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Background Microtubules are dynamic polymers of α/β tubulin heterodimers that play a critical role in cerebral cortical development, by regulating neuronal migration, differentiation, and morphogenesis. Mutations in genes that encode either α- or β-tubulin or a spectrum of proteins involved in the regulation of microtubule dynamics lead to clinically devastating malformations of cortical development, including lissencephaly. Methods This is a single case report or a patient with lissencephaly, developmental delay, nystagmus, persistent hyperplastic primary vitreous, and infantile spasms, and undertook a neurogenetic workup. We include studies of mutant function in Escherichia coli and HeLa cells. Results The patient was found to have a novel de novo mutation in kinesin family member 2A (KIF2A). This mutation results in a substitution of isoleucine at a highly conserved threonine residue within the ATP-binding domain. The KIF2A p.Thr320Ile mutant protein exhibited abnormal solubility, and KIF2A p.Thr320Ile overexpression in cultured cells led to the formation of aberrant microtubule networks. Conclusion Findings support the pathogenic link between KIF2A mutation and lissencephaly, and expand the range of presentation to include infantile spasms and congenital anomalies.

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule associated protein, that is predominantly expressed in post-mitotic neurons, and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.

PubMedID: 28013290 The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the ?/ß-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of b-tubulin to fold or become assembled into the ?/ß-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects. © The Author 2016. Published by Oxford University Press. All rights reserved.

Mutation in a growing spectrum of genes is known to either cause or contribute to primary or secondary microcephaly. In primary microcephaly the genetic determinants frequently involve mutations that contribute to or modulate the microtubule cytoskeleton by causing perturbations of neuronal proliferation and migration. Here we describe four patients from two unrelated families each with an infantile neurodegenerative disorder characterized by loss of developmental milestones at 9–24 months of age followed by seizures, dystonia and acquired microcephaly. The patients harboured homozygous missense mutations (A475T and A586V) in TBCD, a gene encoding one of five tubulin-specific chaperones (termed TBCA-E) that function in concert as a nanomachine required for the de novo assembly of the α/β tubulin heterodimer. The latter is the subunit from which microtubule polymers are assembled. We found a reduced intracellular abundance of TBCD in patient fibroblasts to about 10% (in the case of A475T) or 40% (in the case of A586V) compared to age-matched wild type controls. Functional analyses of the mutant proteins revealed a partially compromised ability to participate in the heterodimer assembly pathway. We show via in utero shRNA-mediated suppression that a balanced supply of tbcd is critical for cortical cell proliferation and radial migration in the developing mouse brain. We conclude that TBCD is a novel functional contributor to the mammalian cerebral cortex development, and that the pathological mechanism resulting from the mutations we describe is likely to involve compromised interactions with one or more TBCD-interacting effectors that influence the dynamics and behaviour of the neuronal cytoskeleton.

Background TUBB8 is a primate-specific β-tubulin isotype whose expression is confined to oocytes and the early embryo. We previously found that mutations in TUBB8 caused oocyte maturation arrest. The objective was to describe newly discovered mutations in TUBB8 and to characterise the accompanying spectrum of phenotypes and modes of inheritance. Methods and results Patients with oocyte maturation arrest were sequenced with respect to TUBB8 . We investigated the effects of identified mutations in vitro, in cultured cells and in mouse oocytes. Seven heterozygous missense and two homozygous mutations were identified. These mutations cause a range of folding defects in vitro, different degrees of microtubule disruption upon expression in cultured cells and interfere to varying extents in the proper assembly of the meiotic spindle in mouse oocytes. Several of the newly discovered TUBB8 mutations result in phenotypic variability. For example, oocytes harbouring any of three missense mutations (I210V, T238M and N348S) could extrude the first polar body. Moreover, they could be fertilised, although the ensuing embryos became developmentally arrested. Surprisingly, oocytes from patients harbouring homozygous TUBB8 mutations that in either case preclude the expression of a functional TUBB8 polypeptide nonetheless contained identifiable spindles. Conclusions Our data substantially expand the range of dysfunctional oocyte phenotypes incurred by mutation in TUBB8 , underscore the independent nature of human oocyte meiosis and differentiation, extend the class of genetic diseases known as the tubulinopathies and provide new criteria for the qualitative evaluation of meiosis II (MII) oocytes for in vitro fertilization (IVF).

Copyright © 2016 Massachusetts Medical Society. All rights reserved. BACKGROUND: Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.

Summary The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of α-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders.

