped end, or when MTs had two similar ends, e.g. flared/flared. MTs with two open ends, e.g. flared/blunt, were considered of unknown polarity. The graph shows the percentage of plus-end morphologies found in untreated, MBC-treated and MBC-released cells. Flared plus ends predominate on growing MTs. (F) A gallery of short MTs with one capped end and one flared end. Scale bars: 50 nm (B), 10 nm (D,F).
Fig. 3.
Growing MT plus ends are mostly funnel-like structures. (A) Cartoon of a short sheet structure, as well as a line drawing representative of what would be seen in different orientations in a tomographic reconstruction of such a sheet. (B) A new funnel-like growing MT structure, which would be seen as a flared end imaged from all angles. (C) 4-nm-thick tomographic slices showing the same MT over a range of 30° angles, with a flared end in each view, showing that it is a structure as visualised in B.
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