Available via license: CC BY 4.0
Content may be subject to copyright.
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
Received: 14 July 2018
Revised: 05 September 2018
Accepted: 13 September 2018
Version of Record published:
12 October 2018
Review Article
Microtubule nucleation by γ-tubulin complexes and
beyond
Corinne A. Tovey and Paul T. Conduit
Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U.K.
Correspondence: Paul T. Conduit (ptc29@cam.ac.uk)
In this short review, we give an overview of microtubule nucleation within cells. It is nearly
30 years since the discovery of γ-tubulin, a member of the tubulin superfamily essential for
proper microtubule nucleation in all eukaryotes. γ-tubulin associates with other proteins to
form multiprotein γ-tubulin ring complexes (γ-TuRCs) that template and catalyse the oth-
erwise kinetically unfavourable assembly of microtubule laments. These laments can be
dynamic or stable and they perform diverse functions, such as chromosome separation
during mitosis and intracellular transport in neurons. The eld has come a long way in un-
derstanding γ-TuRC biology but several important and unanswered questions remain, and
we are still far from understanding the regulation of microtubule nucleation in a multicellular
context. Here, we review the current literature on γ-TuRC assembly, recruitment, and activa-
tion and discuss the potential importance of γ-TuRC heterogeneity, the role of non-γ-TuRC
proteins in microtubule nucleation, and whether γ-TuRCs could serve as good drug targets
for cancer therapy.
Introduction
Microtubules are polarisedpolymers involvedin a widerangeofcellular processes including chromosome
separation, intracellular transport, organellepositioning,cell–cell signalling andcell motility [1].Tight
spatialandtemporalregulation of the formation, organisation, anddynamic behaviour of the microtubule
cytoskeleton is extremelyimportant.Consequently, cellshavedevelopedcomplex mechanisms to regu-
late microtubulenucleation, polymerisation andcatastrophe, severing, stabilisation andtransportation.
Collectively, these processes establish diverse microtubulenetworkswithhighlyspecialisedfunctions in
different cellsoratdifferent times during the cell cycle. Multiprotein γ-tubulin ring complexes (γ-TuR Cs)
template microtubulenucleation within cells(Figure1)[2-5].Theγ-tubulin molecules within this com-
plex are positionedin a single-turn helicalpattern [6]andthe end-on interactions between γ-tubulin and
α/β-tubulin dimers most likelyhelptosupportthelateralassociation between α/β-tubulin dimers dur-
ing their assemblyintoprotofilaments;this is thought to promote the otherwise kineticallyunfavourable
formation of a short tubular structure (the microtubuleseed), which, once beyonda certain size threshold,
can rapidlypolymerise into a microtubulefilament ([3,7]; Figure 1). γ-Tu R Csappeartoregulate micro-
tubulepolarity, as they are always positionedat the α-tubulin-containing ‘minus end’ with the highly
dynamic β-tubulin-exposedplus endextending outwards(Figure1). Microtubulenucleation must be
highlyregulatedin space andtime to ensure the correct formation of microtubulenetworks[8,9].Thus,
γ-TuRCsarenormallyactivatedonlyoncerecruitedto specific sites within the cell known as microtubule
organising centres (MTOCs). How γ-TuR Csareassembled,recruitedandactivatedare still key areas of
interest in the field.
c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
1
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
Figure 1. Templated microtubule nucleation
(A)γ-tubulin molecules (yellow) within the γ-TuRC are positioned in a single-turn helix via their binding to GCP proteins (blue). (B)
γ-tubulin molecules bind to incoming α/β-tubulin dimers from the cytosol and this is thought to promote the lateral interaction
between the α/β-tubulin dimers as they grow into protolaments (a protolament is a single end-to-end chain of tubulin dimers).
(C) Microtubule assembly progresses slowly through an unstable stage where disassembly is more likely than continued assembly
(as indicated by the thickness of the two-way arrows). (D) Assembly is thought to reach a stable stage, where a microtubule seed
containing sufcient tubulin dimers has formed (although the size of this stable seed remains unclear). (E) Once the stable seed
has formed, microtubule polymerisation is favoured and can progress rapidly. Abbreviation: GCP, γ-tubulin complex protein.
γ-TuRC composition
The γ-tubulin small complex
The core subunit of the γ-TuRCis the γ-Tubu lin Small Complex (γ-TuSC), a heterotetramer of ∼300 kDa containing
two molecules of γ-tubulin andone each of γ-tubulin complex protein 2 (GCP2) andGCP3 [10-14] (Figure 2A). In
budding yeast, seven γ-TuSCsformasingle-turn helix template with approximatelythesamepitchanddiameter as a
microtubule[6].Asimilar process likely occurs in higher eukaryotes, but other types of GCPmolecules are predicted
to replace some of the GCP2/3molecules within the ring [15] (see below). GCP2 andGCP3 are conservedin all
eukaryotes andare essentialfor cell viability in all organisms in which they have been studied[10,12,16-22].Thereis
even some degree of functionalconservation between species, as the homologues of GCP2 andGCP3 in fission yeast
canbereplacedto some extent by their human or budding yeast counterparts [23].
GCP proteins and the γ-TuRC
Many species possess three additionalGCPproteins,GCP4,5and6(Table1), which share sequence andstructural
similarity with GCP2 andGCP3 [5,14,24,25].GCPproteinshavespecies-specific names, so for simplicity we will use
the human nomenclature for all species. The structuralsimilarity of GCP4,5and6withGCP2/3, together with their
low stoichiometry within the γ-Tu R C[25-27],suggeststhatGCP4,5and6replace some of the GCP2/3molecules in
the γ-TuRCring ([3,5,24]; Table1andFigure 2A). This hypothesis is supportedby the co-fractionation of GCP4and
GCP5with the γ-TuSC[28] anddomain swapping andFRET-basedinteraction experiments [29].GCP2–6possessa
Grip1andaGrip2 domain (Figure 2B);the N-terminalGrip1domain appears to mediate lateralinteractions between
GCPproteins,whiletheC-terminalGrip2 domain associates with γ-tubulin [21,22,30,31] andcan be swappedbe-
tween GCPproteins[29].Although the precise function andposition of GCP4,5and6remainunclear (Figure 2C),
their removalin different systems reduces γ-TuRCabundance in cytosolic extracts [19,28,32,33],suggestingarole
in γ-TuRCassembly. Chargedregions in GCP4may prevent bidirectionallateralcontacts with other GCPproteins,
suggesting that GCP4is involvedin ring initiation or termination [15].Nevertheless, unlike GCP2 and3, GCP4,5
2c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
(A)
(B)
(C)
(D)
Figure 2. Non-core γ-TuRC components
(A) The canonical γ-TuSC comprises two molecules of γ-tubulin and one each of GCP2 and GCP3, but alternative γ-TuSCs may
exist in which GCP2, GCP3, or both are replaced by either GCP4, 5, or 6. (B) GCP2–6 all contain a Grip1 and a Grip2 domain.
The Grip1 domain mediates interactions between GCP proteins, while the Grip2 domain mediates interactions with γ-tubulin. In
addition, GCP6 contains an expanded central region that includes nine 27-aa repeats of unknown function. (C) GCP4, 5 and 6
(depicted here in purple) are predicted to replace some of the GCP2 and GCP3 molecules within the ring, but their exact positions
remain unknown. MZT1 binds to the N-terminal regions of GCP proteins and acts as an adapter protein for the binding of the
tethering protein NEDD1 (left) and tethering proteins that contain an N-terminal CM1-domain, such as CDK5RAP2 (right). NEDD1
contains putative WD40 repeats that form a β-propeller structure known to mediate protein–protein interactions (presumably with
proteins at MTOCs). The structure of the C-terminus of NEDD1 is currently unknown but is required for binding to the γ-TuRC. The
positions of NME7 kinase, MZT2 and LGALS3BP remain unknown. (D) The function of the extra sequence in GCP6 is unknown,
but may provide a binding site for a GCP6-specic tethering protein (top) or may function in ring assembly by forming interactions
with other γ-TuRC components (bottom). Abbreviations: CM1, centrosomin motif 1; MZT1, MOZART1; MZT2, MOZART 2.
c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
3
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
Tab l e 1 Orthologues of proteins involved in microtubule nucleation in selected species
Category Homo sapiens
Drosophila
melanogaster
Arabidopsis
thaliana
Caenorhabditis
elegans
Candida
albicans
Aspergillus
nidulans
Schizosaccharomyces
pombe
Saccharomyces
cerevisiae
γ-TuSC γ-tubulin γ-tubulin
23C/37C
TUBG1/2 Tbg1 Tub1 mipA Tug1/Tubg1 Tub4
GCP2 Grip84 Spc97p/GCP2 Grip1/Gip1 Spc97 GCP2/B Alp4 Spc97
GCP3 Grip91 Spc98p/GCP3 Grip2/Gip2 Spc98 GCP3/C Alp6 Spc98
γ-TuRC GCPs GCP4 Grip75 GCP4 ? - GCP4/D Gfh1 -
GCP5 Grip128 GCP5 ? - GCP5/E Mod21 -
GCP6 Grip163 GCP6 ? - GCP6/F Alp16 -
γ-TuRC other NEDD1/GCP-WD Grip71 Nedd1 ? - - - -
MOZART1 Mozart1 GIP1a/b Mzt1 Mzt1 Mzt1/MztA Mzt1 -
MOZART2A --? --- -
MOZART2B
NME7 Nmdyn-D7 ? - ? ? ? ?
LGALS3BP ? ? ? ? ? ? ?
