vacuolar atpase in phagosome-lysosome fusion · abservice). experimental animals - mice with...

v-ATPase in membrane fusion

1

Vacuolar ATPase in Phagosome-Lysosome Fusion

Sandra Kissing* 1, Christina Hermsen*

2, Urska Repnik

3, Cecilie Kåsi Nesset

3, Kristine von

Bargen2, Gareth Griffiths

3, Atsuhiro Ichihara

4, Beth S. Lee

5, Michael Schwake

6, Jef De

Brabander7, Albert Haas

2§ and Paul Saftig

1§

1 Institute of Biochemistry, Christian-Albrechts-University of Kiel, Kiel, Germany.

2 Institute for Cell Biology, Friedrich-Wilhelms University, Bonn, Germany

3 Department of Biosciences, University of Oslo, Oslo, Norway

4 Department of Medicine II, Tokyo Women´s Medical University, Tokyo, Japan

5 Department of Physiology and Cell Biology, The Ohio State University College of Medicine,

Columbus, USA

6 Department of Chemistry, Biochemistry III, University of Bielefeld, Bielefeld, Germany

7 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas TX, USA

Running title: v-ATPase in membrane fusion

§ To whom correspondence should be addressed: [email protected]; albert.haas@uni-

bonn.de

* These authors equally contributed to this work

Keywords: vacuolar ATPase; phagocytosis; lysosome; membrane fusion; ATP6AP2; phagosome

maturation; lysosomal acidification

Background: The vacuolar H+-ATPase complex

is thought to contribute to membrane fusion.

Results: v-ATPase complex knockout

experiments in mice revealed that its absence

does not affect phagosome-lysosome fusion.

Conclusion: Participation of v-ATPase in

phagosome-lysosome fusion is unlikely.

Significance: Fusion between lysosomes/late

endosomes and phagosomes is not controlled by

the v-ATPase.

ABSTRACT

The vacuolar H+-ATPase (v-ATPase)

complex is instrumental to establish and

maintain acidification of some cellular

compartments, thereby ensuring their

functionality. Recently, it has been proposed

that the transmembrane V0

sector of v-

ATPase and its a-subunits promote

membrane fusion in the endocytic and

exocytic pathways independent of their

acidification functions. Here, we tested if such

proton-pumping independent role of v-

ATPase also applies to phagosome-lysosome

fusion. Surprisingly, endo(lyso)somes in

mouse embryonic fibroblasts (MEF) lacking

the V0 a3 subunit of the v-ATPase acidified

normally and endosome and lysosome marker

proteins were recruited to phagosomes with

similar kinetics in the presence or absence of

the a3 subunit. Further experiments used

macrophages with a knockdown of v-ATPase

accessory protein 2 (ATP6AP2) expression,

resulting in a strongly reduced level of the V0

sector of the v-ATPase. However, acidification

appeared undisturbed and fusion between

latex bead-containing phagosomes and

lysosomes, as analyzed by electron

microscopy, was even slightly enhanced, as

was killing of non-pathogenic bacteria by V0

http://www.jbc.org/cgi/doi/10.1074/jbc.M114.628891The latest version is at JBC Papers in Press. Published on April 22, 2015 as Manuscript M114.628891

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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mutant macrophages. Pharmacologically

neutralized lysosome pH did not affect

maturation of phagosomes in MEF cells or

macrophages. Finally, locking the two large

parts of the v-ATPase complex together by

the drug saliphenylhalamide A did not inhibit

in vitro- and in cellulo- fusion of phagosomes

with lysosomes. Hence, our data do not

suggest a fusion-promoting role of the v-

ATPase in the formation of phagolysosomes.

