for many years , the identity of components of microtubule - organizing centers ( mtocs ) that nucleate microtubule assembly and establish microtubule polarity was a central unanswered question in the field of mitosis and the cytoskeleton . the discovery of -tubulin ( oakley and oakley , 1989 ) , the key finding that allowed this question to be answered , came from a genetic screen in the fungus aspergillus nidulans designed to identify genes important for microtubule function ( weil et  al . , the existence of members of the tubulin superfamily beyond the microtubule proteins - and -tubulin was completely unexpected at the time , and the discovery of -tubulin is a powerful validation of the value of forward genetics , in which well - designed screens let the cell tell us what is important rather than us imposing our preconceived notions on the cell . -tubulin is ubiquitous in eukaryotes , and genome - sequencing projects have revealed that there are one to three -tubulin genes in eukaryotic genomes ( findeisen et  al . , 2014 ) . it localizes to structurally diverse mtocs and , with few exceptions , is required for microtubule nucleation ( reviewed by job et  al . , 2003 ) . ring - shaped structures called turcs ( -tubulin ring complexes ) that contain -tubulin and associated proteins nucleate microtubule assembly in vitro ( zheng et  al . , 1995 ) and in vivo ( figure 1 , a d ) ( reviewed by teixido - travesa et  al . , 2010 ; kollman et  al . , 2011 ; lin et  al . , 2014 ) . proteins that associate with -tubulin have been identified and designated grips ( -tubulin ring proteins ) or gcps ( -tubulin complex proteins ) ( figure 1d ) . five of these proteins , gcp26 , are structurally related and contain conserved regions called grip motifs ( gunawardane et  al . , 2000 ; the turc consists of gcps that bind to -tubulin and to each other , forming a complex in which a ring of -tubulin molecules effectively mimics the plus end of a microtubule . it forms a pre - existing nucleus for microtubule assembly . at physiological tubulin concentrations , spontaneous assembly of tubulin into microtubules instead , almost all microtubule growth occurs from preformed nucleating structures , principally the turc . ( c ) the finding that gcp26 all bind to -tubulin raises the possibility that gcps and -tubulin may assemble into alternative tusc - like structures ( kollman et  al . , 2011 ) that assemble along with tuscs to form the turc . ( d ) designations for gcps in various organisms in which they have been studied extensively . -tubulin complexes nucleate microtubules from the centrosome and other mtocs in both interphase and mitosis or from microtubule surfaces in mitosis . in interphase and mitosis , -tubulin has been shown to regulate microtubule plus - end dynamics . in mitosis , it plays a role in the spindle assembly checkpoint ( sac ) and the control of mitotic exit . in interphase , -tubulin plays an important role in inhibiting apc / c , thereby promoting the transition from the g1 to the s phase . it also appears to regulate e2f1-mediated gene expression and complexes with rad51 , suggesting a role in the rad51-mediated dna damage response . beyond gcp26 , turcs contain three additional , more recently discovered , core subunit proteins , mozart1 ( mitotic spindle - organizing protein associated with a ring of -tubulin , or gcp9 ) and mozart2a and mozart2b ( gcp8a and gcp8b ) , which are closely related to each other ( hutchins et  al . , 2010 ) . mozart1 is highly conserved in eukaryotes , except in saccharomyces cerevisiae and close relatives ( hutchins et  al . , 2010 ; lin et  al . , 2014 ) , and it is essential for mitotic spindle assembly and function in humans and schizosaccharomyces pombe ( hutchins et  al . arabidopsis thaliana has two mozart1 homologues , gip1 and gip2 , that interact with gcp3 . they are not essential individually , but gip1/2 double mutants are embryonic lethal ( nakamura et  al . , 2012 ) . mozart2a and mozart2b are apparently restricted to vertebrates , in which they play a role in recruitment of the turc to the centrosome ( teixido - travesa et  al . in addition to the core turc components , various phyla have proteins , unrelated in sequence to gcp29 , that are involved in targeting -tubulin complexes to mtocs ( reviewed extensively by lin et  al . , 2014 ) . high - resolution structures of human -tubulin ( aldaz et  al . , 2005 ; rice et  al . , 2008 ) and gcp4 ( guillet et  al . , 2011 ) and lower - resolution structures of small -tubulin complexes ( tuscs , containing two molecules of -tubulin and one molecule each of gcp2 and 3 ) from s. cerevisiae ( kollman et  al . , 2008 , 2010 ) have been determined , and these and other findings have led to a persuasive model for the structure of the turc ( kollman et  al . , 2011 ) . in this model , gcp26 each binds directly to -tubulin and assemble into a structure that contains 13 -tubulin molecules ( perhaps containing five tuscs and one molecule each of gcp4 - 6 bound to -tubulin ; figure 1a ) . gcp3 contains a hinge region , and in the current model , movement about this hinge would alter the positioning of -tubulin molecules . in one position , the -tubulin molecules would be too far apart to nucleate microtubule assembly , but in the other position , the straight position , they would have exactly the same spacing as microtubule protofilaments and would form a perfect template for microtubule assembly . control of the conformation of gcp3 could therefore regulate the ability of the turc to nucleate microtubule assembly . important unanswered questions in this area are the exact arrangement of gcp26 in the turc and the positioning of mozart1 , 2a , and 2b in the complex . the hinge model , although persuasive , needs to be tested rigorously , and if it is correct , it raises the question of what controls the rotation of gcp3 around its hinge . although microtubule nucleation from mtocs is extremely important , in many phyla mtocs are not apparent . even in vertebrate mitosis , centrosomes are not always required for the formation of a functional spindle ( khodjakov et  al . , 2000 ) , and when centrosomes are present , many microtubules may be nucleated independent of the centrosome . ( 2005 ) showed convincingly that cortical microtubules in higher plant cells are nucleated from the sides of existing microtubules at a characteristic angle of 42 with respect to existing microtubules , that -tubulin is at the branch points , and that lateral microtubule nucleation is -tubulin dependent . ( 2005 ) demonstrated that microtubules are nucleated from -tubulin complexes at the sides of cytoplasmic microtubules in s. pombe . these data established the principle that -tubulin complexes function in nucleation of microtubules from the sides of existing microtubules . data obtained over the intervening decade have identified the augmin complex as a key player in lateral microtubule nucleation . augmin is an eight - subunit complex that is important for localizing -tubulin to mitotic spindles and for centrosome - independent microtubule generation in mitotic and meiotic spindles ( goshima et  al . , 2008 ; ho et  al . , 2011 ; petry et  al . , 2011 ; hsia et  al . , 2014 ) . in higher plants , augmin colocalizes with gcps , and they are corecruited to cortical microtubules . knockdown of augmin with a microrna reduces branching lateral nucleation of microtubules ( liu et  al . , 2014 ) . in xenopus extracts , augmin and turcs along with the microtubule assembly factor tpx2 and the gtp - bound rangtpase nucleate microtubule assembly , forming fan - like structures consistent with the possibility that the microtubules are in branched arrays ( petry et  al . , microtubule nucleation persists if augmin or tpx2 are depleted , but fan - like arrays do not form . monitoring the plus - end microtubule - tracking protein eb-1 revealed that new microtubules are nucleated from the sides of existing microtubules and that the newly nucleated microtubules have the same polarity as the microtubules from which they are nucleated . such lateral nucleation of microtubule assembly in a mitotic apparatus would result in the proliferation of spindle microtubules but , importantly , would not disrupt the polarity of microtubules in the spindle . these and related findings support a persuasive although not completely proven model that the augmin complex binds to microtubule walls and recruits the turc through nedd1 or other proteins to initiate new microtubule nucleation ( uehara et  al . , 2009 ; zhu et  al . , 2009 ; johmura et  al . , the fact that augmin apparently plays a role in lateral microtubule nucleation in both plants and animal suggests that augmin - mediated lateral microtubule nucleation is widespread in eukaryotes . however , many organisms do not have homologues of all augmin subunits ( lin et  al . , 2014 ) , and the fact that the polarity of microtubules nucleated from the sides of microtubules