27 research outputs found
Ectopic A-lattice seams destabilize microtubules
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
Mechanochemical modeling of dynamic microtubule growth involving sheet-to-tube transition
Microtubule dynamics is largely influenced by nucleotide hydrolysis and the
resultant tubulin configuration changes. The GTP cap model has been proposed to
interpret the stabilizing mechanism of microtubule growth from the view of
hydrolysis effects. Besides, the microtubule growth involves the closure of a
curved sheet at its growing end. The curvature conversion also helps to
stabilize the successive growth, and the curved sheet is referred to as the
conformational cap. However, there still lacks theoretical investigation on the
mechanical-chemical coupling growth process of microtubules. In this paper, we
study the growth mechanisms of microtubules by using a coarse-grained molecular
method. Firstly, the closure process involving a sheet-to-tube transition is
simulated. The results verify the stabilizing effect of the sheet structure,
and the minimum conformational cap length that can stabilize the growth is
demonstrated to be two dimers. Then, we show that the conformational cap can
function independently of the GTP cap, signifying the pivotal role of
mechanical factors. Furthermore, based on our theoretical results, we describe
a Tetris-like growth style of microtubules: the stochastic tubulin assembly is
regulated by energy and harmonized with the seam zipping such that the sheet
keeps a practically constant length during growth.Comment: 23 pages, 7 figures. 2 supporting movies have not been uploaded due
to the file type restriction
Overexpression of mutant cell division cycle 25 homolog B (CDC25B) enhances the efficiency of selection in Chinese hamster ovary cells
Targeted mutation of Cyln2 in the Williams syndrome critical region links CLIP-115 haploinsufficiency toneurodevelopmental abnormalities in mice : erratum
Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly
Bipolar spindle assembly critically depends on the microtubule plus-end-directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus-end-directed motor dynein, or the dynein-binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5-dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus-end-tracking protein CLIP-170. This function of CLIP-170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP-115, a highly homologous protein that lacks the dynein–dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP-170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together
EB1 interacts with outwardly curved and straight regions of the microtubule lattice
EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice. Microtubules assembled in the presence of GTP, different GTP analogues or cell extracts display similarly curved sheets at their growing ends, which gradually straighten as their protofilament number increases until they close into a tube. Together, our data provide unique structural information on the interaction of EB1 with growing microtubule ends. They further offer insights into the conformational changes that tubulin dimers undergo during microtubule assembly and the architecture of the GTP-cap region