Proximity labeling reveals non-centrosomal microtubule-organizing center components required for microtubule growth and localization

Sanchez, Ariana D.; Branon, Tess C; Cote, Lauren E.; Papagiannakis, Alexandros; Liang, Xing; Pickett, Melissa A.; Shen, Kang; Jacobs‐Wagner, Christine; Ting, Alice Y.; Feldman, Jessica L.

doi:10.1016/j.cub.2021.06.021

Cited by 44 publications

(49 citation statements)

References 89 publications

(126 reference statements)

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“…This property may help to explain how acentrosomal MTOCs form in cells where both PCM proteins and PCM-independent microtubule stabilizers such as CAMSAPs are present. Two well studied examples of such systems are the MTOCs at the Golgi membranes and the apical cortex of epithelial cells, where CAMSAPs and PCM proteins are present simultaneously and may cooperate with each other or play redundant roles (Goldspink et al, 2017; Nashchekin et al, 2016; Noordstra et al, 2016; Sanchez et al, 2021; Toya et al, 2016; Wang et al, 2015; Wu et al, 2016; Zhu and Kaverina, 2013). We found that CAMSAP2stabilized minus ends can exert a highly dominant effect on PCM organization, and this property likely explains how the Golgi, which anchors CAMSAP2-stabilized microtubules, recruits PCM and becomes the major MTOC in cells lacking centrosomes.…”

Section: Discussionmentioning

confidence: 99%

Self-assembly of pericentriolar material in interphase cells lacking centrioles

Chen

Iwanski

et al. 2021

Preprint

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The major microtubule-organizing center (MTOC) in animal cells, the centrosome, comprises a pair of centrioles surrounded by pericentriolar material (PCM), which nucleates and anchors microtubules. Centrosome assembly depends on the interactions of PCM with centrioles, PCM self-association and dynein-mediated transport. Here, we show that if centrioles are lost due to PLK4 depletion or inhibition, PCM still forms a single centrally located MTOC when non-centrosomal microtubule minus end organization pathways are disabled. Acentriolar MTOC assembly depends on dynein-driven coalescence of PCM clusters with attached microtubule minus ends and requires γ-tubulin, pericentrin, CDK5RAP2 and ninein, but not NEDD1, CEP152 or CEP192. PCM self-assembly is inhibited by AKAP450-dependent PCM recruitment to the Golgi and by CAMSAP2-mediated microtubule minus end stabilization. However, if CAMSAP2 is linked to a minus-end-directed motor, a single MTOC containing PCM components can still form, and its organization depends on the presence of pericentrin. Our results reveal that the formation of a single central MTOC in interphase mammalian cells is not strictly centriole dependent but can be driven by self-organization of PCM and microtubule minus ends.

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Section: Discussionmentioning

confidence: 99%

Self-assembly of pericentriolar material in interphase cells lacking centrioles

Chen

Iwanski

et al. 2021

Preprint

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“…In polarized epithelial cells in flies, worms and mammals, CAMSAP homologs were shown to contribute to the organization of apico-basal microtubule arrays that have their minus-ends anchored at non-centrosomal, apical MTOCs ( Meng et al, 2008 ; Tanaka et al, 2012 ; Wang et al, 2015 ; Nashchekin et al, 2016 ; Ning et al, 2016 ; Noordstra et al, 2016 ; Toya et al, 2016 ). The contribution of CAMSAPs to apical minus-end anchoring may involve their ability to decorate microtubule minus-ends and to interact with spectraplakins that tether microtubules to the cortical actin network ( Khanal et al, 2016 ; Nashchekin et al, 2016 ; Sanchez et al, 2021 ). In the larval epidermis in C. elegans , the CAMSAP homolog PTRN-1 functions redundantly with NOCA-1, a worm ninein homolog.…”

Section: Microtubule Anchoring Factors and Mechanismsmentioning

confidence: 99%

Microtubule Anchoring: Attaching Dynamic Polymers to Cellular Structures

Vineethakumari

Lüders

2022

Front. Cell Dev. Biol.

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Microtubules are dynamic, filamentous polymers composed of α- and β-tubulin. Arrays of microtubules that have a specific polarity and distribution mediate essential processes such as intracellular transport and mitotic chromosome segregation. Microtubule arrays are generated with the help of microtubule organizing centers (MTOC). MTOCs typically combine two principal activities, the de novo formation of microtubules, termed nucleation, and the immobilization of one of the two ends of microtubules, termed anchoring. Nucleation is mediated by the γ-tubulin ring complex (γTuRC), which, in cooperation with its recruitment and activation factors, provides a template for α- and β-tubulin assembly, facilitating formation of microtubule polymer. In contrast, the molecules and mechanisms that anchor newly formed microtubules at MTOCs are less well characterized. Here we discuss the mechanistic challenges underlying microtubule anchoring, how this is linked with the molecular activities of known and proposed anchoring factors, and what consequences defective microtubule anchoring has at the cellular and organismal level.

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“…This protocol describes the steps for conducting biotin-ligase based proximity labeling in living C. elegans using TurboID in intestinal cells ( Sanchez et al, 2021 ). We have also previously shown examples of proximity labeling using miniTurbo ( Branon et al, 2018 ).…”

Section: Before You Beginmentioning

confidence: 99%

“…This protocol is useful for targeted in vivo protein network profiling. For complete details on the use and execution of this protocol, please refer to Sanchez et al (2021) .…”

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confidence: 99%

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A proximity labeling protocol to probe proximity interactions in C. elegans

Sanchez

Feldman

2021

STAR Protocols

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Summary Enzyme-catalyzed proximity labeling (PL) has emerged as a critical approach for identifying protein-protein proximity interactions in cells; however, PL techniques were not historically practical in living multicellular organisms due to technical limitations. Here, we present a protocol for applying PL to living C. elegans using the biotin ligase mutant enzyme TurboID. We demonstrated PL in a tissue-specific and region-specific manner by focusing on non-centrosomal MTOCs (ncMTOCs) of intestinal cells. This protocol is useful for targeted in vivo protein network profiling. For complete details on the use and execution of this protocol, please refer to Sanchez et al. (2021) .

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Proximity labeling reveals non-centrosomal microtubule-organizing center components required for microtubule growth and localization

Cited by 44 publications

References 89 publications

Self-assembly of pericentriolar material in interphase cells lacking centrioles

Self-assembly of pericentriolar material in interphase cells lacking centrioles

Microtubule Anchoring: Attaching Dynamic Polymers to Cellular Structures

A proximity labeling protocol to probe proximity interactions in C. elegans

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