betaTubulin56D (ß1 tubulin)


A rapid increase in ecdysteroid hormone synthesis results when the insect prothoracic gland is stimulated with prothoracicotropic hormone (PTTH: see Bombyx and Manduca prothoracicotropic hormone), a brain neuropeptide hormone. PTTH also stimulates the specific synthesis of several proteins, one of which is a beta tubulin. To further understand the possible roles of beta tubulin in the prothoracic gland, beta tubulin cDNA clones were isolated from a tobacco hornworm (Manduca sexta) gland cDNA library. Sequence analysis indicates that these clones were assignable to the beta1 tubulin isoform. Gland beta1 tubulin mRNA levels during the last larval instar and early pupal-adult development exhibit peaks that coincided with peaks in ecdysteroid synthesis. Manipulations of the glands hormonal milieu show that beta1 tubulin mRNA levels respond to 20 hydroxyecdysone and PTTH. The data support the idea that the prothoracic gland beta1 tubulin gene is ubiquitously expressed but exhibits tissue- and developmental-specific regulation of transcription and translation (Rybczynski, 1998).

Microtubule dynamic instability underlies many cellular functions, including spindle morphogenesis and chromosome movement. The role of guanosine triphosphate (GTP) hydrolysis in dynamic instability was investigated by introduction of four mutations into yeast ß-tubulin at amino acids 103 to 109, a site thought to participate in GTP hydrolysis. Three of the mutations increased both the assembly-dependent rate of GTP hydrolysis and the average length of steady-state microtubules over time (a measure of dynamic instability). The fourth mutation did not substantially affect the rate of GTP hydrolysis or the steady-state microtubule lengths. These results demonstrate that the rate of GTP hydrolysis can modulate microtubule length and hence dynamic instability (Davis, 1994).

The exchangeable GTP-binding site on ß-tubulin has been studied extensively, but the primary sequence elements forming the binding site on ß-tubulin remain unknown. Site-directed mutagenesis of the single ß-tubulin gene of Saccharomyces cerevisiae has been used to test a model for the GTP-binding site on beta-tubulin, based on sequence comparisons with members of the GTPase superfamily. The results do not support the proposal that two conserved motifs are cognate to motifs found in GTPase superfamily members. Instead, the data argue that the primary sequence elements of beta-tubulins that interact with bound nucleotide, and presumably also those of the alpha- and gamma-tubulin family members, are different from those of "typical" GTPase superfamily members, such as p21ras. The GTPase superfamily should thus be broadened to include not just the typical GTPases that show strong conservation of primary sequence consensus motifs (GxxxxGK, T, DxxG, DxKN) but also "atypical" GTPases, exemplified by the tubulins and other recently identified GTPases, that do not show the consensus motifs of typical GTPases and that also show no obvious primary sequence relationships among themselves. The tubulins and other atypical GTPases thus appear to represent convergent solutions to the GTP-binding and hydrolysis problem (Sage, 1995).

Microtubule dynamics in vitro are regulated by the tubulin isotype composition. Microtubules assembled from the purified alpha beta III heterodimers are considerably more dynamic than microtubules made from the alpha beta II or alpha beta IV heterodimers or from unfractionated phosphocellulose-purified tubulin. Furthermore, increasing the proportion of the alpha beta II heterodimer in a mixture of the alpha beta II and alpha beta III heterodimers suppresses microtubule dynamics, demonstrating that microtubule dynamics can be influenced by the tubulin isotype composition. The data support the hypothesis that cells might determine the dynamic properties and functions of their microtubules in part by altering the relative amounts of the different tubulin isotypes (Panda, 1994).

Tubulin is a heterodimer of alpha- and beta-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors. The characterization of Alf1p, the yeast ortholog of mammalian cofactor B, is reported. Alf1p interacts with alpha-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with alpha-tubulin. Mutations in alpha-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of alpha-tubulin; this domain is distinct from the region of interaction between alpha-tubulin and beta-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in alpha-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2 (which encodes the other yeast alpha-tubulin cofactor) suggests that Alf1p and Pac2p act in the same pathway leading to functional alpha-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester alpha-tubulin from interaction with beta-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer (Feierbach, 1999).

