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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02439

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Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells

¹ Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
² Medical Clinic and Polyclinic I, Dresden University of Technology, Fetscherstrasse 74, 01307 Dresden, Germany

View larger version (89K): [in a new window]   Fig. 1. Occurrence of prominin-1-containing particles in the ventricular fluid of the embryonic mouse brain. Double immunofluorescence of a cryosection (20 µm) of an E11.5 mouse embryo using mAb 13A4 (b,d,f, red) and antiserum E3 (a,c,f, green) directed against distinct extracellular epitopes of prominin-1. Single 5-µm optical sections of the midbrain were obtained by confocal microscopy in the middle of the cryosection (a,b) and at the level of the glass slide (c,d,f), as outlined in e. (f) Merged images of the area indicated by the white squares in c and d; note the co-localisation of the 13A4 and E3 immunoreactivities. Inset in f is a higher magnification of the particle indicated by the arrowhead in f. Bars, 10 µm.

View larger version (129K): [in a new window]   Fig. 2. Isolation of large and small prominin-1-containing membrane particles from the embryonic ventricular fluid. (a) Ventricular fluid of E10.5-E11 mouse embryos was subjected to differential centrifugation for 5 minutes at 300 g (P1), 20 minutes at 1,200 g (P2), 30 minutes at 10,000 g (P3) and 1 hour at 110,000 g (P4). The entire P1-P4 pellets were analysed by immunoblotting for prominin-1. (b,c) Immunofluorescence of prominin-1 of P2 and P4 particles obtained by differential centrifugation of E13 ventricular fluid for 15 minutes at 10,000 g (b) and 1 hour at 200,000 g (c), respectively. The inset in b shows a P2 particle at higher magnification, which is very similar to the one shown in the inset of Fig. 1f. (d-f) Negative staining EM of prominin-1 immunogold labelled P4 (d) and P2 (e,f) particles obtained by differential centrifugation of E11.5 ventricular fluid as in a.

View larger version (24K): [in a new window]   Fig. 3. Time course of appearance of P2 and P4 prominin-1-containing membrane particles in the ventricular fluid during embryonic mouse brain development. (a) Ventricular fluid of E10.5-E13.5 mouse embryos was subjected to differential centrifugation as in Fig. 2a followed by immunoblotting of the pellets for prominin-1. Total prominin-1 immunoreactivity (sum of P1-P4 pellets, triangles) and prominin-1 immunoreactivity in the P2 pellet (squares) and the P4 pellet (circles) at the various developmental stages was determined. Data are the mean of three independent experiments; bars indicate s.d. For any given experiment, the same volume of ventricular fluid was analysed at the various developmental stages. (b) Cryosections of E7-E12.5 mouse embryos were subjected to immunofluorescence for prominin-1. The number of the ring-like prominin-1-containing particles (see inset in Fig. 1f) in the ventricular fluid was quantified per unit area in single optical sections at the level of the glass slide. E7, amniotic fluid near the neural plate (19x104 µm2); E9.5-E12.5, mean of forebrain and hindbrain ventricular fluids, bars indicate the variation of the forebrain/hindbrain values from the mean (E9.5, 66x104 µm2; E10.5, 139x104 µm2; E12.5, 134x104 µm2).