INTRODUCTION

A phagocytic compartment (phagosome)

is formed when a particle is ingested through

receptor-ligand interaction into a plasma

membrane invagination. Newly formed

phagosomes are not static compartments but

rather acquire degradative and microbicidal

properties through a complex series of

interactions with endomembranes. This process,

collectively termed phagosome maturation,

culminates in the fusion of phagosomes with

lysosomes, yielding a strongly acidic (pH 4.5-

5.0) hybrid organelle enriched in hydrolytic

enzymes and antimicrobial peptides, which

promote killing and degradation of internalized

microorganisms (1,2). The main role of

phagocytosis is the delivery of microbial

invaders and apoptotic bodies to

phagolysosomes. For most microbes, the acidic,

hydrolytically competent environment of the

eventually formed phagolysosome is sufficient to

kill them. The strong acidification of (phago-)

lysosomes is a hallmark feature of the endocytic

and phagocytic pathways and is generated by a

large multiprotein complex, the vacuolar ATPase

(v-ATPase) (3,4). Acidification is required for

trafficking of endosomes (5) and for killing of

ingested microorganisms (6). The precise

subcellular origin of all vesicles which can

deliver v-ATPases to phagosomes is, however,

not clear. The v-ATPase complex is formed by at

least 14 subunits which are organized in two

large sub-particles; of these the V0 sector

contains trans-membrane proteins that form the

proton channel while the V1 sector is cytosolic

and is responsible for the ATPase activity (7).

How exactly the subunits are assembled within

the endoplasmic reticulum and delivered to

lysosomes is not understood. However, it has

been shown that ‘accessory subunits’, such as the

v-ATPase accessory protein 2 (ATP6AP2, (8)),

are important and that a failure to properly

assemble the V0 portion leads to its degradation.

Hence, it is not surprising, that mutated v-

ATPase subunits cause many severe illnesses

such as the progression of cancer and bone

disorder (9,10).

In the past few years, evidence has

accumulated that the transmembrane V0 part of

the v-ATPase, without participation of the V1

sector, can play active and critical roles in

membrane fusion along the endocytic and

exocytic pathways, independent of its proton-

translocating activities. Such evidence stems

from experiments on synaptic vesicle exocytosis

in Drosophila (13), secretion in Caenorhabditis

(14), osteoclast fusion (15) and vesicle fusion in

zebrafish microglia cells after ingestion of

neuron-derived apoptotic bodies (16). The

original observation of an acidification-

independent role in membrane fusion arose from

studies on yeast homotypic vacuole fusion

(11,12). However, a recent study using the same

model organelle proposed a model in which the

major role of the v-ATPase complex would be

for vacuolar acidification (17).

To address the question in how far

fusion events depend on the presence of the v-

ATPase in mammalian cells, we made use of

phagosome-lysosome fusion, as lysosomes are

highly acidic, contain large quantities of v-

ATPase and fuse with endocytic vesicles. As

tools we used the Tcirg1oc/oc

mouse line and

conditionally deleted Atp6ap2 knockout mice.

Cells derived from these mice either specifically

lacked the v-ATPase subunit a3 or showed an

almost complete absence of the v-ATPase V0

sector, yet allowed phagosome-lysosome as well

as endosome-lysosome fusion to progress. Also

locking the V0 and V1 sectors of v-ATPase

together did not affect phagosome maturation

and neither did pharmacologic alkalization of

lysosome pH.

EXPERIMENTAL PROCEDURES

Antibodies - The following antibodies

were used for protein detection in western blot

and immunofluorescence analyses: anti-

ATP6AP2 N-terminal (HPA003156, Sigma-

Aldrich), anti-β-actin (A2066, Sigma-Aldrich),

anti-EEA1 (#C45B10, Cell Signaling

Technology or #AV30074, Sigma-Aldrich), anti-

cathepsin D (clone sII-10, kind gift from Dr. S.