Microtubules (MTs) are organized into distinct systems essential for cell shape, movement, intracellular transport, and division. Electron crystallographic analyses provide little information about how MTs produce diverse structures and functions, perhaps because they failed to visualize the last 10 residues of the alpha- and the last 18 of the beta-tubulin C-terminal tails (CTTs), which likely play a role in MT diversity. CTTs define conserved, nonallelic isotypes in mammals, are major sites of posttranslational modifications (PTMs), are binding sites for microtubule-associated proteins (MAPs), and determine MT motor processivity. Using mutagenesis and homologous gene replacement in Tetrahymena thermophila, mutations, deletions, tail switches, and tail duplications of alpha- and beta-tubulin CTTs have been analyzed. A tail is required for the essential function of both alpha- and beta-tubulin. However, the two tails are interchangeable, and cells grow normally with either an alpha or a beta tail on both tubulins. In addition, an alpha gene containing a duplicated alpha C terminus rescues a lethal mutant lacking all known posttranslational modification sites on the beta C terminus but cannot rescue deletion of the beta tail. Thus, tubulin tails have a second essential function that is not associated with posttranslational modification (Duan, 2002).

Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, a green fluorescent protein (GFP)-tubulin fusion protein was used to observe microtubules in living yeast cells. GFP-tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules reveals distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells have defects in these cortical interactions, resulting in misoriented spindles. Microtubule dynamics are altered in the absence of dynein. These results indicate that microtubules and dynein act together to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation (Carminati, 1997).

The microtubules (MTs) within neuronal processes are highly organized with regard to their polarity and yet are not attached to any detectable nucleating structure. Axonal MTs are uniformly oriented with their plus ends distal to the cell body, whereas dendritic MTs are of both orientations. A test was made of the capacity of motor-driven MT transport to organize distinct MT patterns during process outgrowth. CHO1/MKLP1 is a kinesin-related protein present in the midzonal region of the mitotic spindle where MTs of opposite orientation overlap. Nascent processes, in cells induced to express the N-terminal portion of the molecule, contain uniformly plus-end-distal MTs, but these are joined by minus-end-distal MTs as the processes continue to develop. Thus, this CHO1/MKLP1 fragment establishes a nonuniform MT polarity pattern and does so by a similar sequence of events as occurs with the dendrite, the antecedent of which is a short process with a uniform MT polarity orientation. Two lines of evidence suggest that these results are elicited by motor-driven MT transport. First, there is a depletion of MTs from the cell body during process outgrowth. Second, the same polarity pattern is obtained when net MT assembly is suppressed pharmacologically during process formation. Collectively, these findings provide precedent for the idea that motor-driven transport can organize MTs into distinct patterns of polarity orientation during process outgrowth (Sharp, 1996).

There is controversy concerning the mechanisms by which the axonal microtubule (MT) array is elaborated, with some models focusing on MT assembly and other models focusing on MT transport. Biotinylated tubulin was microinjected into cultured neurons that had already grown short axons. The axons were then permitted to grow longer, after which the cells were prepared for immunoelectron microscopic analyses. Any polymer that assembled or turned over subunits after the introduction of the probe should label for biotin, while any polymer that was already assembled but did not turnover should not label. Therefore, the presence in the newly grown region of the axon of any unlabeled MT polymer is indicative of MT transport. In sampled regions, the majority of the polymer was labeled, indicating that MT assembly events are active during axon growth. Varying amounts of unlabeled polymer were also present in the newly grown regions, indicating that MT transport also occurs. Together these findings demonstrate that MT assembly and transport both contribute to the elaboration of the axonal MT array (Yu, 1996).