View larger version (164K): [in a new window]   Fig. 4. Prominin-1-containing protrusions on the apical plasma membrane of neuroepithelial cells at various stages of embryonic mouse brain development. (a-d) Prominin-1 immunofluorescence at the apical surface of neuroepithelial cells at E8.5 (a,c; future hindbrain) and E11.5 (b,d; dorsal telencephalon), viewed from the lateral (a,b) and lumenal (c,d) side of the neuroepithelium. Scale bar in c: 10 µm. (e-n) Prominin-1 immunogold EM of the apical plasma membrane of neuroepithelial cells at E8.5 (e-g) and E11.5 (h-n) (h,i,k,l,n: midbrain; j,m: forebrain). Scale bars: 300 nm. Some of the gold particles (12 nm) are indicated by arrowheads and junctional complexes by asterisks. (e,f) Microvilli. (g-k) Large pleiomorphic protuberances; note the thin stalks (g,j), the vicinity of the junctional complexes (j,k), the abundant prominin-1 immunoreactivity (h), and the small prominin-1-labelled membrane buds (arrows) in (k); (i) shows the prominin-1-labelled cup-shape surface structure of (n) at higher magnification. (l) Short, thin protrusions; note the concentration of prominin-1 immunoreactivity at the tips (arrows). (m,n) Overviews of the apical plasma membrane of a single (m) and several (n) neuroepithelial cells; note the prominin-1-labelled protuberance in m and the cilium (arrow, note the basal body), but lack of microvilli, in n. (o,p) Scanning EM of the apical plasma membrane of E11.5 neuroepithelial cells in the forebrain. The boxed region in o indicates the area shown at higher magnification in p. Open arrow, cilium; double-headed arrow, cauliflower-like protuberance; triple-headed arrow, cup-shape protuberance. Scale bar: 5 µm (o); 1 µm (p). (q-t) Floorplate of the E11.5 spinal cord. (q) Prominin-1 immunofluorescence at relatively low exposure to show the more intense immunoreactivity of the floorplate cells (FP). Scale bar: 10 µm. (r) Overview of prominin-1 immunogold EM; note the microvilli of the floorplate cells (FP) and the pleiomorphic protuberances but lack of microvilli on the neuroepithelial cells adjacent to the floor plate (double-asterisks); the bracket indicates the area shown at higher magnification in s. Scale bar: 300 nm. (s) Prominin-1-labelled microvilli of the floorplate (12-nm gold). Scale bar: 300 nm. (t) Scanning EM of the apical plasma membrane. Note the bending of the lumenal surface, resulting in a lateral view of the microvilli-bearing floorplate cells and a lumenal view of the adjacent neuroepithelial cells. The black triangle and white bar at the left margin indicate the transition from floorplate to adjacent neuroepithelial cells and an apparent boundary between neuroepithelial cells with abundant vs. sparse protrusions, respectively. Bar, 10 µm.

View larger version (43K): [in a new window]   Fig. 5. Occurrence of prominin-1-containing membrane particles in various human body fluids. Various human body fluids were subjected to differential centrifugation for 5 minutes at 300 g (300 g), 30 minutes at 10,000 g (10,000 g), 1 hour at 200,000 g (200,000 g) and 1 hour at 400,000 g (400,000 g). The resulting pellets were analysed by immunoblotting for prominin-1.

View larger version (159K): [in a new window]   Fig. 6. The P4 prominin-1-containing membrane particles are distinct from exosomes carrying the tetraspanin CD63. (a) 24-hour-conditioned medium of Caco-2 cells grown for 10 days post-confluency was subjected to differential centrifugation as in Fig. 2a, and the resulting pellets were analysed by immunoblotting for prominin-1. (b) 24-hours-conditioned medium of Caco-2 cells grown for 14 days post-confluency was centrifuged for 30 minutes at 10,000 g, and the resulting supernatant, containing the P4 particles, was concentrated and subjected to equilibrium sucrose gradient centrifugation. Aliquots of the fractions (1.5%) and the pellet (50%) were analysed by immunoblotting for prominin-1. (c,d) Negative staining and prominin-1 immunogold (12 nm) labelling EM of the P4 particles present in fraction 7 of the sucrose gradient. Note the presence of both prominin-1-positive (arrows, indicated only in c) and prominin-1-negative (open arrows) small vesicles. (e,f) Negative staining EM of prominin-1 (6-nm gold) and CD63 (12-nm gold) double-immunogold labelled particles present in fraction 7 of the sucrose gradient. Note that the small vesicles showing prominin-1 immunoreactivity are distinct from those showing CD63 immunoreactivity.

View larger version (48K): [in a new window]   Fig. 7. Appearance of prominin-1-containing membrane particles in the culture medium upon differentiation of Caco-2 cells. Caco-2 cells were grown for up to 21 days post-confluency. Cells received fresh medium 24 hours before analysis. At the indicated time points, cells and the P4 pellet obtained from the conditioned medium were analysed by immunoblotting. (a) Immunoblots. Top, prominin-1 in the P4 pellet of the medium; middle, prominin-1 in the cells; bottom, -tubulin in the cells. (b,c) Quantitation of immunoblots for day 1 and day 8 post-confluency. (b) Prominin-1 immunoreactivity in the P4 pellet of the medium is expressed as percentage of total (sum of cells plus P4). Data are the mean of three independent experiments; bars indicate s.d. (c) Prominin-1 immunoreactivity in the cells was normalized to that of -tubulin. Normalized prominin-1 immunoreactivity in the cells at day 8 post-confluency is expressed relative to that at day 1, which was arbitrarily set to 100%. Data are the mean of two independent experiments; bars indicate the variation of the individual values from the mean.

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