ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway

The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks. Here, because of the similarities between AT and NBS, we evaluated the functional interactions between ATM and p95/nbs1. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicated that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 was phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogated an S-phase checkpoint induced by ionizing radiation in normal cells and failed to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases.     

Genomic analysis of metastasis reveals an essential role for RhoC

The most damaging change during cancer progression is the switch from a locally growing tumour to a metastatic killer. This switch is believed to involve numerous alterations that allow tumour cells to complete the complex series of events needed for metastasis. Relatively few genes have been implicated in these events. Here we use an in vivo selection scheme to select highly metastatic melanoma cells. By analysing these cells on DNA arrays, we define a pattern of gene expression that correlates with progression to a metastatic phenotype. In particular, we show enhanced expression of several genes involved in extracellular matrix assembly and of a second set of genes that regulate, either directly or indirectly, the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis. Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important in tumour cell invasion. The genomic approach allows us to identify families of genes involved in a process, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes such as metastasis.     

Cursoriality in bipedal archosaurs

Modern birds have markedly foreshortened tails and their body mass is centred anteriorly, near the wings. To provide stability during powered flight, the avian centre of mass is far from the pelvis, which poses potential balance problems for cursorial birds. To compensate, avians adapted to running maintain the femur subhorizontally, with its distal end situated anteriorly, close to the animal's centre of mass; stride generation stems largely from parasagittal rotation of the lower leg about the knee joint. In contrast, bipedal dinosaurs had a centre of mass near the hip joint and rotated the entire hindlimb during stride generation. Here we show that these contrasting styles of cursoriality are tightly linked to longer relative total hindlimb length in cursorial birds than in bipedal dinosaurs. Surprisingly, Caudipteryx, described as a theropod dinosaur, possessed an anterior centre of mass and hindlimb proportions resembling those of cursorial birds. Accordingly, Caudipteryx probably used a running mechanism more similar to that of modern cursorial birds than to that of all other bipedal dinosaurs. These observations provide valuable clues about cursoriality in Caudipteryx, but may also have implications for interpreting the locomotory status of its ancestors.     

Cortex-restricted disruption of NMDAR1 impairs neuronal patterns in the barrel cortex

In the rodent primary somatosensory cortex, the configuration of whiskers and sinus hairs on the snout and of receptor-dense zones on the paws is topographically represented as discrete modules of layer IV granule cells (barrels) and thalamocortical afferent terminals. The role of neural activity, particularly activity mediated by NMDARs (N-methyl-D-aspartate receptors), in patterning of the somatosensory cortex has been a subject of debate. We have generated mice in which deletion of the NMDAR1 (NR1) gene is restricted to excitatory cortical neurons, and here we show that sensory periphery-related patterns develop normally in the brainstem and thalamic somatosensory relay stations of these mice. In the somatosensory cortex, thalamocortical afferents corresponding to large whiskers form patterns and display critical period plasticity, but their patterning is not as distinct as that seen in the cortex of normal mice. Other thalamocortical patterns corresponding to sinus hairs and digits are mostly absent. The cellular aggregates known as barrels and barrel boundaries do not develop even at sites where thalamocortical afferents cluster. Our findings indicate that cortical NMDARs are essential for the aggregation of layer IV cells into barrels and for development of the full complement of thalamocortical patterns.     

Functional architecture of an intracellular membrane t-SNARE

Lipid bilayer fusion is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) located on the vesicle membrane (v-SNAREs) and the target membrane (t-SNAREs). The assembled v-SNARE/t-SNARE complex consists of a bundle of four helices, of which one is supplied by the v-SNARE and the other three by the t-SNARE. For t-SNAREs on the plasma membrane, the protein syntaxin supplies one helix and a SNAP-25 protein contributes the other two. Although there are numerous homologues of syntaxin on intracellular membranes, there are only two SNAP-25-related proteins in yeast, Sec9 and Spo20, both of which are localized to the plasma membrane and function in secretion and sporulation, respectively. What replaces SNAP-25 in t-SNAREs of intracellular membranes? Here we show that an intracellular t-SNARE is built from a 'heavy chain' homologous to syntaxin and two separate non-syntaxin 'light chains'. SNAP-25 may thus be the exception rather than the rule, having been derived from genes that encoded separate light chains that fused during evolution to produce a single gene encoding one protein with two helices.     

Myosin V orientates the mitotic spindle in yeast

Coordination of spindle orientation with the axis of cell division is an essential process in all eukaryotes. In addition to ensuring accurate chromosomal segregation, proper spindle orientation also establishes differential cell fates and proper morphogenesis. In both animal and yeast cells, this process is dependent on cytoplasmic microtubules interacting with the cortical actin-based cytoskeleton, although the motive force was unknown. Here we show that yeast Myo2, a myosin V that translocates along polarized actin cables into the bud, orientates the spindle early in the cell cycle by binding and polarizing the microtubule-associated protein Kar9 (refs 7-9). The tail domain of Myo2 that binds Kar9 also interacts with secretory vesicles and vacuolar elements, making it a pivotal component of yeast cell polarization.