It is well established that axons regulate Schwann cell phenotype. Do axons influence the arrangement of Schwann cell microtubules (MTs)? MTs in undifferentiated Schwann cells are nucleated from and attached to a single MT organizing center (MTOC) that is associated with the centrosome. Physical contact with appropriate axons initiates a myelin-forming phenotype that disperses MT minus ends and induces multiple MT-nucleating sites in Schwann cell perinuclear cytoplasm. The axonal signal that initiates myelin breakdown during Wallerian degeneration induces multiple MTOCs and MT bundles in Schwann cell perinuclear cytoplasm and in cytoplasm between degenerating myelin ovoids. These results establish that axons influence Schwann cell MT distribution by regulating the location and number of MT-nucleation sites (Kidd, 1996).

Neuronal function is dependent on the transport of materials from the cell body to the synapse via anterograde axonal transport. Anterograde axonal transport consists of several components that differ in both rate and protein composition. In fast transport, membranous organelles are moved along microtubules by the motor protein kinesin. The cytoskeleton and the cytomatrix proteins move in the two components of slow transport. While the mechanisms underlying slow transport are unknown, it has been hypothesized that the movement of microtubules in slow transport is generated by sliding. To determine whether dynein, a motor protein that causes microtubule sliding in flagella, may play a role in slow axonal transport, the transport rate components were examined with which cytoplasmic dynein is associated in rat optic nerve. Nearly 80% of the anterogradely moving dynein was associated with slow transport, whereas only approximately 15% of the dynein was associated with the membranous organelles of anterograde fast axonal transport. A segmental analysis of the transport of dynein through contiguous regions of the optic nerve and tract showed that dynein is associated with the microfilaments and other proteins of slow component b. Dynein from this transport component has the capacity to bind microtubules in vitro. These results are consistent with the hypothesis that cytoplasmic dynein generates the movement of microtubules in slow axonal transport. A model is presented to illustrate how dynein attached to the slow component b complex of proteins is appropriately positioned to generate force of the correct polarity to slide microtubules down the axon (Dillman, 1996).

A large body of evidence indicates that microtubules (MTs) conduct organelle transport in axons, but recent studies on extruded squid axoplasm have suggested that actin microfilaments (MFs) may also play a role in this process. To investigate the separate contributions to transport of each class of cytoskeletal element in intact vertebrate axons, mitochondrial movements were monotered in chick sympathetic neurons experimentally manipulated to eliminate MTs, MFs, or both. Mitochondria moved bidirectionally at normal velocities along the length of neurites which contained MTs and lacked MFs, but did not even enter neurites grown without MTs but containing MFs. In cytochalasin-treated cells, which retained MTs but lacked MFs, average mitochondrial velocity increased in both directions, but net directional transport decreased. In vinblastine-treated cells, which lacked MTs but retained essentially normal levels of MFs, mitochondria continued to move bidirectionally but the average mitochondrial velocity and excursion length were reduced for both directions of movement, and the mitochondria spent threefold as much time moving in the retrograde as in the anterograde direction, resulting in net retrograde transport. Treatment of established cultures with both drugs produced neurites lacking MTs and MFs but still rich in neurofilaments; these showed a striking absence of any mitochondrial motility. These data indicate that axonal organelle transport can occur along both MTs and MFs in vivo, but with different velocities and net transport properties (Morris, 1995).

Stathmin is a highly conserved ubiquitous cytoplasmic protein, phosphorylated in response to extracellular signals and during the cell cycle. Stathmin has recently been shown to destabilize microtubules, but the molecular mechanisms of this function remained unclear. Stathmin directly interacts with tubulin. The stathmin/tubulin interaction leads to the formation of a 7.7 S complex with a 60-A Stokes radius, associating one stathmin with two tubulin heterodimer molecules as determined by direct quantification using Western blotting. This interaction is sensitive to pH and ionic environment. The affinity is lowered with a fully "pseudophosphorylated" 4-Glu mutant form of stathmin, suggesting that it is modulated in vivo by stathmin phosphorylation. Stathmin reduces the growth rate of microtubules with no effect on the catastrophe frequency. Overall, these results suggest that the stathmin destabilizing activity on microtubules is related to tubulin sequestration by stathmin (Curmi, 1997).