Höning, University Cologne, Germany), anti-

cathepsin L (#AF1515, R&D Systems), anti-

glyceraldehyde-3-phosphate-dehygrogenase

(GAPDH, clone FL335, #sc-25778, Santa Cruz

Biotechnology), anti-LAMP1 (1D4B, DSHB),

anti-LAMP-2 (Abl93, DSHB), anti-myc (clone


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9B11, #2276, Cell Signaling Technology), anti-

transferrin receptor (TIB-219, ATCC), anti-V0 a1

(18), anti-V0 a2 (#ab96803, Abcam), anti-V0 a3

(generous gift from Dr. T. Jentsch, FMP Berlin,

Germany), anti-V0 d1 (#18274-1-AP, Proteintech

group), anti-V1 A (kindly supplied by Dr. Shoji

Ohkuma, Kanazawa University, Japan (19); anti-

V1 E1 (20), rabbit polyclonal antibody to bovine

B-subunit of vacuolar ATPase (Yoshinori

Moriyama, Okayama University, Japan) and

anti-V1 B2 (clone D2F9R, #14617, Cell

Signaling Technology). Secondary antibodies

conjugated to horseradish peroxidase (HPO),

AlexaFluor488, AlexaFluor594 or

AlexaFluor647 were purchased from Dianova

and Life Technologies. An anti-V0 c antibody

was raised in rabbit against a synthetic peptide

corresponding to residues 26-44 of the murine

protein (CSAMGAAYGTAKSGTGIAAM) and

purified by affinity chromatography against the

immobilized peptide (Pineda abservice). To

generate an anti-ATP6AP2 C-terminal antibody,

rabbits were immunized with a synthetic peptide

corresponding to residues 332-345 of the murine

protein (DPGYDSIIYRMTNQ, Pineda

abservice).

Experimental animals - Mice with loxP-

sites flanking exon 2 of the Atp6ap2-gene have

been described previously (21). Atp6ap2Flox/Flox

female mice were bred with male mice,

expressing the Cre recombinase under the

control of an inducible Mx1 promotor (22) or the

LysM promotor (23) to yield Atp6ap2Flox/Y

Cretg/+

mice. Further breeding with homozygous

Atp6ap2Flox/Flox

female mice resulted in

Atp6ap2Flox/Flox

/Cretg/+

or Atp6ap2Flox/Y

/Cretg/+

animals (cKO). Littermates negative for Mx1-

Cre or LysM-Cre, respectively, served as control

(wild-type).

Cre-expression was induced in 6-weeks-

old Mx1-Cre transgenic and control mice by i.p.

injection of 3 doses of 250 μg polyinosinic–

polycytidylic acid (pI-pC, Sigma-Aldrich) within

5 days. Mice were kept for further 10 days and

sacrificed for experimental analysis. When

required i.p. injection of 0.5 ml 4 % (w/v)

Brewer’s thioglycolate solution (Difco/BD

Biosciences) was performed to enrich peritoneal

macrophages and cells harvested by peritoneal

lavage 3 days later.

We are grateful to Dr. Uwe Kornak,

Charité Berlin, Germany, for providing mice

carrying the osteosclerotic mutation (oc/oc) in

the Tcirg1 locus (24). All animal experiments

were conducted in agreement with local

guidelines for the use of animals and their care.

Cell lines and primary cell culture - All

cell types were grown in Dulbecco’s Modified

Eagle’s Medium (DMEM) with 4 mM L-

glutamine and 4.5 g/l glucose (PAA Laboratories

or Sigma-Aldrich), supplemented with 10 %

(v/v) fetal bovine serum (FBS, PAA

Laboratories or Biochrom AG). For maintenance

of primary cells, 100 U/ml penicillin and

100 µg/ml streptomycin (pen/strep, PAA

Laboratories or Sigma-Aldrich) were added to

the growth media. Cultures were grown at 37 °C

in a humidified 5 % CO2 atmosphere conditions,

unless stated otherwise.

Murine embryonic fibroblast were

generated from 13 – 14 day old embryos of

breeding pairs yielding Tcirg1oc/oc

(a3-deficient)

and Tcirg1+/+

(wild-type) MEFs. Embryos were

decapitated and inner organs removed before

single cells were obtained by trypsin digest. All

MEF lines were immortalized after 3 – 4

passages by transfection with the SV40 Large T

antigen. Where indicated, MEFs were further

cotransfected with FcγRII (kind gift of Dr.

Sergio Grinstein) and pcDNA4/TO (Addgene)

and selected for stable protein expression in the

presence of 250 µg/ml Zeocin (InvivoGen).

