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Ectopic A-lattice seams destabilize microtubules.

Katsuki M, Drummond DR, Cross RA - Nat Commun (2014)

Bottom Line: It is currently unclear how A-lattice seams influence microtubule dynamic instability.Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly.On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK [2].

ABSTRACT
Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

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Effect of ectopic A-lattice seams on MT dynamics.B-lattice single-seam MT seeds were assembled using pig tubulin labelled with Alexa-488 fluorescent dye and the GTP analogue GMPCPP. A-lattice-enriched seeds were co-assembled from Alexa-488 pig tubulin, GMPCPP and 5 μM or 50 μM Mal3FL and pelleted through a glycerol cushion to remove excess Mal3 (a). Seeds were attached to a flow cell surface using Anti-Alexa-488 antibody, the flow cell flushed with buffer to remove any residual Mal3 and then free S. pombe tubulin and GTP were added to enable growth of dynamic GTP MTs (b). The location of the Alexa-488-labelled seeds (green) was visualized using epifluorescence microscopy and the dynamic unlabelled MT ends by dark-field microscopy (grey) (c). Kymographs of MTs were recorded from B-lattice (d, red) and A-lattice-enriched MTs (e, green), the end location of the MTs determined automatically, and the MT dynamic parameters of growth (f) and shrinkage (g) rates together with catastrophe frequency (h) determined and plotted for the fast plus end (purple) and slow minus end (blue) of MTs nucleated by GMPCPP tubulin seeds (red) or seeds co-assembled with 5 μM (light green) or 50 μM (dark green) Mal3FL. Error bars in (f) and (g) show s.e.m. and in (h) the Poisson confidence limits (P=0.05). Mean rates and frequencies are shown in Table 1. (e) is an example of plus end enhanced catastrophe frequency often observed in A-lattice-enriched MTs that was infrequently seen in B-lattice single-seam MTs (d). The distributions of catastrophe frequencies for MTs growing from individual seeds were also plotted (i). The minus end shrinkage rate for individual seeds was then plotted against the plus end catastrophe frequency for the same seed using the pooled data from the 0, 5 and 50 μM seeds (j). The minus end shrinkage and plus end catastrophe of the pooled data have a significant correlation (Spearman coefficient r=0.6039, P=0.0037). Scale bar in (c) is 4 μm.
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f4: Effect of ectopic A-lattice seams on MT dynamics.B-lattice single-seam MT seeds were assembled using pig tubulin labelled with Alexa-488 fluorescent dye and the GTP analogue GMPCPP. A-lattice-enriched seeds were co-assembled from Alexa-488 pig tubulin, GMPCPP and 5 μM or 50 μM Mal3FL and pelleted through a glycerol cushion to remove excess Mal3 (a). Seeds were attached to a flow cell surface using Anti-Alexa-488 antibody, the flow cell flushed with buffer to remove any residual Mal3 and then free S. pombe tubulin and GTP were added to enable growth of dynamic GTP MTs (b). The location of the Alexa-488-labelled seeds (green) was visualized using epifluorescence microscopy and the dynamic unlabelled MT ends by dark-field microscopy (grey) (c). Kymographs of MTs were recorded from B-lattice (d, red) and A-lattice-enriched MTs (e, green), the end location of the MTs determined automatically, and the MT dynamic parameters of growth (f) and shrinkage (g) rates together with catastrophe frequency (h) determined and plotted for the fast plus end (purple) and slow minus end (blue) of MTs nucleated by GMPCPP tubulin seeds (red) or seeds co-assembled with 5 μM (light green) or 50 μM (dark green) Mal3FL. Error bars in (f) and (g) show s.e.m. and in (h) the Poisson confidence limits (P=0.05). Mean rates and frequencies are shown in Table 1. (e) is an example of plus end enhanced catastrophe frequency often observed in A-lattice-enriched MTs that was infrequently seen in B-lattice single-seam MTs (d). The distributions of catastrophe frequencies for MTs growing from individual seeds were also plotted (i). The minus end shrinkage rate for individual seeds was then plotted against the plus end catastrophe frequency for the same seed using the pooled data from the 0, 5 and 50 μM seeds (j). The minus end shrinkage and plus end catastrophe of the pooled data have a significant correlation (Spearman coefficient r=0.6039, P=0.0037). Scale bar in (c) is 4 μm.