Specific alpha- and beta-tubulin isotypes optimize the functions of sensory cilia in Caenorhabditis elegans

Primary cilia have essential roles in transducing signals in eukaryotes. At their core is the ciliary axoneme, a microtubule-based structure that defines cilium morphology and provides a substrate for intraflagellar transport. However, the extent to which axonemal microtubules are specialized for sensory cilium function is unknown. In the nematode C. elegans, primary cilia are present at the dendritic ends of most sensory neurons, where they provide a specialized environment for the transduction of particular stimuli. Three tubulin isotypes, the alpha-tubulins TBA-6 and TBA-9 and the beta-tubulin TBB-4, are specifically expressed in overlapping sets of C. elegans sensory neurons and localize to the sensory cilia of these cells. Although cilia still form in mutants lacking tba-6, tba-9, and tbb-4, ciliary function is often compromised: these mutants exhibit a variety of sensory deficits as well as the mislocalization of signaling components. In at least one case, that of the CEM cephalic sensory neurons, cilium architecture is disrupted in mutants lacking specific ciliary tubulins. While there is likely to be some functional redundancy among C. elegans tubulin genes, the results indicate that specific tubulins optimize the functional properties of C. elegans sensory cilia (Hurd, 2010).

These studies do not allow determination whether TBA-6, TBA-9 and TBB-4 are the exclusive components of ciliary microtubules in wild-type animals. It is possible that TBA-6:TBB-4 or TBA-9:TBB-4 dimers contribute only to certain domains of the cilium, with the rest of the axoneme composed of more general 'housekeeping' tubulins such as TBA-1/2 and TBB-1/2. It is also possible that the entire axoneme is built from a complex mixture of tubulin dimers using both cilium-specific and more general subunits. Either of these possibilities is consistent with observations that the efficient localization of ciliary tubulins to sensory endings, particularly in the ray neurons, usually depends on the co-expressed ciliary tubulin partner. However, the fact that cilia can be built in the absence of these partners indicates that there are not absolute isoform requirements either for heterodimerization or for incorporation into a growing microtubule (Hurd, 2010)

At the functional level, however, these studies clearly indicate that the sensory cilia generated in tba-6, tba-9, and tbb-4 mutants are abnormal. Several behaviors dependent on sensory input -- male mating, nose touch, and basal locomotion -- were compromised in tubulin mutants. Defects were found at the molecular level; the localization of the TRP channels TRP-4 and PKD-2 in the cilia of the RnA and RnB neurons, respectively, were disrupted in tba-6, tba-9, and tbb-4 mutants. Interestingly, these experiments revealed that the consequences of losing an α-tubulin (either tba-6 or tba-9) were often different from those of losing the β-tubulin tbb-4, indicating some cell-type specificity in the functions of ciliary microtubules. Together, these data show that the functional redundancy exhibited by tubulin isoforms in building the basic components of cilia does not extend to their more subtle functional properties and that α- and β-tubulins may play distinct roles in optimizing cilium specialization (Hurd, 2010)

Microtubules interact with Rac

Members of the Ras-related Rho family are involved in controlling actin-based changes in cell morphology. Microinjection of Rac1 (see Drosophila Rac), RhoA, and Cdc42Hs into Swiss 3T3 cells induces pinocytosis and membrane ruffling, stress fiber formation, and filopodia formation, respectively. To identify target proteins involved in these signaling pathways, cell extracts immobilized on nitrocellulose have been probed with [gamma-32P]GTP-labeled Rac1, RhoA, and Cdc42Hs. Two 55-kDa brain proteins that bind Rac1 but not RhoA or Cdc42Hs have been identified. These 55-kDa proteins are abundant, have pI values of around 5.5, and can be purified by Q-Sepharose chromatography. The characteristics evident upon two-dimensional gel analysis suggest that the proteins comprise alpha- and beta-tubulin. Indeed, beta-tubulin specific antibodies detect one of the purified 55-kDa proteins. Rac1 binds pure tubulin (purified by cycles of polymerization and depolymerization) only in the GTP-bound state. The GTPase negative Rac1 point mutants, G12V and Q61L, do not significantly affect the ability of Rac1 to interact with tubulin while the "effector-site" mutant D38A prevents interaction. These results suggest that the Rac1-tubulin interaction may play a role in Rac1 function (Best, 1996).

