A mathematical understanding of how cytoplasmic dynein walks on microtubules.

Cytoplasmic dynein 1 (hereafter referred to simply as dynein) is a dimeric motor protein that walks and transports intracellular cargos towards the minus end of microtubules. In this article, we formulate, based on physical principles, a mechanical model to describe the stepping behaviour of cytoplasmic dynein walking on microtubules from the cell membrane towards the nucleus. Unlike previous studies on physical models of this nature, we base our formulation on the whole structure of dynein to include the temporal dynamics of the individual subunits such as the cargo (for example, an endosome, vesicle or bead), two rings of six ATPase domains associated with diverse cellular activities (AAA+ rings) and the microtubule-binding domains which allow dynein to bind to microtubules. This mathematical framework allows us to examine experimental observations on dynein across a wide range of different species, as well as being able to make predictions on the temporal behaviour of the individual components of dynein not currently experimentally measured. Furthermore, we extend the model framework to include backward stepping, variable step size and dwelling. The power of our model is in its predictive nature; first it reflects recent experimental observations that dynein walks on microtubules using a weakly coordinated stepping pattern with predominantly not passing steps. Second, the model predicts that interhead coordination in the ATP cycle of cytoplasmic dynein is important in order to obtain the alternating stepping patterns and long run lengths seen in experiments.

Dynein-dynactin complex subunits are differentially localized in brain and spinal cord, with selective involvement in pathological features of neurodegenerative disease.

AIMS: The dynein-dynactin complex, mostly recognized for axonal retrograde transport in neurones, has an ever growing list of essential subcellular functions. Here, the distribution of complex subunits in human central nervous system (CNS) has been assessed using immunohistochemistry in order to test the hypothesis that this may be altered in neurodegenerative disease. METHODS: Three dynactin and two dynein subunits were immunolocalized in the CNS of human post mortem sections from motor neurone disease, Alzheimer's disease and patients with no neurological disease. RESULTS: Unexpectedly, coordinated distribution of complex subunits was not evident, even in normal tissues. Complex subunits were differentially localized in brain and spinal cord, and localization of certain subunits, but not others, occurred in pathological structures of motor neurone and Alzheimer's diseases. CONCLUSIONS: These results suggest that dynein-dynactin complex subunits may have specific subcellular roles, and primary events that disturb the function of individual components may result in disequilibrium of subunit pools, with the possibility that availability for normal cytoplasmic functions becomes impaired, with consequent organelle and axonal transport misfunction.

SHIRPA, a protocol for behavioral assessment: validation for longitudinal study of neurological dysfunction in mice.

Mouse models of neurological abnormalities are only valuable if accurately assessed. The three-stage SHIRPA procedure is used for the standardised assessment of mouse phenotype and has been reported in a high throughput experiment in which different mutants were ascertained at one age point using stage 1 of the protocol. In this study we have validated SHIRPA using a large cohort with one single mutation, 'legs at odd angles that causes neurological dysfunction. The cohort aged from 1 to 16 months during this study and this is the first longitudinal SHIRPA analysis.

Mice, the motor system, and human motor neuron pathology.

Motor neurons are among some of the most unusual cells in the body becaue of their immense size and their role as the critical link between the motor centers of the brain and the muscles. In addition to their intrinsic biological interest, it is vital that we gain a better understanding of these cells and their pathology, since motor neuron degenerative diseases are lethal disorders that affect young and old and are relatively common. For example, one form of spinal muscular atrophy (SMA) is the most common genetic killer of children in the developed world. Amyotrophic lateral sclerosis (ALS), another form of motor neuron degeneration, is the third most common neurodegenerative cause of adult death, after Alzheimer's disease and Parkinson's disease, and is significantly more common than multiple sclerosis (Motor Neurone Disease Association 1998). Currently, approximately 1 in 500 people in England and Wales who die have a form of motor neuron disease (Motor Neurone Disease Association 1998). Each year, 5000 Americans are diagnosed with ALS, and of these, 10% are under 40 years old. Mouse models of motor neuron degeneration are essential for understanding the causes and mechanisms of motor neuron pathology. These mice are yielding important information that will ultimately lead to treatments and potentially cures for these diseases.

Genealogies of mouse inbred strains.