CM1-domain
γ-TuRC tethering
CDK5RAP2,
Myomegalin,
Pericentrin
Cnn, (Plp - no
CM1 domain)
? ? Spc110,
Spc72
PcpA, ApsB Pcp1, Mto1/Mto2 Spc110, Spc72
γ-TuRC-
independent
microtubule
nucleators
chTog/CKAP5 Msps ? Zyg9 ? ? Alp14 Stu2
TPX2 Mei38 Tpx2 Tpxl-1 - - - -
The table shows orthologues of γ-TuRC proteins, CM1-domain-containing γ-TuRC tethering proteins and γ-TuRC-independent proteins involved in
microtubule nucleation across a selection of species, as indicated. ‘-’ refers to cases where attempts in the literature have failed to identify an orthologue
and ‘?’ refers to cases where we are unaware of any attempts to identify an orthologue. Abbreviation: CM1, centrosomin motif 1.
and6arenotessentialfor viability in Drosophila,fissionyeastorAspergillus [19,32,34,35],showingthatproper
assemblyofγ-TuRCs(atleast in the cytosol)isnotrequiredfor a large proportion of γ-TuRCfunction.Thisismost
likelybecauseγ-TuRCscanassembleintheabsenceofGCP4,5and6atMTOCs(assuggestedin [32]), similar to the
naturalsituation in yeast cells(seebelow). One key function of GCP4,5and6 in higher eukaryotes may, therefore, be
to providegreaterγ-TuRCdiversity in the presence of a wider range of MTOCs(suggestedin [5]). For example, GCP6
is involvedin the localisation of γ-TuR Cs to keratin fibres in epithelialcells [36],andGCP4andGCP6 are required
in both maleandfemalegermlines in Drosophila [32,35,37,38].Very littleisknownabouttheadditionalsequences
foundin GCP5andGCP6 (which, in GCP6, contains nine 27-aa repeats) andit is possible that these regions form
important interactions either within the γ-TuRCstructure or with tethering or modifying proteins at MTOCs(Figure
2D). Elucidating exactly how the extra GCP proteins integrate into γ-TuR Cswill shedlight on the relevance of these
non-essentialproteins.
Other γ-TuRC proteins
In organisms other than budding yeast, γ-Tu R Cscontainadditionalproteins. The first was discoveredin Drosophila
andnamedDgrip71WD [39](now known simplyasGrip71). Despite its name, Grip71 lacks Grip domains andin-
steadcontains N-terminalWD40repeats (predictedto form a β-propeller structure that mediates protein–protein
interactions) andaC-terminaldomain with unknown structure requiredfor Grip71’s association with the γ-TuR C
(Figure 2C)[40-42].Grip71 andits mammalian homologues (NEDD1,alternativelynamedas GCP-WD)havebeen
extensivelystudied[32,39-56],collectively showing that these proteins are dispensableforγ-TuRCassemblyand
insteadfunction in γ-TuR Crecruitment to different MTOCs.
More recently, additionalcomponents of the γ-Tu R Cwere identified: MOZART1(MZT1), MOZART2 (MZT2),
LGALS3BP andthekinaseNME7( [26,27,57-59]; Ta b le1andFigure 2C). Ofthese,MZT1is the most widelycon-
served(Table1)andhas been the most extensivelystudied[28,38,58,60-67].MZT1homologues are small (∼8.5kDa)
andcomprise just three α-helices [65,66]; studies in plants, yeast andculturedhuman cellshaveshownthatMZT1
interacts directlywiththeN-terminalregion of GCP proteins (i.e. at the base of the γ-TuRC)[28,60-65] andthat they
regulate the interaction between the γ-Tu R Candγ-Tu R C-tethering proteins [28,64].Inall three systems, removalor
knockdown of MZT1impairs γ-TuR Crecruitment to various MTOCsandcellsfailin mitosis [28,58,60-64,66,67].
4c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
Surprisingly, how e ver, MZT1is not essentialin Drosophila andis expressedonly in the testes, where it is impor-
tant for γ-TuRCrecruitment to basalbodies, but not to mitochondria, in developing sperm cells[38].Itremainsto
be testedwhether MZT1also defines specific subsets of γ-TuRCsinothermulticellular animalsystems, andwhat
impact this might have on γ-TuR Crecruitment andfunction. Although MZT1is clearlyinvolvedin γ-Tu R Cre-
cruitment in cells where it is expressed,thereareconflicting reports regarding a possibleroleforMZT1in γ-Tu R C
assembly[28,64].
Less is known about the other newlydiscoveredγ-TuRCproteins. MOZART2A and2B are foundonlyindeuteros-
tomes;they have no sequence relation to MZT1but are also small (∼16.5kDa) andare involvedin γ-TuRCrecruit-
ment, although potentiallyonlyduring interphase [26].ThekinaseNME7is requiredfor efficient nucleation from
centrosomes in human cellsandincreases the in vitro nucleation activity of purifiedhuman γ-TuRCs[68].Thisin-
crease,however,isrelativelysmall andso other mechanisms may work synergisticallywithNME7phosphorylation.
Whether NME7kinases function at γ-TuR Cs in other systems remains to be tested.Nostudyhasyetinvestigateda
potentialroleforLGALS3BP at γ-TuR Cs, but its expression levelsaffectcentrosomenumberandstructure [59].In
summary, more work is neededbefore we have a full understanding of these additionalγ-TuRCcomponents.
γ-TuRC assembly, recruitment and activation
Assembly of the γ-TuRC ring
Inthepast,different modelsofγ-tubulin complex-mediatedmicrotubulenucleation (template comparedwith
protofilament) hadbeen proposed[69,70]. The consensus now is that the template modelis correct andthat micro-
tubulenucleation is catalysedafter multipleγ-TuSCsassemble, often with other proteins, into ring-like structures.
These structures can be regardedas γ-TuRCs, regardless of whether they contain γ-TuRC-specific components. One
key question is how γ-Tu R Csassemble. This is complex because assemblymayoccurinacell- or MTOC-specific
manner. Inhighereukaryotes,suchashumans,Xenopus andDrosophila,γ-Tu RCscanassembleinthecytosolbe-
fore being recruitedto an MTOC.Thiscytosolic assemblydepends, to potentiallydiffering degrees, on GCP4,5and6
[28,29,32,33,35] and,insomecell types, on Mzt1[64].Infissionyeast,despitethepresenceofaMzt1homologue and
GCP4,5and6homologues, γ-Tu R Csarelargelyabsentfromthecytosol[34] andso presumablyformspecificallyat
MTOCs;here, assemblyiscatalysedby the binding of the Mto1/Mto2 complex (see below) [71].Similarly, in bu dding
yeast, which contains onlyγ-tubulin, GCP2 andGCP3, γ-Tu R Csareabsentfromthecytosolandassemblyoccurs
exclusivelyatSpindlePoleBodies (SPBs, the yeast equivalent of centrosomes), stimulatedby the binding of Spc110
(see below) [6].Thus,atleast two classes of γ-TuRCassembly exist:cytosolic assemblyan
dMTOC-specific assembly,
andit remains possiblethatbothclasses coexist within the same species or cell type.
Recruitment to MTOCs
γ-TuRCscanberecruitedto different MTOCssuchasthecentrosome,Golgi, nuclear envelope, cell cortex, mito-
chondria, andeven the sides of pre-existing microtubules. This dependsontheorganism,cell type andcell cycle
stage, andneedstobetightlyregulatedto ensure correct microtubulenetworkformation.Consequently, t h ere ar e
avarietyofproteinsthatcanrecruitγ-TuRCstodifferent MTOCs(thatwetermγ-TuRC-tethering proteins), in-
cluding yeast Spc110,Spc72, Mto1andPcp1,Drosophila Cnn andGrip71 andmammalian CDK5RAP2, Myome-
galin, Pericentrin andNEDD1.γ-TuRCscanalso be recruitedto different MTOCs by the same tethering protein.
For example, NEDD1 recruits γ-TuRCsbothtocentrosomesandto the sides of pre-existing microtubules (via the
multiprotein Augmin/HAUScomplex) in human cells[40,41,46,72-75],and,whilethemajor isoform of Drosophila
Cnn recruits γ-TuRCstocentrosomesindividing cells[76-80],testes
-specific isoforms of Cnn recruit γ-Tu R Csto
mitochondria in sperm cells[81].Therearealso differences between species because the Drosophila homologue of
NEDD1,Grip71,isimportantforγ-Tu R Crecruitment to spindles (via Augmin) but not to centrosomes [32,47,82].
Most γ-TuR C-tethering proteins (except the Grip71/NEDD1 homologues) contain an N-terminalcentrosomin mo-
tif 1(CM1)domain of ∼60amino acids[83,84],whichisnecessaryforbinding andrecruiting γ-Tu R CstoMTOCs
[27,64,76,85-87].Evidence from the structure of Spc110-boundγ-TuRCssuggeststheCM1 domain bindsdirectly
to the ring of GCPproteins[6],although this remains unproven. The CM1 domain is also necessary for γ-TuR C
assemblyinyeast[71,87] andfor ectopic activation of γ-TuRCs in human andfission yeast cells[27,71].Thus,the
CM1 domain is capableoflinking the processes of γ-Tu R Crecruitment, assembly, andactivation.
Given the roleoftheCM1 domain in γ-Tu R Cactivation (see also below), it seems sensiblethatCM1-domain
proteins bindγ-Tu R CsonlyatMTOCs, which presumably explains why endogenous CM1-domain proteins donot
readilyco-purify with γ-Tu RCsfromcytosolic extracts [14,25,39,58].Incontrast,Grip71/NEDD1 homologues do
c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
5
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
co-purify with γ-TuRCsandso dobindγ-Tu R Csinthecytosol[39-41].Thereasonforthisdifference is unclear, es-
peciallyasGrip71 andNEDD1 are not requiredfor cytosolic γ-TuRCassembly [32,40,41].Intriguingly, the b inding
of CM1-domain proteins andGrip71/NEDD1 homologues is mutuallyexclusive, at least in some cell types [27,49],
suggesting that they bindto the γ-TuR Cin a similar region. It is therefore possiblethatGrip71/NEDD1 homologues
boundto γ-TuR Csmaskthebinding of CM1-domainproteinsinthecytosolin order to prevent premature γ-TuRC
activation. Alternatively, the CM1-domain proteins may be unabletobindγ-Tu R Csinthecytosoluntilthey undergo
MTOC-specific post-translationalmodifications. Importantly, bot h p o ssibi lities would helpavoidthe premature ac-
tivation of γ-TuRCs. Moreover, it may be important to regulate closely which tethering protein bindstheγ-Tu R C,
as NEDD1-boundγ-TuRCsservetoanchormicrotubu
les whileCDK5RAP2-boundγ-TuR Csnucleate microtubules
in mouse keratinocytes [49].Itwill be important in future to understandmore about how the binding of different
γ-TuRC-tethering proteins can regulate γ-TuRCrecruitment andfunction.