in s. pombe is opposite to that of augmin - mediated nucleation ( janson et  al . , 2005 ) suggests there are at least two lateral nucleation mechanisms . the theme that has emerged from two decades of research is that -tubulin complexes are almost universally involved in microtubule nucleation . mechanisms have evolved , however , to control and target these complexes , such that microtubule nucleation occurs when and where it is needed . the traditional view has been that each protein has a single function , but increasingly , proteins have been shown to be multifunctional . in this regard , increasing evidence indicates that -tubulin has functions beyond microtubule nucleation ( cuschieri et  al . , 2007 ) . in many ways , this makes sense , because -tubulin is at the hub of the microtubule cytoskeleton and , along with other mtoc components , is in a unique position to receive and send signal molecules transported by microtubule motors . the problem with interpreting some of these data , however , is that , because -tubulin has a critical role in mitosis and disruption of mitosis has many consequences , it is difficult to distinguish putative non  microtubule - nucleation functions of -tubulin from consequences of mitotic failure or alteration of microtubule nucleation . nevertheless , we believe that there is ample evidence that -tubulin has important functions beyond microtubule nucleation ( summarized in figure 1e ) . there have been repeated reports from multiple phyla that mutations or deficiencies in -tubulin or gcps alter plus - end microtubule dynamics ( paluh et  al . , 2000 ; , 2001 ; zimmerman and chang , 2005 ; bouissou et  al . , 2009 ) and that -tubulin alters the distribution of the plus end tracking protein bim1 in s. cerevisiae ( cuschieri et  al . , 2006 ) and its homologue , eb1 , in drosophila ( bouissou et  al . , 2014 ) . considering that there is constant bidirectional transport along microtubules , one idea is that -tubulin complexes at mtocs could bind catastrophe or rescue factors or the motor molecules that transport them . this would facilitate loading of molecules that affect microtubule dynamics onto motor molecules , thereby affecting their transport to microtubule plus ends and , consequently , affecting microtubule dynamics at plus ends . consistent with this idea , -tubulin mutations alter binding of motor proteins and/or microtubule stability factors ( vogel et  al . , 2001 ; zimmerman and chang , 2005 ; cuschieri et  al . , 2006 ) . for example , some -tubulin mutations alter the binding of kinesin-14 and kinesin-5 to -tubulin complexes , while others alter the interaction of cytoplasmic dynein with spindle pole bodies ( spbs ; li et  al . , 2005 ; rodriguez et  al . , 2008 ; olmsted et  al . , 2014 ) , although the binding of type 5 and 14 kinesins may be involved in regulating microtubule nucleation rather than plus - end microtubule dynamics ( olmsted et  al . , 2014 ) . the second proposed explanation comes from work in drosophila , in which turc proteins localize along astral microtubules and are proposed to act as rescue factors that arrest microtubule disassembly ( bouissou et  al . , it is worth noting that the proposed rescue activity of turcs distal to mtocs might not be intrinsic to the turc but a function of its binding to rescue factors , as discussed above . another possibility that is unfortunately difficult to test is that -tubulin mutations alter the microtubule lattice and thereby alter microtubule dynamics . there have also been persuasive reports of perturbation of the regulation of mitotic exit by alterations of -tubulin or gcps . alanine - scanning mutants of human -tubulin expressed as the sole -tubulin in s. pombe allowed cells to go through anaphase and cytokinesis when spindle formation was disrupted ( hendrickson et  al . , 2001 ) . mutations in the gcp4 and gcp5 homologues of s. pombe caused a failure of mitotic arrest in the presence of the antimicrotubule agent thiabendazole ( vardy and toda , 2000 ) . similarly , a -tubulin mutation in a. nidulans caused mitotic exit before successful completion of mitosis in a strain in which the establishment of spindle bipolarity was delayed by a type 14 kinesin deletion ( prigozhina et  al . another allele caused a premature mitotic exit even when the microtubule cytoskeleton was completely disassembled with benomyl ( prigozhina et  al . likewise , rna interference ( rnai ) of dgrip84 ( gcp2 ) in drosophila caused an untimely