Ezrin is an important membrane/actin cytoskeleton linker protein, especially in epithelia. membrane surface projections, forming the basis for a novel theory for ezrin as an organizer and regulator of membrane recruitment. 223445-75-8 A model simulating the cellular distribution of ezrin and its associated membrane- and F-actin-binding forms is usually given to forecast redistributions observed with phosphorylation and mutant overexpression, and it can easily be altered as more specific information regarding binding constants and specific sites becomes available. for 5 min, 223445-75-8 followed by resuspension in fresh (5-AGG TGT GGC ATG CGG AAC ACC GTG GGA TGC-3) and (5-CTC GCC CTT GCT CAC CAT TAA CTT ATC ATC TTT CTC-3). YFP sequence was PCR amplified from pEYFP-N1 (Clontech) with (5-GAG AAA GAT GAT AAG TTA ATG GTG AGC AAG GGC GAG-3) and (5-GCC AAT CTT TGG GGT CTT GTA CAG CTC GTC-3). Using the mixture of the above PCR products as template, a fusion gene of ezrin-YFP (920 bp) was PCR amplified with and (5-GAC GAG CTG TAC AAG ACC CCA AAG ATT GGC-3) and (5-C CCG CGG TGC GGC CGC TTA CAG GGC CTC GAA CTC-3). In the final PCR reaction, ezrin sequence aa176Caa586 fused with YFP was amplified with and I and I and ligated with similarly digested pDC311/WT-ezrin-CFP (provided DNA sequence for aa1C175 of ezrin), producing pDC311/WT-ezrin-YFP, the plasmid conveying full-length wild-type ezrin fused with a YFP tag. The open-reading frame of the construct was fully sequenced to make sure the accuracy of PCR and enzymatic operations. Recombinant adenovirus rAd-ezrin-WT-YFP was generated by cotransfecting human embryonic kidney 293 cells with pDC311/WT-ezrin-YFP 223445-75-8 and pBHGloxE1,3Cre (Microbix Biosystems, made up of altered adenovirus type-5 genome) using the CellPhect Transfection kit (Amersham Biosciences). A single viral colony was isolated, amplified, and titrated. Aliquots of computer virus were stored at ?80C. Immunofluorescence. Parietal cells produced on Matrigel-coated coverslips were infected with rAd-ezrin-WT-YFP, rAd-ezrin-TA-CFP mutant, or rAd-ezrin-TD-CFP mutant. Infected cells were fixed with 3.7% formaldehyde at the postplating time points specified in the figure legends. Fixed cells were permeabilized with 0.5% Triton X-100 in PBS. To stain exogenous ezrin and H-K-ATPase, cells were incubated with rabbit anti-green fluorescent protein (GFP) (which also recognizes GFP derivatives YFP and CFP, from Immunology SLC2A3 Consultants Laboratory, Newberg, OR) and mouse monoclonal anti-H-K-ATPase (2G11, Affinity Bioreagents, Boulder, CO). To stain for F-actin, Alexa 546-Phalloidin (Invitrogen) was used. The 223445-75-8 secondary antibodies were Alexa 488-conjugated goat-anti-rabbit IgG and Alexa 555-conjugated goat-anti-mouse IgG from Invitrogen (Eugene, OR). In some instances the secondary antibody for H-K-ATPase staining was cy5-donkey anti-mouse (Jackson ImmunoResearch), excitation 633 nm and emission 640C704 nm. Images of Alexa 555 (excitation with 543-nm laser, emission from 590C655 nm) and Alexa 488 (excitation with 488-nm laser, emission from 505C580 nm) were collected in the summary Fig. 5) is usually indicated for each time point and ezrin construct. The total number of individual experiments (i.at the., = rabbits) is usually indicated in Fig. 5 story. Images were collected by two investigators and were independently counted and scored by a different investigator. Cell phenotypic categories for manifestation of rAd-fluorescent protein (FP)-labeled ezrin were as follows: At the, and the rate of dephosphorylation = EP, where and are rate constants for the kinase and phosphatase reactions at T567, applying equally to ezrin that is usually free or bound to membrane. (These constants 223445-75-8 are both set to zero for the accumulation of exogenous TA or TD, which are treated in the model as At the that cannot be phosphorylated or EP that cannot be dephosphorylated.) Reactions for binding of At the and EP to cellular membranes and to F-actin were as follows: Ezrin-membrane and ezrin-actin equilibrium binding reactions: and and and that they are not shown here but are.