munication between cells and the extracellular matrix (ECM) is critical for regulation of cell growth, survival, migration, and differentiation. Remodeling of the ECM can occur under normal physiological conditions, as a result of tissue injury, and in certain pathological conditions. ECM remodeling leads to alterations in ECM composition and organization that can alter many aspects of cell behavior, including cell migration. The cell migratory response varies depending on the type, amount, and organization of ECM molecules present, as well as the integrin and proteoglycan repertoire of the cells. We and others have shown that the deposition of several ECM molecules, including collagen types I and III, depends on the presence and stability of ECM fibronectin. Hence, the effect of fibronectin and fibronectin matrix on cell function may partially depend on its ability to direct the deposition of collagen in the ECM. In this study, we used collagen-binding fibronectin mutants and recombinant peptides that interfere with fibronectin-collagen binding to show that fibronectindependent collagen I deposition regulates the cell migratory response to fibronectin. These data show that the ability of fibronectin to organize other proteins in the ECM is an important aspect of fibronectin function and highlight the importance of understanding how interactions between ECM proteins influence cell behavior. extracellular matrix; contractility CELL FATE DECISIONS involving cell growth, differentiation, and survival rely on the ability of cells to coordinate diverse input from cytokines, growth factors, and extracellular matrix (ECM) molecules (3,89,94). The effects of ECM molecules on cell behavior are particularly complicated, since they depend on the mixture of ECM molecules that are present, the way the ECM proteins are organized and presented to cells, and the presence of proteases, protease inhibitors, and endocytic mechanisms that can alter the levels of ECM proteins and ECM degradation products. Understanding how ECM proteins act in concert to elicit biological effects is key to understanding how cell-ECM interactions maintain normal tissue function and influence the cell response to tissue injury.There is much data showing that mixtures of different ECM molecules can have effects distinct from that of a single ECM molecule. For example, coating dishes with a combination of tenascin C and fibronectin results in altered expression of matrix metalloproteinases (MMPs) (86), whereas addition of tenascin C to dishes coated with fibrin and fibronectin results in altered cytoskeletal organization (90) compared with cells seeded in the absence of tenascin C. The ability of fibronectin null cells to produce a fibronectin matrix is also dependent on the combination of matrix proteins present on the substrate (4). Furthermore, mixed collagen and fibronectin substrates have been shown to alter the response of endothelial cells to shear stress compared with their response to fibronectin alone (59). Similarly, addition of soluble matric...

Extracellular matrix remodeling occurs during development, tissue repair, and in a number of pathologies, including fibrotic disorders, hypertension, and atherosclerosis. Extracellular matrix remodeling involves the complex interplay between extracellular matrix synthesis, deposition, and degradation. Factors that control these processes are likely to play key roles in regulating physiological and pathological extracellular matrix remodeling. Our data show that fibronectin polymerization into the extracellular matrix regulates the deposition and stability of other extracellular matrix proteins, including collagen I and thrombospondin-1 (Sottile and Hocking, 2002. Mol. Biol. Cell 13, 3546). In the absence of continual fibronectin polymerization, there is a loss of fibronectin matrix fibrils, and increased levels of fibronectin degradation. Fibronectin degradation occurs intracellularly after endocytosis and can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and by caveolae-disrupting agents. Down-regulation of caveolin-1 by RNAi inhibits loss of fibronectin matrix fibrils, fibronectin internalization, and fibronectin degradation; these processes can be restored by reexpression of caveolin-1. These data show that fibronectin matrix turnover occurs through a caveolin-1-dependent process. Caveolin-1 regulation of fibronectin matrix turnover is a novel mechanism regulating extracellular matrix remodeling. INTRODUCTIONExtracellular matrix (ECM) remodeling is a dynamic, cellmediated process that occurs during development, tissue repair, and in a variety of pathological events including atherosclerosis, hypertension, and ischemic injury (Clark, 1996;Prescott et al., 1999;Streuli, 1999;Newby and Zaltsman, 2000;Intengan and Schiffrin, 2001). Furthermore, abnormal matrix remodeling is associated with fibrosis, arthritis, reduced angiogenesis, and developmental abnormalities (Liu et al., 1995;Vu et al., 1998;Holmbeck et al., 1999). During tissue repair, the precise deposition of ECM molecules, including collagen I and fibronectin, is required to preserve the structural and functional integrity of tissues (Clark, 1996). Excessive or inappropriate deposition of ECM molecules, as occurs during fibrosis, disrupts normal tissue architecture, leading to impaired organ function (Mutsaers et al., 1997;Zeisberg et al., 2000). The mechanisms that control ECM organization and homeostasis are incompletely understood. We have recently shown that fibronectin matrix polymerization is essential for the organization, as well as the maintenance of ECM architecture . Our data show that the cell-dependent process of polymerizing fibronectin into the ECM is required for the deposition and maintenance of fibrillar fibronectin, collagen-I, and thrombosponin-1 . These data are consistent with other studies showing that collagen I and collagen III deposition into the ECM are regulated by fibronectin (McDonald et al., 1982;Velling et al., 2002). Fibronectin has also been implicated in regulating the deposition of tenascin C (Chu...