mitotic exit in the presence of colchicine ( colombie et  al . thus genetic or rnai perturbations of -tubulin or gcps in three organisms and four laboratories all point to a microtubule nucleation - independent role for -tubulin complexes in the control of mitotic exit . a possibly related area in which results from several labs have indicated a non  microtubule nucleation function for -tubulin is coordination of mitotic events and/or the spindle assembly checkpoint ( sac ) . in a systematic alanine mutagenesis screen , a number of -tubulin mutants were shown to cause premature anaphase entry and defective cytokinesis , suggesting that -tubulin regulates the sac and orderly mitotic exit ( hendrickson et  al . , 2001 ) . an a. nidulans -tubulin allele caused late mitotic events ( chromosomal disjunction , spindle elongation , and mitotic exit ) to become disordered ( prigozhina et  al . , 2004 ) . the failure of chromosomal disjunction in this instance was not due to lack of microtubule assembly or force generation , because chromosomes were often stretched dramatically in anaphase , even though they did not disjoin . with respect to mitotic regulation and the sac , it is worth noting that there are reports that the sac / mitotic regulatory proteins cdc20 and bubr1 are in a complex with -tubulin in mammalian cells ( muller et  al . , 2006 ) and that mad2 binds to alp4p ( gcp2 ) in s. pombe ( mayer et  al . , 2006 ) . finally , there is incontrovertible evidence that -tubulin plays a role in the regulation of cell cycle progression in interphase . in a. nidulans , a -tubulin allele , mipad159 , causes failure of inactivation of the anaphase - promoting complex / cyclosome ( apc / c ) at the g1/s boundary ( nayak et  al . , 2010 ) . this failure correlates with failure of the apc / c activator cdh1 to dissociate from the spb , and it can be overridden by deletion of cdh1 ( edgerton - morgan and oakley , 2012 ) . importantly , this failure occurs if microtubules are present or if they are completely depolymerized , so it is not a microtubule nucleation phenomenon ( nayak et . ( a. nidulans has a single gene with functional domains of bub1 and bubr1 ) and mps1 , thereby abrogating the sac ( edgerton et  al . , 2015 ) . note also that analyses of an alp4 ( gcp2 ) mutation indicate alp4 has an essential function in g1 in s. pombe ( vardy and toda , 2000 ) . this mutation , moreover , can cause septation , even when mitosis is arrested , by allowing inappropriate recruitment of the sid1 kinase to the spb ( vardy et  al . , 2002 ) . there are also reports that -tubulin modulates the activity of e2f transcription factors , which control the expression of a number of genes that are necessary for dna replication and centrosome duplication ( hoog et  al . the key challenge in understanding the various nonnucleation functions of -tubulin will be to move from phenomenology to molecular mechanism . although the functional significance is not yet clear , there is strong and intriguing evidence of the association of -tubulin with tumor suppressor proteins , particularly those involved in dna repair . it binds to rad51 in nuclei in response to dna damage ( lesca et  al . , 2005 ) and coimmunoprecipitates with atr , brca1 , and c53 from nuclei ( zhang et  al . , 2007 ; hubert et  al . , 2011 ; horejsi et  al . , brca1/bard1 binds to and monoubiquitinates -tubulin , and there is evidence that this ubiquitination is important in the regulation of centrosome number ( hsu et  al . , 2001 ; starita et  al . , 2004 ) , a particularly interesting finding , since centrosome amplification is important to cancer progression . -tubulin expression or localization pattern is altered in a variety of cancers , including some multiple myelomas , non  small cell lung cancers ( maounis et  al . , 2012 ; dementyeva et  al . , 2013 ) , ductal hyperplasia and breast cancer ( niu et  al . , 2009 ; cho et  al . , 2010 ) , gliomas and glioblastoma cell lines ( katsetos et  al . , 2006 ) , and medulloblastomas and medulloblastoma cell lines ( caracciolo et  al . , 2010 ) . spontaneous mutations in one of the two human -tubulin genes , tubg1 , are associated with lissencephaly and microcephaly ( poirier et  al . 2014 ) . further effort devoted to studies of both the microtubule nucleation - dependent and nucleation - independent functions of -tubulin in different cell and tissue contexts should shed light on the relevance of this cytoskeleton protein in health and disease .