Circumferential skin creases Kunze type (CSC-KT) is a specific congenital entity with an unknown genetic cause. The disease phenotype comprises characteristic circumferential skin creases accompanied by intellectual disability, a cleft palate, short stature, and dysmorphic features. Here, we report that mutations in either MAPRE2 or TUBB underlie the genetic origin of this syndrome. MAPRE2 encodes a member of the microtubule end-binding family of proteins that bind to the guanosine triphosphate cap at growing microtubule plus ends, and TUBB encodes a β-tubulin isotype that is expressed abundantly in the developing brain. Functional analyses of the TUBB mutants show multiple defects in the chaperone-dependent tubulin heterodimer folding and assembly pathway that leads to a compromised yield of native heterodimers. The TUBB mutations also have an impact on microtubule dynamics. For MAPRE2, we show that the mutations result in enhanced MAPRE2 binding to microtubules, implying an increased dwell time at microtubule plus ends. Further, in vivo analysis of MAPRE2 mutations in a zebrafish model of craniofacial development shows that the variants most likely perturb the patterning of branchial arches, either through excessive activity (under a recessive paradigm) or through haploinsufficiency (dominant de novo paradigm). Taken together, our data add CSC-KT to the growing list of tubulinopathies and highlight how multiple inheritance paradigms can affect dosage-sensitive biological systems so as to result in the same clinical defect.

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the α/β- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a β-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.

Chaperonins participate in the facilitated folding of a variety of proteins in vivo. To see whether the same spectrum of target proteins can be productively folded by the double-ring prokaryotic chaperonin GroEL-GroES and its single-ring human mitochondrial homolog, Hsp60-Hsp10, we expressed the latter in an Escherichia coli strain engineered so that the groE operon is under strict regulatory control. We found that expression of Hsp60-Hsp10 restores viability to cells that no longer express GroEL-GroES, formally demonstrating that Hsp60-Hsp10 can carry out all essential in vivo functions of GroEL-GroES.

In vivo, many proteins must interact with molecular chaperones to attain their native conformation. In the case of tubulin, newly synthesized α- and β-subunits are partially folded by cytosolic chaperonin, a double-toroidal ATPase with homologs in all kingdoms of life and in most cellular compartments. α- and β-tubulin folding intermediates are then brought together by tubulin-specific chaperone proteins (named cofactors A–E) in a cofactor-containing supercomplex with GTPase activity. Here we show that tubulin subunit exchange can only occur by passage through this supercomplex, thus defining it as a dimer-making machine. We also show that hydrolysis of GTP by β-tubulin in the supercomplex acts as a switch for the release of native tubulin heterodimer. In this folding reaction and in the related reaction of tubulin-folding cofactors with native tubulin, the cofactors behave as GTPase-activating proteins, stimulating the GTP-binding protein β-tubulin to hydrolyze its GTP.

In vitro transcription/translation of actin cDNA and analysis of the translation products by native-PAGE was used to study the maturation pathway of actin. During the course of actin synthesis, several distinct actin-containing species were observed and the composition of each determined by immunological procedures. After synthesis of the first approximately 145 amino acids, the nascent ribosome-associated actin chain binds to the recently identified heteromeric chaperone protein, prefoldin (PFD). PFD remains bound to the relatively unfolded actin polypeptide until its posttranslational delivery to cytosolic chaperonin (CCT). We show that alpha- and beta-tubulin follow a similar maturation pathway, but to date find no evidence for an interaction between PFD and several noncytoskeletal proteins. We conclude that PFD functions by selectively targeting nascent actin and tubulin chains pending their transfer to CCT for final folding and/or assembly.

Facilitated protein folding by the double toroidal bacterial chaperonin, GroEL/GroES, proceeds by a "two-stroke engine" mechanism in which an allosteric interaction between the two rings synchronizes the reaction cycle by controlling the binding and release of cochaperonin. Using chimeric chaperonin molecules assembled by fusing equatorial and apical domains derived from GroEL and its mammalian mitochondrial homolog, Hsp60, we show that productive folding by Hsp60 and its cognate cochaperonin, Hsp10, proceeds in vitro and in vivo without the formation of a two-ring structure. This simpler "one-stroke" engine works because Hsp60 has a different mechanism for the release of its cochaperonin cap and bound target protein.

The α—β tubulin heterodimer is the subunit from which microtubules are assembled. The pathway leading to correctly folded α- and β-tubulins is unusually complex: it involves cycles of ATP-dependent interaction of newly synthesized tubulin subunits with cytosolic chaperonin, resulting in the production of quasi-native folding intermediates, which must then be acted upon by additional protein cofactors. These cofactors form a supercomplex containing both α- and β-tubulin polypeptides, from which native heterodimer is released in a GTP-dependent reaction. Here, we discuss the current state of our understanding of the function of cytosolic chaperonin and cofactors in tubulin folding.

