Alterations to nuclear architecture and genome behavior in senescent cells.

The organization of the genome within interphase nuclei, and how it interacts with nuclear structures is important for the regulation of nuclear functions. Many of the studies researching the importance of genome organization and nuclear structure are performed in young, proliferating, and often transformed cells. These studies do not reveal anything about the nucleus or genome in nonproliferating cells, which may be relevant for the regulation of both proliferation and replicative senescence. Here, we provide an overview of what is known about the genome and nuclear structure in senescent cells. We review the evidence that nuclear structures, such as the nuclear lamina, nucleoli, the nuclear matrix, nuclear bodies (such as promyelocytic leukemia bodies), and nuclear morphology all become altered within growth-arrested or senescent cells. Specific alterations to the genome in senescent cells, as compared to young proliferating cells, are described, including aneuploidy, chromatin modifications, chromosome positioning, relocation of heterochromatin, and changes to telomeres

α4β1 Integrin Regulates Lamellipodia Protrusion via a Focal Complex/Focal Adhesion-independent Mechanism

α4β1 integrin plays an important role in cell migration. We show that when ectopically expressed in Chinese hamster ovary cells, α4β1 is sufficient and required for promoting protrusion of broad lamellipodia in response to scratch-wounding, whereas α5β1 does not have this effect. By time-lapse microscopy of cells expressing an α4/green fluorescent protein fusion protein, we show that α4β1 forms transient puncta at the leading edge of cells that begin to protrude lamellipodia in response to scratch-wounding. The cells expressing a mutant α4/green fluorescent protein that binds paxillin at a reduced level had a faster response to scratch-wounding, forming α4-positive puncta and protruding lamellipodia much earlier. While enhancing lamellipodia protrusion, this mutation reduces random motility of the cells in Transwell assays, indicating that lamellipodia protrusion and random motility are distinct types of motile activities that are differentially regulated by interactions between α4β1 and paxillin. Finally, we show that, at the leading edge, α4-positive puncta and paxillin-positive focal complexes/adhesions do not colocalize, but α4β1 and paxillin colocalize partially in ruffles. These findings provide evidence for a specific role of α4β1 in lamellipodia protrusion that is distinct from the motility-promoting functions of α5β1 and other integrins that mediate cell adhesion and signaling events through focal complexes and focal adhesions

Modulation of Fibroblast Morphology and Adhesion during Collagen Matrix Remodeling

When fibroblasts are placed within a three-dimensional collagen matrix, cell locomotion results in translocation of the flexible collagen fibrils of the matrix, a remodeling process that has been implicated in matrix morphogenesis during development and wound repair. In the current experiments, we studied formation and maturation of cell–matrix interactions under conditions in which we could distinguish local from global matrix remodeling. Local remodeling was measured by the movement of collagen-embedded beads towards the cells. Global remodeling was measured by matrix contraction. Our observations show that no direct relationship occurs between protrusion and retraction of cell extensions and collagen matrix remodeling. As fibroblasts globally remodel the collagen matrix, however, their overall morphology changes from dendritic to stellate/bipolar, and cell–matrix interactions mature from punctate to focal adhesion organization. The less well organized sites of cell–matrix interaction are sufficient for translocating collagen fibrils, and focal adhesions only form after a high degree of global remodeling occurs in the presence of growth factors. Rho kinase activity is required for maturation of fibroblast morphology and formation of focal adhesions but not for translocation of collagen fibrils

Dynamics of the α6β4 Integrin in Keratinocytes

The integrin α6β4 has been implicated in two apparently contrasting processes, i.e., the formation of stable adhesions, and cell migration and invasion. To study the dynamic properties of α6β4 in live cells two different β4-chimeras were stably expressed in β4-deficient PA-JEB keratinocytes. One chimera consisted of full-length β4 fused to EGFP at its carboxy terminus (β4-EGFP). In a second chimera the extracellular part of β4 was replaced by EGFP (EGFP-β4), thereby rendering it incapable of associating with α6 and thus of binding to laminin-5. Both chimeras induce the formation of hemidesmosome-like structures, which contain plectin and often also BP180 and BP230. During cell migration and division, the β4-EGFP and EGFP-β4 hemidesmosomes disappear, and a proportion of the β4-EGFP, but not of the EGFP-β4 molecules, become part of retraction fibers, which are occasionally ripped from the cell membrane, thereby leaving “footprints” of the migrating cell. PA-JEB cells expressing β4-EGFP migrate considerably more slowly than those that express EGFP-β4. Studies with a β4-EGFP mutant that is unable to interact with plectin and thus with the cytoskeleton (β4(R1281W)-EGFP) suggest that the stabilization of the interaction between α6β4 and LN-5, rather than the increased adhesion to LN-5, is responsible for the inhibition of migration. Consistent with this, photobleaching and recovery experiments revealed that the interaction of β4 with plectin renders the bond between α6β4 and laminin-5 more stable, i.e., β4-EGFP is less dynamic than β4(R1281W)-EGFP. On the other hand, when α6β4 is bound to laminin-5, the binding dynamics of β4 to plectin are increased, i.e., β4-EGFP is more dynamic than EGFP-β4. We suggest that the stability of the interaction between α6β4 and laminin-5 is influenced by the clustering of α6β4 through the deposition of laminin-5 underneath the cells. This clustering ultimately determines whether α6β4 will inhibit cell migration or not

The Yeast<i>TEL1</i>Gene Partially Substitutes for Human<i>ATM</i>in Suppressing Hyperrecombination, Radiation-Induced Apoptosis and Telomere Shortening in A-T Cells

Homozygous mutations in the human ATM gene lead to a pleiotropic clinical phenotype of ataxia-telangiectasia (A-T) patients and correlating cellular deficiencies in cells derived from A-T donors. Saccharomyces cerevisiae tel1 mutants lacking Tel1p, which is the closest sequence homologue to the ATM protein, share some of the cellular defects with A-T. Through genetic complementation of A-T cells with the yeast TEL1 gene, we provide evidence that Tel1p can partially compensate for ATM in suppressing hyperrecombination, radiation-induced apoptosis, and telomere shortening. Complementation appears to be independent of p53 activation. The data provided suggest that TEL1 is a functional homologue of human ATM in yeast, and they help to elucidate different cellular and biochemical pathways in human cells regulated by the ATM protein.</jats:p