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IN THIS ISSUE |
The many facets of Smad regulation (p. 4359) Commentary
Smads are intracellular signalling molecules activated following binding of members of the TGF-ß superfamily to receptor serine/threonine kinases. Phosphorylation of R-Smads by the receptors allows them to form complexes with Co-Smad, enter the nucleus and regulate gene expression. A third class of Smad, I-Smads, plays an inhibitory role, competing with R-Smads for receptors and marking receptors for degradation. Carl-Henrik Heldin and co-workers discuss our understanding of Smad phosphorylation and nucleoplasmic shuttling, as well as the importance of oligomerization and ubiquitination in Smad signalling. Recent work has shown that phosphorylation of the R-Smad C-terminus allows it to interact with other Smads, leading to formation of homo-oligomers and subsequently Co-Smad-containing hetero-oligomers. Moreover, phosphorylation appears to induce a conformational change that exposes the R-Smad nuclear localization sequence, allowing importin ß1 to bind and mediate Ran-dependent nuclear import. Once in the nucleus, Smads can stimulate gene expression directly, by binding to DNA or cooperating with transcription factors, and indirectly, by regulating other factors through ubiquitination.
Cell adhesion in Dictyostelium development (p. 4349) Commentary
Cell-cell adhesion is essential during animal development: not only does it have an architectural role, but junctional proteins such as ß-catenin also link cell adhesion to signalling pathways that regulate cell proliferation, differentiation and apoptosis. But how important is cell adhesion lower down the evolutionary ladder? Juliet Coates and Adrian Harwood examine the importance of cell-cell adhesion in Dictyostelium - a unicellular amoeba that, when food is scarce, aggregates and differentiates to form a complex fruiting body. The cadherin-related soluble molecule DdCAD-1 and the N-CAM-related molecule csA mediate cell-cell adhesion and control aggregate size during the early stages of Dictyostelium development. A third adhesion molecule, gp150, functions at the mound stage and is required for expression of genes that control differentiation and morphogenesis. Likewise, the ß-catenin homologue Aardvark, an essential component of the adherens junctions that maintain fruiting-body integrity, regulates expression of prespore-specific genes such as psA. In Dictyostelium, as in animals, cell-cell adhesion thus appears both to have mechanical roles and to function in signalling pathways that control morphogenesis.
Identifying essential genes by RNAi (p. 4557)
RNA interference (RNAi) is a post-transcriptional gene silencing mechanism in which an mRNA is specifically degraded following introduction of a homologous double-stranded RNA. The phenomenon has immense potential as a tool for ‘knock down’ experiments that interfere with expression of specific genes; however, application of the technique to mammalian cells has been hampered by global, non-specific cellular responses and negative results with certain genes. Klaus Weber and co-workers now show that, using 21-nucleotide small interfering RNAs, RNAi can be successfully applied to a host of genes in mammalian cultured cells. These include those encoding major cellular proteins, such as actin, vimentin and the mitotic kinase Cdc2. The authors have used the technique to define essential and nonessential genes, finding, for example, that lamins B1 and B2 are essential but lamin A/C is not. Significantly, they demonstrate that, in cases in which a gene has previously been knocked out, the RNAi knock down produces identical results. These experiments thus pave the way for widespread use of this revolutionary technique in cell biology.
Membrane fusion in SNARE helix-bundle mutants (p. 4397)
Fusion of intracellular membranes is driven by interactions between highly conserved SNARE proteins in the two bilayers. Structural studies suggest that formation of a highly thermostable 4-helix bundle containing helices donated by each SNARE drives membrane fusion. The formation of such a stable structure cannot, however, be reconciled with the observed reversibility of fusion. Robert Burgoyne and co-workers have assessed the effects of mutations that disrupt the thermostability of the 4-helix bundle during bilayer fusion in regulated exocytosis. The authors used a sensitive cellular approach in which an endogenous SNARE, SNAP-25, is removed by botulinum neurotoxin E (BoNT/E) and cells are transfected with BoNT/E-resistant SNAP-25 constructs. They demonstrate that the helix-bundle mutations affect neither the time course of exocytosis nor the kinetics of single exocytic release events. Burgoyne and co-workers conclude that the lack of effect of these stability-reducing mutations indicates that formation of a highly stable SNARE complex does not in fact drive membrane fusion but instead takes place after fusion has occurred.
Nuclear defects associated with human lamin A/C mutations (p. 4435, p. 4447 & p. 4459)
Mutations in the LMNA gene, which encodes the nuclear lamina components lamin A and lamin C, cause distinct inherited forms of muscular dystrophy, cardiomyopathy and lipodystrophy. But the nature of the nuclear abnormalities the different mutations produce and why they result in distinct tissue-restricted diseases are unclear. Three papers in this issue examine the effects of different lamin A/C mutations in cultured cells. Howard Worman and co-workers have transfected myoblasts with disease-linked lamin A/C mutants (see p. 4435); Brian Burke and co-workers have performed similar experiments in HeLa cells and LMNA-null fibroblasts (see p. 4447). Brigitte Buendia and co-workers have taken a slightly different approach: analysis of primary cultures of fibroblasts from individuals who have Dunnigan-type familial partial lypodystrophy (FPLD). The Worman group show that a subset of the lamin A/C mutations - those affecting the rod domain - generate intranuclear inclusions and significantly disrupt the endogenous lamina. The Burke group demonstrate that point mutations that cause dilated cardiomyopathy or Emery-Dreifuss muscular dystrophy modify assembly properties of lamins A and C and interfere with the organisation of endogenous lamins. Finally, Buendia and co-workers find that the R482Q and R482W lamin A/C mutations present in the lipodystrophy patients cause nuclear envelope defects, disrupt lamin arrangements and produce abnormal chromatin organization. Together these findings indicate that disease-associated LMNA mutations produce distinct defects in nuclear architecture and nuclear envelope organization. They thus represent a first step in our attempts to unravel the cellular pathophysiology of inherited lamin A/C disorders.
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