The production of native alpha/beta tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on beta-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from alpha-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of alpha-tubulin folding in vitro. This enabled an integrated study of alpha- and beta-tubulin folding: we find that the pathways leading to the formation of native alpha- and beta-tubulin converge in that the folding of the alpha subunit requires the participation of cofactor complexes containing the beta subunit and vice versa. We also show that sequestration of native alpha-or beta-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining alpha-and beta-tubulin polypeptides in an activated conformational state required for the formation of native alpha/beta heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.

T HE central dogma of molecular biology holds that proteins contain within their primary amino acid sequence all the information that is required to dictate their three-dimensional structure. In principle, therefore, proteins can fold spontaneously (2). It is now clear, however, that the proper folding of many proteins requires facilitation. The facilitation reaction is accomplished via interaction with chaperonins, a class of multisubunit toroidal complexes which hydrolyzes ATP as part of the mechanism whereby its members contribute to the productive folding of their target polypeptides (4, 5, 11, 12, 15, 16, 20). Chaperonins are both structurally and functionally distinct from a class of molecules (typified by the heat shock protein hsp70) that is also thought to participate in the overall protein folding pathway by maintaining polypeptides in a partially unfolded conformation pending their translocation or presentation to chaperonin (for a review see reference 15). Bacteria, chloroplasts, mitochondria, and the cytosol of eukaryotes each contain a single kind of chaperonin molecule. These are different from one other, but homologous. No chaperonin has so far been found in the endoplasmic reticulum. Here we present our view of how the eukaryotic cytosolic chaperonin functions, particularly in relation to what is known about the mechanism of facilitated folding by chaperonin from prokaryotes.

The tubulin heterodimer consists of one α- and one β-tubulin polypeptide. Neither protein can partition to the native state or assemble into polymerization competent heterodimers without the concerted action of a series of chaperone proteins including five tubulin-specific chaperones (TBCs) termed TBCA-TBCE. TBCA and TBCB bind to and stabilize newly synthesized quasi-native β- and α-tubulin polypeptides, respectively, following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin. There is free exchange of β-tubulin between TBCA and TBCD, and of α-tubulin between TBCB and TBCE, resulting in the formation of TBCD/β and TBCE/α, respectively. The latter two complexes interact, forming a supercomplex (TBCE/α/TBCD/β). Discharge of the native α/β heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound β-tubulin (E-site) GTP hydrolysis. This reaction acts as a switch for disassembly of the supercomplex and the release of E-site GDP-bound heterodimer, which becomes polymerization competent following spontaneous exchange with GTP. The tubulin-specific chaperones thus function together as a tubulin assembly machine, marrying the α- and β-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate α- or β-tubulin as stable independent entities in the absence of their cognate partners, and implies that each exists and is maintained in the heterodimer in a nonminimal energy state. Here, we describe methods for the purification of recombinant TBCs as biologically active proteins following their expression in a variety of host/vector systems.

Chaperonins are ubiquitous multisubunit toroidal complexes that aid protein folding in an ATP-dependent manner. Current models of folding by the bacterial chaperonin GroEL depict its role as unfolding and releasing molecules that have misfolded, so that they can return to a potentially productive folding pathway in solution. Accordingly, a given target polypeptide might require several cycles of binding and ATP-driven release from different chaperonin complexes before reaching the native state. Surprisingly, cycling of a target protein does not guarantee its folding, and we report here that unfolded beta-actin or alpha-tubulin both form tight complexes when presented to either GroEL or its mitochondrial homologue, and both undergo cycles of release and rebinding upon incubation with ATP, but no native protein is produced. We conclude that different chaperonins produce distinctive spectra of folding intermediates.

Androgen receptor (AR)-mediated transcription is modulated by interaction with coregulatory proteins. We demonstrate that the unconventional prefoldin RPB5 interactor (URI) is a new regulator of AR transcription and is critical for antagonist (bicalutamide) action. URI is phosphorylated upon androgen treatment, suggesting communication between the URI and AR signaling pathways. Whereas depletion of URI enhances AR-mediated gene transcription, overexpression of URI suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth. Repression of AR-mediated transcription is achieved, in part, by URI binding and regulation of androgen receptor trapped clone 27 (Art-27), a previously characterized AR corepressor. Consistent with this idea, genome-wide expression profiling in prostate cancer cells upon depletion of URI or Art-27 reveals substantially overlapping patterns of gene expression. Further, depletion of URI increases the expression of the AR target gene NKX-3.1, decreases the recruitment of Art-27, and increases AR occupancy at the NKX-3.1 promoter. While Art-27 can bind AR directly, URI is bound to chromatin prior to hormone-dependent recruitment of AR, suggesting a role for URI in modulating AR recruitment to target genes.

