Budding yeast Sec16 is a large peripheral endoplasmic reticulum (ER) membrane protein that functions in generating COPII transport vesicles and in clustering COPII components at transitional ER (tER) sites. Sec16 interacts with multiple COPII components. Although the COPII assembly pathway is evolutionarily conserved, Sec16 homologues have not been described in higher eukaryotes. Here, we show that mammalian cells contain two distinct Sec16 homologues: a large protein that we term Sec16L and a smaller protein that we term Sec16S. These proteins localize to tER sites, and an N-terminal region of each protein is necessary and sufficient for tER localization. The Sec16L and Sec16S genes are both expressed in every tissue examined, and both proteins are required in HeLa cells for ER export and for normal tER organization. Sec16L resembles yeast Sec16 in having a C-terminal conserved domain that interacts with the COPII coat protein Sec23, but Sec16S lacks such a C-terminal conserved domain. Immunoprecipitation data indicate that Sec16L and Sec16S are each present at multiple copies in a heteromeric complex. We infer that mammalian cells have preserved and extended the function of Sec16.

A common application of fluorescent proteins is to label whole cells, but many red fluorescent proteins are cytotoxic when used with standard high-level expression systems. We engineered a rapidly maturing tetrameric fluorescent protein called DsRed-Express2 that shows minimal cytotoxicity. DsRed-Express2 exhibits strong and stable expression in bacterial and mammalian cells, and it outperforms other available red fluorescent proteins with regard to photostability and phototoxicity.

Fluorescent proteins (FPs) with far-red excitation and emission are desirable for multicolor labeling and live-animal imaging. We describe E2-Crimson, a far-red derivative of the tetrameric FP DsRed-Express2. Unlike other far-red FPs, E2-Crimson is noncytotoxic in bacterial and mammalian cells. E2-Crimson is brighter than other far-red FPs and matures substantially faster than other red and far-red FPs. Approximately 40% of the E2-Crimson fluorescence signal is remarkably photostable. With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers. We show that E2-Crimson has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy.

Previous studies have shown that haploinsufficiency of the splanchnic and septum transversum mesoderm Forkhead Box (Fox) f1 transcriptional factor caused defects in lung and gallbladder development and that Foxf1 heterozygous (؉/؊) mice exhibited defective lung repair in response to injury. In this study, we show that Foxf1 is expressed in hepatic stellate cells in developing and adult liver, suggesting that a subset of stellate cells originates from septum transversum mesenchyme during mouse embryonic development. Because liver regeneration requires a transient differentiation of stellate cells into myofibroblasts, which secrete type I collagen into the extracellular matrix, we examined Foxf1 ؉/؊ liver repair following carbon tetrachloride injury, a known model for stellate cell activation. We found that regenerating Foxf1 ؉/؊ liver exhibited defective stellate cell activation following CCl 4 liver injury, which was associated with diminished induction of type I collagen, ␣-smooth muscle actin, and Notch-2 protein and resulted in severe hepatic apoptosis despite normal cellular proliferation rates. Furthermore, regenerating Foxf1 ؉/؊ livers exhibited decreased levels of interferon-inducible protein 10 (IP-10), delayed induction of monocyte chemoattractant protein 1 (MCP-1) levels, and aberrantly elevated expression of transforming growth factor ␤1. T he Forkhead Box (Fox) family of transcription factors shares homology in the winged helix DNA binding domain, 1 and its members play important roles in cellular proliferation, differentiation, and metabolic homeostasis. [2][3][4][5][6] Haploinsufficiency of the Foxf1 gene (previously known as HFH-8 or Freac-1) in heterozygous (ϩ/Ϫ) mice causes perinatal lethality from pulmonary hemorrhage and severe defects in alveolarization, vascularization, and fusion of lung lobes. 7-9 Lung hemorrhage was observed in one half of newborn Foxf1 ϩ/Ϫ mice that had an 80% reduction in pulmonary Foxf1 levels (low Foxf1 ϩ/Ϫ) and reduced expression of genes involved in lung morphogenesis. 7 Interestingly, expression of these lung developmental genes was unchanged in 40% of the newborn Foxf1 ϩ/Ϫ mice that had near wildtype (WT) pulmonary levels of Foxf1 messenger RNA (mRNA) (high Foxf1 ϩ/Ϫ mice) without pulmonary hemorrhage, but they exhibited diminished alveolar septation. 7 Moreover, the high Foxf1 ϩ/Ϫ mice had normal life spans and adult lung morphology, suggesting that these mice compensated for the alveolar septation defect but exhibited defective lung repair in response to injury. 10 Liver development initiates at 9 days postcoitum (dpc) of mouse embryogenesis, when the cardiac mesenchyme induces the hepatic primordium to emerge from the foregut endoderm that invades the septum transversum mesenchyme. 6 Previous expression studies have shown