Migrating fibroblasts in culture exhibit an elongated, polarized shape, with a wide flat lamella that terminates in a ruffling lamellipodium at the leading edge, facing the direction of migration, and a trailing cell body tapering back to an extended tail. The tapering cell sides are relatively inactive. Membrane protrusions and ruffles are continuously initiated at the leading edge and undergo a smooth centripetal movement, known as retrograde flow, towards the cell body. Localized actin polymerization, nucleated at the leading edge, is required for ruffling activity, retrograde flow and protrusion of the leading edge to drive directed cell migration. Microtubules emanate out from the cell center toward the leading edge, aligned with the direction of migration, where their plus ends exhibit random changes between periods of growth and shortening; such fluctuations are termed dynamic instability. When treated with agents that cause microtubule disassembly, fibroblasts lose their extended shape, and the protrusive lamellipodial activity that is normally confined to the leading edge is reduced and distributed to random sites around the cell periphery. Thus, microtubules are thought to promote lamellipodial protrusion and direct sites of actin polymerization. Indeed, correlative video and immunofluorescence microscopy has shown that when microtubules enter a protrusion of the leading edge, the edge becomes stabilized against retraction, thereby promoting the cell's forward advance (Waterman-Storer, 1999 and references therein).

The prevailing hypothesis is that microtubules, extending from the cell center to the leading edge, serve as tracks for the directed delivery of membrane vesicles to the lamellipodium, where the membrane of the vesicles is inserted into the plasma membrane at the leading edge to drive lamellipodial protrusion and retrograde flow during cell migration. However, cell migration is stopped by pharmacological stabilization of microtubule dynamic instability without disassembly of microtubule tracks, indicating that migration requires some aspect of microtubule dynamic instability. In contrast to the prevailing view, the growth of microtubules induced in fibroblasts by removal of the microtubule destabilizer nocodazole has been shown to activate Rac1 GTPase, leading to the polymerization of actin in lamellipodial protrusions. Lamellipodial protrusions are also activated by the rapid growth of a disorganized array of very short microtubules induced by the microtubule-stabilizing drug taxol. Thus, neither microtubule shortening nor long-range microtubule-based intracellular transport is required for activating protrusion. It is suggested that the growth phase of microtubule dynamic instability at leading-edge lamellipodia locally activates Rac1 to drive actin polymerization and lamellipodial protrusion required for cell migration. Thus, protrusion of the leading edge can occur independently of microtubule-based organelle transport. However, the long-term maintenance of protrusive activity and the establishment of fibroblast polarity may also require that microtubules are organized into a radial array (Waterman-Storer, 1999).

How does the growth phase of microtubule dynamic instability activate Rac1? One possibility is that microtubule growth directly generates cytoplasmic Rac1-GTP. Rac1-GTP, but not Rac1-GDP, is known to bind to tubulin dimers. Thus, if Rac1-GTP and tubulin were to compete for the same binding site on a microtubule, it is possible that Rac1-GTP could be displaced from tubulin and released into the cytoplasm when the tubulin is added to the end of a microtubule during growth. This would explain how growing microtubule ends could provide active Rac1 to the leading cell edge. Alternatively, there is biochemical evidence to indicate that microtubules may enhance Rac1 activity by mediating the assembly of microtubule-bound Rac1 signaling complexes. Indeed, both upstream guanine-nucleotide-exchange-factor activators of Rac1, including GEF-H1, Vav and Lfc, and the downstream Rac1 effectors MLK2 and JNK either bind to tubulin or localize to microtubules in cells. It is also possible that these signaling complexes could associate preferentially with growing microtubule plus ends in vivo, making their assembly dependent on microtubule growth, similar to the behaviour observed for the microtubule-endosome linking protein, CLIP-170. Finally, it may be that the activity of some other Rho-family member is responsible for microtubule-growth-dependent Rac1 activity. RhoG activates both Rac1 and the Rho-family member Cdc42Hs in a microtubule-dependent way. Thus, microtubule-growth-induced lamellipodial protrusions could be due to RhoG activity. However, it is not clear how RhoG activity could depend on fresh microtubule growth. Another member of the Rho family of GTPases, RhoA, is also important in the relationship between microtubules and F-actin in cell contractility and adhesion. RhoA inhibitors block the assembly of F-actin stress fibers and the formation of focal adhesions that are induced by microtubule depolymerization. Not surprisingly, direct measurement of RhoA-GTP levels in cells has also shown that depolymerization of microtubules with colchicine activates RhoA. This observation, together with the results presented here, indicates that Rho-family members may underlie the elusive crosstalk between microtubules and actin that is required for the regulation of cell motility and cytokinesis (Waterman-Storer, 1999).

