Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein

Duellberg, Christian; Trokter, Martina; Jha, Rupam; Sen, Indrani; Steinmetz, Michel O.; Surrey, Thomas

doi:10.1038/ncb2999

Cited by 104 publications

(130 citation statements)

References 45 publications

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“…The individual dynactin and dynein subunits are labelled. (The double band of mCherry‐Lis1 originates from different mCherry maturation states, as previously observed for other mCherry tagged proteins; Duellberg et al , 2014. )…”

Section: Resultssupporting

confidence: 81%

“…Together, these results suggest that in the context of the entire dynactin complex, the CAP‐Gly domain of p150 can be exposed, to allow end tracking of dynein without the requirement of other regulators. These findings go beyond previous work with a p150 fragment (Duellberg et al , 2014) providing insight into the ability of the dynactin complex to mediate dynein end tracking.…”

Section: Resultssupporting

confidence: 54%

“…Purity of both complexes was demonstrated by SDS–PAGE, and the subunit composition of the dynactin complex was verified by mass spectrometry (Fig EV1A). To be able to study simultaneously dynamic microtubule end tracking and processive motility initiation of dynein/dynactin, we adjusted the conditions of previous in vitro reconstitution experiments, using here a buffer that was intermediate in ionic strength compared to previous dynein motility studies on static microtubules (Trokter et al , 2012; McKenney et al , 2014; Schlager et al , 2014) and microtubule end tracking experiments (Duellberg et al , 2014; Roberts et al , 2014) (Materials and Methods). …”

Section: Resultsmentioning

confidence: 99%

“…Its N‐terminal CAP‐Gly domain protrudes from the shoulder of the dynactin complex (Chowdhury et al , 2015; Urnavicius et al , 2015) and binds directly to microtubules (Culver‐Hanlon et al , 2006; Peris et al , 1998; Ross et al , 2006), as well as to EBs (Honnappa et al , 2006). In vitro reconstitution experiments with a p150 fragment showed that the p150 CAP‐Gly domain and the first coiled‐coil of p150, which interacts with the intermediate chain of the dynein complex (Karki & Holzbaur, 1995; Vaughan & Vallee, 1995; King et al , 2003), were sufficient for mediating EB‐dependent end tracking of the human dynein complex (Duellberg et al , 2014). The regulation of the balance between dynein microtubule end tracking and processive motility was, however, not studied in these experiments as this requires the presence of the entire dynactin complex.…”

Section: Introductionmentioning

confidence: 99%

“…Lis1 has been reported to regulate both initiation of minus‐end‐directed motility (Egan et al , 2012; Splinter et al , 2012; Moughamian et al , 2013) and plus‐end tracking of dynein (Coquelle et al , 2002; Splinter et al , 2012) in cells. However, Lis1 was not required for dynein end tracking in a minimal in vitro reconstitution (Duellberg et al , 2014), raising the question as to why Lis1 is needed for dynein end tracking in cells. Lis1 is a homodimeric 45 kDa protein that binds directly to the dynein motor domain (Mateja et al , 2006; Kardon & Vale, 2009), and was reported to induce a more strongly microtubule‐bound state of dynein (Yamada et al , 2008; McKenney et al , 2010; Torisawa et al , 2011), thereby increasing the force produced by dynein (McKenney et al , 2010; Reddy et al , 2016), and slowing down microtubule transport by surface‐immobilised dynein motors (Yamada et al , 2008; Torisawa et al , 2011; Wang et al , 2013).…”

Section: Introductionmentioning

confidence: 99%

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Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

et al. 2017

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Cytoplasmic dynein is involved in a multitude of essential cellular functions. Dynein's activity is controlled by the combinatorial action of several regulatory proteins. The molecular mechanism of this regulation is still poorly understood. Using purified proteins, we reconstitute the regulation of the human dynein complex by three prominent regulators on dynamic microtubules in the presence of end binding proteins (EBs). We find that dynein can be in biochemically and functionally distinct pools: either tracking dynamic microtubule plus‐ends in an EB‐dependent manner or moving processively towards minus ends in an adaptor protein‐dependent manner. Whereas both dynein pools share the dynactin complex, they have opposite preferences for binding other regulators, either the adaptor protein Bicaudal‐D2 (BicD2) or the multifunctional regulator Lissencephaly‐1 (Lis1). BicD2 and Lis1 together control the overall efficiency of motility initiation. Remarkably, dynactin can bias motility initiation locally from microtubule plus ends by autonomous plus‐end recognition. This bias is further enhanced by EBs and Lis1. Our study provides insight into the mechanism of dynein regulation by dissecting the distinct functional contributions of the individual members of a dynein regulatory network.

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Section: Resultssupporting

confidence: 81%

Section: Resultssupporting

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

et al. 2017

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Tubulin mutations in neurodevelopmental disorders as a tool to decipher microtubule function

Fourel

Boscheron

2020

FEBS Letters

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Malformations of cortical development (MCDs) are a group of severe brain malformations associated with intellectual disability and refractory childhood epilepsy. Human missense heterozygous mutations in the 9 α‐tubulin and 10 β‐tubulin isoforms forming the heterodimers that assemble into microtubules (MTs) were found to cause MCDs. However, how a single mutated residue in a given tubulin isoform can perturb the entire microtubule population in a neuronal cell remains a crucial question. Here, we examined 85 MCD‐associated tubulin mutations occurring in TUBA1A, TUBB2, and TUBB3 and their location in a three‐dimensional (3D) microtubule cylinder. Mutations hitting residues exposed on the outer microtubule surface are likely to alter microtubule association with partners, while alteration of intradimer contacts may impair dimer stability and straightness. Other types of mutations are predicted to alter interdimer and lateral contacts, which are responsible for microtubule cohesion, rigidity, and dynamics. MCD‐associated tubulin mutations surprisingly fall into all categories, thus providing unexpected insights into how a single mutation may impair microtubule function and elicit dominant effects in neurons.

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SLK/LOSK kinase regulates cell motility independently of microtubule organization and Golgi polarization

et al. 2016

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Cell motility is an essential complex process that requires actin and microtubule cytoskeleton reorganization and polarization. Such extensive rearrangement is closely related to cell polarization as a whole. The serine/threonine kinase SLK/LOSK is a potential regulator of cell motility, as it phosphorylates a series of cytoskeleton-bound proteins that collectively participate in the remodeling of migratory cell architecture. In this work, we report that SLK/LOSK is an indispensable regulator of cell locomotion that primarily acts through the small GTPase RhoA and the dynactin subunit p150(Glued). Both RhoA and dynactin affect cytoskeleton organization, polarization, and general cell locomotory activity to various extents. However, it seems that these events are independent of each other. Thus, SLK/LOSK kinase effectively functions as a switch that links all of the processes underlying cell motility to provide robust directional movement.

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Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein

Cited by 104 publications

References 45 publications

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

Tubulin mutations in neurodevelopmental disorders as a tool to decipher microtubule function

SLK/LOSK kinase regulates cell motility independently of microtubule organization and Golgi polarization

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