NMR studies of the delta subunit of the Escherichia coli F1F0-ATPsynthase reveal that it consists of an N-terminal six alpha-helix bundle and a less well ordered C terminus. Both domains are part of one of two separate connections between F1 and F0.

The proton-translocating ATP synthases couple the generation of ATP to the protonmotive force present across membranes involved in energy transduction (for reviews, see Refs. 1-4). These complex enzymes consist of a peripheral F 1 sector, which catalyzes ATP synthesis and hydrolysis, and an integral F 0 sector, which catalyzes movements of protons across the membrane. In the relatively simple ATP synthase of Escherichia coli, F 1 contains five types of subunits in a stoichiometry of ␣ 3 ␤ 3 ␥␦⑀, while F 0 contains three types of subunits in a stoichiometry of ab 2 c 9 -12 . Subunit interactions at the interface of the two sectors are responsible for coupling their catalytic activities.Recent work has strongly indicated that hydrolysis of ATP by F 1 is accompanied by rotation of the ␥ and ⑀ subunits relative to the ␣ 3 ␤ 3 hexameric ring (5-11), consistent with proposals from Paul Boyer's laboratory (12). The high resolution structure of the mitochondrial F 1 (13) reveals that the N and C termini of ␥ form an antiparallel coiled-coil running up the center of the ␣ 3 ␤ 3 ring; this structure appears to function as an asymmetric spindle, which, by rotating, plays the major role in directing conformational changes at the catalytic sites. In the intact ATP synthase, the rotation of ␥ and ⑀ should be coupled to proton conduction through F 0 . The a and c subunits provide those residues that are essential for proton conduction.In all systems, ␦ or the analogous mitochondrial protein called oligomycin sensitivity conferral protein (OSCP), 1 is essential for the coupling of the catalytic activities of the two sectors. The ␦ subunit (reviewed in Ref. 14) has no significant effect on steady-state ATP hydrolysis rates by isolated F 1 -ATPase but does alter unisite hydrolysis (15). ␦ binds to F 1 through interactions with the external surface of the N-terminal third of the ␣ subunit (16 -20). In some systems, ␦ alters the proton permeability of F 0 (21). This effect is not seen in E. coli, but here ␦ is essential for the interaction of F 1 and F 0 , implying a link between ␦ and F 0 (22). The physical and functional nature of the ␦-F 0 interaction is currently the subject of intense interest. In recent work, an interaction of ␦ or OSCP with the b subunit of F 0 has been demonstrated (23-25). Nearest neighbor analysis by chemical cross-linking had not revealed crosslinks between b and ␦ in the E. coli system (26), but they had been reported for the corresponding subunits in the chloroplast (27) and mitochondrial (28) enzymes. The importance of the cytoplasmic domain of b to the F 1 -F 0 interaction has also been demonstrated through proteolysis (29 -31) and direct binding (32) studies.E. coli ␦ purified following pyridine treatment of F 1 -ATPase was shown to be an elongated monomer (33), but the low yield of the preparation limited the scope of work that could be carried out. The current studies were undertaken to produce recombinant ␦ in quantities appropriate for high resolution structural analysis and to permit the charac...

95616 (J. Chandler, J. Callis) Deubiquitinating enzymes are essential to the ubiquitin (Ub)/26S proteasome system where they release Ub monomers from the primary translation products of poly-Ub and Ub extension genes, recycle Ubs from polyubiquitinated proteins, and reverse the effects of ubiquitination by releasing bound Ubs from individual targets. The Ub-specific proteases (UBPs) are one large family of deubiquitinating enzymes that bear signature cysteine and histidine motifs. Here, we genetically characterize a UBP subfamily in Arabidopsis (Arabidopsis thaliana) encoded by paralogous UBP3 and UBP4 genes. Whereas homozygous ubp3 and ubp4 single mutants do not display obvious phenotypic abnormalities, double-homozygous mutant individuals could not be created due to a defect in pollen development and/or transmission. This pollen defect was rescued with a transgene encoding wild-type UBP3 or UBP4, but not with a transgene encoding an active-site mutant of UBP3, indicating that deubiquitination activity of UBP3/UBP4 is required. Nuclear DNA staining revealed that ubp3 ubp4 pollen often fail to undergo mitosis II, which generates the two sperm cells needed for double fertilization. Substantial changes in vacuolar morphology were also evident in mutant grains at the time of pollen dehiscence, suggesting defects in vacuole and endomembrane organization. Even though some ubp3 ubp4 pollen could germinate in vitro, they failed to fertilize wild-type ovules even in the absence of competing wildtype pollen. These studies provide additional evidence that the Ub/26S proteasome system is important for male gametogenesis in plants and suggest that deubiquitination of one or more targets by UBP3/UBP4 is critical for the development of functional pollen.