Microtubules interact with APC

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating ß-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization. The microtubule binding domain is deleted in the transforming mutations of APC; however, the direct effect of APC protein on microtubules has never been examined. Binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3ß. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation (Zumbrunn, 2001).

INCENP interacts with tubulin

Inner centromere protein (INCENP; see Drosophila Inner centromere protein) is a chromosomal passenger protein with an essential role in mitosis. At the metaphase/anaphase transition, some INCENP transfers from the centromeres to the central spindle; the remainder then transfers to the equatorial cortex prior to cleavage furrow formation. The molecular associations dictating INCENP behavior during mitosis are currently unknown. Targeting INCENP to the cleavage plane requires dynamic microtubules, but not F-actin. When microtubules are eliminated, INCENP is dispersed across the entire cell cortex. Yeast two-hybrid and in vitro binding data demonstrate that INCENP binds directly to beta-tubulin via a conserved domain encompassing residues 48-85. Furthermore, INCENP binds to microtubules polymerized from purified tubulin in vitro and appears to bundle microtubules when expressed in the interphase cytoplasm. These data indicate that INCENP is a microtubule-binding protein that targets to the equatorial cortex through interactions requiring microtubules (Wheatley, 2001).

CRMP-2 binds to tubulin heterodimers to promote microtubule assembly

Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2: Drosophila homolog Collapsin Response Mediator Protein) is a mammalian homologue of UNC-33, mutations in which result in abnormal axon termination. CRMP-2 is critical for axonal differentiation. Two activities of CRMP-2 have been identified: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 binds tubulin dimers with higher affinity than it binds microtubules. Association of CRMP-2 with microtubules is enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin is apparently distinct from that of Tau, which preferentially binds microtubules. In neurons, overexpression of CRMP-2 promotes axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibits axonal growth and branching in a dominant-negative manner. Taken together, these results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs (Fukata, 2002).

Tubulin interaction with Parkin

Mutations of parkin, a protein-ubiquitin isopeptide ligase (E3), appear to be the most frequent cause of familial Parkinson's disease (PD). Parkin binds strongly to alpha/beta tubulin heterodimers and microtubules. The strong binding between parkin and tubulin, as well as that between parkin and microtubules, is mediated by three independent domains: linker, RING1, and RING2. These redundant strong interactions made it virtually impossible to separate parkin from microtubules by high concentrations of salt (3.8 m) or urea (0.5 m). Parkin co-purified with tubulin and was found in a highly purified tubulin preparation. Expression of either full-length parkin or any of its three microtubule-binding domains significantly attenuates colchicine-induced microtubule depolymerization. The abilities of parkin to bind to and stabilize microtubules are not affected by PD-linked mutations that abrogate its E3 ligase activity. Thus, the tubulin/microtubule-binding activity of parkin and its E3 ligase activity are independent. The strong binding between parkin and tubulin/microtubules through three redundant interaction domains may not only stabilize microtubules but also guarantee the anchorage of this E3 ligase on microtubules. Because many misfolded proteins are transported on microtubules, the localization of parkin on microtubules may provide an important environment for its E3 ligase activity toward misfolded substrates (Yang, 2005).