The mouse is a prime organism of choice for modelling human disease. Over 450 inbred strains of mice have been described, providing a wealth of different genotypes and phenotypes for genetic and other studies. As new strains are generated and others become extinct, it is useful to review periodically what strains are available and how they are related to each other, particularly in the light of available DNA polymorphism data from microsatellite and other markers. We describe the origins and relationships of inbred mouse strains, 90 years after the generation of the first inbred strain. Given the large collection of inbred strains available, and that published information on these strains is incomplete, we propose that all genealogical and genetic data on inbred strains be submitted to a common electronic database to ensure this valuable information resource is preserved and used efficiently.

The kinesin light chain gene: its mapping and exclusion in mouse and human forms of inherited motor neuron degeneration.

The underlying genetic cause is known for only 10-20% of familial motor neuron disease (MND). Thus the genes involved in the aetiology of 80-90% of familial MND remain to be determined, and animal models are powerful tools for undertaking this task. We have mapped a heritable form of motor neuron degeneration in the mouse to a region that has homology to human chromosome 14q32.1-qter. This region contains the kinesin light chain gene (KLC1), which is a candidate for involvement in motor neuron degeneration because of its function in the motor-protein kinesin, and its neuronal expression. To investigate the role of KLC1 in a mouse motor neuron degeneration mutant that we are studying, we have identified mouse Klc1 gene sequences and mapped them with respect to our mutant locus. We have also investigated KLC1 in human patients with familial MND. Based on recombination and the absence of mutations in the coding region of KLC1, this gene can be excluded as a candidate gene in our mouse mutation and, where we have investigated, it is normal in human familial MND.

Complementation analysis of testis tumor cells.

Testis tumors are cured using cisplatin-based chemotherapy in over 80% of patients, and sensitivity to cisplatin is retained by testis tumor cells in vitro. The aim of this study was to use complementation analysis to determine how many genes control sensitivity to cisplatin in testis tumor cells. Four testis tumor cell lines were transfected with pSV2NEO and pBABE plasmids, conferring G418 and puromycin resistance, respectively. Self-crosses were generated to control for gene dosage, and the parentage of the hybrids was confirmed by PCR amplification of VNTR regions. Karyotyping confirmed that all the hybrids retained at least 88% of the combined number of chromosomes of the two parental cell lines. Cisplatin sensitivity was measured by clonogenic assay and complementation was not observed. This finding provides evidence that there is a single common mechanism controlling cisplatin sensitivity in testis tumor cells.

Genetic basis of drug sensitivity in human testis tumour cells.

Testicular germ cell tumours (TGCT) are cured in over 80% of patients by using combination chemotherapy. However, the mechanism regulating this sensitivity has not been defined. Because cells derived from patients with DNA repair syndromes are similar to TCGT in their sensitivity to certain DNA-damaging agents and some of the genes involved have been cloned by functional complementation, the purpose of our study was to determine whether drug sensitivity in TGCT also has a genetic basis. Three testis tumour cell lines (cisplatin-sensitive) and 3 bladder cancer cell lines (cisplatin-resistant) were fused with a cisplatin-sensitive cell line (D98orC1). The authenticity of the hybrids was confirmed by karyotyping and PCR analysis of locus-specific sites, and sensitivities to cisplatin were measured by colony forming assays. The hybrids between sensitive cell lines were more resistant to cisplatin than the parental cells, indicating that functional complementation had occurred. The hybrids between the cisplatin-resistant and sensitive cells were intermediate in their cisplatin sensitivity, indicating that resistance is incompletely dominant. We conclude that cisplatin sensitivity has a genetic basis in TGCT and that resistance to cisplatin can be conferred by somatic cell fusion. Our data indicate that gene(s) controlling sensitivity to chemotherapy in TGCT might be identified by expression cloning.

A YAC contig encompassing the XRCC5 (Ku80) DNA repair gene and complementation of defective cells by YAC protoplast fusion.