Regulation by phosphorylation and isoform expression
Many phosphorylation sites have been identifiedin various γ-Tu R Ccomponents [36,88-95] andγ-TuRCtether-
ing proteins, including Spc110 [84,91,96-100],NEDD1 [41,46,50-54,94,101],Grip71 [95], Pericentrin [102,103],
CDK5RAP2 [94,103],Cnn [95,104-107] andMto2 [108].Severalof these sites have been characterisedandhave
been shown to play roles in γ-TuR Cassembly, recruitment or activation, including at specific MTOCs. For exam-
ple, phosphorylation of Ser405 in NEDD1 is requiredfor chromosomalmicrotubulenucleation, but not centrosomal
nucleation, in Xenopus egg extracts [50],while other phosphorylation sites in NEDD1 regulate its binding to the
γ-TuRC[51,54]. Phosphorylation can also negativelyregulate γ-TuRCrecruitment andactivity, as phosphorylation
of GCP6 by CDK1inhibits its binding to intermediate filaments in culturedhuman epithelialcells[36],andhyper-
phosphorylation of Mto2 in fission yeast during mitosis leadstothedisassemblyoftheMto1/Mto2 complex and
subsequent inactivation of γ-TuRCsatnon-SPB sites [108].It is now important to try andunderstandexactlywhat
effects these, andother, phosphorylationeventshaveontheγ-Tu RCto induce recruitment, activity or assembly, for
examplebyinducing structuralchanges in the γ-Tu R C.Itwill also be important to understandhow phosphorylation
can finely tune nucleation events in a cell- andMTOC-specific manner.
As mentionedabove, the γ-TuRC-tethering proteins that contain a CM1 domain typicallyassociatewithγ-Tu RCs
onlyatMTOCs, andphosphorylation is a plausiblemechanismtocontrolthis. For example, phosphorylation of
Spc110 regulates its binding to γ-TuRCstosomedegree [84] (although Spc110 oligomerisation is clearlyalso impor-
tant [87]). Whether phosphorylation regulates the binding of other CM1-domain proteins remains unclear, but there
is circumstantialevidence that this occurs in Drosophila.Drosophila Cnn is phosphorylatedspecificallyatcentro-
somes [104] where it is important for γ-TuR Crecruitment [76-80],andunphosphorylatedCnn does not associate
with γ-TuRCsinin vitro binding assays [38].How phosphorylation might regulate binding is unclear;it has been sug-
gestedthat the extreme N-terminalregion of Cnn foldsbackandinhibits the CM1 domain, as this N-terminalregion
is absent from testes-specific isoforms of Cnn that are abletobindγ-Tu R Csin vitro [38].Thus,theaddition of nega-
tivelychargedphosphate groups may help expose the CM1 domain to allow γ-Tu R Cbinding. A similar region in yeast
Spc110 could also be regulatory, as it is absent from the structure of Spc110-boundγ-TuR Cs[6]andit is not essential
for γ-TuRCbinding [87].Thissuggeststhatsimilar regulatory regions may exist in other CM1-domain proteins, but
more work is neededto test these modelsandrevealany conservation across species. Given the recent findings that
different Cnn isoforms binddifferentlytoγ-TuRCs[38] andrecruit γ-TuRCstodifferent MTOCs[81],itwill be
important to explore potentialisoform differences in other γ-TuRC-tethering proteins andin Cnn homologues in
different species, particularlyasdifferent CDK5RAP2 isoforms do exist [109-111].Itislikely that a combination of
phosphorylation andisoform differences contributes to the tight spatiotemporalregulation of γ-TuRCrecruitment
andactivation.
Activation of γ-TuRCs
The assemblyoftheγ-Tu R Calone is thought to be insufficient for it to promote microtubulenucleation;it must also
be activated. Purifiedγ-Tu R Cshaverelativelylow nucleating activity, but this increases when they are mixedwith
fragments of tethering proteins that contain the CM1 domain [27].When these truncatedfragments are expressedin
cells, ectopic microtubules are nucleatedthroughout the cytoplasm [27].How the binding of CM1-domain proteins
induces γ-TuRCactivity is not known, but the structure of the budding yeast γ-TuR Csuggests that a flexiblelinker
region in GCP3 must move in order to position the γ-tubulin molecules correctlyformicrotubuleassembly[6].This
structure, however, was generatedfrom Spc110-boundγ-TuR Cs, suggesting that binding of a CM1-domain protein
6c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
is not sufficient to move the GCP3 linker, at least in budding yeast. Moreover, artificiallyinducing structuralrear-
rangements that better positionedthe γ-tubulin molecules resultedin onlyapproximatelytwo-fold enhancement of
nucleation activity [112]. Thus, other mechanisms must exist, one of which is phosphorylation. Mimicking phospho-
rylation of Saccharomyces cerevisiae Spc110 increases nucleation activity approximatelythree-fold [84],andNME7
kinase increases the nucleation activity of human γ-TuRCsbyapproximately2.5-fold [68].Whether these phospho-
rylation events leadto a conformationalchange in the γ-Tu R C, or bring about some other process, is not known. In
summary, it is likelythat,in vivo, a combination of allosteric regulation by CM1-domain proteins andphosphory-
lation helps to activate γ-TuRCs. Itispossiblethatdifferent activation mechanisms function in different cellsandat
different MTOCsandhowthisisregulatedremains an exciting area for future research.
γ-TuRC heterogeneity
All eukaryotes express the core γ-TuR Ccomponents γ-tubulin, GCP2 andGCP3, but not necessarilyall of the ad-
ditionalcomponents. For example, the Candida albicans genome contains onlyMZT1in addition to the core com-
ponents [64],whileDrosophila additionallycontainsGCP4,GCP5,GCP6, NEDD1/Grip71,MZT1andNME7[5].
The classicalview is that all γ-TuR Cs within the same organism have the same protein composition, but this has now
been disproved.Thefirstevidence came from studies showing that not all mammalian γ-TuR CscontainNEDD1
[27,62].Thisledto the suggestion that subpopulations of γ-Tu R Cs may exist [5].ItwasthenshownthatNEDD1
andCDK5RAP2 boundmutuallyexclusivelytoγ-Tu R Csinmousekeratinocytesandthat NEDD1-boundγ-TuR Cs
functionedto anchor microtubules whileCDK5RAP2-boundγ-TuRCsnucleatedmicrotubules (Figure 3A) [49].
Most recently, it wa s s hown t h at Drosophila MZT1is expressedonlyinthetestesandis present in, andrequiredfor,
the γ-TuRCs that are recruitedto basalbodies, but not mitochondria, in sperm cells (Figure 3B) [38].Thedifferential
association of Mzt1with onlyasubsetofγ-Tu R Csmayalso occur in Arabidopsis thaliana [62],although this anal-
ysis is complicatedbythepresenceoftwoMZT1 genes in plants. Collectively, these st u dies have now shown beyond
doubt that γ-Tu R Ccomposition can vary between tissues andeven between MTOCswithinthesamecell,andthat
this can influence γ-TuR Crecruitment andfunction.
What about other γ-TuR Ccomponents?Current data does not rule out that they could also confer γ-TuR Chetero-
geneity (Figure 3C). This can be difficult to test, as experiments typicallyassaythewholepopulation of γ-TuRCs. For
example, whileGCP4,5or 6 can co-immunoprecipitate with each other in all pairwise combinations from human cell
extracts [25],itremainspossiblethatdifferent subsets coexist, e.g. GCP4/5-onlycomplexes, GCP5/6-onlycomplexes
andGCP4/6-onlycomplexes. Recent FRET andcross-linking experiments in HeLa cellsdetecteddirect interactions
between GCP4andGCP5,suggestingtheseproteinsareadjacent within γ-TuRCs, but the data cannot ruleoutthat
these interactions took place in onlyasubsetofcomplexes [29].Indeed, a comparison of protein levelsafterimmuno-
precipitating GCP6 or γ-tubulin from HeLa cellsshowsthatsimilar amounts of GCP5are co-immunoprecipitated
but that much less GCP4is co-immunoprecipitatedwith GCP6 [58], suggesting that some complexes contain GCP5
andGCP6 but not GCP4.Moreover, GCP6 can localisetoSPBsintheabsenceofGCP4andGCP5in both fission
yeast [67] andAspergillus nidulans [19]andGCP6-containing γ-TuR Cscanbedetectedin extracts that have been
depletedof GCP4or GCP5[28],suggestingthatGCP6-onlycomplexes can exist. Most data highlight GCP6 as being
more important for γ-TuR CassemblythanGCP4or GCP5[19,28,67],butγ-Tu R Cs purifiedfrom human embryonic
kidney cellsusingCDK5RAP2 fragments contain sub-stoichiometric levelsofGCP6, i.e. <1moleculeperγ-TuR C
[27],suggestingthatnotall γ-Tu R CscontainGCP6. This may reflect differences in the composition of γ-TuRCs
between cell types or between γ-TuR Csboundby different tethering proteins or may simplyreflect the difficulty of
measuring the stoichiometry of the γ-Tu R Ccomponents. Clearly more work is requiredto see whether other types
of γ-TuRCheterogeneity reallydo exist andwhat functionalrelevance this might have.
