Assembly and regulation of γ-tubulin complexes

Farache, Dorian; Emorine, Laurent J.; Haren, Laurence; Merdes, Andreas

doi:10.1098/rsob.170266

Cited by 57 publications

(56 citation statements)

References 72 publications

(133 reference statements)

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“…In fission yeast, filamentous fungi and metazoans, some GCP2s and GCP3s are replaced in the complex by an additional GCP4, GCP5 and GCP6 proteins and in metazoan γTuRC is a stable complex whose assembly is independent of the recruitment to target structures such as centrosomes (Farache et al, 2018;Lin et al, 2015;Murphy et al, 2001;Oegema et al, 1999;Tovey and Conduit, 2018). The exact stoichiometry and subunit order of human γTuRC is not known.…”

Section: Introductionmentioning

confidence: 99%

Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

Consolati

Locke

Roostalu

et al. 2019

Preprint

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The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. However, the mechanism of its regulation is not understood. Here, we purified human γTuRC and quantitatively characterized its nucleation properties in a TIRF microscopy-based real-time nucleation assay. We find that microtubule nucleation by γTuRC is kinetically inhibited compared to microtubule elongation. Determining the cryo-EM structure of γTuRC at 4 Å resolution reveals an asymmetric conformation with only part of the complex in a 'closed' conformation matching the microtubule geometry. Several factors stabilise the closed conformation. One is actin in the core of the complex and others, likely MZT1 or MZT2, line the outer perimeter of the closed part of γTuRC. The opposed side of γTuRC is in an 'open', nucleation-incompetent conformation, leading to a structural asymmetry, explaining the kinetic inhibition of nucleation by human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.

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Section: Introductionmentioning

confidence: 99%

Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

Consolati

Locke

Roostalu

et al. 2019

Preprint

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“…g-tubulin is not required for MT assembly at the fat body ncMTOC g-tubulin, partnering with g-tubulin complex proteins (GCPs) to form the g-tubulin ring complex (g-TuRC), is widely employed as a MT nucleator or anchor at centrosomes and other MTOCs (Kollman et al, 2011;Lin et al, 2015;Oakley et al, 2015;Muroyama and Lechler, 2017; Sanchez and Feldman, 2017;Farache et al, 2018;Sallee et al, 2018;Tillery et al, 2018). Compared to the larval brain, the fat body expresses significantly less g-tubulin, about 120-fold less ( Figure 9A), but is detectable by IF and is localized to the ncMTOC throughout larval developmental stages ( Figure 9C, D), together with other g-TuRC components including GCP2 and GCP3 ( Figure 6).…”

Section: Centrosomal Proteins Are Localized At the Fat Body Ncmtoc Bumentioning

confidence: 99%

“…Compared to the relatively large number of MT plus-end proteins, few MT minus-end proteins have been identified. Gammatubulin (g-tubulin), a conserved and essential MT minus-end protein that nucleates centrosomal MTs (Kollman et al, 2011;Oakley et al, 2015;Farache et al, 2018), also nucleates and anchors non-centrosomal MTs in many differentiated cell types (Chen et al, 2017;Muroyama and Lechler, 2017;Sanchez and Feldman, 2017;Tillery et al, 2018). The XMAP215/Msps/Stu2/ Dis1/Alp14/ZYG-9/ch-TOG/MOR1 is an ancient family of MT polymerases that was recently identified as a new in vivo MT nucleator at centrosomes and spindle pole bodies through its association with g-tubulin (Flor-Parra et al, 2018;Gunzelmann et al, 2018;Thawani et al, 2018); a role for the MT polymerase at nucleating MTs at ncMTOCs has not been reported.…”

Section: Introductionmentioning

confidence: 99%

A perinuclear microtubule-organizing center controls nuclear positioning and basement membrane secretion

