reverse transcribed using random oligo-dT primers and a Verso cDNA Synthesis Kit (Thermo Fisher, AB1453) according to the manufacturer's instructions. Real-time PCR was performed using SsoAdvanced SYBR Green (Bio-Rad, 6090), and Rpl7 expression was used for normalization. The following primer sets were used to identify transcripts: collagen 1a1, 5′-AATGGCACGGCTGTGTGCGA and 5′-AACGGGTCCCCTTG-GGCCTT; collagen 3a1, 5′-TCCCCTGGAATCTGTGAATC and 5′-TGAGTCGAATTGGGGAGAAT; periostin, 5′-ACGGAGCTCAGG-GCTGAAGATG and 5′-GTTTGGGCCCTGATCCCGAC.Cell death analysis. At 90% confluence, primary skin fibroblasts were treated with 200 nM staurosporine for 36 hours or vehicle (DMSO). Cell death was determined by the Muse Count & Viability Assay (Millipore, MCH100102) as previously described (62). Briefly, the medium was collected with the trypsin-liberated cells, which were centrifuged and washed twice with PBS and then incubated with the Muse Count & Viability reagent. The cells were then quantified on a Muse cell analyzer (Millipore) at 5,000 counts per sample.Statistics. One-way ANOVA with post hoc Tukey's honest significant difference (HSD) or Student's t test was used to determine statistical significance, depending on the type of data analyzed and number of comparisons. P values of less than 0.05 were considered statistically significant. Averaged data are presented with SEM to indicate variability.Study approval. Mice were observed daily and cages changed weekly by certified veterinary technicians at Cincinnati Children's Hospital Medical Center. Mice were also closely assessed for their well-being, monitored by adequate physical activity and food intake on a daily basis. Housing conditions and husbandry conformed to AAALAC standards as well as the standard guidelines from the NIH Office of Laboratory Animal Welfare (http://grants.nih.gov/grants/olaw/animal_use. htm). The institution also retains ongoing certification by AAALAC.

Fibroblasts are a dynamic cell type that achieve selective differentiated states to mediate acute wound healing and long-term tissue remodeling with scarring. With myocardial infarction injury, cardiomyocytes are replaced by secreted extracellular matrix proteins produced by proliferating and differentiating fibroblasts. Here, we employed 3 different mouse lineage-tracing models and stage-specific gene profiling to phenotypically analyze and classify resident cardiac fibroblast dynamics during myocardial infarction injury and stable scar formation. Fibroblasts were activated and highly proliferative, reaching a maximum rate within 2 to 4 days after infarction injury, at which point they expanded 3.5-fold and were maintained long term. By 3 to 7 days, these cells differentiated into myofibroblasts that secreted abundant extracellular matrix proteins and expressed smooth muscle α-actin to structurally support the necrotic area. By 7 to 10 days, myofibroblasts lost proliferative ability and smooth muscle α-actin expression as the collagen-containing extracellular matrix and scar fully matured. However, these same lineage-traced initial fibroblasts persisted within the scar, achieving a new molecular and stable differentiated state referred to as a matrifibrocyte, which was also observed in the scars of human hearts. These cells express common and unique extracellular matrix and tendon genes that are more specialized to support the mature scar.

The relative importance of plasma membrane Ca 2؉ -ATPase (PMCA) 1 and PMCA4 was assessed in mice carrying null mutations in their genes (Atp2b1 and Atp2b4). Loss of both copies of the gene encoding PMCA1 caused embryolethality, whereas heterozygous mutants had no overt disease phenotype. Despite widespread and abundant expression of PMCA4, PMCA4 null (Pmca4 ؊/؊ ) mutants exhibited no embryolethality and appeared outwardly normal. Loss of PMCA4 impaired phasic contractions and caused apoptosis in portal vein smooth muscle in vitro; however, this phenotype was dependent on the mouse strain being employed. Pmca4 ؊/؊ mice on a Black Swiss background did not exhibit the phenotype unless they also carried a null mutation in one copy of the Pmca1 gene. Pmca4 ؊/؊ male mice were infertile but had normal spermatogenesis and mating behavior. Pmca4؊/؊ sperm that had not undergone capacitation exhibited normal motility but could not achieve hyperactivated motility needed to traverse the female genital tract. Ultrastructure of the motility apparatus in Pmca4 ؊/؊ sperm tails was normal, but an increased incidence of mitochondrial condensation indicated Ca 2؉ overload. Immunoblotting and immuno-histochemistry showed that PMCA4 is the most abundant isoform in testis and sperm and that it is localized to the principle piece of the sperm tail, which is also the location of the major Ca 2؉ channel (CatSper) required for sperm motility. These results are consistent with an essential housekeeping or developmental function for PMCA1, but not PMCA4, and show that PMCA4 expression in the principle piece of the sperm tail is essential for hyperactivated motility and male fertility.

Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compound (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. We demonstrate that iTOP is a useful tool in systems in which transient cell manipulation drives permanent cellular changes. As an example, we demonstrate that iTOP can mediate the delivery of recombinant Cas9 protein and short guide RNA, driving efficient gene targeting in a non-integrative manner.