Mentions: Dynamic MTs were polymerized from GMPCPP-stabilized MT seeds to determine whether the MT dynamics were altered by A-lattice enrichment of the seeds. We had previously studied the effect of Mal3 on the dynamics of S. pombe MTs nucleated by B-lattice single-seam pig tubulin seeds37, therefore we utilized this well-characterized system. Seeds containing extra A-lattice were assembled by including 5 or 50 μM of Mal3FL at the nucleation step29. Excess Mal3 was then removed before dynamics were measured by pelleting through a glycerol cushion (Fig. 4a). Alexa-488-labelled seeds were attached to a flow cell surface using anti-alexa-488 antibodies, which stabilize the GMPCPP seeds similarly to rigor binding of kinesin. Flow cells were flushed with buffer to remove any residual Mal3 or glycerol and removal of residual Mal3 was confirmed in control experiments using GFP–Mal3 fusion protein, which was undetectable after the flushing steps. Purified unlabelled single-isoform S. pombe tubulin and GTP were then flowed in. The added tubulin polymerizes from the seeds to form dynamic MTs, which are only anchored to the surface through the antibody-bound seed (Fig. 4b). Seed position was recorded using epifluorescence microscopy, and the entire dynamic MT was imaged by dark-field microscopy using an illumination wavelength to which the fluorophore in the seeds is insensitive (Fig. 4b,c, Supplementary Movie 3). Kymographs were created and the MT ends automatically tracked (Fig. 4d,e). The data show that the plus-end catastrophe frequency of MTs growing from A-lattice rich MT seeds is enhanced at least 1.5-fold over those growing from B-lattice single-seam seeds with a single A-lattice seam. The minus-end catastrophe frequency is also enhanced, by threefold (Fig 4h, Table 1). We examined the catastrophe frequency of dynamic MTs grown from individual seeds. The catastrophe frequencies of MTs grown from B-lattice single-seam seeds were more tightly clustered than with the 5 or 50 μM Mal3FL seeds, for which higher catastrophe frequencies were more common (Fig. 4i). Plus-end shrinkage rates following catastrophe were not significantly different. The absence of an effect on shrinkage is not a detection artefact, since shrinkage of the MTs in Fig. 4e would still be easily detectable over several data points at more than twice the actual rates measured. Minus end shrinkage is 1.5 times faster in A-lattice-rich MTs than in B-lattice MTs (Fig. 4g, Table 1). MTs with faster minus-end shrinkage rates were more likely to grow from seeds with higher plus-end catastrophe frequencies (Fig. 4j).


Ectopic A-lattice seams destabilize microtubules.

Katsuki M, Drummond DR, Cross RA - Nat Commun (2014)