Centrobin-tubulin interaction is required for centriole elongation and stability

Centrobin is a daughter centriole protein that is essential for centrosome duplication. However, the molecular mechanism by which centrobin functions during centriole duplication remains undefined. This study, carried out in mammalian cultured cells, shows that centrobin interacts with tubulin directly, and centrobin-tubulin interaction is pivotal for the function of centrobin during centriole duplication. Centrobin was found to be recruited to the centriole biogenesis site via its interaction with tubulins during the early stage of centriole biogenesis, and its recruitment is dependent on hSAS-6 but not centrosomal P4.1-associated protein (CPAP) and CP110. The function of centrobin is also required for the elongation of centrioles, which is likely mediated by its interaction with tubulin. Furthermore, disruption of centrobin-tubulin interaction led to destabilization of existing centrioles and the preformed procentriole-like structures induced by CPAP expression, indicating that centrobin-tubulin interaction is critical for the stability of centrioles. Together, this study demonstrates that centrobin facilitates the elongation and stability of centrioles via its interaction with tubulins (Gudi, 2011).

The coiled-coil protein centrobin is preferentially localized to the daughter centriole and is required for centriole duplication. Centrobin is recruited to the procentrioles at the beginning of S phase. During S, G2, and M phases, there are two centrobin-positive centrioles, the newly assembled procentrioles. After cell division, most G1 phase cells have one centrobin-positive centriole, the daughter centriole assembled in the previous cell cycle. Upon reentering S phase, centrobin on the daughter centriole assembled in the previous cycle becomes undetectable in the majority of the cells. In the absence of centrobin, no discernible centriole structures were assembled as demonstrated by EM analysis (Zou, 2005). Centrobin has also been reported to be a substrate of the kinase Nek2 and plays a role in stabilizing the microtubule network (Jeong, 2007). In addition, centrobin was found to regulate the assembly of functional mitotic spindles (Jeffery, 2010; Gudi, 2011 and references therein).

Centrioles are predominantly composed of α/β-tubulins. Centriole elongation, at least visually, is the process of assembling the nine microtubule triplets by adding α/β-tubulin dimers to an undefined initiating template structure. This study demonstrates that centrobin interacts directly with the core components of the centriole, α-tubulins via its C-terminal 139 residues (centrobin-TuBD). Centrobin-TuBD exhibited a clear centrosomal localization in addition to a diffused cytoplasmic and nuclear localization. It is noticeable that although centrobin-TuBD can bind strongly to tubulins, no detectable microtubular localization of centrobin-TuBD or adverse effect on microtubule nucleation was observed. Although surprising, this finding correlates with previous observations that endogenous centrobin is not clearly detectable on microtubules (Zou, 2005). Jeong (2007) had reported that centrobin is detectable in association with the roots or initiating points of microtubules in U2OS and MCF7 cells but not in HeLa cells. It is very likely that the tubulins at the initiating points of microtubules and tubulins at the centrosomes share similar conformation with the centrosomal tubulins, in which their centrobin-binding domain is exposed. When the tubulins are assembled into microtubules, the centrobin-binding domain is no longer accessible (Gudi, 2011).