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.

The folding of actin and tubulin is mediated via interaction with a heteromeric toroidal complex (cytoplasmic chaperonin) that hydrolyzes ATP as part of the reaction whereby native proteins are ultimately released. Vertebrate actin-related protein (actin-RPV) (also termed centractin) and gamma-tubulin are two proteins that are distantly related to actin and tubulin, respectively: gamma-tubulin is exclusively located at the centrosome, while actin-RPV is conspicuously abundant at the same site. Here we show that actin-RPV and gamma-tubulin are both folded via interaction with the same chaperonin that mediates the folding of beta-actin and alpha- and beta-tubulin. In each case, the unfolded polypeptide forms a binary complex with cytoplasmic chaperonin and is released as a soluble, monomeric protein in the presence of Mg-ATP and the presence or absence of Mg-GTP. In contrast to alpha- and beta-tubulin, the folding of gamma-tubulin does not require the presence of cofactors in addition to chaperonin itself. Monomeric actin-RPV produced in in vitro folding reactions cocycles efficiently with native brain actin, while in vitro folded gamma-tubulin binds to polymerized microtubules in a manner consistent with interaction with microtubule ends. Both monomeric actin-RPV and gamma-tubulin bind to columns of immobilized nucleotide: monomeric actin-RPV has no marked preference for ATP or GTP, while gamma-tubulin shows some preference for GTP binding. We show that actin-RPV and gamma-tubulin compete with one another, and with beta-actin or alpha-tubulin, for binary complex formation with cytoplasmic chaperonin.

The mechanism of binding of microtubule-associated protein 2 (MAP2) to taxol-stabilized microtubules (MTs) was examined through Scatchard analysis of equilibrium binding and by immunoelectron microscopy. We demonstrate the following. 1) Binding is a cooperative process as indicated by sigmoidal binding curves, prominent humps in Scatchard plots, and an all-or-none response in binding during ligand titrations. At high tubulin/MAP2 ratios, the Kd for noncontiguous binding (5-25 microM) is estimated to be 100-1500 times greater than that predicted for contiguous binding, suggesting a high degree of cooperativity. 2) Cooperativity is indicated independently by a highly clustered or patchy distribution of MAP2 on MTs as revealed by immunoelectron microscopy. 3) The binding of truncated constructs of mouse MAP2 protein suggests that a domain of MAP2 conferring cooperativity is located in or near the MT binding site near the carboxyl terminus. We speculate that in the cell, cooperativity may generate MTs with uniform biochemical properties and contribute to the segregation of MAPs in neuronal cell processes.

Assembly of the α/β tubulin heterodimer requires the participation of a series of chaperone proteins (TBCA-E) that function downstream of the cytosolic chaperonin (CCT) as a heterodimer assembly machine. TBCD and TBCE are also capable of acting in a reverse reaction in which they disrupt native heterodimers. Homologs of TBCA-E exist in all eukaryotes, and the amino acid sequences of α- and β-tubulin isotypes are rigidly conserved among vertebrates. However, the efficiency with which TBCD effects tubulin disruption in vivo depends on its origin: bovine (but not human) TBCD efficiently destroys tubulin and microtubules upon overexpression in cultured cells. Here we show that recombinant bovine TBCD is produced in HeLa cells as a stoichiometric cocomplex with β-tubulin, consistent with its behavior in vitro and in vivo. In contrast, expression of human TBCD using the same host/vector system results in the generation of TBCD that is not complexed with β-tubulin. We show that recombinant human TBCD functions indistinguishably from its nonrecombinant bovine counterpart in in vitro CCT-driven folding reactions, in tubulin disruption reactions, and in tubulin GTPase activating protein assays in which TBCD and TBCC stimulate GTP hydrolysis by β-tubulin at a heterodimer concentration far below that required for polymerization into microtubules. We conclude that bovine and human TBCD have functionally identical roles in de novo tubulin heterodimer assembly, and show that the inability of human TBCD to disrupt microtubule integrity upon overexpression in vivo can be overcome by siRNA-mediated suppression of expression of the TBCD regulator Arl2 (ADP ribosylation factor-like protein).