MEFs deficient for cathepsin D (CtsD-/-

) and

cathepsin L (CtsL-/-

) have been described before

(25,26).

Murine primary peritoneal macrophages

were collected by peritoneal lavage with 8 ml

ice-cold phosphate-buffered saline (PBS),

centrifuged at 210 x g for 10 min at 4 °C and

resuspended in DMEM with FBS and pen/strep

for plating. Non-adherent cells were removed

after 3 h incubation at 37 °C and 5 % CO2 and

experiments conducted the following day.

To obtain bone marrow-derived

macrophages (BMDMs), long bones (tibia,

femur, scapula) from Atp6ap2 cKO and control

animals were dissected and the bone marrow

flushed through an 100 µm nylon cell strainer

(BD) with PBS. BMDMs were cultured in

DMEM containing 10 mM HEPES, 10 mM L-

Glutamin, 1 mM Na-Pyruvate, pen/strep, 1 %

GlutaMAX, 10 % (v/v) FCS, 5 % horse serum,

and 30 % spent L929 culture supernatant at

9.5 % CO2 and cells were used in experimental

setups after 7-10 days of differentiation.

The murine macrophage-like cell lines

RAW264.7 (TIB-71) was from ATCC, the

murine macrophage-like cell line J774E (27) was


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kindly donated by Philip Stahl (Washington

University, St. Louis, USA).

RNA extraction, reverse transcription

and quantitative real-time PCR (qRT-PCR) -

Total RNA was extracted using the NucleoSpin

RNA II kit (Macherey-Nagel) and 0.2 – 1 µg

RNA spent for reverse transcription using the

RevertAid RT Kit and random hexamer primers

(Thermo Fisher Scientific). Specific assays for

qRT-PCR were created with the Universal Probe

Library Assay Design Center and PCR

performed in a Lightcycler 480 II (Roche).

Relative mRNA expression was calculated by

normalizing Cp values to the logarithmic average

Cp of the most stable house keeping genes

(Tuba1a, Hprt1 and Sdha). Resulting DCp values

were compared between genotypes for statistical

analyses. Primer efficiency (E) was determined

for each PCR by co-measurement of a set of

serial cDNA dilutions to obtain E(-DCp)

plots

describing relative mRNA expression levels.

Western blotting - Total cell lysates were

generated by adding PBS containing 1 % (v/v)

Triton-X-100 and 1x cOmplete Protease

Inhibitor Cocktail (Roche) to cells on ice for

20 min, followed by sonication for 2 x 10 s using

a Branson Sonifier 450 (level 7 in a cup horn,

Emerson Industrial Automation) and

centrifugation at 16,000 x g for 10 min at 4 °C.

Protein concentrations of the resulting

supernatants were measured with the Pierce

BCA protein assay kit (Thermo Fisher Scientific)

and samples adjusted to 2 µg protein/ml. Twenty

– 40 µg protein were subjected to SDS-PAGE

and analyzed by immunoblotting. Lysosomes

were purified and immunoblotted as described

(28) and used at 10 µg protein per lane.

Subcellular fractionation – Confluent

RAW264.7 macrophage cultures were incubated

for 2 h in the presence of 10 µM

saliphenylhalamide A (SaliPhe) or DMSO, and

lysed in a dounce homogenizer with 15 strokes

in HB (8.6 % (w/v) sucrose, 20 mM

HEPES/KOH pH 7.2, 0.5 mM EGTA)

containing 1x cOmplete Protease Inhibitor

Cocktail (Roche). Total lysates (fraction T) were

separated into nuclei (fraction P1) and post

nuclear supernatant (PNS, fraction S1) by a 15

min centrifugation step at 960 x g. For further

fractionation, PNS were centrifuged at 128,000 x

g for 60 min to yield fractions enriched in

cytosolic (fraction S2) and membrane-bound

(fraction P2) proteins. Pellets and supernatants

were handled in equal volumina. Samples of 10

µl were taken from each fraction and subjected

to western blotting.