The forkhead box f1 (Foxf1) transcription factor is expressed in the visceral (splanchnic) mesoderm, which is involved in mesenchymal-epithelial signaling required for development of organs derived from foregut endoderm such as lung, liver, gall bladder, and pancreas. Our previous studies demonstrated that haploinsufficiency of the Foxf1 gene caused pulmonary abnormalities with perinatal lethality from lung hemorrhage in a subset of Foxf1؉/؊ newborn mice. During mouse embryonic development, the liver and biliary primordium emerges from the foregut endoderm, invades the septum transversum mesenchyme, and receives inductive signaling originating from both the septum transversum and cardiac mesenchyme. In this study, we show that Foxf1 is expressed in embryonic septum transversum and gall bladder mesenchyme. Foxf1؉/؊ gall bladders were significantly smaller and had severe structural abnormalities characterized by a deficient external smooth muscle cell layer, reduction in mesenchymal cell number, and in some cases, lack of a discernible biliary epithelial cell layer. This Foxf1؉/؊ phenotype correlates with decreased expression of vascular cell adhesion molecule-1 (VCAM-1), ␣ 5 integrin, plateletderived growth factor receptor ␣ (PDGFR␣) and hepatocyte growth factor (HGF) genes, all of which are critical for cell adhesion, migration, and mesenchymal cell differentiation.At 9-days postcoitum (dpc) 1 during mouse embryogenesis, the liver primordium emerges from the foregut endoderm, invades the septum transversum mesenchyme, and receives bone morphogenetic protein 4 (Bmp-4) and fibroblast growth factor 2 (Fgf2) signaling originating from the septum transversum and cardiac mesenchyme, respectively (1, 2). This hepatic specification is associated with expression of the Foxa2 (HNF-3␤) and Gata4 transcription factors (3). Liver morphogenesis involves a proliferative expansion period and development of the bipotential hepatoblasts that begin to differentiate into hepatocytes and bile duct epithelial cells of the intrahepatic bile ducts (IHBD) at 13.5 dpc (3,4). Interestingly, targeted disruption of either the homeodomain Hex gene or the Hgf gene allows normal development of the mouse hepatic diverticulum, but these cells fail to migrate into the septum transversum and undergo liver morphogenesis (5-7).In the adult liver, bile is synthesized in hepatocytes from cholesterol, secreted into the bile canaliculi and transported through the intrahepatic and extrahepatic biliary system to the gall bladder, where it is stored for secretion into the digestive tract to emulsify lipids (8). The gall bladder and extrahepatic bile ducts (EHBD) develop from the caudal portion of the liver primordium at 10 dpc of mouse embryogenesis (9). However, little is known regarding visceral (splanchnic) mesoderm transcription factors that regulate expression of genes involved in mesenchymal-epithelial induction of gall bladder development from the liver primordium. Visceral mesenchymal expression of the homeodomain Hlx gene is required for the proli...

In mammalian cells, the 'Golgi reassembly and stacking protein' (GRASP) family has been implicated in Golgi stacking, but the broader functions of GRASP proteins are still unclear. The yeast Saccharomyces cerevisiae contains a single non-essential GRASP homolog called Grh1. However, Golgi cisternae in S. cerevisiae are not organized into stacks, so a possible structural role for Grh1 has been difficult to test. Here, we examined the localization and function of Grh1 in S. cerevisiae and in the related yeast Pichia pastoris, which has stacked Golgi cisternae. In agreement with earlier studies indicating that Grh1 interacts with coat protein II (COPII) vesicle coat proteins, we find that Grh1 colocalizes with COPII at transitional endoplasmic reticulum (tER) sites in both yeasts. Deletion of P. pastoris Grh1 had no obvious effect on the structure of tER-Golgi units. To test the role of S. cerevisiae Grh1, we exploited the observation that inhibiting ER export in S. cerevisiae generates enlarged tER sites that are often associated with the cis Golgi. This tER-Golgi association was preserved in the absence of Grh1. The combined data suggest that Grh1 acts early in the secretory pathway, but is dispensable for the organization of secretory compartments.

The nuclear division takes place in the daughter cell in the basidiomycetous budding yeast Cryptococcus neoformans . Unclustered kinetochores gradually cluster and the nucleus moves to the daughter bud as cells enter mitosis. Here, we show that the evolutionarily conserved Aurora B kinase Ipl1 localizes to the nucleus upon the breakdown of the nuclear envelope during mitosis in C . neoformans . Ipl1 is shown to be required for timely breakdown of the nuclear envelope as well. Ipl1 is essential for viability and regulates structural integrity of microtubules. The compromised stability of cytoplasmic microtubules upon Ipl1 depletion results in a significant delay in kinetochore clustering and nuclear migration. By generating an in silico model of mitosis, we previously proposed that cytoplasmic microtubules and cortical dyneins promote atypical nuclear division in C . neoformans . Improving the previous in silico model by introducing additional parameters, here we predict that an effective cortical bias generated by cytosolic Bim1 and dynein regulates dynamics of kinetochore clustering and nuclear migration. Indeed, in vivo alterations of Bim1 or dynein cellular levels delay nuclear migration. Results from in silico model and localization dynamics by live cell imaging suggests that Ipl1 spatio-temporally influences Bim1 or/and dynein activity along with microtubule stability to ensure timely onset of nuclear division. Together, we propose that the timely breakdown of the nuclear envelope by Ipl1 allows its own nuclear entry that helps in spatio-temporal regulation of nuclear division during semi-open mitosis in C . neoformans .

A mechanistic in silico model predicts mitotic events and effects of perturbation in budding yeasts belonging to Ascomycota and Basidiomycota. The model identifies distinct pathways based on the population of cytoplasmic microtubules and cortical dyneins as determinants of nuclear and spindle positioning in these phyla.