Malformations of cortical development are characteristic of a plethora of diseases that includes polymicrogyria, periventricular and subcortical heterotopia and lissencephaly. Mutations in TUBA1A and TUBB2B, each a member of the multigene families that encode alpha- and beta-tubulins, have recently been implicated in these diseases. Here we examine the defects that result from nine disease-causing mutations (I188L, I238V, P263T, L286F, V303G, L397P, R402C, 402H, S419L) in TUBA1A. We show that the expression of all the mutant proteins in vitro results in the generation of tubulin heterodimers in varying yield and that these can co-polymerize with microtubules in vitro. We identify several kinds of defects that result from these mutations. Among these are various defects in the chaperone-dependent pathway leading to de novo tubulin heterodimer formation. These include a defective interaction with the chaperone prefoldin, a reduced efficiency in the generation of productive folding intermediates as a result of inefficient interaction with the cytosolic chaperonin, CCT, and, in several cases, a failure to stably interact with TBCB, one of five tubulin-specific chaperones that act downstream of CCT in the tubulin heterodimer assembly pathway. Other defects include structural instability in vitro, diminished stability in vivo, a compromised ability to co-assemble with microtubules in vivo and a suppression of microtubule growth rate in the neurites (but not the soma) of cultured neurons. Our data are consistent with the notion that some mutations in TUBA1A result in tubulin deficit, whereas others reflect compromised interactions with one or more MAPs that are essential to proper neuronal migration.

To the Editor: Sheridan and colleagues recently reported that mutations in the tubulin gene TUBA8 result in polymicrogyria with optic nerve hypoplasia (PMGOH [MIM 613180]).1 This conclusion is based on the mapping of two consanguineous families of Pakistani origin to a 7.42 Mb region on chromosome 22q11.2 that contains ∼230 genes including TUBA8. Drawing on our previous finding that mutations in TUBA1A cause lissencephaly2 and that mutations in TUBB2B cause asymmetric polymicrogyria,3 Sheridan and colleagues sequenced TUBA8 and found a 14 bp deletion in intron 1 that affects splicing. They provide further evidence that TUBA8 is involved in the disease state by analyzing its expression in the developing mouse brain by in situ hybridization. They report that Tuba8 is widely expressed in developing neural structures, with strongest expression in the cortical plate at E15.5 and E18.5 and in the cortical plate, subplate, and hippocampus at P0.

Neurons develop a highly polarized morphology consisting of dendrites and a long axon. Both axons and dendrites contain microtubules and microtubule-associated proteins (MAPs) with characteristic structures. Among MAPs, MAP2 is specifically expressed in dendrites whereas MAP2C and tau are abundant in the axon. But the influence of MAP2, MAP2C and tau on the organization of microtubule domains in dendrites versus axons is unknown. Both MAP2 and tau induce microtubule bundle formation in fibroblasts after transfection of complementary DNAs, and a long process resembling an axon is extended in Sf9 cells infected with recombinant baculovirus expressing tau. We have now expressed MAP2 and MAP2C in Sf9 cells in order to compare their morphology and the arrangement of their microtubules to that found in Sf9 cells expressing tau. We report here that the spacing between microtubules depends on the MAP expressed: in cells expressing MAP2, the distance is similar to that found in dendrites, whereas the spacing between microtubules in cells expressing MAP2C or tau is similar to that found in axons.

The functional subunit of microtubules is a heterodimer consisting of α- and β-tubulin. An understanding of tubulin dimerization has been hampered because it has not proved possible to purify native tubulin monomers. To study the process whereby tubulin dimers are formed, we made use of tubulins synthesized by in vitro transcription and translation. We present evidence that the in vitro synthesis of different mouse α-tubulin isotypes involves a multimolecular complex. The synthesis of mouse β-tubulin isotypes also involves the formation of multimolecular complexes, though different isotypes behave somewhat differently from one another. The properties of in vitro synthesized α- and β-tubulin multimolecular complexes strongly suggest that they are intermediates in the biosynthesis of tubulin monomers. Upon release, these monomers can exchange with pre-existing tubulin heterodimers. © 1992 Wiley-Liss, Inc.

Polymicrogyria is a relatively common but poorly understood defect of cortical development characterized by numerous small gyri and a thick disorganized cortical plate lacking normal lamination. Here we report de novo mutations in a b-tubulin gene, TUBB2B, in four individuals and a 27-gestational-week fetus with bilateral asymmetrical polymicrogyria. Neuropathological examination of the fetus revealed an absence of cortical lamination associated with the presence of ectopic neuronal cells in the white matter and in the leptomeningeal spaces due to breaches in the pial basement membrane. In utero RNAi-based inactivation demonstrates that TUBB2B is required for neuronal migration. We also show that two disease-associated mutations lead to impaired formation of tubulin heterodimers. These observations, together with previous data, show that disruption of microtubule-based processes underlies a large spectrum of neuronal migration disorders that includes not only lissencephaly and pachygyria, but also polymicrogyria malformations. The crucial role of the tubulin superfamily in diverse cellular processes 1 and the association of TUBA1A mutations with a broad

We show that the expression of the gene encoding glial fibrillary acidic protein (GFAP) gene is affected by at least three cis-acting elements. A positive regulatory element that is located between nucleotides -1,631 and -1,479 can confer cell type-specific expression on a heterologous gene. A second regulatory element is located between nucleotides -97 and -80. The third is a negative regulatory element that is located within the first intron of the gene. Deletion of this element activates GFAP expression in HeLa cells, and affects promoter function in glioma cells.