Co-immunoprecipitation - For co-

immunoprecipitation analyses, RAW264.7 cells

were grown to almost complete confluence.

Where indicated, cells were pre-incubated for 2 h

with 10 µM SaliPhe or DMSO as carrier control

and treatment continued throughout the whole

immunoprecipitation protocol. Cell lysates were

generated using lysis buffer (40 mM HEPES pH

7.4, 12.5 mM EDTA, 2.5 mM MgCl2, 10 mM

β-glycerophosphate, 10 mM NaF, 0.3 % (w/v)

CHAPS, 1x cOmplete Protease Inhibitor

Cocktail (Roche)) and centrifuged at 16,000 x g

and 4 °C for 10 min. Dynabeads Protein G (Life

Technologies) were pre-incubated with rotation

in 5 % (w/v) BSA in lysis buffer for 2 h at 4 °C

to block unspecific binding. Three mg of soluble

protein were pre-cleared of proteins with affinity

to the bead matrix material by a 2 h incubation

with 25 µl Dynabeads Protein G at 4 °C, beads

removed and the lysates incubated in presence of

primary antibody overnight at 4 °C with rotation.

50 µl of fresh, blocked Dynabeads were added to

the lysate-antibody mix for 30 min at room

temperature to capture immune complexes.

Immunoprecipitated proteins bound to beads

were then rinsed 3x in lysis buffer containing

150 mM NaCl. 50 µl SDS-sample buffer was

added per sample and samples were heated for

5 min at 95 °C. Ten µl of each denatured

immunoprecipitate were separated using 4 – 12%

NuPAGE Novex Bis- Tris Protein Gels (Life

Technologies) and immunoblotted. For

detection of v-ATPase V1 subunit B2, Clean Blot

IP Detection Reagent (Life Technologies) was

used instead of regular anti-rabbit-HPO to avoid

interference of denatured IP antibody fragments.

Immunofluorescence analysis - Semi-

confluent cultures of cells grown on coverslips

were fixed in 4 % (w/v) paraformaldehyde

solution for 20 min at room temperature and

permeabilized with 0.2 % (w/v) saponin in PBS.

Unspecific antibody binding was blocked by

preincubating the fixed and permeabilized

samples with 10 % (v/v) FBS in 0.2 % (w/v)

saponin in PBS (blocking solution). Antibody-

staining was performed in blocking solution

overnight at 4 °C (primary antibody) and for 1 h

at room temperature (secondary antibody) in a

humidified chamber. Coverslips were embedded

in 17 % (w/v) Mowiol 4–88 (Calbiochem), 33 %

(v/v) glycerol, 20 mg/ml DABCO (1,4-diaza-

bicyclo-[2,2,2]-octane, Sigma-Aldrich) and

5 µg/ml DAPI (4-,6-diamidino-2-phenylindole,


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Sigma-Aldrich) in PBS. An Olympus FV1000

confocal laser scanning microscope was used for

image acquisition.

Visualization of the endocytic pathway

was achieved with dextran-Texas Red (MW

70,000, Life Technologies). Cells grown on

coverslips were incubated with 0.5 mg/ml

dextran-Texas Red in DMEM containing

1 mg/ml bovine serum albumin (BSA) for

30 min. After rinsing 3 times with PBS and

incubation in DMEM + 1 mg/ml BSA for further

3 h, samples were processed for

immunofluorescence analysis as described

above. In a similar approach, the self-quenched

fluorochrome DQ-BSA Red (Life Technologies)

was used to assess proteolytic activities.

Therefore, cells were cultured overnight in the

presence of 100 µg/ml DQ-BSA Red in DMEM

containing 1 mg/ml BSA, rinsed 3 times with

PBS and processed for fluorescence labeling.

Analysis of lysosome pH - Acidic cellular

compartments were detected using the

acidotropic dye LysoTracker Red DND-99 (Life

Techologies). Cells were incubated with 333 nM

LysoTracker Red in DMEM only for 20 min at

37 °C, fixed in 4 % (w/v) paraformaldehyde

solution and embedded or directly analyzed by

live cell fluorescence microscopy.

