Probing protein interactions in living mammalian cells on a microtubule bench

Boca, Mirela; Kretov, Dmitry A.; Desforges, Bénédicte; Mephon-Gaspard, Alix; Curmi, Patrick A.; Pastré, David

doi:10.1038/srep17304

Cited by 14 publications

(30 citation statements)

References 29 publications

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“…To confine RBPs on microtubules, they were fused to tau (also known as MAPT) ( Boca et al, 2015 ) ( Fig. 1 A,B), a microtubule-associated protein, labeled with either RFP or GFP.…”

Section: Resultsmentioning

confidence: 99%

“…Tau has a higher affinity for polymerized tubulin than for free tubulin, which favors its presence on microtubules rather than in the cytosol ( Lee et al, 1989 ). In addition, its unstructured projection domain serves as a spacer ( Lee et al, 1989 ) to preserve RBP accessibility ( Boca et al, 2015 ). As required in this approach, none of the RBPs investigated in the present study interact by themselves with microtubules.…”

Section: Resultsmentioning

confidence: 99%

“…Vectors for mammalian expression of tau–RFP–RBPs and tau–GFP–RBPs were engineered using the gateway strategy as previously described in detail in Boca et al (2015) . Briefly, all RBPs were fused to the longest isoform of the human tau protein (accession number: ), which has the longest N-terminal projection domain.…”

Section: Methodsmentioning

confidence: 99%

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Microtubules as platforms for probing liquid–liquid phase separation in cells – application to RNA-binding proteins

Maucuer

Desforges

Joshi

et al. 2018

Journal of Cell Science

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Liquid–liquid phase separation enables compartmentalization of biomolecules in cells, notably RNA and associated proteins in the nucleus. Besides having critical functions in RNA processing, there is a major interest in deciphering the molecular mechanisms of compartmentalization orchestrated by RNA-binding proteins such as TDP-43 (also known as TARDBP) and FUS because of their link to neuron diseases. However, tools for probing compartmentalization in cells are lacking. Here, we developed a method to analyze the mixing and demixing of two different phases in a cellular context. The principle is the following: RNA-binding proteins are confined on microtubules and quantitative parameters defining their spatial segregation are measured along the microtubule network. Through this approach, we found that four mRNA-binding proteins, HuR (also known as ELAVL1), G3BP1, TDP-43 and FUS form mRNA-rich liquid-like compartments on microtubules. TDP-43 is partly miscible with FUS but immiscible with either HuR or G3BP1. We also demonstrate that mRNA is essential to capture the mixing and demixing behavior of mRNA-binding proteins in cells. Taken together, we show that microtubules can be used as platforms to understand the mechanisms underlying liquid–liquid phase separation and their deregulation in human diseases.

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“…To confine RBPs on microtubules, they were fused to tau (also known as MAPT) ( Boca et al, 2015 ) ( Fig. 1 A,B), a microtubule-associated protein, labeled with either RFP or GFP.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Microtubules as platforms for probing liquid–liquid phase separation in cells – application to RNA-binding proteins

Maucuer

Desforges

Joshi

et al. 2018

Journal of Cell Science

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“…MAP7 17–282 comprises MAP7’s microtubule binding domain) 57 and then asked whether 3xStrep-GFP-tagged STIL would follow CEP85 to the microtubule cytoskeleton. A comparable system based on the microtubule binding protein Tau was used previously to probe protein–protein interactions in vivo 58 . The results shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Direct binding of CEP85 to STIL ensures robust PLK4 activation and efficient centriole assembly

et al. 2018

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Centrosomes are required for faithful chromosome segregation during mitosis. They are composed of a centriole pair that recruits and organizes the microtubule-nucleating pericentriolar material. Centriole duplication is tightly controlled in vivo and aberrations in this process are associated with several human diseases, including cancer and microcephaly. Although factors essential for centriole assembly, such as STIL and PLK4, have been identified, the underlying molecular mechanisms that drive this process are incompletely understood. Combining protein proximity mapping with high-resolution structural methods, we identify CEP85 as a centriole duplication factor that directly interacts with STIL through a highly conserved interaction interface involving a previously uncharacterised domain of STIL. Structure-guided mutational analyses in vivo demonstrate that this interaction is essential for efficient centriolar targeting of STIL, PLK4 activation and faithful daughter centriole assembly. Taken together, our results illuminate a molecular mechanism underpinning the spatiotemporal regulation of the early stages of centriole duplication.

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“…17 The microtubule-assembly domain, which contains four imperfect sequence repeats, is responsible for the binding of tau to microtubules. Indeed, the microtubule-assembly domain fused with GFP is able to trace microtubules in cells; 18,19 however, it has some adverse effects on microtubule dynamics due to the large size of the fusion protein. To avoid this issue, here we sought to develop a fluorescent tau-derived peptide with small size and delivered it into cells via a cell-penetrating peptide strategy.…”

Section: Introductionmentioning

confidence: 99%

Modified heptapeptide from tau binds both tubulin and microtubules

Liu

et al. 2020

Thoracic Cancer

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Background: Microtubules are the major cytoskeletal component in eukaryotes which are essential for a large spectrum of cellular activities. Monitoring the behavior of microtubules is helpful for a better understanding of the regulatory mechanism governing microtubule architecture and microtubule-based activities. Here, we characterized the binding capability of a modified heptapeptide from tau to both tubulin and microtubules and sought to develop it as a fluorescent peptide for monitoring microtubules. Methods: To deliver the fluorescent peptide into the cells, a cell-penetrating peptide was conjugated to the modified heptapeptide from tau and synthesized. The affinity of the modified heptapeptide was determined by microscale thermophoresis. The microtubule labeling ability was determined by adding the peptide into the polymerized microtubule solutions or cultured HeLa cells.; Results: Affinity determination revealed that the tau-derived peptide specifically bound to tubulin. In addition, the peptide was able to label polymerized microtubules in solution, although no obvious microtubule filaments were observed clearly in living cells, probably due to the inadequate affinity. Conclusions: These results suggest that using a peptide-based strategy for imaging microtubules might be plausible and attempts to improve its affinity is warranted in the future.

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Probing protein interactions in living mammalian cells on a microtubule bench

Cited by 14 publications

References 29 publications

Microtubules as platforms for probing liquid–liquid phase separation in cells – application to RNA-binding proteins

Microtubules as platforms for probing liquid–liquid phase separation in cells – application to RNA-binding proteins

Direct binding of CEP85 to STIL ensures robust PLK4 activation and efficient centriole assembly

Modified heptapeptide from tau binds both tubulin and microtubules

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