Two members of the beige/CHS (BEACH) family are involved at different stages in the organization of the endocytic pathway in Dictyostelium
Sophie Cornillon1,
Annick Dubois2,
Franz Brückert3,
Yaya Lefkir2,
Anna Marchetti1,
Mohammed Benghezal1,
Arturo De Lozanne4,
François Letourneur2 and
Pierre Cosson1,*
1 Université de
Genève, Centre
Médical Universitaire,
Département de morphologie, 1 Rue Michel
Servet, CH1211 Genève 4, Switzerland
2 Institut de Biologie et de Chimie des
Protéines, UMR5086-CNRS,
Université Lyon I, 7 Passage du Vercors, 69367
Lyon cedex 07, France
3 Laboratoire de Biochimie et Biophysique des
Systèmes
Intégrés,
UMR314 CNRS, CEA, 38054 Grenoble, France
4 Section of Molecular Cell and Developmental Biology and Institute for Cellular
and Molecular Biology, 241 Patterson Bldg., Mail code C0930, University of
Texas, Austin, TX 78712, USA
View larger version (13K):
[in a new window]
|
Fig. 1. The BEACH family of proteins. (A) A schematic drawing of four proteins of
the BEACH family: Dictyostelium LvsA and LvsB and human LYST and FAN.
The BEACH domains are indicated by dark gray boxes, the putative WD repeats by
vertical black bars. The long N-terminal extension of LYST exhibits no
homology with that of Lvs proteins. (B) Southern blot of the lvsB
mutant. Genomic DNA was digested with EcoRI, migrated in an agarose
gel, blotted and hybridized with an LVSB probe. The 1.5 Kb band in
wild type was replaced by a 3 Kb band in the mutant clone owing to the
integration of the bsr cassette (1.5 Kb).
|
|
View larger version (15K):
[in a new window]
|
Fig. 2. Secretion of lysosomal enzymes in lvsA and lvsB mutant
cells. Cells were incubated in SB medium to induce secretion of lysosomal
enzymes. The activity of three lysosomal enzymes was determined in the
cellular pellet and the supernatant at the indicated times. Enzymes tested
were  -mannosidase (A), acid phosphatase (B) and N-acetylglucosaminidase
(C). Over the course of the experiment, the total amount of the enzymatic
activity did not vary significantly. The percentage of the total activity
secreted in the medium is indicated. Filled circles, wild-type cells; open
circles, lvsA cells; open triangles, lvsB cells.
|
|
View larger version (70K):
[in a new window]
|
Fig. 3. Localization of the vacuolar H+-ATPase in lvsA cells.
Wild-type (A,C) or lvsA mutant cells (B,D) were fixed and labeled by
immunofluorescence with the indicated antibodies. The top panel in (A)
represents labeling in a plane close to the substrate, whereas in other panels
pictures correspond to a plane towards the middle of the cell body. The
circles indicate the contractile vacuole (Rh50+,
HATPase+, p80-), the arrows the early endocytic
compartment (Rh50-, HATPase+, p80low) and the
arrowheads the late endocytic compartment (Rh50-,
HATPase-, p80high). Scale bar, 10 µm.
|
|
View larger version (95K):
[in a new window]
|
Fig. 4. Localization of vacuolar H+-ATPase in phagosomes. Wild-type (A)
and lvsA mutant cells (B) were allowed to phagocytose
rhodamine-labeled yeast particles for 15 minutes. Cells were then fixed and
the vacuolar H+-ATPase revealed by immunofluorescence. More
vacuolar H+-ATPase was seen in phagosomes in lvsA mutant
cells (B) than in wild-type cells (A). For electron microscopy, wild-type (C)
and mutant lvsA cells (D) were allowed to phagocytose
Klebsiella bacteria (K.a.) for 1 hour. Cells were then fixed
and processed for immunoelectron microscopy using an antibody to vacuolar
H+-ATPase and a secondary antibody coupled to 10 nm gold particles.
Gold particles are indicated by arrows. The early phagosomes contain more
vacuolar H+-ATPase in lvsA mutant cells than in wild-type
cells (Table 1). A small number
of late phagosomes (spacious and containing several bacteria) were also seen
and were not considered in this study. Scale bar, 1 µm.
|
|
View larger version (24K):
[in a new window]
|
Fig. 5. Internalization of particles and fluid phase in lvsA mutant cells.
Wild-type (filled circles) or lvsA mutant cells (open circles) were
incubated for various times in the presence of fluorescent latex beads (A),
rhodamine-labeled Klebsiella aerogenes (B) or FITC-dextran (C). The
amount of internalized fluorescence was analyzed using a fluorescent-activated
cell sorter (FACS). The results are expressed as a percentage of the
internalization by wild-type cells after 90 minutes. Insert in (A), to check
for fluorescent latex beads internalization at early time-points, cells were
incubated with beads for various times up to 20 minutes, then fixed and
analyzed by FACS. (D) To measure recycling of internalized fluid phase to the
extracellular medium, cells were allowed to internalize FITC-dextran for 1
hour, then washed and incubated for the indicated times. The fluorescence
remaining in the cells was then analyzed by FACS.
|
|
View larger version (78K):
[in a new window]
|
Fig. 6. Formation of macropinosomes is normal in lvsA mutant cells.
Wild-type (A) and lvsA mutant cells (B) grown in HL5 medium were
observed in phase contrast with a Zeiss Axiovert 100 microscope. Pictures were
recorded every 10 seconds. In both series of pictures the cells can be seen to
extend pseudopods and form macropinosomes with similar morphologies and
kinetics.
|
|
View larger version (21K):
[in a new window]
|
Fig. 7. Phagocytosis of different particles by lvsA and lvsB
mutant cells. Cells were incubated for 1 hour with the indicated particles.
The amount of internalized fluorescence was determined by FACS. The results
are expressed as a percentage of the internalization by wild-type cells in the
same experiment. The indicated values are the mean and standard deviation of
seven independent experiments. Beads, latex beads, K.a., Klebsiella
aerogenes, E. coli, Escherichia coli, P.a., Pneumophila aeruginosa, S.b.,
Streptococcus bovis.
|
|
View larger version (53K):
[in a new window]
|
Fig. 8. Formation of the phagocytic cup in lvsA mutant cells. Wild-type
(A) or lvsA mutant cells (B) expressing CRAC-GFP fusion protein were
incubated with rhodamine-labeled yeast particles for 1 hour. Cells were then
fixed and observed with a confocal fluorescence microscope. CRAC-GFP
accumulates on the cytosolic face of the phagocytic cup but is not observed on
mature phagosomes.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2002
