No abstract

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.

The genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple disease-causing missense mutations in TUBG1, DYNC1H1 and KIF2A, as well as a single germline mosaic mutation in KIF5C. We find a frequent recurrence of mutations in DYNC1H1, implying that this gene is a major locus implicated in unexplained MCD. The mutations in KIF5C, KIF2A and DYNC1H1 drastically affect ATP hydrolysis, productive protein folding or microtubule binding, while suppression of Tubg1 expression in vivo interferes with proper neuronal migration and expression of Tubg1 mutations in S. cerevisiae results in disruption of normal microtubule behaviour. Our data reinforce the importance of centrosome- and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and post-mitotic processes are major contributors to the pathogenesis of MCD.

Tau protein is a family of microtubule binding proteins, heterogeneous in molecular weight, that are induced during neurite outgrowth and are found prominently in neurofibrillary tangles in Alzheimer's disease. The predicted amino acid sequences of two forms of tau protein from mouse brain were determined from complementary DNA clones. These forms are identical in their amino-terminal sequences but differ in their carboxyl-terminal domains. Both proteins contain repeated sequences that may be tubulin binding sites. The sequence suggests that tau is an elongated molecule with no extensive alpha-helical or beta-sheet domains. These complementary DNAs should enable the study of various functional domains of tau and the study of tau expression in normal and pathological states.

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.

No abstract

Within the wide spectrum of axonal diameters occurring in mammalian nerve fibers, each class of neurons has a relatively restricted range of axonal calibers. The control of caliber has functional significance because diameter is the principal determinant of conduction velocity in myelinated nerve fibers. Previous observations support the hypothesis that neurofilaments (NF) are major intrinsic determinants of axonal caliber in large myelinated nerve fibers. Following interruption of axons (axotomy) by crushing or cutting a peripheral nerve, caliber is reduced in the proximal axonal stumps, which extend from the cell bodies to the site of axotomy. (The distal axonal stumps, which are disconnected from the cell bodies, degenerate and are replaced by the outgrowth of regenerating axonal sprouts arising from the proximal stump.) This reduction in axonal caliber in the proximal stumps is associated with a selective diminution in the amount of NF protein undergoing slow axonal transport in these axons, with a decrease in axonal NF content, and with reduced conduction velocity. The present report demonstrates that changes in axonal caliber after axotomy correlate with a selective alteration in NF gene expression. Hybridization with specific cDNAs was used to measure levels of mRNA encoding the 68-kDa neurofilament protein (NF68), ,3-tubulin, and actin in lumbar sensory neurons of rat at various times after crushing the sciatic nerve. Between 4 and 42 days after axotomy by nerve crush, the levels of NF68 mRNA were reduced 2-to 3-fold. At the same times, the levels of tubulin and actin mRNAs were increased several-fold. These findings support the hypothesis that the expression of a single set of neuron-specific genes (encoding NF) directly determines axonal caliber, a feature of neuronal morphology with important consequences for physiology and behavior.The synthesis and axonal transport of neurofilament (NF) proteins are thought to play a major role in the control of axonal caliber in large myelinated nerve fibers (1, 2). This concept is based on several observations: in normal nerve fibers NF are the most numerous cytoskeletal elements, NF density remains constant over a wide range of calibers, and NF number correlates closely with axonal area (1,(3)(4)(5). The relatively constant density of axonal NF is closely related to the presence of interfilament cross-bridges that appear to determine the spacing between adjacent NF (4,(6)(7)(8). The observation that the 200-kDa NF protein (NF200) is directly associated with these cross-bridges (8) raises the possibility that cross-bridge formation is an intrinsic property of NF. Thus, the volume of axoplasm occupied by the three-dimensional network of interconnected NF correlates closely with the number of NF profiles per axonal cross-section. Nevertheless, it should be noted that the close relationship between axonal caliber and NF content present in normal fibers is altered in a variety of pathological situations-e.g., NF density is markedly increased in giant axonal ...

SummaryThe 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.