First published online August 4, 2005
Journal of Experimental Biology 208, 3093-3101 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01749
Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tick Boophilus microplus
F. A. Lara1,
U. Lins2,
G. H. Bechara3 and
P. L. Oliveira1,*
1 Departamento de Bioquímica Médica, ICB, Universidade Federal
do Rio de Janeiro, Brazil
2 Departamento de Virologia, IMPPG, Universidade Federal do Rio de Janeiro,
Brazil
3 Departamento de Patologia Veterinária, Universidade Estadual
Paulista UNESP, Campus Jaboticabal, São Paulo, Brazil
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Fig. 1. Uptake of albumin and hemoglobin by tick midgut digest cells. Digest cells
were incubated for 4 h in culture medium containing albumin or hemoglobin
labeled with rhodamine. Incubation conditions were (A,B) 1 µmol
l–1 rhodamine–hemoglobin; (C,D) 1 µmol
l–1 rhodamine–hemoglobin plus 100 µmol
l–1 unlabeled hemoglobin; (E,F) 1 µmol
l–1 rhodamine–albumin; (G,H) 1 µmol
l–1 rhodamine–albumin plus 100 µmol
l–1 unlabeled albumin. (A,C,E,G) Differential interference
contrast (DIC) images; (B,D,F,H) rhodamine fluorescence. Scale bar, 10 µm.
Large hemoglobin-accumulating vesicles (asterisks) and small
albumin-accumulating vesicles (arrows) are indicated in the figure. Hemosomes
are seen as brown-colored structures on DIC panels and are not labeled by any
of the proteins conjugated to rhodamine.
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Fig. 2. Acidification of digestive vesicles. Digest cells were incubated with 100
µmol l–1 of globin for 2 h, and then exposed to 50 µmol
l–1 of Acridine Orange for 1 min, washed and observed by
fluorescence microscopy. Small (arrows) and large (asterisks) acid digestive
vesicles are indicated. Scale bar, 10 µm.
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Fig. 3. Light absorption and fluorescence spectra of Pd-mesoporphyrin IX. Spectra
of Pd-mP (5 µmol l–1) were recorded in water, methanol,
phosphatidylcholine liposomes or bound to 5 µmol l–1
globin. (A) Light absorption spectra; (B) fluorescence emission spectra,
 ex=400 nm.
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Fig. 4. Time course of proteolytic digestion of Pd-mP-globin. Fluorescence emission
of Pd-mP (5 µmol l–1) in water was recorded at 545 nm and
665 nm with excitation at 400 nm. Emission spectra A–D (insets) using
the same excitation wavelength were acquired at the times indicated by black
arrows, after addition of 5 µmol l–1 globin (asterisks)
and 1% bovine trypsin (red arrow).
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Fig. 5. Digestion of Pd-mP-globin in vivo. Partially engorged females were
fed with serum enriched with 100 µmol l–1 Pd-mP-globin for
12 h, as described in Materials and methods. Anterior midguts were fixed and
embedded in methacrylate, and semi-thin sections were observed under a
fluorescence microscope, as described under Materials and methods. (A) Two
digest cells 2 h after feeding; (B) two digest cells 6 h after feeding.
*Digestive vesicles; arrows, hemosomes; LU, intestinal lumen. Scale bar, 30
µm.
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Fig. 6. Time course of degradation of Pd-mP-globin by digest cells in
vitro. Digest cells were incubated in culture medium with 100 µmol
l–1 Pd-mP-globin for (A) 2 h, (B) 4 h. (C–E) Pulse (4 h
incubation with Pd-mP-globin) and chase experiments: (C) 4 h; (D) 8 h; (E) 20
h; (F) 44 h. Yellow arrows, digestive vesicles; green arrows, hemosomes.
Fluorescence of Pd-mP was observed using a Zeiss-09 filter set. Scale bars, 40
µm.
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Fig. 7. Confocal fluoroscopy of Pd-mP emission spectra in digest cells in
vitro. Digest cells were incubated for 4 h with Pd-mP-globin 100 µmol
l–1, and observed 8 h later using laser excitation at 514 nm.
Images were collected from 530 to 700 nm, with ±10 nm intervals between
each consecutive image. (A) Digest cell total emission image. Arrows indicate
areas of the image selected for spectral analysis: (1) cytosol, (2) hemosome
and (3) digest vesicle. Scale bar, 20 µm. (B) Emission spectra obtained
from image analyses. (C) Pd-mP 5 µmol l–1 fluorescence
spectra with ex=514 nm in water, methanol,
phosphatidylcholine liposomes, or bound to 5 µmol l–1
globin.
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Fig. 8. Pd-mP uptake by the hemosomes. Digest cells were incubated for 2 h in
culture medium with 100 µmol l–1 Pd-mP-globin, followed by
6 h of chase. Arrows indicate labeled hemosomes; asterisks, digestive
vesicles. Inset shows labeled hemosomes at higher magnification. Fluorescence
of Pd-mP was observed using a Zeiss-02 filter set. Scale bar, 20 µm (10
µm in inset).
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© The Company of Biologists Ltd 2005