Other microtubule nucleation factors
Itisnowbecomingclear that two types of non-γ-TuR Cproteins can promote microtubulenucleation:Tog domain
proteins, such as XMAP215,andhomologues of TPX2[113-117].Togdomain proteins are microtubulepolymerases
that regulate microtubule growth by promoting the longitudinaladdition of tubulin dimers via interactions between
tubulin andthe Tog domains, andit is now thought that this also occurs during the earlystagesofmicrotubulenucle-
ation [113-118].Strongevidence suggests that this involves interactions between Tog domain proteins andγ-Tu R Cs,
as budding yeast Stu2 forms a complex with Spc72-boundγ-TuRCs[119],fissionyeastAlp14 co-immunoprecipitates
with γ-TuRCcomponents [116],andthe C-terminaldomain of XMAP215 bindsγ-tubulin [117].Anelegant model
has been proposed[118] in which the γ-Tu R Cmediates the lateralinteractions between tubulin dimers andsets the
13-protofilament lattice structure, whileTogdomain proteins, boundto the γ-Tu R Cvia their C-terminaldomain,
c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
7
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
Figure 3. Known and potential forms of γ-TuRC heterogeneity
(A) In mouse keratinocytes, γ-TuRCs are bound mutually exclusively by either NEDD1 or CDK5RAP2, suggesting that both tethering
proteins bind to a similar region of the γ-TuRC. NEDD1-bound γ-TuRCs serve to anchor microtubules while CDK5RAP2-bound
γ-TuRCs nucleate microtubules. The position of GCP4, 5 and 6 within γ-TuRCs in these cells remains unknown. (B)InDrosophila,
most γ-TuRCs do not contain MZT1 (red), which is expressed only in the testes. Within the testes, MZT1 is predominantly expressed
in sperm cells but in early elongating sperm is only present in γ-TuRCs that are recruited to the basal body, and is not present in
γ-TuRCs recruited to mitochondria. The position of GCP4, 5 and 6 within Drosophila γ-TuRCs remains unknown. (C) While the
position of GCP4, 5 and 6 within γ-TuRCs remains unknown, positive FRET data in HeLa cells suggest that GCP4 and GCP5
are adjacent to each other within the ring, while negative FRET data suggest GCP6 is not adjacent to either GCP4 or GCP5 (left)
[29]. Stoichiometry measurements from HEK293T cells and immunoprecipitation experiments from HeLa cells suggest that some
complexes do not contain GCP6 (middle left) [27] and that some complexes do not contain GCP4 (middle right) [58] respectively.
GCP6 can still associate with γ-TuRCs in the absence of GCP4 or GCP5, suggesting that some complexes can form with only
GCP6 (right) [28].
8c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
(A) (B) (C)
Figure 4. Different modes of microtubule nucleation
(A)Theγ-TuRC templates the addition of tubulin dimers to form a microtubule, but is predicted to promote only lateral and not lon-
gitudinal interactions between tubulin dimers. (B) Under certain conditions, the combination of tubulin dimers and microtubule-as-
sociated proteins (MAPs) is sufcient to promote microtubule nucleation. Tog domain proteins help polymerise the microtubule
by promoting the longitudinal addition of tubulin dimers. TPX2 homologues bind across tubulin dimers within the lattice and help
prevent catastrophe of the nascent microtubule seed. (C)In vivo, it is likely that a combination of a γ-TuRC and these MAPs
drive highly efcient microtubule nucleation. This presumably occurs at centrosomes, where all of these proteins concentrate, but
whether other MTOCs (that are less-efcient microtubule nucleators) use specic mechanisms remains unknown.
promote the longitudinaladdition of tubulin dimers. TPX2homologues, however, are also important for microtubule
nucleation. ItwasshownthatXenopus TPX2 promotes the phosphorylation of NEDD1 by Aurora A to stimulate
microtubulenucleation [101],butithasrecentlyemergedthat TPX2homologues also help prevent catastrophe of
nascent microtubuleseeds[113-115,120].Recentstructuraldata show that TPX2 proteins bindacross longitudinal
andlateraltubulin interfaces within the microtubulelattice [121]. Thus, microtubulenucleation is likelymostrobust
inthepresenceoftheγ-Tu R C,aTogdomain protein andaTPX2homologue (Figure 4).
WhileXMAP215 andTPX2homologues appear to work synergisticallywithatemplate to promote microtubule
nucleation [113,114,116,117], there is strong evidence that microtubulescanbenucleatedboth in vitro andin vivo
in the absence of γ-TuRCs[113,115,122-127].Consistent with this, in vitro studies have shown that XMAP215 and
TPX2homologues are sufficient for microtubulenucleation at relativelylow tubulin concentrations [113,115],and
the Tog domain protein Stu2, but not the γ-Tu RC,isrequiredfor kinetochore-driven microtubule formation in bud-
ding yeast [127].Incontrast,however,XMAP215-mediatednucleation is inefficient in the absence of γ-TuRCsin
Xenopus egg extracts [117],andZyg9 andMsps are not requiredfor γ-TuRC-independent microtubulenucleation
from centrosomes in Caenorhabditis elegans embryos [124] or from acentrosomalsites in culturedDrosophila cells
[126]respectively. Th us, mec hanis ms of mi c rotu bulenucleation may vary andmight be relatedto the particular cell
type or MTOC; it is also possiblethatotherregulators of microtubulenucleation remain to be discovered.
Given that at least some microtubulepopulationscanbenucleatedindependentlyofγ-Tu R Cs, it is worth con-
templating why cellsneedγ-TuRCsatall.Webelieve there are at least three reasons:firstly, tem platedmicrotubule
nucleation is more efficient [13,27,49,69,84,87,112-114],presumablyasitpromotesthelateralinteractions between
tubulin dimers. Secondly, γ-TuRCscandefine the 13protofilament arrangement foundin most cell types, which may
c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
9
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
(or may not) allow molecular motors a more efficient route along straight protofilaments [128].Thirdly, γ-TuRCscan
helpregulate microtubulepolarity by defining the position of the minus end.Collectively, these functions presumably
explain why γ-TuRCsareessentialfor cell andorganism viability [10,12,16-22].
The γ-TuRC as a drug target
γ-TuRCsmayoffergoodanticancer targets given their roles during cell division. Currently, some of the most com-
mon andmost effective chemotherapy agents, such as Taxanes andVinca Alkaloids, bindmicrotubules directly. These
compounds, however, often leadto a condition known as chemotherapy-inducedperipheralneuropathy (CIPN)
[129,130].CIPN presents as numbness, pain, tingling, andheightenedsensitivity in the extremities;it limits drug
dosage and/or duration andcan persist after chemotherapy, andit is a major cause of cancer survivor disability
[129,130].Thecellular mechanism by which CIPN occurs is not fullyunderstood,butdying back of axonalpro-
jections in the epidermis has been observedin patients andin modelsystems andthis could be causedby axonal
transport defects [131-133]; however, alternative mechanisms have been suggested,including mitotoxicity anddis-
ruption to calcium homoeostasis [134].Targetingγ-TuRCs, insteadof microtubules directly, may of fer a viab leal-
ternative [44,135,136]because inhibiting γ-TuRCswould perturb cancer cells[44,135] but may not have a dramatic
effect on mature neurons, which would already have generatedandstabilisedtheir microtubulenetworks.Forex-
ample, axonaltransport along stablepre-existing microtubules in neurons might remain unperturbedafter γ-TuRC
inhibition. Moreover, microtubule severing in neurons may be able to compensate for any reduction in microtubule
generation via the γ-Tu R Cpathway. That said,thereisevidence that γ-TuRCsbindto the sides of pre-existing micro-
tubules andregulate microtubuledynamics [125,137],andso it will first be important to assess the roleofγ-TuRCs
in neurons. The challenge will then be to develop drugs that can inhibit γ-TuRCfunction in a highlyspecificman-
ner. Currently, th e onlyγ-TuRC-inhibiting drug is Gatastatin, which was identifiedby testing derivatives of drugs
known to bindα/β-tubulin andwas foundto bindγ-tubulin with a 12-fold greater affinity than α/β-tubulin [138].
Itmayalso be important, however, to consider γ-Tu R Cheterogeneity, as this may help increase specificity. Thus,
the non-core γ-TuRCcomponents may providegoodtargets for anticancer drugs, as their inhibition may affect only
subsets of γ-TuRCs. Of course, we first needto understandmore about γ-TuRCheterogeneity andthe roleofeach
γ-TuRCcomponent within the complex.
Summary
•Structural data from yeast has established that the template model for γ-TuRC-mediated mi-
crotubule nucleation is correct. In budding yeast, γ-TuRCs comprise a single-turn helical ring of
seven γ-TuSCs. In higher eukaryotes, it is likely that GCP4, 5 and 6 replace some of the GCP2/3
molecules within the helical ring. How this occurs, and the precise function of GCP4, 5 and 6,
remains unclear.
•Several γ-TuRC components have been discovered only recently. Of these, MOZART1 is the most
conserved through evolution and is the best studied, functioning in γ-TuRC recruitment (and pos-
sibly γ-TuRC assembly) in several systems.
•γ-TuRCs are recruited to various microtubule organising centres (MTOCs) in cells via
γ-TuRC-tethering proteins that normally contain an N-terminal CM1 domain. Binding of these CM1
domain proteins to γ-TuRCs is important for γ-TuRC recruitment, but can also inuence γ-TuRC
assembly and activation.
•The composition of γ-TuRCs varies between species, but can also vary within the same species
and even within the same cell. More work is needed to understand the extent of this heterogeneity
and its functional relevance.
•There is an emerging role for non-γ-TuRC proteins in microtubule nucleation. Several recent stud-
ies have shown that chTOG domain proteins and TPX2 homologues work synergistically with
γ-TuRCs (or articial templates) for efcient microtubule nucleation.
10 c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
•γ-TuRCs have been identied as potential anti-cancer targets and the rst γ-tubulin inhibitor,
gatastatin, has recently been developed. γ-TuRC-inhibiting drugs could in theory lead to a re-
duction in the occurrence of chemotherapy-induced peripheral neuropathy (CIPN), although this
remains to be explored.
Competing interests
The authors declare that there are no competing interests associated with the manuscript.
Abbreviations
CIPN, chemotherapy-induced peripheral neuropathy; CM1, centrosomin motif 1; GCP, γ-tubulin complex protein; MTOC, mi-
crotubule organising centre; MZT1, MOZART1; MZT2, MOZART2; SPB, spindle pole body; γ-TuRC, γ-tubulin ring complex;
γ-TuSC, γ-tubulin small complex.
