Changes in the Composition of Plasma Membrane Proteins During Differentiation of Embryonic Chick Erythroid Cell
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Proc. NatI. Acad. Sci. USA Vol. 74, No. 3, pp. 1062-1066, March 1977 Cell Biology Changes in the composition of plasma membrane proteins during differentiation of embryonic chick erythroid cell (red blood cell/development/membrane isolation and characterization/sodium dodecyl sulfate-polyacrylamide gels) LEE-NIEN'L. CHAN Physiology Department, University of Connecticut Health Center, Farmington, Conn. 06032 Communicated by George E. Palade, November 29,1976 ABSTRACT Erythroid cells which are homogeneous with protein composition of plasma membranes from embryonic regard to sta e of maturation are naturally available from the chick erythroid cells at various stages of maturation. Significant circulation of chick embryos at various times of development. This provides a convenient system for examining the changes changes both in quality and in quantity of membrane proteins in plasma membrane protein composition during red cell mat- occur during embryonic erythroid differentiation. uration, Plasma membranes are isolated from chick embryonic erythroid cells at various stages of maturation. Extensive char- MATERIALS AND METHODS acterization of the isolated membranes show that they are pure Materials. Fertilized White Leghorn eggs are obtained from and their roteins undegraded. Analyses by sodium dodecyl sulfate/polyacrylamide gel electrophoresis show that both the Spafas Co., Norwich, Conn. The eggs are incubated in a qualitative and quantitative changes occur in membrane protein Humidaire Incubator (model no. 50) at 380 (dry bulb) and 290 composition during the early stage of erythroid differentiation. (wet bulb). Red blood cells are collected from the circulation Specific proteins of red cell membrane such as "spectrin" and of the embryos after various times of incubation by cutting open band three proteins are present in low levels in early erythro- the main blood vessels and allowing the blood cells to be blasts but increase in their relative amounts with maturation. A pumped or drained out. The cells are washed extensively with steady-state membrane protein composition seems to be es- cold Howard Ringer's saline before use (7). Contamination by tablished by the late polychromatophilic erythroblast stage. other cell types is negligible in red cell samples collected in this The erythrocyte membrane is a most useful system for the study manner. Furthermore, the white blood cell counts in the em- of cell membrane structure and membrane-associated func- bryonic circulation is insignificant until hatching (8). The stages tions. The protein composition of erythrocyte plasma mem- of maturation of erythroid cells from embryos at various days branes has been extensively studied (see ref. 1). In general, the of incubation are as follows: basophilic erythroblast, 2.5 days; major erythrocyte membrane polypeptides, as resolved by so- midpolychromatophilic erythroblast, 3.5 days; late poly- dium dodecyl sulfate (NaDodSO4/polyacrylamide gel elec- chromatophilic erythroblast, 4.5 days; reticulocyte, 6 days; trophoresis), are common in all mammalian species as well as proerythrocyte and erythrocyte, 8 days; erythrocyte, 18 in certain avian species (1-5). days. Since the morphology and cellular functions of erythroid cells 125I-Labeled concanavalin A (Con A) is the generous gift of change dramatically during differentiation, it is of importance M. Sheetz, Physiology Department, University of Connecticut to know the specific developmental changes that occur in the Health Center, Farmington, Conn. Nonidet P-40 is obtained plasma membrane. However, very little is known about the from Imperial Shell. Sodium tetrathionate is from Pfaltz and plasma membrane of immature erythroid cells (6). This is Bauer, and NaDodSO4 is from BDH. All other chemicals are presumably due to the technical difficulties in obtaining from Sigma or Baker. erythroid cells at early stages of maturation in sufficient Isolation of Plasma Membranes. All operations are carried quantity and homogeneity. Erythropoiesis in the chick embryo, out at 40. The red blood cells are suspended in a hypotonic however, provides an exceptionally suitable system for such buffer [10mM Tris-HCl at pH 7.5, 10mM KCl, 1.5mM MgCl2, developmental studies. The series of primitive chick erythroid with 5 mM Na2S406 present as a proteolytic enzyme inhibitor cells naturally develop as a synchronized cohort in the em- (4)] and then homogenized in a tight-fitting Dounce homoge- bryonic circulation, such that erythroid cells taken from em- nizer. An appropriate volume of 2 M sucrose is added imme- bryos between 2 and 6 days of incubation are at each of the diately to the homogenate to restore isotonicity. The