Na ؉ /H؉ exchangers (NHEs) mediate electroneutral exchange of Na ؉ for H ؉ and thereby play a central role in pH regulation and Na ؉ homeostasis. NHE3, the predominant epithelial isoform, is found in apical membranes of renal and intestinal epithelial cells, where it contributes to NaCl (re)absorption. NHE activity has been detected in endomembrane vesicles of epithelial cells, but the precise compartment involved and its functional role have not been defined. Many aspects of the targeting machinery that defines the compartmentation and polarity of epithelia are also functional in nonepithelial cells. We therefore compared the targeting of NHE1, the basolateral isoform, with that of NHE3 in Chinese hamster ovary cells. To circumvent the confounding effects of endogenous exchangers, epitope-tagged constructs of NHE1 and NHE3 were stably expressed in antiport-deficient (AP-1) cells. While NHE1 was found almost exclusively in the surface membrane, NHE3 was also found intracellularly, accumulating in a juxtanuclear compartment. Confocal microscopy showed this compartment to be distinct from the Golgi, trans-Golgi network, and lysosomes. Instead, NHE3 colocalized with transferrin receptors and with cellubrevin, markers of recycling endosomes. The activity of NHE3 in endomembranes was assessed by targeting pH-sensitive probes to the recycling endosomes using a chimeric cellubrevin construct with an accessible extracellular epitope. Fluorescence ratio imaging indicated that cellubrevin resides intracellularly in an acidic compartment. In AP-1 cells, endosomal acidification was unaffected by omission of Na ؉ but was dissipated entirely by concanamycin, a blocker of H ؉ -ATPases. In contrast, the cellubrevin compartment was more acidic in NHE3 transfectants, and the acidification was only partially reduced by concanamycin. Moreover, removal of extracellular Na ؉ resulted in a significant alkalization of the endocytic compartment. These results indicate that NHE3 is present and active in recycling endosomes. By recruiting NHE3 to the plasma membrane, modulation of vesicular traffic could contribute to the regulation of Na ؉ reabsorption across epithelia.

Assembly of collagen into fibrils is widely studied as a spontaneous and entropy-driven process. To determine whether vascular smooth muscle cells (SMCs) impact the formation of collagen fibrils, we microscopically tracked the conversion of soluble to insoluble collagen in human SMC cultures, using fluorescent type I collagen at concentrations less than that which supported self-assembly. Collagen microaggregates were found to form on the cell surface, initially as punctate collections and then as an increasingly intricate network of fibrils. These fibrils displayed 67-nm periodicity and were found in membrane-delimited cellular invaginations. Fibril assembly was inhibited by an anti-alpha2beta1 integrin antibody and accelerated by an alpha2beta1 integrin antibody that stimulates a high-affinity binding state. Newly assembled collagen fibrils were also found to co-localize with newly assembled fibronectin fibrils. Moreover, inhibition of fibronectin assembly with an anti-alpha5beta1 integrin antibody completely inhibited collagen assembly. Collagen fibril formation was also linked to the cytoskeleton. Fibrils formed on the stretched tails of SMCs, ran parallel to actin microfilament bundles, and formed poorly on SMCs transduced with retrovirus containing cDNA for dominant-negative RhoA and robustly on SMCs expressing constitutively active RhoA. Lysophosphatidic acid, which activates RhoA and stimulates fibronectin assembly, stimulated collagen fibril formation, establishing for the first time that collagen polymerization can be regulated by soluble agonists of cell function. Thus, collagen fibril formation is under close cellular control and is dynamically integrated with fibronectin assembly, opening new possibilities for modifying collagen deposition.

Mitogen-activated protein kinase (MAPK) pathways mediate some important cellular processes and are likely to also regulate preimplantation development. The role of p38 MAP kinase signaling during murine preimplantation development was investigated in the present study. p38 MAPK, p38-regulated or -activated kinase (PRAK; MK5), map kinase-activated protein kinase 2 (MK2), and heat shock protein 25 (hsp25) mRNAs and proteins were detected throughout preimplantation development. Two-cell stage embryos cultured in the presence of SB220025 and SB203580 (specific inhibitors of p38 MAPK alpha/beta), progressed to the eight-cell stage with the same frequency as controls; however, treated embryos halted their development at the 8- to 16-cell stage. In addition, embryos treated with p38 MAPK inhibitors displayed a complete loss of MK2 and hsp25 phosphorylation and also a complete loss of filamentous actin as indicated by the absence of rhodamine-phalloidin staining. In these inhibitor-treated groups, the embryos were composed of a mixture of compacting and noncompacting cells, and the embryos were one to two cell divisions behind controls. Treated embryos remained viable as the developmental blockade was rescued by removing embryos from the drug treatment and placing them in drug-free medium until they progressed to the blastocyst stage. This study demonstrates that p38 MAPK activity is required to support development through the murine preimplantation interval.

The epithelial isoform of the Na ؉ /H ؉ exchanger, NHE3, associates with at least two related regulatory factors called NHERF1/EBP50 and NHERF2/TKA-1/ E3KARP. These factors in addition interact with the cytoskeletal protein ezrin, which in turn binds to actin. The possible linkage of NHE3 with the cytoskeleton prompted us to test the effect of actin-modifying agents on NHE3 activity. Cytochalasins B and D and latrunculin B, which interfere with actin polymerization, induced a profound inhibition of NHE3 activity. The effect was isoform-specific inasmuch as the "housekeeping" exchanger NHE1 was virtually unaffected. Cytoskeletal disorganization was associated with a subcellular redistribution of NHE3, which accumulated at sites where actin aggregated, suggesting a physical interaction of exchangers with the cytoskeleton. An interaction was further suggested by the co-sedimentation of a detergent-insoluble fraction of NHE3 with the actin cytoskeleton. Inhibition of transport was not due to diminution in the number of transporters at the plasmalemma. Functional analyses of NHE1/NHE3 chimeras revealed that the cytoplasmic domain of NHE3 conferred sensitivity to cytochalasin B. Progressive carboxyl-terminal and internal deletions of the cytoplasmic region of NHE3 indicated that the region between residues 650 and 684 is critical for this response. This region overlaps with the domain reported to interact with NHERF and also contains a putative ezrin-binding site; hence, it likely plays a role in interactions with the cytoskeleton.

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.