Despite the importance of cell fusion for mammalian development and physiology, the factors critical for this process remain to be fully defined, which has severely limited our ability to reconstitute cell fusion. Myomaker (Tmem8c) is a muscle-specific protein required for myoblast fusion. Expression of myomaker in fibroblasts drives their fusion with myoblasts, but not with other myomaker-expressing fibroblasts, highlighting the requirement of additional myoblast-derived factors for fusion. Here we show that Gm7325, which we name myomerger, induces the fusion of myomaker-expressing fibroblasts. Thus, myomaker and myomerger together confer fusogenic activity to otherwise non-fusogenic cells. Myomerger is skeletal muscle-specific and genetic deletion in mice results in a paucity of muscle fibres demonstrating its requirement for normal muscle formation. Myomerger deficient myocytes differentiate and harbour organized sarcomeres but are fusion-incompetent. Our findings identify myomerger as a fundamental myoblast fusion protein and establish a system that begins to reconstitute mammalian cell fusion.

The NBC1 Na ؉ /HCO 3 ؊ cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO 3 ؊ absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO 3 ؊ secretion in the intestinal tract, null mutant (NBC1 /HCO 3 Ϫ cotransporters (1-3). NBC1 has two protein variants, which localize to basolateral membranes (4) and mediate electrogenic Na ϩ /HCO 3 Ϫ cotransport (2, 3). The kNBC1 variant is expressed in kidney epithelia and eye (4, 5), and the pNBC1 variant is expressed in pancreas, duodenum, colon, and several other tissues (4 -8). The stoichiometry of the transporter can be altered from 1NaϪ by phosphorylation of a residue near the carboxyl terminus (9). In the kidney, the ion stoichiometry and electrochemical driving forces for NBC1 result in Na ϩ and HCO 3 Ϫ extrusion across the basolateral membrane (2, 3, 9, 10); thus, in the kidney, NBC1 functions in HCO 3 Ϫ reabsorption in the proximal tubule (2, 3). In pancreas and the intestinal tract, the ion stoichiometry and driving forces for NBC1 appear to result in Na ϩ and HCO 3 Ϫ entry into the cell (2, 8); thus, in intestine and colon, NBC1 has been proposed to mediate HCO 3 Ϫ uptake across the basolateral membrane to support transepithelial anion secretion (8, 11).Human patients with proximal renal tubular acidosis resulting from mutations in NBC1 have been reported (12-16), thereby confirming a bicarbonate-absorptive role for NBC1 in kidney. The primary mutations were single amino acid substitutions (R298S, T485S, R510H, A799V, R881C, and S427L), which appeared to cause decreased function of the cotransporter rather than loss of function (12)(13)(14). One patient had an inactivating mutation in the unique N terminus of the kidney NBC1 variant (Q29X), but the pancreatic variant, which is expressed in many other tissues and at low levels in kidney (4), was intact (15). Only a single patient has been identified with a complete inactivating mutation, a nucleotide deletion that causes a frameshift at codon 721 (16). The pRTA resulting from NBC1 mutations clearly shows that this transporter is essential for renal HCO 3 Ϫ absorption; however, clinically significant intestinal disease has not been reported.NBC1 has been localized to the basolateral membrane of epithelial cells lining both the small and large intestine (8,17,18). In the colon, its expression was greatest in crypt cells, consist-* This work was supported by National Institutes of Health (NIH) Grants DK50594 and HL61974 (to G. E. S.), DK67749 (to L. R. G.), DK57552 (to J. N. L.), DK48816 (to L. L. C.), and T32-RR-07004 (to J. E. S.) and NIEHS, NIH, Grant ES06096 (to the Center for Environmental Genetics, Alvaro Puga PI). The costs of publication of this article were defrayed in part by the payment of page charges. This article mus...

The AE2 Cl ؊ /HCO 3 ؊ exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for HCO 3 ؊ efflux and Cl ؊ influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2 ؊/؊ ) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2 ؊/؊ mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2 ؊/؊ mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2 ؊/؊ gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.

embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1 ؊/؊ embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca 2؉ pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer. and Mn 2ϩ (10). Loss of PMR1 affects outer chain glycosylation, proteolytic processing, and trafficking of proteins in the secretory pathway (9). In mammals, SPCA1 is expressed in all tissues (1), whereas SPCA2 is expressed in only a limited set of tissues (3). Like PMR1, both SPCA1 and SPCA2 are localized to the Golgi and transport Ca 2ϩ and Mn 2ϩ (3, 11). There is evidence that the cell biological functions of SPCA1 are also similar to those of PMR1 (12).Loss of one copy of the human ATP2C1 gene, encoding SPCA1, causes Hailey-Hailey disease (HHD), an autosomal dominant skin disorder (13,14). SPCA1 protein levels in HHD keratinocytes are reduced to about half of normal levels, and Golgi Ca 2ϩ handling is impaired (15). HHD is similar to Darier disease, which is caused by null mutations in one copy of the human ATP2A2 gene, encoding SERCA2 (16). Both diseases are characterized by acantholysis (a disruption of cell-cell contacts) in the suprabasal layers of the skin. As the major ER Ca 2ϩ pump in most tissues, including keratinocytes, the function of SERCA2 is similar to that of SPCA1 in that it maintains luminal Ca 2ϩ concentrations in a major compartment of the secretory pathway. In mice, SERCA2 haploinsufficiency does not cause Darier disease but does lead to squamous cell tumors of keratinized epithelial cells (17, 18), the same cell type affected in Darier disease. In humans, a low incidence of squamous cell tumors has been reported in both Darier disease (19) and HHD (20, 21), but it is unclear whether this is a chance association or is caused by the reduction in Ca 2ϩ pump levels and activity. In the current study, we developed a gene-targeted mouse model for SPCA1 and analyzed the phenotype resulting from heterozygous and homozygous null mutations. The results sho...