References
1Goodson, H.V. and Jonasson, E.M. (2018) Microtubules and microtubule-associated proteins. Cold Spring Harb. Perspect. Biol. 10,a022608,
https://doi.org/10.1101/cshperspect.a022608
2 Sulimenko, V., H´
ajkov ´
a, Z., Klebanovych, A. and Dr ´
aber, P. (2017) Regulation of microtubule nucleation mediated by γ-tubulin complexes. Protoplasma
254, 1–13, https://doi.org/10.1007/s00709-016-1070-z
3 Kollman, J.M., Merdes, A., Mourey, L. and Agard, D.A. (2011) Microtubule nucleation by γ-tubulin complexes. Nat. Rev. Mol. Cell Biol. 12, 709–721,
https://doi.org/10.1038/nrm3209
4 Farache, D., Emorine, L., Haren, L. and Merdes, A. (2018) Assembly and regulation of γ-tubulin complexes. Open Biol. 8, 170266,
https://doi.org/10.1098/rsob.170266
5Teixid
´
o-Travesa, N., Roig, J. and L¨
uders, J. (2012) The where, when and how of microtubule nucleation - one ring to rule them all. J. Cell Sci. 125,
4445–4456,https://doi.org/10.1242/jcs.106971
6Kollman, J.M., Polka, J.K., Zelter, A., Davis, T.N. and Agard, D.A. (2010) Microtubule nucleating gamma-TuSC assembles structures with 13-fold
microtubule-like symmetry. Nature 466, 879–882, https://doi.org/10.1038/nature09207
7 Roostalu, J. and Surrey, T. (2017) Microtubule nucleation: beyond the template. Nat. Rev. Mol. Cell Biol. 91, 321
8 Sanchez, A.D. and Feldman, J.L. (2016) Microtubule-organizing centers: from the centrosome to non-centrosomal sites. Curr. Opin. Cell Biol. 44,
93–101, https://doi.org/10.1016/j.ceb.2016.09.003
9 Petry, S. and Vale, R.D. (2015) Microtubule nucleation at the centrosome and beyond. Nat. Cell Biol. 17, 1089–1093,
https://doi.org/10.1038/ncb3220
10 Knop, M. and Schiebel, E. (1997) Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their
interaction with Spc110p. EMBO J. 16,6985–6995, https://doi.org/10.1093/emboj/16.23.6985
11 Murphy, S.M., Urbani, L. and Stearns, T. (1998) The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body
components spc97p and spc98p. J. Cell Biol. 141,663–674, https://doi.org/10.1083/jcb.141.3.663
12 Vardy, L. and Toda, T. (2000) The fission yeast gamma-tubulin complex is required in G(1) phase and is a component of the spindle assembly
checkpoint. EMBO J. 19,6098–6111, https://doi.org/10.1093/emboj/19.22.6098
13 Oegema, K., Wiese, C., Martin, O.C., Milligan, R.A., Iwamatsu, A., Mitchison, T.J. et al. (1999) Characterization of two related Drosophila
gamma-tubulin complexes that differ in their ability to nucleate microtubules. J. Cell Biol. 144, 721–733, https://doi.org/10.1083/jcb.144.4.721
14 Gunawardane, R.N., Martin, O.C., Cao, K., Zhang, L., Dej, K., Iwamatsu, A. et al. (2000) Characterization and reconstitution of Drosophila
gamma-tubulin ring complex subunits. J. Cell Biol. 151, 1513–1524, https://doi.org/10.1083/jcb.151.7.1513
15 Guillet, V., Knibiehler, M., Gregory-Pauron, L., Remy, M.-H., Chemin, C., Raynaud-Messina, B. et al. (2011) Crystal structure of γ-tubulin complex
protein GCP4 provides insight into microtubule nucleation. Nat. Struct. Mol. Biol. 18, 915–919, https://doi.org/10.1038/nsmb.2083
16Martin, O.C., Gunawardane, R.N., Iwamatsu, A. and Zheng, Y. (1998) Xgrip109: a gamma tubulin-associated protein with an essential role in gamma
tubulin ring complex (gammaTuRC) assembly and centrosome function. J. Cell Biol. 141,675–687, https://doi.org/10.1083/jcb.141.3.675
17 Liu, J. and Lessman, C.A. (2007) Soluble tubulin complexes, gamma-tubulin, and their changing distribution in the zebrafish (Danio rerio) ovary,
oocyte and embryo. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 147,56–73, https://doi.org/10.1016/j.cbpb.2006.12.014
18 Seltzer, V., Janski, N., Canaday, J., Herzog, E., Erhardt, M., Evrard, J.-L. et al. (2007) Arabidopsis GCP2 and GCP3 are part of a soluble gamma-tubulin
complex and have nuclear envelope targeting domains. Plant J. 52, 322–331, https://doi.org/10.1111/j.1365-313X.2007.03240.x
19 Xiong, Y. and Oakley, B.R. (2009) In vivo analysis of the functions of gamma-tubulin-complex proteins. J. Cell Sci. 122, 4218–4227,
https://doi.org/10.1242/jcs.059196
20 Colombi´
e, N., V ´
erollet, C., Sampaio, P., Moisand, A., Sunkel, C., Bourbon, H.-M. et al. (2006) The Drosophila gamma-tubulin small complex subunit
Dgrip84 is required for structural and functional integrity of the spindle apparatus. Mol. Biol. Cell 17, 272–282,
https://doi.org/10.1091/mbc.e05-08-0722
c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4.0 (CC BY).
11
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
21 Knop, M., Pereira, G., Geissler, S., Grein, K. and Schiebel, E. (1997) The spindle pole body component Spc97p interacts with the gamma-tubulin of
Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication. EMBO J. 16, 1550–1564,
https://doi.org/10.1093/emboj/16.7.1550
22 Geissler, S., Pereira, G., Spang, A., Knop, M., Sou`
es, S., Kilmartin, J. et al. (1996) The spindle pole body component Spc98p interacts with the
gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. EMBO J. 15, 3899–3911,
https://doi.org/10.1002/j.1460-2075.1996.tb00764.x
23 Riehlman, T.D., Olmsted, Z.T., Branca, C.N., Winnie, A.M., Seo, L., Cruz, L.O. et al. (2013) Functional replacement of fission yeast γ-tubulin small
complex proteins Alp4 and Alp6by human GCP2 and GCP3. J. Cell Sci. 126,4406–4413, https://doi.org/10.1242/jcs.128173
24 Guillet, V., Knibiehler, M., Gregory-Pauron, L., Remy, M.-H., Chemin, C., Raynaud-Messina, B. et al. (2011) Crystal structure of γ-tubulin complex
protein GCP4 provides insight into microtubule nucleation. Nat. Struct. Mol. Biol. 18, 915–919, https://doi.org/10.1038/nsmb.2083
25 Murphy, S.M., Preble, A.M., Patel, U.K., O’Connell, K.L., Dias, D.P., Moritz, M. et al. (2001) GCP5 and GCP6: two new members of the human
gamma-tubulin complex. Mol. Biol. Cell 12, 3340–3352, https://doi.org/10.1091/mbc.12.11.3340
26Teixid´
o-Travesa, N., Vill´
en, J., Lacasa, C., Bertran, M.T., Archinti, M., Gygi, S.P. et al. (2010) The gammaTuRC revisited: a comparative analysis of
interphase and mitotic human gammaTuRC redefines the set of core components and identifies the novel subunit GCP8. Mol. Biol. Cell 21,
3963–3972, https://doi.org/10.1091/mbc.e10-05-0408
27 Choi, Y.-K., Liu, P., Sze, S.K., Dai, C. and Qi, R.Z. (2010) CDK5RAP2 stimulates microtubule nucleation by the gamma-tubulin ring complex. J. Cell Biol.
191, 1089–1095, https://doi.org/10.1083/jcb.201007030
28 Cota, R.R., Teixid´
o-Travesa, N., Ezquerra, A., Eibes, S., Lacasa, C., Roig, J. et al. (2017) MZT1 regulates microtubule nucleation by linking γTuRC
assembly to adapter-mediated targeting and activation. J. Cell Sci. 130,406–419, https://doi.org/10.1242/jcs.195321
29 Farache, D., Jauneau, A., Chemin, C., Chartrain, M., Remy, M.-H., Merdes, A. et al. (2016) Functional analysis of gamma-tubulin complex proteins
indicates specific lateral association via their N-terminal domains. J. Biol. Chem. 291, 23112–23125, https://doi.org/10.1074/jbc.M116.744862
30 Kollman, J.M., Zelter, A., Muller, EGD, Fox, B., Rice, L.M., Davis, T.N. et al. (2008) The structure of the gamma-tubulin small complex: implications of
its architecture and flexibility for microtubule nucleation. Mol. Biol. Cell 19, 207–215, https://doi.org/10.1091/mbc.e07-09-0879
31 Nguyen, T., Vinh, D.B., Crawford, D.K. and Davis, T.N. (1998) A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and
Spc98p, components of the yeast gamma-tubulin complex. Mol. Biol. Cell 9, 2201–2216,https://doi.org/10.1091/mbc.9.8.2201
32 V´
erollet, C., Colombi ´
e, N., Daubon, T., Bourbon, H.-M., Wright, M. and Raynaud-Messina, B. (2006) Drosophila melanogaster gamma-TuRC is
dispensable for targeting gamma-tubulin to the centrosome and microtubule nucleation. J. Cell Biol. 172, 517–528,
https://doi.org/10.1083/jcb.200511071
33 Zhang, L., Keating, T.J., Wilde, A., Borisy, G.G. and Zheng, Y. (2000) The role of Xgrip210 in gamma-tubulin ring complex assembly and centrosome
recruitment. J. Cell Biol. 151, 1525–1536,https://doi.org/10.1083/jcb.151.7.1525
34 Anders, A., Lourenc¸o, P.C.C. and Sawin, K.E. (2006) Noncore components of the fission yeast gamma-tubulin complex. Mol. Biol. Cell 17, 5075–5093,
https://doi.org/10.1091/mbc.e05-11-1009
35 Vogt, N., Koch, I., Schwarz, H., Schnorrer, F. and N ¨
usslein-Volhard, C. (2006) The gammaTuRC components Grip75 and Grip128 have an essential
microtubule-anchoring function in the Drosophila germline. Development 133,3963–3972, https://doi.org/10.1242/dev.02570
36Oriolo, A.S., Wald, F.A., Canessa, G. and Salas, P.J.I. (2007) GCP6binds to intermediate filaments: a novel function of keratins in the organization of
microtubules in epithelial cells. Mol. Biol. Cell 18, 781–794, https://doi.org/10.1091/mbc.e06-03-0201
37 Schnorrer, F., Luschnig, S., Koch, I. and N ¨
usslein-Volhard, C. (2002) Gamma-tubulin37C and gamma-tubulin ring complex protein 75 are essential for
bicoid RNA localization during drosophila oogenesis. Dev. Cell 3,685–696,https://doi.org/10.1016/S1534-5807(02)00301-5
38 Tovey, C.A., Tubman, C.E., Hamrud, E., Zhu, Z., Dyas, A.E., Butterfield, A.N. et al. (2018) γ-TuRC heterogeneity revealed by analysis of Mozart1. Curr.