homoge- stages of maturation between basophilic erythroblasts and re- nate is then layered over a sucrose step gradient [3 volumes of ticulocytes, respectively (7). The definitive erythroid cell series, 28% sucrose (wt/vol) over 1 volume of 50% sucrose (wt/vol) in which predominate in the circulation after 7 days of incubation, a buffer containing 5 mM Tris-HCl at pH 7.4, 2.4 mM is relatively more heterogeneous because they are stem-cell Mg(OAc)2, 0.14 M NaCl, and 5 mM Na2S406)] and centrifuged derived. However, because progressively more mature cells are in swinging buckets (Beckman SW 27 rotor) at 117,000 X g for released into the circulation, the majority of the definitive cells 40-45 min. The membrane fraction at the 28% and 50% sucrose are also at a similar state of maturation at any one embryonic interphase is collected, resuspended in 10 ml 0.02 M Tris-HCl age (7). Thus, homogeneous populations of erythroid cells at at pH 7.4 and spun at 15,000 X g for 10 min. The pellet is each stage of maturation between basophilic erythroblasts and washed once more before it is taken up in a buffer containing mature erythrocytes can be easily obtained by harvesting 10mM Tris.HCI at pH 8,1 mM EDTA, and 2% NaDodSO4. The erythroid cells from the circulation of embryos at the appro- protein concentration is determined and then adjusted to 1 priate ages of development. mg/ml in Fairbanks loading buffer (3) before storage at The present paper describes a systematic examination of the -20°. Electron Microscopy. The isolated membranes are fixed Abbreviations: NaDodSO4, sodium dodecyl sulfate; Con A, concana- chemically in 3% glutaraldehyde buffered with 0.1 M sodium valin A. phosphate at pH 7.3, postfixed with 2% osmium tetroxide 1062 Downloaded by guest on September 27, 2021 Cell Biology: Chan Proc. Natl. Acad. Sci. USA 74 (1977) 1063 buffered with 0.1 M S-collidine (pH 7.3), stained intact in a 2% uranyl acetate solution at 60°, dehydrated in a graded series of ethanol dilutions, and embedded in an epoxy resin. Thin sections of the membrane pellets are stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron microscope. Characterization of Membrane Preparation. For protein determinations, the method of Lowry et al. (9) is used. The RNA and DNA contents of the membrane preparations are measured by means of the orcinol and the diphenylamine techniques, respectively (10). The specific activity of cytochrome oxidase in the whole cell homogenate, all fractions of the sucrose step gradient, and the isolated membranes is assayed by the method of Cooperstein and Lazarow (11). The amount of membrane protein per ghost is determined by using '25I-Con A binding as an indirect measure of the number of membrane ghosts per preparation. Packed cells (0.1 ml) are incubated at 4° in 1 ml of Howard Ringer's saline containing a specific amount of '25I-Con A (6 X 105 cpm/ml is routinely used). After 30 min, the cells are washed extensively with ice-cold saline and aliquots are removed to determine cell number as well as the amount of 125I-Con A bound per cell. X. Membranes are then prepared from the remaining cells and the amount of '25I-Con A bound per ,ug of membrane protein is determined. From these values, the amount of membrane protein per ghost is estimated. NaDodSO4/Polyacrylamide Gel Electrophoresis. The FIG. 1. Electron micrographs of a typical plasma membrane isolated membranes are solubilized in buffer containing 2% preparation. This particular sample was prepared from 7-day-old NaDodSO4 and are analyzed on NaDodSO4/polyacrylamide erythroid cells. Magnification X10,790; bar, 1 gsm. Inset magnification slab gels using a modified version of the system of Fairbanks X166,000; bar, 100 A. et al. (3). These slab gels contain an exponential gradient of 4% acrylamide at the top and 10% acrylamide at the bottom and Contamination by membranes from three nonplasma provide good resolution of the membrane polypeptides. The membrane sources are of concern; namely, nuclear membrane, gels are stained with Coomassie brilliant blue and the stained endoplasmic reticulum, and mitochondrial membrane. The protein patterns are scanned with a spectrodensitometer breakage of nuclei during the isolation procedure is minimized (Schoeffel Instruments Corp.) at 550 nm. The relative amounts by the presence of magnesium in the buffers used and by the of proteins per band are estimated by measuring the area under speed of the process. Also, the immediate addition of sucrose each peak. to restore isotonicity to the homogenate helps in keeping the To check the possibility of losing certain protein components nuclei intact. Electron micrographs of typical membrane from the membrane during isolation, we analyzed proteins in samples show virtually no detectable contamination by nuclei, the supernatant fractions from the sucrose