The Chinese hamster ovary xrs mutants are sensitive to ionizing radiation, defective in DNA double-strand break rejoining, and unable to carry out V(D)J recombination effectively. Recently, the gene defective in these mutants, XRCC5, has been shown to encode Ku80, a component of the Ku protein and DNA-dependent protein kinase. We present here a YAC contig involving 25 YACs mapping to the region 2q33-q34, which encompasses the XRCC5 gene. Eight new markers for this region of chromosome 2 are identified. YACs encoding the Ku80 gene were transferred to xrs cells by protoplast fusion, and complementation of all the defective phenotypes has been obtained with two YACs. We discuss the advantages and disadvantages of this approach as a strategy for cloning human genes complementing defective rodent cell lines.

An extended panel of hamster-human hybrids for chromosome 2q.

A hamster-human hybrid containing only the q arm of chromosome 2 has been used to construct a panel of hybrids bearing reduced regions of chromosome 2 using the technique of irradiation fusion gene transfer. The human chromosome 2 carried the Ecogpt gene and all hybrids were selected using this marker. The integrated Ecogpt gene was localized to the region 2q33-34, resulting in the selective retention of this region in the hybrids. These data were combined with another previously constructed panel of hybrids containing regions of 2q, which were enriched for the region 2q36-37. The combined hybrid panel is useful for the mapping of new markers to defined regions of chromosome 2 and for the cloning of genes located on 2q by a positional strategy.

Subchromosomal localization of a gene (XRCC5) involved in double strand break repair to the region 2q34-36.

We have previously shown that human chromosome 2 can complement both the radiation sensitivity and the defect in double strand break rejoining characteristic of ionizing radiation (IR) group 5 mutants. A number of human-hamster hybrids containing segments of human chromosome 2 were obtained by microcell transfer into two group 5 mutants. In most, but not all, of these hybrids, the repair defect was complemented by the human chromosomal DNA. Two complementing microcell hybrids were irradiated and fused to XR-V15B, an IR group 5 mutant, to generate further hybrids bearing smaller regions of chromosome 2. All hybrids were examined for complementation of the repair defect. The region of chromosome 2 present was determined using PCR with primers specific for various human genes located on chromosome 2. A complementing hybrid bearing only a small region of chromosome 2 was finally generated. From this analysis we deduced that the XRCC5 gene was tightly linked to the marker, TNP1, which is located in the region 2q35.

A hamster-human subchromosomal hybrid cell panel for chromosome 2.

We have constructed hamster-human hybrid cell lines containing fragments of human chromosome 2 as their only source of human DNA. Microcell-mediated chromosome transfer was used to transfer human chromosome 2 from a monochromosomal mouse-human hybrid line to a radiation-sensitive hamster mutant (XR-V15B) defective in double-strand break rejoining. The human chromosome 2 carried the Ecogpt gene and hybrids were selected using this marker. The transferred human chromosome was frequently broken, and the resulting microcell hybrids contained different sized segments of the q arm of chromosome 2. Two microcell hybrids were irradiated and fused to XR-V15B to generate additional hybrids bearing reduced amounts of human DNA. All hybrids were analyzed by PCR using primers specific for 27 human genes located on chromosome 2. From these data we have localized the integrated gpt gene on the human chromosome 2 to the region q36-37 and present a gene order for chromosome 2 markers.

Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2.

Complementation of the repair defect in hamster xrs mutants has been achieved by transfer of human chromosome 2 using the method of microcell-mediated chromosome transfer. The xrs mutants belong to ionizing radiation complementation group 5, are highly sensitive to ionizing radiation, and have an impaired ability to rejoin radiation-induced DNA double-strand breaks. Both phenotypes were corrected by chromosome 2, although the correction of radiation sensitivity was only partial. Complementation was achieved in two members of this complementation group, xrs6 and XR-V15B, derived independently from the CHO and V79 cell lines, respectively. The presence of human chromosome 2 in complemented clones was examined cytogenetically and by PCR analysis with primers directed at a human-specific long interspersed repetitive sequence or chromosome 2-specific genes. Complementation was observed in 25/27 hybrids, one of which contained only the q arm of chromosome 2. The two noncomplementing hybrids were missing segments of chromosome 2. The use of a back-selection system enabled the isolation of clones that had lost the human chromosome and these regained radiation sensitivity. Transfer of several other human chromosomes did not result in complementation of the repair defect in XR-V15B. These data show that the gene defective in xrs cells, XRCC5, which is involved in double-strand break rejoining, is located on human chromosome 2q.