Biol. 28, 2314–2323, https://doi.org/10.1016/j.cub.2018.05.044
39 Gunawardane, R.N., Martin, O.C. and Zheng, Y. (2003) Characterization of a new gammaTuRC subunit with WD repeats. Mol. Biol. Cell 14,
1017–1026,https://doi.org/10.1091/mbc.e02-01-0034
40 Haren, L., Remy, M.-H., Bazin, I., Callebaut, I., Wright, M. and Merdes, A. (2006) NEDD1-dependent recruitment of the gamma-tubulin ring complex to
the centrosome is necessary for centriole duplication and spindle assembly. J. Cell Biol. 172, 505–515, https://doi.org/10.1083/jcb.200510028
41 L ¨
uders, J., Patel, U.K. and Stearns, T. (2006)GCP-WD is a gamma-tubulin targeting factor required for centrosomal and chromatin-mediated
microtubule nucleation. Nat. Cell Biol. 8, 137–147, https://doi.org/10.1038/ncb1349
42 Manning, J.A., Shalini, S., Risk, J.M., Day, C.L. and Kumar, S. (2010) A direct interaction with NEDD1 regulates gamma-tubulin recruitment to the
centrosome. PLoS ONE 5,e9618, https://doi.org/10.1371/journal.pone.0009618
43 Liu, L. and Wiese, C. (2008) Xenopus NEDD1 is required for microtubule organization in Xenopus egg extracts. J. Cell Sci. 121, 578–589,
https://doi.org/10.1242/jcs.018937
44 Tillement, V., Haren, L., Roullet, N., Etievant, C. and Merdes, A. (2009) The centrosome protein NEDD1 as a potential pharmacological target to induce
cell cycle arrest. Mol. Cancer 8, 10, https://doi.org/10.1186/1476-4598-8-10
45 Manning, J.A., Lewis, M., Koblar, S.A. and Kumar, S. (2010) An essential function for the centrosomal protein NEDD1 in zebrafish development. Cell
Death Differ. 17, 1302–1314, https://doi.org/10.1038/cdd.2010.12
46Johmura, Y., Soung, N.-K., Park, J.-E., Yu, L.-R., Zhou, M., Bang, J.K. et al. (2011) Regulation of microtubule-based microtubule nucleation by
mammalian polo-like kinase 1. Proc. Natl Acad. Sci. U.S.A. 108, 11446–11451, https://doi.org/10.1073/pnas.1106223108
47 Reschen, R.F., Colombi´
e, N., Wheatley, L., Dobbelaere, J., St Johnston, D., Ohkura, H. et al. (2012) Dgp71WD is required for the assembly of the
acentrosomal Meiosis I spindle, and is not a general targeting factor for the γ-TuRC. Biol. Open 1, 422–429, https://doi.org/10.1242/bio.2012596
48 Walia, A., Nakamura, M., Moss, D., Kirik, V., Hashimoto, T. and Ehrhardt, D.W. (2014) GCP-WD mediates γ-TuRC recruitment and the geometry of
microtubule nucleation in interphase arrays of Arabidopsis.Curr. Biol. 24, 2548–2555, https://doi.org/10.1016/j.cub.2014.09.013
12 c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4.0 (CC BY).
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
49 Muroyama, A., Seldin, L. and Lechler, T. (2016) Divergent regulation of functionally distinct γ-tubulin complexes during differentiation. J. Cell Biol.
213,679–692, https://doi.org/10.1083/jcb.201601099
50 Pinyol, R., Scrofani, J. and Vernos, I. (2013) The role of NEDD1 phosphorylation by Aurora A in chromosomal microtubule nucleation and spindle
function. Curr. Biol. 23, 143–149, https://doi.org/10.1016/j.cub.2012.11.046
51 Gomez-Ferreria, M.A., Bashkurov, M., Helbig, A.O., Larsen, B., Pawson, T., Gingras, A.-C. et al. (2012) Novel NEDD1 phosphorylation sites regulate
γ-tubulin binding and mitotic spindle assembly. J. Cell Sci. 125, 3745–3751, https://doi.org/10.1242/jcs.105130
52 Haren, L., Stearns, T. and L¨
uders, J. (2009) Plk1-dependent recruitment of gamma-tubulin complexes to mitotic centrosomes involves multiple PCM
components. PLoS ONE 4, e5976,https://doi.org/10.1371/journal.pone.0005976
53 Sdelci, S., Sch ¨
utz, M., Pinyol, R., Bertran, M.T., Regu´
e, L., Caelles, C. et al. (2012) Nek9 phosphorylation of NEDD1/GCP-WD contributes to Plk1 control
of γ-tubulin recruitment to the mitotic centrosome. Curr. Biol. 22, 1516–1523, https://doi.org/10.1016/j.cub.2012.06.027
54 Zhang, X., Chen, Q., Feng, J., Hou, J., Yang, F., Liu, J. et al. (2009) Sequential phosphorylation of Nedd1 by Cdk1 and Plk1 is required for targeting of
the gammaTuRC to the centrosome. J. Cell Sci. 122, 2240–2251, https://doi.org/10.1242/jcs.042747
55 Zeng, C.J.T., Lee, Y.-R.J. and Liu, B. (2009) The WD40 repeat protein NEDD1 functions in microtubule organization during cell division in Arabidopsis
thaliana.Plant Cell 21, 1129–1140, https://doi.org/10.1105/tpc.109.065953
56Ma, W., Baumann, C. and Viveiros, M.M. (2010) NEDD1 is crucial for meiotic spindle stability and accurate chromosome segregation in mammalian
oocytes. Dev. Biol. 339, 439–450, https://doi.org/10.1016/j.ydbio.2010.01.009
57 Janski, N., Herzog, E. and Schmit, A.-C. (2008) Identification of a novel small Arabidopsis protein interacting with gamma-tubulin complex protein 3.
Cell Biol. Int. 32,546–548, https://doi.org/10.1016/j.cellbi.2007.11.006
58 Hutchins, J.R.A., Toyoda, Y., Hegemann, B., Poser, I., H´
erich´
e, J.-K., Sykora, M.M. et al. (2010) Systematic analysis of human protein complexes
identifies chromosome segregation proteins. Science 328, 593–599, https://doi.org/10.1126/science.1181348
59 Fogeron, M.-L., M ¨
uller, H., Schade, S., Dreher, F., Lehmann, V., K¨
uhnel, A. et al. (2013) LGALS3BP regulates centriole biogenesis and centrosome
hypertrophy in cancer cells. Nat. Commun. 4, 1531, https://doi.org/10.1038/ncomms2517
60 Janski, N., Masoud, K., Batzenschlager, M., Herzog, E., Evrard, J.-L., Houln´
e, G. et al. (2012) The GCP3-interacting proteins GIP1 and GIP2 are
required for γ-tubulin complex protein localization, spindle integrity, and chromosomal stability. Plant Cell 24, 1171–1187,
https://doi.org/10.1105/tpc.111.094904
61 Masuda, H., Mori, R., Yukawa, M. and Toda, T. (2013) Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex
recruitment to the MTOC, but not its assembly. Mol. Biol. Cell 24, 2894–2906,https://doi.org/10.1091/mbc.e13-05-0235
62 Nakamura, M., Yagi, N., Kato, T., Fujita, S., Kawashima, N., Ehrhardt, D.W. et al. (2012) Arabidopsis GCP3-interacting protein 1/MOZART 1 is an
integral component of the γ-tubulin-containing microtubule nucleating complex. Plant J. 71,216–225,
https://doi.org/10.1111/j.1365-313X.2012.04988.x
63 Dhani, D.K., Goult, B.T., George, G.M., Rogerson, D.T., Bitton, D.A., Miller, C.J. et al. (2013) Mzt1/Tam4, a fission yeast MOZART1 homologue, is an
essential component of the γ-tubulin complex and directly interacts with GCP3Alp6.Mol. Biol. Cell 24, 3337–3349,
https://doi.org/10.1091/mbc.e13-05-0253
64 Lin, T.-C., Neuner, A., Flemming, D., Liu, P., Chinen, T., J¨
akle, U. et al. (2016) MOZART1 and γ-tubulin complex receptors are both required to turn
γ-TuSC into an active microtubule nucleation template. J. Cell Biol. 215, 823–840
65 Cukier, C.D., Tourdes, A., El-Mazouni, D., Guillet, V., Nomme, J., Mourey, L. et al. (2017) NMR secondary structure and interactions of recombinant
human MOZART1 protein, a component of the gamma-tubulin complex. Protein Sci. 26, 2240–2248, https://doi.org/10.1002/pro.3282
66 Batzenschlager, M., Masoud, K., Janski, N., Houln´
e, G., Herzog, E., Evrard, J.-L. et al. (2013) The GIP gamma-tubulin complex-associated proteins are
involved in nuclear architecture in Arabidopsis thaliana. Front. Plant Sci. 4, 480, https://doi.org/10.3389/fpls.2013.00480
67 Masuda, H. and Toda, T. (2016) Synergistic role of fission yeast Alp16GCP6and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle
pole bodies and spindle assembly. Mol. Biol. Cell 27, 1753–1763, https://doi.org/10.1091/mbc.e15-08-0577
68 Liu, P., Choi, Y.-K. and Qi, R.Z. (2014) NME7 is a functional component of the γ-tubulin ring complex. Mol. Biol. Cell 25, 2017–2025,
https://doi.org/10.1091/mbc.e13-06-0339
69 Zheng, Y., Wong, M.L., Alberts, B. and Mitchison, T. (1995) Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. Nature
378, 578–583, https://doi.org/10.1038/378578a0
70 Erickson, H.P. and Stoffler, D. (1996) Protofilaments and rings, two conformations of the tubulin family conserved from bacterial FtsZ to alpha/beta and
gamma tubulin. J. Cell Biol. 135,5–8, https://doi.org/10.1083/jcb.135.1.5
71 Lynch, E.M., Groocock, L.M., Borek, W.E. and Sawin, K.E. (2014) Activation of the γ-tubulin complex by the Mto1/2 complex. Curr. Biol. 24,896–903,
https://doi.org/10.1016/j.cub.2014.03.006
72 Lawo, S., Bashkurov, M., Mullin, M., Ferreria, M.G., Kittler, R., Habermann, B. et al. (2009) HAUS, the 8-subunit human Augmin complex, regulates
centrosome and spindle integrity. Curr. Biol. 19,816–826,https://doi.org/10.1016/j.cub.2009.04.033
73 Uehara, R., Nozawa, R.-S., Tomioka, A., Petry, S., Vale, R.D., Obuse, C. et al. (2009) The augmin complex plays a critical role in spindle microtubule
generation for mitotic progression and cytokinesis in human cells. Proc. Natl Acad. Sci. U.S.A. 106,6998–7003,
https://doi.org/10.1073/pnas.0901587106
74 Zhu, H., Coppinger, J.A., Jang, C.-Y., Yates, J.R. and Fang, G. (2008) FAM29A promotes microtubule amplification via recruitment of the
NEDD1-gamma-tubulin complex to the mitotic spindle. J. Cell Biol. 183, 835–848, https://doi.org/10.1083/jcb.200807046
75 Zhu, H., Fang, K. and Fang, G. (2009) FAM29A, a target of Plk1 regulation, controls the partitioning of NEDD1 between the mitotic spindle and the
centrosomes. J. Cell Sci. 122, 2750–2759, https://doi.org/10.1242/jcs.048223
76Zhang, J. and Megraw, T.L. (2007) Proper recruitment of gamma-tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires
Centrosomin Motif 1. Mol. Biol. Cell 18, 4037–4049, https://doi.org/10.1091/mbc.e07-05-0474
c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4.0 (CC BY).