The agyria (lissencephaly)/pachygyria phenotypes are catastrophic developmental diseases characterized by abnormal folds on the surface of the brain and disorganized cortical layering. In addition to mutations in at least four genes—LIS1, DCX, ARX and RELN—mutations in a human α-tubulin gene, TUBA1A, have recently been identified that cause these diseases. Here, we show that one such mutation, R264C, leads to a diminished capacity of de novo tubulin heterodimer formation. We identify the mechanisms that contribute to this defect. First, there is a reduced efficiency whereby quasinative α-tubulin folding intermediates are generated via ATP-dependent interaction with the cytosolic chaperonin CCT. Second, there is a failure of CCT-generated folding intermediates to stably interact with TBCB, one of the five tubulin chaperones (TBCA–E) that participate in the pathway leading to the de novo assembly of the tubulin heterodimer. We describe the behavior of the R264C mutation in terms of its effect on the structural integrity of α-tubulin and its interaction with TBCB. In spite of its compromised folding efficiency, R264C molecules that do productively assemble into heterodimers are capable of copolymerizing into dynamic microtubules in vivo. The diminished production of TUBA1A tubulin in R264C individuals is consistent with haploinsufficiency as a cause of the disease phenotype.

Microtubules are indispensable dynamic structures that contribute to many essential biological functions. Assembly of the native α/β tubulin heterodimer, the subunit that polymerizes to form microtubules, requires the participation of several molecular chaperones, namely prefoldin, the cytosolic chaperonin CCT, and a series of five tubulin-specific chaperones termed cofactors A–E (TBCA–E). Among these, TBCC, TBCD, and TBCE are essential in higher eukaryotes; they function together as a multimolecular machine that assembles quasinative CCT-generated α- and β-tubulin polypeptides into new heterodimers. Deletion and truncation mutations in the gene encoding TBCE have been shown to cause the rare autosomal recessive syndrome known as HRD, a devastating disorder characterized by congenital hypoparathyroidism, mental retardation, facial dysmorphism, and extreme growth failure. Here we identify cryptic translational initiation at each of three out-of-frame AUG codons upstream of the genetic lesion as a unique mechanism that rescues a mutant HRD allele by producing a functional TBCE protein. Our data explain how afflicted individuals, who would otherwise lack the capacity to make functional TBCE, can survive and point to a limiting capacity to fold tubulin heterodimers de novo as a contributing factor to disease pathogenesis.

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.

Factors that regulate the microtubule cytoskeleton are critical in determining cell behavior. Here we describe the function of a novel protein that we term E-like based on its sequence similarity to the tubulin-specific chaperone cofactor E. We find that upon overexpression, E-like depolymerizes microtubules by committing tubulin to proteosomal degradation. Our data suggest that this function is direct and is based on the ability of E-like to disrupt the tubulin heterodimer in vitro. Suppression of E-like expression results in an increase in the number of stable microtubules and a tight clustering of endocellular membranes around the microtubule-organizing center, while the properties of dynamic microtubules are unaffected. These observations define E-like as a novel regulator of tubulin stability, and provide a link between tubulin turnover and vesicle transport.

Eukaryotic prefoldin (PFD) is a heterohexameric chaperone with a jellyfish-like structure whose function is to deliver nonnative target proteins, principally actins and tubulins, to the eukaryotic cytosolic chaperonin for facilitated folding. Here we demonstrate that functional PFD can spontaneously assemble from its six constituent individual subunits (PFD1-PFD6), each expressed as a recombinant protein. Using engineered forms of PFD assembled in vitro, we show that the tips of the PFD tentacles are required to form binary complexes with authentic target proteins. We show that PFD uses the distal ends of different but overlapping sets of subunits to form stable binary complexes with different target proteins, namely actin and alpha- and beta-tubulin. We also present data that suggest a model for the order of these six subunits within the hexamer. Our data are consistent with the hypothesis that PFD, like the eukaryotic cytosolic chaperonin, has co-evolved specifically to facilitate the folding of its target proteins.