Effect of ectopic A-lattice seams on MT dynamics.B-lattice single-seam MT seeds were assembled using pig tubulin labelled with Alexa-488 fluorescent dye and the GTP analogue GMPCPP. A-lattice-enriched seeds were co-assembled from Alexa-488 pig tubulin, GMPCPP and 5 μM or 50 μM Mal3FL and pelleted through a glycerol cushion to remove excess Mal3 (a). Seeds were attached to a flow cell surface using Anti-Alexa-488 antibody, the flow cell flushed with buffer to remove any residual Mal3 and then free S. pombe tubulin and GTP were added to enable growth of dynamic GTP MTs (b). The location of the Alexa-488-labelled seeds (green) was visualized using epifluorescence microscopy and the dynamic unlabelled MT ends by dark-field microscopy (grey) (c). Kymographs of MTs were recorded from B-lattice (d, red) and A-lattice-enriched MTs (e, green), the end location of the MTs determined automatically, and the MT dynamic parameters of growth (f) and shrinkage (g) rates together with catastrophe frequency (h) determined and plotted for the fast plus end (purple) and slow minus end (blue) of MTs nucleated by GMPCPP tubulin seeds (red) or seeds co-assembled with 5 μM (light green) or 50 μM (dark green) Mal3FL. Error bars in (f) and (g) show s.e.m. and in (h) the Poisson confidence limits (P=0.05). Mean rates and frequencies are shown in Table 1. (e) is an example of plus end enhanced catastrophe frequency often observed in A-lattice-enriched MTs that was infrequently seen in B-lattice single-seam MTs (d). The distributions of catastrophe frequencies for MTs growing from individual seeds were also plotted (i). The minus end shrinkage rate for individual seeds was then plotted against the plus end catastrophe frequency for the same seed using the pooled data from the 0, 5 and 50 μM seeds (j). The minus end shrinkage and plus end catastrophe of the pooled data have a significant correlation (Spearman coefficient r=0.6039, P=0.0037). Scale bar in (c) is 4 μm.
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f4: Effect of ectopic A-lattice seams on MT dynamics.B-lattice single-seam MT seeds were assembled using pig tubulin labelled with Alexa-488 fluorescent dye and the GTP analogue GMPCPP. A-lattice-enriched seeds were co-assembled from Alexa-488 pig tubulin, GMPCPP and 5 μM or 50 μM Mal3FL and pelleted through a glycerol cushion to remove excess Mal3 (a). Seeds were attached to a flow cell surface using Anti-Alexa-488 antibody, the flow cell flushed with buffer to remove any residual Mal3 and then free S. pombe tubulin and GTP were added to enable growth of dynamic GTP MTs (b). The location of the Alexa-488-labelled seeds (green) was visualized using epifluorescence microscopy and the dynamic unlabelled MT ends by dark-field microscopy (grey) (c). Kymographs of MTs were recorded from B-lattice (d, red) and A-lattice-enriched MTs (e, green), the end location of the MTs determined automatically, and the MT dynamic parameters of growth (f) and shrinkage (g) rates together with catastrophe frequency (h) determined and plotted for the fast plus end (purple) and slow minus end (blue) of MTs nucleated by GMPCPP tubulin seeds (red) or seeds co-assembled with 5 μM (light green) or 50 μM (dark green) Mal3FL. Error bars in (f) and (g) show s.e.m. and in (h) the Poisson confidence limits (P=0.05). Mean rates and frequencies are shown in Table 1. (e) is an example of plus end enhanced catastrophe frequency often observed in A-lattice-enriched MTs that was infrequently seen in B-lattice single-seam MTs (d). The distributions of catastrophe frequencies for MTs growing from individual seeds were also plotted (i). The minus end shrinkage rate for individual seeds was then plotted against the plus end catastrophe frequency for the same seed using the pooled data from the 0, 5 and 50 μM seeds (j). The minus end shrinkage and plus end catastrophe of the pooled data have a significant correlation (Spearman coefficient r=0.6039, P=0.0037). Scale bar in (c) is 4 μm.
Mentions: Dynamic MTs were polymerized from GMPCPP-stabilized MT seeds to determine whether the MT dynamics were altered by A-lattice enrichment of the seeds. We had previously studied the effect of Mal3 on the dynamics of S. pombe MTs nucleated by B-lattice single-seam pig tubulin seeds37, therefore we utilized this well-characterized system. Seeds containing extra A-lattice were assembled by including 5 or 50 μM of Mal3FL at the nucleation step29. Excess Mal3 was then removed before dynamics were measured by pelleting through a glycerol cushion (Fig. 4a). Alexa-488-labelled seeds were attached to a flow cell surface using anti-alexa-488 antibodies, which stabilize the GMPCPP seeds similarly to rigor binding of kinesin. Flow cells were flushed with buffer to remove any residual Mal3 or glycerol and removal of residual Mal3 was confirmed in control experiments using GFP–Mal3 fusion protein, which was undetectable after the flushing steps. Purified unlabelled single-isoform S. pombe tubulin and GTP were then flowed in. The added tubulin polymerizes from the seeds to form dynamic MTs, which are only anchored to the surface through the antibody-bound seed (Fig. 4b). Seed position was recorded using epifluorescence microscopy, and the entire dynamic MT was imaged by dark-field microscopy using an illumination wavelength to which the fluorophore in the seeds is insensitive (Fig. 4b,c, Supplementary Movie 3). Kymographs were created and the MT ends automatically tracked (Fig. 4d,e). The data show that the plus-end catastrophe frequency of MTs growing from A-lattice rich MT seeds is enhanced at least 1.5-fold over those growing from B-lattice single-seam seeds with a single A-lattice seam. The minus-end catastrophe frequency is also enhanced, by threefold (Fig 4h, Table 1). We examined the catastrophe frequency of dynamic MTs grown from individual seeds. The catastrophe frequencies of MTs grown from B-lattice single-seam seeds were more tightly clustered than with the 5 or 50 μM Mal3FL seeds, for which higher catastrophe frequencies were more common (Fig. 4i). Plus-end shrinkage rates following catastrophe were not significantly different. The absence of an effect on shrinkage is not a detection artefact, since shrinkage of the MTs in Fig. 4e would still be easily detectable over several data points at more than twice the actual rates measured. Minus end shrinkage is 1.5 times faster in A-lattice-rich MTs than in B-lattice MTs (Fig. 4g, Table 1). MTs with faster minus-end shrinkage rates were more likely to grow from seeds with higher plus-end catastrophe frequencies (Fig. 4j).

Bottom Line: It is currently unclear how A-lattice seams influence microtubule dynamic instability.Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly.On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK [2].

ABSTRACT
Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

Show MeSH
Related in: MedlinePlus