Importantly, overexpression of centrobin-TuBD can disrupt the endogenous full-length centrobin-tubulin interaction, providing an a valuable tool to dissect the function of centrobin during centrobin duplication. Using centrobin-TuBD, it was demonstrated that centrobin is recruited to centrioles and facilitates centriole elongation via its interaction with centrosomal tubulins. Moreover, it was found that centrobin-TuBD overexpression destabilized the existing mother centrioles in addition to inhibiting the assembly of new centrioles, indicating that centrobin is required for the stability of centrioles. Previous findings (Zou, 2005) indicated that, in asynchronized cells, centrobin is mainly found on the daughter centrioles. Even in HU-treated cells, in which the degradation or displacement of centrobin from the daughter centriole is inhibited, there is at least one or two mother centrioles exhibiting no centrobin staining, which will suggest that centrobin should not be required for the stability of these mature centrioles. Two possible scenarios are proposed to explain these conflicting findings. First, centrobin is indeed required for the stability of both daughter and mature mother centrioles. On the mature mother centrioles, centrobin is still there to maintain their stability but becomes undetectable because additional mother centriole proteins block the access of centrobin antibody. Second, centrobin is only required to maintain the stability of the daughter centrioles. Once the daughter centrioles mature to become mother centrioles by recruiting additional mother centriole proteins and extensive modification of centriolar tubulins, centrobin is no longer required for their stability. Centrobin is then either degraded or displaced from the mother centrioles by the mother centriole proteins or the tubulin modifications. However, because of the small size of centrobin-TuBD and its presence at high concentration, it can still access the centrobin-binding domain on the tubulins and competes with the mother centriole proteins. Consequently, centrobin-TuBD will displace the mother centriole proteins and lead to destabilization of the mother centrioles. Although current data cannot distinguish between these two scenarios, it is speculated that the first scenario is more plausible because centrobin can indeed be present on the mother centriole as is evident in HU-treated cells. One seemingly conflicting piece of evidence against this hypothesis is that centrobin depletion inhibited the centriole duplication but did not destabilize the mother centrioles. The likely explanation is that centrobin assembled into the mother centrioles is stabilized and is impervious to depletion, as are most cellular structural proteins. Hence, centrobin depletion cannot destabilize the mother centriole, whereas centrobin-TuBD can displace the centrobin on the mother centrioles and lead to their destabilization. However, no direct evidence for this is available, and further studies are required to prove this scenario. Furthermore, in the rescue experiments, the existing centrioles were still destabilized in 5% of cells and were not rescued by the wild-type centrobin expression; therefore, it is possible that both scenarios can coexist and account for the observed destabilization of existing centrioles (Gudi, 2011).

The key findings from this study suggest that centrobin functions at least at three stages of the centriole duplication pathway (see Model of centrobin function during the centriole duplication process). At the beginning of centriole biogenesis, hSAS-6 is first recruited to the proximal end of the mother centrioles in the G1/S phase. Centrobin is then recruited and likely participates in the undefined centriole initiation structure formation along with the other proposed centriole initiation proteins, including CPAP, CEP135, γ-tubulin, and CP110. During the elongation of procentrioles, centrobin may function as a scaffold protein via its interaction with tubulins to facilitate the addition of tubulin dimers to the centriole initiation complex. Centrobin also acts to stabilize the newly assembled daughter centrioles before their maturation. Whether centrobin is required to maintain the stability of the mature mother centriole remains to be determined. The function of centrobin during centriole elongation and its function to maintain the stability of the centriole are likely both mediated by its ability to bind to tubulins (Gudi, 2011).

CPAP also has the ability to bind to microtubules, and regulation of its cellular levels is required to maintain the centriole length. It has been suggested that as a result of its tubulin binding property, CPAP might act as a scaffold for tubulin addition during procentriole biogenesis. Because the property of tubulin binding is shared by CPAP and centrobin and both are required for centriole biogenesis, it would be interesting to study whether centrobin and CPAP cooperate to facilitate the assembly of the microtubule triplets that form the main structure of centrioles. So far, there is no convincing evidence indicating the existence of a lower eukaryotic centrobin homologue. If centrobin indeed cooperates with CPAP for centriole elongation, it will indicate that centrobin is functionally similar to C. elegans SAS-5. In summary, it is concluded that centrobin-tubulin interaction is pivotal for centrobin recruitment to the centriole biogenesis site, centriole elongation, and stabilization of nascent centrioles until maturation (Gudi, 2011).

Regulation of microtubule motors by tubulin isotypes and post-translational modifications

The 'tubulin-code' hypothesis proposes that different tubulin genes or post-translational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogeneous tubulin species has hindered biochemical testing of this hypothesis. This study engineered 25 alpha/beta-tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single-molecule assays. The results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, kinesin-1 motility on neuronal beta-tubulin (TUBB3) was shown to be increased by polyglutamylation, and robust kinesin-2 motility was shown to require detyrosination of alpha-tubulin. The results also show that different molecular motors recognize distinctive tubulin 'signatures', which supports the premise of the tubulin-code hypothesis (Sirajuddin, 2014).

betaTubulin56D (ß1 tubulin): Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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