Shafinaz Akhter

Intestinal growth and differentiation in zebrafish

Intestinal development in amniotes is driven by interactions between progenitor cells derived from the three primary germ layers. Genetic analyses and gene targeting experiments in zebrafish offer a novel approach to dissect such interactions at a molecular level. Here we show that intestinal anatomy and architecture in zebrafish closely resembles the anatomy and architecture of the mammalian small intestine. The zebrafish intestine is regionalized and the various segments can be identified by epithelial markers whose expression is already segregated at the onset of intestinal differentiation. Differentiation of cells derived from the three primary germ layers begins more or less contemporaneously, and is preceded by a stage in which there is rapid cell proliferation and maturation of epithelial cell polarization. Analysis of zebrafish mutants with altered epithelial survival reveals that seemingly related single gene defects have different effects on epithelial differentiation and smooth muscle and enteric nervous system development.

Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts.

We developed a confocal morphometric analysis to quantitate the relative plasma membrane (PM) expression of the Na/H exchanger NHE3 in living PS120 fibroblasts. NHE3 is a membrane transport protein that is acutely regulated by changes in the number of molecules expressed at the PM. To quantitate the PM expression of NHE3 under various experimental conditions, we stably expressed a chimera of rabbit NHE3 and green fluorescent protein (NHE3–GFP) in PS120 fibroblasts. A three-dimensional (3D) map of the intracellular distribution of NHE3–GFP was obtained by confocal laser scanning microscopy (CLSM) of cells superfused with a styryl dye, FM 4–64. This fluorophore rapidly and reversibly labeled the outer lipid layer of the PM, which allowed generation of a digital mask of the PM and calculation of the fraction of a total cellular NHE3–GFP expressed at the PM. This analysis was successfully used to quantitate the relative PM expression of NHE3–GFP in control cells (25%) and a decrease in the expression caused by subsequent exposure of cells to wortmannin (5.1%). Reliability of the method was confirmed by cell surface biotinylation, which yielded very similar results. Confocal morphometric analysis is fast and reproducible and could potentially be used for investigations on regulation of expression of other membrane proteins.

Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface

NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian Na+/H+exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHE1V, NHE2V, or NHE3V: 1) maximal reaction velocity ( V max) by22Na+uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 ± 3.5, 64.6 ± 3.3, 20.0 ± 2.6, and 14.0 ± 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 ± 9.6, 92.1 ± 8.6, and 99.2 ± 9.1 s-1, when V max was determined using 22Na uptake at 22°C and 742 ± 47, 459 ± 16, and 609 ± 39 s-1 when V max was determined using fluorometry at 37°C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3.

Separate functions for brush border membrane and endosomal NHE3 in a polarized epithelial cell line

AN UPDATE OF CANCER RISK IN HNPCC: A STUDY OF 101 FAMILIES INCLUDING 58 FAMILIES ASSOCIATED WITH MISMATCH REPAIR MUTATIONS. Hans F. Vasen, Astrid Stonnorken, Fokko N. Nagengast, Jan Kleibeuker, Gerrit G. Griffioen, Fred H. Menko, Riccardo Fodde, Babs G. Taal, Pal Moller, Juul Th Wijnen, Dept of Gastroenterology, LUMC, Leiden, Netherlands; Unit of Med Genetics, Norwegian Radium Hosp, Oslo, Norway; Dept of Gastroenterology, Nijmegen Univ Hosp, Nijmegen, Netherlands; Dept of Gastroenterology, Groningen Univ Hosp, Groningen, Netherlands; Clin Genetic Ctr, Free Univ Hosp, Amsterdam, Netherlands; Lab for Anthropogenetics, Leiden, Netherlands; Dept of Gastroenterology, Netherlands Cancer Institute, Amsterdam, Netherlands; Lab of Anthropogenetics, Leiden, Netherlands.