Zheng¹,

Buchwalter²,

Zheng³

et al. 2019

Preprint

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Non-centrosomal microtubule-organizing centers (ncMTOCs) have a variety of roles presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization, and function. Here we report a novel perinuclear MTOC in Drosophila fat body cells that is anchored by Msp300/Nesprin at the cytoplasmic surface of the nucleus. Msp300 recruits the MT minus-end protein Patronin/CAMSAP, which functions redundantly with Ninein to further recruit the MT polymerase Msps/XMAP215 to assemble noncentrosomal MTs and does so independently of the widespread MT nucleation factor g-tubulin.Functionally, the fat body ncMTOC and the radial MT arrays it organizes is essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture and MT regulation properties that serves vital functions. Highlights• A novel perinuclear MTOC in differentiated fat body cells • The predominant nucleator, g-tubulin, is not required at the fat body ncMTOC • Msp300/Nesprin organizes the ncMTOC at the nuclear surface by recruiting Patronin/CAMSAP and the spectraplakin Shot • Patronin cooperates with Ninein to control MT assembly at the fat body ncMTOC by recruiting Msps • Msps, a MT polymerase, is essential for radial MT elongation from the fat body ncMTOC • Patronin and Msps associate • The ncMTOC and radial MTs, but not actin, control nuclear positioning in the fat body • The fat body MTOC controls retrograde endocytic trafficking to regulate plasma membrane growth and secretion of basement membrane proteins Here we report the discovery of an ncMTOC that is assembled on the surface of nuclei in Drosophila larval fat body cells, a differentiated cell type that has critical secretory functions and serves the metabolic needs of the organism. Assembly of this perinuclear ncMTOC requires the Msp300/Nesprin (nuclear envelope spectrin repeat protein) as a primary organizer/anchor. Msp300 recruits Patronin, which functions redundantly with Ninein for MT assembly at the ncMTOC. Patronin and Ninein cooperate to recruit Msps, which is essential for the elongation of MTs from the fat body ncMTOC. Recruitment of Msps and MT assembly at the ncMTOC are independent of g-tubulin. Functionally, this ncMTOC is necessary for: 1) the regulation of nuclear positioning, 2) controlling plasma membrane growth by facilitating dynein/Rab5-mediated retrograde endosomal trafficking of excess plasma membrane, and 3) the secretion of collagen IV and other basement membrane (BM) proteins. Together, this study identifies a unique perinuclear ncMTOC with novel MT assembly mechanisms that controls physiological roles of fat body cells by supporting nuclear positioning and vital secretory functions of fat body cells.

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“…γ-TuRCs assemble in the cytosol but they are typically inactive until recruited to specific sites, such as microtubule organising centres (MTOCs), the cytosol around mitotic chromatin or the sides of pre-existing microtubules via Augmin/HAUS [18][19][20][21][22] . A range of MTOCs exist, including centrosomes, the Golgi apparatus and the nuclear below the epidermis, making them ideal for imaging through the translucent body wall.…”

Section: Introductionmentioning

confidence: 99%

Microtubules originate asymmetrically at the somatic Golgi and are guided via Kinesin2 to maintain polarity within neurons

Mukherjee

Brooks

Bernard

et al. 2019

Preprint

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Microtubules form polarised arrays throughout axons and dendrites necessary for neuronal growth and maintenance. γ-tubulin-ring-complexes (γ-TuRCs) nucleate microtubules at microtubule organising centres (MTOCs), but how microtubules are generated and organised within neurons remains unclear. We show that γ-TuRCs are predominantly recruited to the somatic Golgi, rather than to dendritic Golgi outposts, within Drosophila neurons. Microtubules nucleated from the somatic Golgi grow preferentially towards and into the axon, while growing microtubules that approach dendrites are excluded. Both directed growth and dendritic exclusion depend upon Kinesin-2, which associates with plus ends and directs their growth towards the plus ends of adjacent microtubules. We propose that plus-end-associated Kinesin-2 guides growing microtubules nucleated from the somatic Golgi towards the axon and prevents plus end entry into dendrites when engaging with oppositely polarised microtubules.In summary, microtubules are nucleated from the somatic Golgi within neurons and their guidance is necessary to maintain neuronal microtubule polarity. envelope, and different cells use different MTOCs to help generate and organise their specific microtubule arrays 18 . γ-TuRC recruitment occurs via γ-TuRC "tethering proteins" that simultaneously bind to the MTOC and γ-TuRC, possibly also helping to activate the γ-TuRC. An example is Drosophila Centrosomin (Cnn) and its mammalian homologue CDK5RAP2, which recruit γ-TuRCs to centrosomes during mitosis [23][24][25][26] and whose binding can activate γ-TuRCs 27-29 . γ-TuRCs can be recruited to different MTOCs by different tethering proteins, and it was also recently shown that different isoforms of Cnn can mediate recruitment to different MTOCs 29 . Thus, there is potentially a wide-range of γ-TuRC recruitment mechanisms available and understanding which mechanisms are used in different cells, including neurons, remains poorly understood.Although it is known that γ-tubulin is important 10-14 , it remains unclear how microtubule nucleation is regulated within neurons. During early development of mammalian neurons, the centrosome within the soma nucleates microtubules 30 that are severed and transported into neurites via motor-based microtubule sliding 31 . Microtubules sliding is also important for axon outgrowth in Drosophila cultured neurons 32,33 , and for establishing microtubule polarity [34][35][36][37][38] . Centrosomes are inactivated, however, at later developmental stages 30 and are dispensable for neuronal development in both mammalian and fly neurons 30,39 . No other active MTOCs within the neuronal soma have been described. Nevertheless, microtubules continue to grow within the soma 11,39 , and in mammalian neurons this depends in part on HAUS 11 , which is also important for microtubule growth within axons and dendrites 11,40 . Some MTOCs have been identified within dendrites in different neurons. For example, the basal body, or its surrounding region, within C. elegans ciliated neurons a...

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Assembly and regulation of γ-tubulin complexes

Cited by 57 publications

References 72 publications

Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

A perinuclear microtubule-organizing center controls nuclear positioning and basement membrane secretion

Microtubules originate asymmetrically at the somatic Golgi and are guided via Kinesin2 to maintain polarity within neurons

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