Arl2 is a member of the ADP-ribosylation factor family of 20-kDa GTPases that is highly conserved in eukaryotes. Recent results revealed that a portion of cellular Arl2 and its binding partner, BART, localize to mitochondria. Because approximately 90% of cellular Arl2 is cytosolic, we investigated properties of the soluble protein and found that it is stably bound in a complex that migrates in gel filtration medium with a predicted molecular mass of approximately 300 kDa. This complex was purified approximately 500-fold from the soluble fraction of bovine brain. Protein components were identified by mass spectroscopy and revealed the presence of four other proteins that include the tubulin folding cochaperone cofactor D and all three subunits of at least two protein phosphatase 2A (PP2A) protein phosphatase trimers. The presence of more than one PP2A B-type subunit and the low stoichiometry of Arl2 indicate that the purified preparation still contains a mixture of complexes that cannot currently be completely resolved. Thus, although all the soluble Arl2 in bovine brain is in high molecular mass complexes, only a portion of the total cellular cofactor D and PP2A are associated with the Arl2. We further show that the Arl2 in the complex cannot bind GTP and that complexed cofactor D does not efficiently participate in tubulin refolding reactions in a manner comparable with free cofactor D. Our data suggest functional roles for the cytosolic Arl2 complex in modulating tubulin and microtubule behavior as well as a possible role in apoptosis.

The biogenesis of the cytoskeletal proteins actin and tubulin involves interaction of nascent chains of each of the two proteins with the oligomeric protein prefoldin (PFD) and their subsequent transfer to the cytosolic chaperonin CCT (chaperonin containing TCP-1). Here we show by electron microscopy that eukaryotic PFD, which has a similar structure to its archaeal counterpart, interacts with unfolded actin along the tips of its projecting arms. In its PFD-bound state, actin seems to acquire a conformation similar to that adopted when it is bound to CCT. Three-dimensional reconstruction of the CCT:PFD complex based on cryoelectron microscopy reveals that PFD binds to each of the CCT rings in a unique conformation through two specific CCT subunits that are placed in a 1,4 arrangement. This defines the phasing of the CCT rings and suggests a handoff mechanism for PFD.

Mutations in the retinitis pigmentosa 2 (RP2) gene cause a severe form of X-linked retinal degeneration. RP2 is a ubiquitous 350 amino acid plasma membrane-associated protein, which shares homology with the tubulin-specific chaperone cofactor C. RP2 protein, like cofactor C, stimulates the GTPase activity of tubulin in combination with cofactor D. RP2 has also been shown to interact with ADP ribosylation factor-like 3 (Arl3) in a nucleotide and myristoylation-dependant manner. In this study we have examined the relationship between RP2, cofactor C and Arl3 in patient-derived cell lines and in the retina. Examination of lymphoblastoid cells from patients with an Arg120stop nonsense mutation in RP2 revealed that the expression levels of cofactor C and Arl3 were not affected by the absence of RP2. In human retina, RP2 was localized to the plasma membrane of cells throughout the retina. RP2 was present at the plasma membrane in both rod and cone photoreceptors, extending from the outer segment through the inner segment to the synaptic terminals. There was no enrichment of RP2 staining in any photoreceptor organelle. In contrast, cofactor C and Arl3 localized predominantly to the photoreceptor connecting cilium in rod and cone photoreceptors. Cofactor C was cytoplasmic in distribution, whereas Arl3 localized to other microtubule structures within all cells. Arl3 behaved as a microtubule-associated protein: it co-localized with microtubules in HeLa cells and this was enhanced following microtubule stabilization with taxol. Furthermore, Arl3 co-purified with microtubules from bovine brain. Following microtubule depolymerization with nocodazole, Arl3 relocalized to the nuclear membrane. These data suggest that RP2 functions in concert with Arl3 to link the cell membrane with the cytoskeleton in photoreceptors as part of the cell signaling or vesicular transport machinery.

Mutations in the X-linked retinitis pigmentosa 2 gene cause progressive degeneration of photoreceptor cells. The retinitis pigmentosa 2 protein (RP2) is similar in sequence to the tubulin-specific chaperone cofactor C. Together with cofactors D and E, cofactor C stimulates the GTPase activity of native tubulin, a reaction regulated by ADP-ribosylation factor-like 2 protein. Here we show that in the presence of cofactor D, RP2 protein also stimulates the GTPase activity of tubulin. We find that this function is abolished by mutation in an arginine residue that is conserved in both cofactor C and RP2. Notably, mutations that alter this arginine codon cause familial retinitis pigmentosa. Our data imply that this residue acts as an "arginine finger" to trigger the tubulin GTPase activity and suggest that loss of this function in RP2 contributes to retinal degeneration. We also show that in Saccharomyces cerevisiae, both cofactor C and RP2 partially complement the microtubule phenotype resulting from deletion of the cofactor C homolog, demonstrating their functional overlap in vivo. Finally, we find that RP2 interacts with GTP-bound ADP ribosylation factor-like 3 protein, providing a link between RP2 and several retinal-specific proteins, mutations in which also cause retinitis pigmentosa.

