Faculty Bibliography

The genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple pathogenic missense mutations in TUBG1, DYNC1H1 and KIF2A, as well as a single germline mosaic mutation in KIF5C, in subjects with MCD. We found a frequent recurrence of mutations in DYNC1H1, implying that this gene is a major locus for unexplained MCD. We further show that the mutations in KIF5C, KIF2A and DYNC1H1 affect ATP hydrolysis, productive protein folding and microtubule binding, respectively. In addition, we show that suppression of mouse Tubg1 expression in vivo interferes with proper neuronal migration, whereas expression of altered gamma-tubulin proteins in Saccharomyces cerevisiae disrupts normal microtubule behavior. Our data reinforce the importance of centrosomal and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and postmitotic processes are major contributors to the pathogenesis of MCD.

The tubulin heterodimer consists of one alpha- and one beta-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 beta- and alpha-tubulin polypeptides, respectively, following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin. There is free exchange of beta-tubulin between TBCA and TBCD, and of alpha-tubulin between TBCB and TBCE, resulting in the formation of TBCD/beta and TBCE/alpha, respectively. The latter two complexes interact, forming a supercomplex (TBCE/alpha/TBCD/beta). Discharge of the native alpha/beta heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound beta-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 alpha- and beta-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate alpha- or beta-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.

The formation of the mammalian cortex requires the generation, migration, and differentiation of neurons. The vital role that the microtubule cytoskeleton plays in these cellular processes is reflected by the discovery that mutations in various tubulin isotypes cause different neurodevelopmental diseases, including lissencephaly (TUBA1A), polymicrogyria (TUBA1A, TUBB2B, TUBB3), and an ocular motility disorder (TUBB3). Here, we show that Tubb5 is expressed in neurogenic progenitors in the mouse and that its depletion in vivo perturbs the cell cycle of progenitors and alters the position of migrating neurons. We report the occurrence of three microcephalic patients with structural brain abnormalities harboring de novo mutations in TUBB5 (M299V, V353I, and E401K). These mutant proteins, which affect the chaperone-dependent assembly of tubulin heterodimers in different ways, disrupt neurogenic division and/or migration in vivo. Our results provide insight into the functional repertoire of the tubulin gene family, specifically implicating TUBB5 in embryonic neurogenesis and microcephaly.

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

Assembly of the alpha/beta 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 alpha- and beta-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 beta-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 beta-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 beta-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). (c) 2010 Wiley-Liss, Inc

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

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 beta-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 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 alpha-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 alpha-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 alpha-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

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 alpha-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