IntroductionPericellular proteolysis of cell surface molecules, extracellular factors, and ECM is intrinsically linked to cell function and fate (1, 2). Regulation of these multiple proteolytic processes is primarily achieved through coordinated interactions between proteinase, their biologic inhibitors, and the substrates. Tissue inhibitors of metalloproteinases (TIMP-1 to TIMP-4) are emerging as essential regulatory molecules, given their direct inhibitory function against two metzincin families of zinc-dependent proteinases involved in pericellular proteolysis; namely, the MMPs (matrix metalloproteinases) and the ADAMs (a disintegrin and metalloprotease) (3, 4). Although considerable literature supports the involvement of TIMPs in inhibiting cell migration, invasion, angiogenesis, and metastasis, the direct influence of TIMPs on cell fate remains little explored.Although the four TIMP proteins share similar secondary and tertiary structures and in general are able to inhibit all MMPs albeit with varying degrees of affinity, TIMP-3 stands out as a unique member. For instance, unlike the other TIMPs, which after secretion become freely diffusible within the cellular microenvironment, TIMP-3 becomes tightly bound to the ECM (5-7). In addition, inherited mutations in Timp-3 lead to Sorsby fundus dystrophy, a degenerative eye disease (8). Further, Timp-3 silencing through promoter methylation is often found in cancer specimens (9, 10), whereas other TIMPs often show tumorigenic upregulation (3). TIMP-3 exhibits direct inhibitory activity against several ADAMs that are not inhibited by other TIMPs, including TNF-α convertase (TACE, ADAM-17) (11, 12), ADAM-12S (13), aggrecanase-1 (ADAM-TS4), and aggrecanase-2 (ADAM-TS5) (14). Likely through inhibition of ADAM-mediated ectodomain shedding, TIMP-3 also inhibits cell shedding of L-selectin (15) syndecans-1 and -4 (16), IL-6 receptor (17), c-MET (18), and cleavage of IGF binding proteins-3 and -5 (13). Finally, several reports have found that high levels of TIMP-3 are proapoptotic in both normal and cancer cell lines (19)(20)(21)(22)(23)(24), and that the pro-death domain resides in its N-terminal domain (25). Using mammary gland involution as a model for physiological apoptosis and recently generated TIMP-3-deficient mice The proapoptotic proteinase inhibitor TIMP-3 is the only molecule of this family thought to influence cell death. We examined epithelial apoptosis in TIMP-3-deficient mice during mammary gland involution. Lactation was not affected by the absence of TIMP-3, but glandular function, as measured by gland-to-body weight ratio and production of β-casein, was suppressed earlier during postlactational involution than in controls. Histological examination revealed accelerated lumen collapse, alveolar-epithelial loss, and adipose reconstitution in Timp-3 -/-females. Epithelial apoptosis peaked on the first day of involution in Timp-3-null glands but at day 3 in wild-type littermates. Unscheduled activation of gelatinase-A was evident by zymography and correlat...

Local actin assembly is associated with sites of exocytosis in processes ranging from phagocytosis to compensatory endocytosis. Here, we examine whether the trigger for actin-coat assembly around exocytosing Xenopus egg cortical granules is 'compartment mixing'--the union of the contents of the plasma membrane with that of the secretory granule membrane. Consistent with this model, compartment mixing occurs on cortical granule-plasma membrane fusion and is required for actin assembly. Compartment mixing triggers actin assembly, at least in part, through diacylglycerol (DAG), which incorporates into the cortical granule membranes from the plasma membrane after cortical granule-plasma membrane fusion. DAG, in turn, directs long-term recruitment of protein kinase Cbeta (PKCbeta) to exocytosing cortical granules, where it is required for activation of Cdc42 localized on the cortical granules. The results demonstrate that mixing of two membrane compartments can direct local actin assembly and indicate that this process is harnessed during Xenopus egg cortical granule exocytosis to drive compensatory endocytosis.

Actin is involved in endocytosis in organisms ranging from yeast to mammals. In activated Xenopus eggs, exocytosing cortical granules (CGs) are surrounded by actin "coats," which compress the exocytosing compartments, resulting in compensatory endocytosis. Here, we examined the roles of two myosins in actin coat compression. Myosin-2 is recruited to exocytosing CGs late in coat compression. Inhibition of myosin-2 slows coat compression without affecting actin assembly. This differs from phenotype induced by inhibition of actin assembly, where exocytosing CGs are trapped at the plasma membrane (PM) completely. Thus, coat compression is likely driven in part by actin assembly itself, but it requires myosin-2 for efficient completion. In contrast to myosin-2, the long-tailed myosin-1e is recruited to exocytosing CGs immediately after egg activation. Perturbation of myosin-1e results in partial actin coat assembly and induces CG collapse into the PM. Intriguingly, simultaneous inhibition of actin assembly and myosin-1e prevents CG collapse. Together, the results show that myosin-1e and myosin-2 are part of an intricate machinery that coordinates coat compression at exocytosing CGs.

Cell repair is a conserved and medically important process. Cell damage triggers the rapid accumulation of several different lipids around wounds, and the lipids sort into distinct domains around them. One of these lipids—diacylglycerol—is required for activation of Rho and Cdc42 and healing.