13
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
77 Conduit, P.T., Richens, J.H., Wainman, A., Holder, J., Vicente, C.C., Pratt, M.B. et al. (2014) A molecular mechanism of mitotic centrosome assembly in
Drosophila.Elife 3,https://doi.org/10.7554/eLife.03399
78 Vaizel-Ohayon, D. and Schejter, E.D. (1999) Mutations in centrosomin reveal requirements for centrosomal function during early Drosophila
embryogenesis. Curr. Biol. 9, 889–898, https://doi.org/10.1016/S0960-9822(99)80393-5
79 Megraw, T.L., Li, K., Kao, L.R. and Kaufman, T.C. (1999) The centrosomin protein is required for centrosome assembly and function during cleavage in
Drosophila.Development 126, 2829–2839
80 Eisman, R.C., Phelps, M.A.S. and Kaufman, T. (2015) An amino-terminal polo kinase interaction motif acts in the regulation of centrosome formation
and reveals a novel function for centrosomin (cnn) in Drosophila.Genetics 201,685–706,https://doi.org/10.1534/genetics.115.181842
81 Chen, J.V., Buchwalter, R.A., Kao, L.-R. and Megraw, T.L. (2017) A splice variant of centrosomin converts mitochondria to microtubule-organizing
centers. Curr. Biol. 27, 1928–1940, https://doi.org/10.1016/j.cub.2017.05.090
82 Dobbelaere, J., Josu´
e, F., Suijkerbuijk, S., Baum, B., Tapon, N. and Raff, J. (2008) A genome-wide RNAi screen to dissect centriole duplication and
centrosome maturation in Drosophila.PLoS Biol. 6, e224, https://doi.org/10.1371/journal.pbio.0060224
83 Sawin, K.E., Lourenc¸ o, P.C.C. and Snaith, H.A. (2004) Microtubule nucleation at non-spindle pole body microtubule-organizing centers requires fission
yeast centrosomin-related protein mod20p. Curr. Biol. 14,763–775, https://doi.org/10.1016/j.cub.2004.03.042
84 Lin, T.-C., Neuner, A., Schlosser, Y.T., Schiebel, E., Scharf, A.N. and Weber, L. (2014) Cell-cycle dependent phosphorylation of yeast pericentrin
regulates γ-TuSC-mediated microtubule nucleation. Elife 3, e02208, https://doi.org/10.7554/eLife.02208
85 Fong, K.-W., Choi, Y.-K., Rattner, J.B. and Qi, R.Z. (2008) CDK5RAP2 is a pericentriolar protein that functions in centrosomal attachment of the
gamma-tubulin ring complex. Mol. Biol. Cell 19, 115–125, https://doi.org/10.1091/mbc.e07-04-0371
86Samejima, I., Miller, V.J., Groocock, L.M. and Sawin, K.E. (2008) Two distinct regions of Mto1 are required for normal microtubule nucleation and
efficient association with the gamma-tubulin complex in vivo.J. Cell Sci. 121, 3971–3980, https://doi.org/10.1242/jcs.038414
87 Lyon, A.S., Morin, G., Moritz, M., Yabut, K.C.B., Vojnar, T., Zelter, A. et al. (2016) Higher-order oligomerization of Spc110p drives γ-tubulin ring
complex assembly. Mol. Biol. Cell 27,https://doi.org/10.1091/mbc.e16-02-0072
88 Vogel, J., Drapkin, B., Oomen, J., Beach, D., Bloom, K. and Snyder, M. (2001) Phosphorylation of gamma-tubulin regulates microtubule organizationin
budding yeast. Dev. Cell 1,621–631, https://doi.org/10.1016/S1534-5807(01)00073-9
89 Keck, J.M., Jones, M.H., Wong, C.C.L., Binkley, J., Chen, D., Jaspersen, S.L. et al. (2011) A cell cycle phosphoproteome of the yeast centrosome.
Science 332, 1557–1561, https://doi.org/10.1126/science.1205193
90 Lin, T.-C., Gombos, L., Neuner, A., Sebastian, D., Olsen, J.V., Hrle, A. et al. (2011) Phosphorylation of the yeast γ-tubulin Tub4 regulates microtubule
function. PLoS ONE 6, e19700, https://doi.org/10.1371/journal.pone.0019700
91 Fong, K.K., Zelter, A., Graczyk, B., Hoyt, J.M., Riffle, M., Johnson, R. et al. (2018) Novel phosphorylation states of the yeast spindle pole body. Biol.
Open,https://doi.org/10.1242/bio.033647
92 Bahtz, R., Seidler, J., Arnold, M., Haselmann-Weiß, U., Antony, C., Lehmann, W.D. et al. (2012) GCP6is a substrate of Plk4 and required for centriole
duplication. J. Cell Sci. 125,486–496,https://doi.org/10.1242/jcs.093930
93 Alvarado-Kristensson, M., Rodr´
ıguez, M.J., Sili ´
o, V., Valpuesta, J.M. and Carrera, A.C. (2009) SADB phosphorylation of gamma-tubulin regulates
centrosome duplication. Nat. Cell Biol. 11, 1081–1092, https://doi.org/10.1038/ncb1921
94 Dephoure, N., Zhou, C., Vill´
en, J., Beausoleil, S.A., Bakalarski, C.E., Elledge, S.J. et al. (2008) A quantitative atlas of mitotic phosphorylation. Proc. Natl
Acad. Sci. U.S.A. 105, 10762–10767, https://doi.org/10.1073/pnas.0805139105
95 Zhai, B., Vill´
en, J., Beausoleil, S.A., Mintseris, J. and Gygi, S.P. (2008) Phosphoproteome analysis of Drosophila melanogaster embryos. J. Proteome
Res. 7,1675–1682, https://doi.org/10.1021/pr700696a
96Friedman, D.B., Sundberg, H.A., Huang, E.Y. and Davis, T.N. (1996) The 110-kD spindle pole body component of Saccharomyces cerevisiae is a
phosphoprotein that is modified in a cell cycle-dependent manner. J. Cell Biol. 132, 903–914, https://doi.org/10.1083/jcb.132.5.903
97 Friedman, D.B., Kern, J.W., Huneycutt, B.J., Vinh, D.B., Crawford, D.K., Steiner, E. et al. (2001) Yeast Mps1p phosphorylates the spindle pole
component Spc110p in the N-terminal domain. J. Biol. Chem. 276, 17958–17967, https://doi.org/10.1074/jbc.M010461200
98 Huisman, S.M., Smeets, M.F.M.A. and Segal, M. (2007) Phosphorylation of Spc110p by Cdc28p-Clb5p kinase contributes to correct spindle
morphogenesis in S. cerevisiae.J. Cell Sci. 120, 435–446,https://doi.org/10.1242/jcs.03342
99 Stirling, D.A. and Stark, M.J. (1996) The phosphorylation state of the 110 kDa component of the yeast spindle pole body shows cell cycle dependent
regulation. Biochem. Biophys. Res. Commun. 222,236–242, https://doi.org/10.1006/bbrc.1996.0728
100 Liang, F., Richmond, D. and Wang, Y. (2013) Coordination of chromatid separation and spindle elongation by antagonistic activities of mitotic and
S-phase CDKs. PLoS Genet. 9, e1003319, https://doi.org/10.1371/journal.pgen.1003319
101 Scrofani, J., Sardon, T., Meunier, S. and Vernos, I. (2015) Microtubule nucleation in mitosis by a RanGTP-dependent protein complex. Curr. Biol. 25,
131–140, https://doi.org/10.1016/j.cub.2014.11.025
102 Lee, K. and Rhee, K. (2011) PLK1 phospγ-tubulin ring complexhorylation of pericentrin initiates centrosome maturation at the onset of mitosis. J. Cell
Biol. 195, 1093–1101, https://doi.org/10.1083/jcb.201106093
103 Santamaria, A., Wang, B., Elowe, S., Malik, R., Zhang, F., Bauer, M. et al. (2011) The Plk1-dependent phosphoproteome of the early mitotic spindle.
Mol. Cell. Proteomics 10,https://doi.org/10.1074/mcp.M110.004457
104 Conduit, P.T., Feng, Z., Richens, J.H., Baumbach, J., Wainman, A., Bakshi, S.D. et al. (2014) The centrosome-specific phosphorylation of Cnn by
Polo/Plk1 drives Cnn scaffold assembly and centrosome maturation. Dev. Cell 28,659–669, https://doi.org/10.1016/j.devcel.2014.02.013
105 Bodenmiller, B., Malmstrom, J., Gerrits, B., Campbell, D., Lam, H., Schmidt, A. et al. (2007) PhosphoPep-a phosphoproteome resource for systems
biology research in Drosophila Kc167 cells. Mol. Syst. Biol. 3, 139, https://doi.org/10.1038/msb4100182
106Bodenmiller, B., Campbell, D., Gerrits, B., Lam, H., Jovanovic, M., Picotti, P. et al. (2008) PhosphoPep-a database of protein phosphorylation sites in
model organisms. Nat. Biotechnol. 26, 1339–1340, https://doi.org/10.1038/nbt1208-1339
14 c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4.0 (CC BY).