Publisher Summary This chapter describes several experiments that give a qualitative feel for how Type II chaperonins might facilitate protein folding. Imaging of CCT/actin complexes and experiments on actin and tubulin folding in vitro suggest that target proteins bind to chaperonin in a surprisingly native-like state, probably consisting of only one or a few kinetically trapped folding intermediates. Chaperonins are a specialized but ubiquitous group of molecular chaperones that are characterized by a multisubunit ring structure. Chaperonins exist in all kingdoms of life, in all cases using cycles of ATP binding and hydrolysis to generate allosteric changes that promote the correct folding of bound target protein. Type I chaperonins are present in prokaryotes (exemplified by GroEL/GroES), in mitochondria (exemplified by Hsp60/hspl0), and in chloroplasts (exemplified by Rubisco subunit binding protein), while Type II chaperonins include the chaperonin present in archaea (exemplified by the thermosome) and the chaperonin present in the cytosol of eukaryotes (referred to here as CCT, for cytosolic chaperonin-containing TCP-1, but which is also termed c-cpn or TriC). Indeed, chaperones in general appear to function by releasing and rebinding target proteins, allowing a kinetic partition of folding intermediates among the chaperone proteins present in the cell, and leading to their folding, subcellular sorting, or degradation.

Microtubules are dynamic polymers that are essential to cell division as a major component of the mitotic spindle, and consist largely of two soluble proteins termed a- and 3-tubulin. The biological activity of these proteins depends critically on their proper folding. This is a multi-step process, involving ATP-dependent interaction with cytosolic chaperonin, followed by an obligatory cascade of ATP-independent interactions with several protein cofactors. These proteins (termed cofactors A, B, C, D and E) flinction specifically in the a- and t3-tubulin folding pathways. The goal of the project proposed in this application is to use the tools of contemporary molecular biology to search for compounds that act as specific inhibitors of one or more of these proteins. Because the synthesis of native tubulin is essential for cell division, reagents that specifically prevent the productive folding of tubulin are likely to be useful as novel chemotherapeutic agents for the treatment of breast cancer, either alone or in combination with existing drugs. Because their mode of action would target the de novo production of functional tubulin rather than the biochemical properties of tubulin per se, the development of such drugs would result in a new class of anti-tumor compounds.

We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton. Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin. We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins.

Cytosolic chaperonin, the eukaryotic cytosolic homolog of GroEL, has certain unusual features that make it uniquely useful for studying the mechanism of chaperonin action. It is of particular interest as an essential component in the generation of native actin and tubulin in vivo. We describe a method for the purification of mammalian c-cpn from rabbit reticulocyte lysate via a three-step procedure involving ion-exchange chromatography, affinity selection on ATP-agarose, and gel filtration. We also describe a sensitive in vitro-folding assay for the activity of c-cpn and other chaperone proteins, and a simple nondenaturing gel assay for the analysis of folding reaction products.

We describe the complete beta-tubulin folding pathway. Folding intermediates produced via ATP-dependent interaction with cytosolic chaperonin undergo a sequence of interactions with four proteins (cofactors A, D, E, and C). The postchaperonin steps in the reaction cascade do not depend on ATP or GTP hydrolysis, although GTP plays a structural role in tubulin folding. Cofactors A and D function by capturing and stabilizing beta-tubulin in a quasi-native conformation. Cofactor E binds to the cofactor D-beta-tubulin complex; interaction with cofactor C then causes the release of beta-tubulin polypeptides that are committed to the native state. Sequence analysis identifies yeast homologs of cofactors D (cin1) and E (pac2), characterized by mutations that affect microtubule function.

Microtubules play an important role in establishing cellular architecture. Neuronal microtubules are considered to have a role in dendrite and axon formation. Different portions of the developing and adult brain microtubules are associated with different microtubule-associated proteins (MAPs). The roles of each of the different MAPs are not well understood. One of these proteins, MAP1B, is expressed in different portions of the brain and has been postulated to have a role in neuronal plasticity and brain development. To ascertain the role of MAP1B, we generated mice which carry an insertion in the gene by gene-targeting methods. Mice which are homozygous for the modification die during embryogenesis. The heterozygotes exhibit a spectrum of phenotypes including slower growth rates, lack of visual acuity in one or both eyes, and motor system abnormalities. Histochemical analysis of the severely affected mice revealed that their Purkinje cell dendritic processes are abnormal, do not react with MAP1B antibodies, and show reduced staining with MAP1A antibodies. Similar histological and immunochemical changes were observed in the olfactory bulb, hippocampus, and retina, providing a basis for the observed phenotypes.