Estimating the Microtubule GTP Cap Size In Vivo

Seetapun, Dominique; Castle, Brian T.; McIntyre, Alistair J.; Tran, Phong T.; Odde, David J.

doi:10.1016/j.cub.2012.06.068

Cited by 109 publications

(147 citation statements)

References 39 publications

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“…Comparison of this value to cytosolic concentrations of EB1 suggests that most EB1 is dimeric, consistent with recent measurements in vivo [Seetapun et al, 2012], but raises the possibility that modulation of EB1 dimerization through posttranslational modification or interaction with binding proteins might play a role in the regulation of EB1 activity and thus MT dynamics.…”

Section: Resultssupporting

confidence: 88%

“…Our measured K d of 90 nM for the EB1–EB1 dimerization is near these values and suggests that while most of the EB1 in cytosol is likely dimeric, a portion of the EB1 present in cells may be in the monomeric form, though the exact monomer–dimer ratio is difficult to predict because of differences in buffer conditions and effects such as molecular crowding. Recently, Seetapun et al [2012] concluded that EB1 exists primarily in dimer form in LLCPK1 epithelial cells, in agreement with these conclusions. However, it is interesting to consider the possibility that under some conditions, a shift in the EB1 monomer–dimer ratio could play a role in EB1 regulation, an issue that was also raised by De Groot et al [2010].…”

Section: Resultssupporting

confidence: 69%

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Biochemical evidence that human EB1 does not bind preferentially to the microtubule seam

et al. 2013

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EB1 is a highly conserved microtubule (MT) plus end tracking protein (+TIP) involved in regulating MT dynamics, but the mechanisms of its effects on MT polymerization remain undefined. Resolving this question requires understanding how EB1 interacts with MTs. Previous electron microscopy of the S. pombe EB1 homolog Mal3p suggested that Mal3p binds specifically to the MT seam, implying that EB1 family members promote MT polymerization by stabilizing the seam. However, more recent electron microscopy indicates that Mal3p binds everywhere except the seam. Neither set of experiments investigated the behavior of human EB1, or provided an explanation for why these studies arrived at different answers. To resolve these questions, we have used a combination of MT-binding assays and theoretical modeling with MTBindingSim. Our results indicate that human EB1 binds to the lattice, consistent with the recent Mal3p results, and show that Mal3p-binding assays that were previously interpreted as evidence for preferential seam binding are equally consistent with weak lattice binding. In addition, we used analytical ultracentrifugation to investigate the possibility that the EB1 monomer–dimer equilibrium might contribute to EB1 binding behavior, and determined that the EB1 dimerization dissociation constant is approximately 90 nM. We and others find that the cellular concentration of EB1 is on the order of 200 nM, suggesting that a portion of EB1 may be monomeric at physiological concentrations. These observations lead us to suggest that regulation of EB1 dimerization might play a role in controlling EB1 function.

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Section: Resultssupporting

confidence: 88%

Section: Resultssupporting

confidence: 69%

Biochemical evidence that human EB1 does not bind preferentially to the microtubule seam

et al. 2013

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“…3F), implying a cap of 20-60 tubulin layers long (because 13 tubulin subunits comprise a layer) for our range of experimental conditions. This is in agreement with earlier estimates for microtubules growing in vitro (13,14,19) and in living cells (18). Over the same range of tubulin concentrations the amplitude of the fluctuations increased from 64 to 178 subunits (Fig.…”

Section: Resultssupporting

confidence: 81%

“…These studies revealed that the cap consists of hundreds of tubulins in the stabilizing conformation and that they have a roughly monoexponential distribution starting from the growing microtubule end, giving rise to the comet-shaped appearance of the EB binding region (13,15,17,18). The majority of this EB cap is lost during a period of several seconds before catastrophe occurs (16,19), indicating that the EB binding region is critical for stability.…”

mentioning

confidence: 99%

Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation

Rickman

Duellberg

Cade

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Growing microtubules are protected from depolymerization by the presence of a GTP or GDP/Pi cap. End-binding proteins of the EB1 family bind to the stabilizing cap, allowing monitoring of its size in real time. The cap size has been shown to correlate with instantaneous microtubule stability. Here we have quantitatively characterized the properties of cap size fluctuations during steadystate growth and have developed a theory predicting their timescale and amplitude from the kinetics of microtubule growth and cap maturation. In contrast to growth speed fluctuations, cap size fluctuations show a characteristic timescale, which is defined by the lifetime of the cap sites. Growth fluctuations affect the amplitude of cap size fluctuations; however, cap size does not affect growth speed, indicating that microtubules are far from instability during most of their time of growth. Our theory provides the basis for a quantitative understanding of microtubule stability fluctuations during steady-state growth.microtubules | dynamic instability | GTP cap | EB1 | biochemical network

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“…Trap lifetimes were stochastically set according to timescales typical for microtubule dynamics (on average, τ on ∼ 50 s and τ off ∼ 15 s). It is estimated that, in vivo, up to 200 cytoplasmic Mal3 proteins can bind to a single microtubule tip (28). However, we set a lower maximum to the number of membrane-bound particles that can interact with a simulated microtubule tip in a normal-incidence geometry: we impose that each trap can bind at most 50 membrane-bound particles.…”

Section: Methodsmentioning

confidence: 99%

Chimera proteins with affinity for membranes and microtubule tips polarize in the membrane of fission yeast cells

Recouvreux

Sokolowski

Γραμμουστιάνου

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Cell polarity refers to a functional spatial organization of proteins that is crucial for the control of essential cellular processes such as growth and division. To establish polarity, cells rely on elaborate regulation networks that control the distribution of proteins at the cell membrane. In fission yeast cells, a microtubule-dependent network has been identified that polarizes the distribution of signaling proteins that restricts growth to cell ends and targets the cytokinetic machinery to the middle of the cell. Although many molecular components have been shown to play a role in this network, it remains unknown which molecular functionalities are minimally required to establish a polarized protein distribution in this system. Here we show that a membrane-binding protein fragment, which distributes homogeneously in wild-type fission yeast cells, can be made to concentrate at cell ends by attaching it to a cytoplasmic microtubule end-binding protein. This concentration results in a polarized pattern of chimera proteins with a spatial extension that is very reminiscent of natural polarity patterns in fission yeast. However, chimera levels fluctuate in response to microtubule dynamics, and disruption of microtubules leads to disappearance of the pattern. Numerical simulations confirm that the combined functionality of membrane anchoring and microtubule tip affinity is in principle sufficient to create polarized patterns. Our chimera protein may thus represent a simple molecular functionality that is able to polarize the membrane, onto which additional layers of molecular complexity may be built to provide the temporal robustness that is typical of natural polarity patterns.

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Estimating the Microtubule GTP Cap Size In Vivo

Cited by 109 publications

References 39 publications

Biochemical evidence that human EB1 does not bind preferentially to the microtubule seam

Biochemical evidence that human EB1 does not bind preferentially to the microtubule seam

Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation

Chimera proteins with affinity for membranes and microtubule tips polarize in the membrane of fission yeast cells

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