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
107 Eisman, R.C., Phelps, M.A.S. and Kaufman, T.C. (2009) Centrosomin: a complex mix of long and short isoforms is required for centrosome function
during early development in Drosophila melanogaster .Genetics 182, 979–997, https://doi.org/10.1534/genetics.109.103887
108 Borek, W.E., Groocock, L.M., Samejima, I., Zou, J., de Lima Alves, F., Rappsilber, J. et al. (2015) Mto2 multisite phosphorylation inactivates
non-spindle microtubule nucleation complexes during mitosis. Nat. Commun. 6, 7929, https://doi.org/10.1038/ncomms8929
109 Kraemer, N., Issa-Jahns, L., Neubert, G., Ravindran, E., Mani, S., Ninnemann, O. et al. (2015) Novel alternative splice variants of mouse Cdk5rap2.
PLoS ONE 10, e0136684, https://doi.org/10.1371/journal.pone.0136684
110 Wang, Z., Zhang, C. and Qi, R.Z. (2014) A novel myomegalin isoform functions in Golgi microtubule organization and ER-Golgi transport. J. Cell Sci.
127, 4904–4917, https://doi.org/10.1242/jcs.155408
111 Park, J.S.Y., Lee, M.-K., Kang, S., Jin, Y., Fu, S., Rosales, J.L. et al. (2015) Species-specific expression of full-length and alternatively spliced variant
forms of CDK5RAP2. PLoS ONE 10, e0142577, https://doi.org/10.1371/journal.pone.0142577
112 Kollman, J.M., Greenberg, C.H., Li, S., Moritz, M., Zelter, A., Fong, K.K. et al. (2015) Ring closure activates yeast γTuRC for species-specific
microtubule nucleation. Nat. Struct. Mol. Biol.,https://doi.org/10.1038/nsmb.2953
113 Roostalu, J., Cade, N.I. and Surrey, T. (2015) Complementary activities of TPX2 and chTOGconstitute an efficient importin-regulated microtubule
nucleation module. Nat. Cell Biol. 17, 1422–1434, https://doi.org/10.1038/ncb3241
114 Wieczorek, M., Bechstedt, S., Chaaban, S. and Brouhard, G.J. (2015) Microtubule-associated proteins control the kinetics of microtubule nucleation.
Nat. Cell Biol.,https://doi.org/10.1038/ncb3188
115 Woodruff, J.B., Ferreira Gomes, B., Widlund, P.O., Mahamid, J., Honigmann, A. and Hyman, A.A. (2017) The centrosome is a selective condensate that
nucleates microtubules by concentrating tubulin. Cell 169,1066–1077, https://doi.org/10.1016/j.cell.2017.05.028
116Flor-Parra, I., Iglesias-Romero, A.B. and Chang, F. (2018) The XMAP215 ortholog Alp14 promotes microtubule nucleation in fission yeast. Curr. Biol.
28,1681–1691, https://doi.org/10.1016/j.cub.2018.04.008
117 Thawani, A., Kadzik, R.S. and Petry, S. (2018) XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring
complex. Nat. Cell Biol. 20, 1–18, https://doi.org/10.1038/s41556-018-0091-6
118 L ¨
uders, J. (2018) XMAP215 joins microtubule nucleation team. Nat. Cell Biol. 20, 508–510, https://doi.org/10.1038/s41556-018-0100-9
119 Usui, T., Maekawa, H., Pereira, G. and Schiebel, E. (2003) The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics
and anchorage. EMBO J. 22, 4779–4793, https://doi.org/10.1093/emboj/cdg459
120 Reid, T.A., Schuster, B.M., Mann, B.J., Balchand, S.K., Plooster, M., McClellan, M. et al. (2016) Suppression of microtubule assembly kinetics by the
mitotic protein TPX2. J. Cell Sci. 129, 1319–1328, https://doi.org/10.1242/jcs.178806
121 Zhang, R., Roostalu, J., Surrey, T. and Nogales, E. (2017) Structural insight into TPX2-stimulated microtubule assembly. Elife 6, 1518,
https://doi.org/10.7554/eLife.30959
122 Sampaio, P., Rebollo, E., Varmark, H., Sunkel, C.E. and Gonz ´
alez, C. (2001) Organized microtubule arrays in gamma-tubulin-depleted Drosophila
spermatocytes. Curr. Biol. 11, 1788–1793, https://doi.org/10.1016/S0960-9822(01)00561-9
123 Llamazares, S., Tavosanis, G.andGonz ´
alez, C. (1999) Cytological characterisation of the mutant phenotypes produced during early embryogenesis by
null and loss-of-function alleles of the gammaTub37C gene in Drosophila.J. Cell Sci. 112,659–667
124 Hannak, E., Oegema, K., Kirkham, M., G¨
onczy, P., Habermann, B. and Hyman, A.A. (2002) The kinetically dominant assembly pathway for centrosomal
asters in Caenorhabditis elegans is gamma-tubulin dependent. J. Cell Biol. 157, 591–602, https://doi.org/10.1083/jcb.200202047
125 Bouissou, A., V ´
erollet, C., Sousa, A., Sampaio, P., Wright, M., Sunkel, C.E. et al. (2009) {gamma}-Tubulin ring complexes regulate microtubule plus
end dynamics. J. Cell Biol. 187, 327–334, https://doi.org/10.1083/jcb.200905060
126Rogers, G.C., Rusan, N.M., Peifer, M. and Rogers, S.L. (2008) A multicomponent assembly pathway contributes to the formation of acentrosomal
microtubule arrays in interphase Drosophila cells. Mol. Biol. Cell 19,3163–3178, https://doi.org/10.1091/mbc.e07-10-1069
127 Kitamura, E., Tanaka, K., Komoto, S., Kitamura, Y., Antony, C. and Tanaka, T.U. (2010) Kinetochores generate microtubules with distal plus ends: their
roles and limited lifetime in mitosis. Dev. Cell 18, 248–259, https://doi.org/10.1016/j.devcel.2009.12.018
128 Chaaban, S. and Brouhard, G.J. (2017) A microtubule bestiary: structural diversity in tubulin polymers. Mol. Biol. Cell 28, 2924–2931,
https://doi.org/10.1091/mbc.e16-05-0271
129 Argyriou, A.A., Kyritsis, A.P., Makatsoris, T. and Kalofonos, H.P. (2014) Chemotherapy-induced peripheral neuropathy in adults: a comprehensive
update of the literature. Cancer Manag. Res. 6, 135–147, https://doi.org/10.2147/CMAR.S44261
130 Fukuda, Y., Li, Y. and Segal, R.A. (2017) A mechanistic understanding of axon degeneration in chemotherapy-induced peripheral neuropathy. Front.
Neurosci. 11, 481, https://doi.org/10.3389/fnins.2017.00481
131 LaPointe, N.E., Morfini, G., Brady, S.T., Feinstein, S.C., Wilson, L. and Jordan, M.A. (2013) Effects of eribulin, vincristine, paclitaxel and ixabepilone on
fast axonal transport and kinesin-1 driven microtubule gliding: implications for chemotherapy-induced peripheral neuropathy. Neurotoxicology 37,
231–239, https://doi.org/10.1016/j.neuro.2013.05.008
132 Theiss, C. and Meller, K. (2000) Taxol impairs anterograde axonal transport of microinjected horseradish peroxidase in dorsal root ganglia neurons in
vitro.Cell Tissue Res. 299, 213–224, https://doi.org/10.1007/s004410050019
133 Shemesh, O.A. and Spira, M.E. (2010) Paclitaxel induces axonal microtubules polar reconfiguration and impaired organelle transport: implications for
the pathogenesis of paclitaxel-induced polyneuropathy. Acta Neuropathol. 119, 235–248, https://doi.org/10.1007/s00401-009-0586-0
134 Gornstein, E.L. and Schwarz, T.L. (2017) Neurotoxic mechanisms of paclitaxel are local to the distal axon and independent of transport defects. Exp.
Neurol. 288, 153–166,https://doi.org/10.1016/j.expneurol.2016.11.015
135 Whitehurst, A.W., Bodemann, B.O., Cardenas, J., Ferguson, D., Girard, L., Peyton, M. et al. (2007) Synthetic lethal screen identification of
chemosensitizer loci in cancer cells. Nature 446, 815–819, https://doi.org/10.1038/nature05697
136Cala, O., Remy, M.-H., Guillet, V., Merdes, A., Mourey, L., Milon, A. et al. (2013) Virtual and biophysical screening targeting the γ-tubulin complex - a
new target for the inhibition of microtubule nucleation. PLoS ONE 8,e63908, https://doi.org/10.1371/journal.pone.0063908
c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4.0 (CC BY).
15
Essays in Biochemistry (2018) EBC20180028
https://doi.org/10.1042/EBC20180028
137 Bouissou, A., V ´
erollet, C., de Forges, H., Haren, L., Bellaiche, Y., Perez, F. et al. (2014) γ-Tubulin Ring Complexes and EB1 play antagonistic roles in
microtubule dynamics and spindle positioning. EMBO J. 33, 114–128, https://doi.org/10.1002/embj.201385967
138 Chinen, T., Liu, P., Shioda, S., Pagel, J., Cerikan, B., Lin, T.-C. et al. (2015) The γ-tubulin-specific inhibitor gatastatin reveals temporal requirements of
microtubule nucleation during the cell cycle. Nat. Commun. 6, 8722, https://doi.org/10.1038/ncomms9722
139 Weil, C.F., Oakley, C.E. and Oakley, B.R. (1986) Isolation of mip (microtubule-interacting protein) mutations of Aspergillus nidulans.Mol. Cell. Biol. 6,
2963–2968, https://doi.org/10.1128/MCB.6.8.2963
140 Oakley, C.E. and Oakley, B.R. (1989) Identification of gamma-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus
nidulans.Nature 338,662–664, https://doi.org/10.1038/338662a0
16 c
2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
