Novel proteins linking the actin cytoskeleton to the endocytic machinery in Saccharomyces cerevisiae.

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.

Nesprin-2-dependent ERK1/2 compartmentalisation regulates the DNA damage response in vascular smooth muscle cell ageing.

Prelamin A accumulation and persistent DNA damage response (DDR) are hallmarks of vascular smooth muscle cell (VSMC) ageing and dysfunction. Although prelamin A is proposed to interfere with DNA repair, our understanding of the crosstalk between prelamin A and the repair process remains limited. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have emerged as key players in the DDR and are known to enhance ataxia telangiectasia-mutated protein (ATM) activity at DNA lesions, and in this study, we identified a novel relationship between prelamin A accumulation and ERK1/2 nuclear compartmentalisation during VSMC ageing. We show both prelamin A accumulation and increased DNA damage occur concomitantly, before VSMC replicative senescence, and induce the localisation of ERK1/2 to promyelocytic leukaemia protein nuclear bodies (PML NBs) at the sites of DNA damage via nesprin-2 and lamin A interactions. Importantly, VSMCs treated with DNA damaging agents also displayed prelamin A accumulation and ERK compartmentalisation at PML NBs, suggesting that prelamin A and nesprin-2 are novel components of the DDR. In support of this, disruption of ERK compartmentalisation at PML NBs, by either depletion of nesprin-2 or lamins A/C, resulted in the loss of ATM from DNA lesions. However, ATM signalling and DNA repair remained intact after lamins A/C depletion, whereas nesprin-2 disruption ablated downstream Chk2 activation and induced genomic instability. We conclude that lamins A/C and PML act as scaffolds to organise DNA-repair foci and compartmentalise nesprin-2/ERK signalling. However, nesprin-2/ERK signalling fidelity, but not their compartmentalisation at PML NBs, is essential for efficient DDR in VSMCs.