Ratiometric measurement of lysosome

pH was performed as described previously

(29,30). Briefly, endocytic compartments of the

analyzed cells were loaded with dextran-Oregon

Green 514 (0.5 mg/ml, 70,000 MW, Life

Technologies) overnight. To enable pH analysis

of late endosomes and lysosomes only, early

compartments were cleared of label by a 2 h

chase in the absence of the dextran derivate.

Addition of 200 nM bafilomycin A1 or 10 µg/ml

nigericin for 15 min at the end of this chase

period was used to collapse the lysosomal pH

gradient. Ratiometric imaging was accomplished

by exciting samples alternately at 440 nm or

488 nm. For an in situ calibration cells were

sequentially incubated with K+-rich buffer

solutions (145 mM KCl, 10 mM glucose, 1 mM

MgCl2, and 20 mM of either MES or acetate, pH

4.0 to 6.5) including 10 µg/ml of the K+-

ionophore nigericin at the end of each

experiment. Lysosome pH was then interpolated

from the generated calibration curve fitted to the

Boltzmann equation.

In cellulo latex bead phagocytosis assays

Immunofluorescence analysis - Latex beads (1.1

µm diameter, Sigma-Aldrich) were opsonized

with IgG (human or murine, Sigma-Aldrich)

overnight at 4 °C to allow uptake by hFcγRII-

expressing MEFs or murine macrophages. Cells

were grown to semi-confluence on coverslips

and the media replaced by serum-free DMEM at

the point of latex bead administration. To

synchronize uptake, beads were briefly spun

down onto the cells at 300 x g for 1 min and

bead uptake allowed for 15 min at 37 °C, 5 %

CO2. Latex bead excess was washed away with

PBS and fresh DMEM was added. Beads that

have not been taken up were stained with anti-

IgG antibodies coupled to AlexaFluor594 for 1

min and the samples processed for

immunofluorescence staining of the marker

proteins EEA1 or LAMP-2, respectively. In case

of primary murine macrophages, co-staining

with an anti-V0 a3 antibody was included to

assess v-ATPase knockdown efficiency. Where

indicated, 200 nM bafilomycin A1 or 10 µg/ml

nigericin were used to alkalinize lysosomes

during the 15 min incubation with latex beads

and during the following chase periods.

A second approach to follow fusion

between latex bead-containing phagosomes

(LBPs) and lysosomes in cells was done in

RAW264.7 cells. Briefly, endocytic

compartments of RAW264.7 macrophages were

loaded with 50 µg/ml BSA-rhodamine overnight.

Cells were rinsed with PBS and incubated in the

absence of BSA-rhodamine for 2 h to ensure

labeling of late endosomes and lysosomes only.

Treatment with 10 µM SaliPhe or DMSO,

respectively, was started concurrently with the

2 h chase period and continued until cell lysis.

Latex beads (1 µm diameter, Polysciences)

opsonized with murine IgG Fc-fragments

(Thermo Fisher Scientific) were then added in

DMEM for 10 min. After washing away the

excessive latex beads, RAW264.7 macrophages

were incubated for further 0 – 80 min. Cells were

homogenized and LBPs purified similar as in

(28) but placing the density gradient in a 2 ml

minifuge tube which is centrifuged for 30 min

and 1,250 x g at 4 °C in a swing out table top

centrifuge rotor. Colocalization between BSA-

rhodamine and latex beads was assessed after

fixation and mounting on glass slides using an

Axioplan microscope (Zeiss).

Electron microscopic analysis - 1x 106

bone marrow-derived macrophages were labeled

with ferrous nanoparticles (10 nm) for 30 min

followed by 3 h of incubation in fresh media to

ensure labeling of lysosomes only. 1 µm murine

IgG-Fc-fragment-coated latex beads were added

for 10 min (MOI = 13) followed by three times


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rinsing of the cells to remove non-ingested latex

beads. Cells were incubated for 10 min or 120

min at 37 °C in fresh media to allow phagosome

maturation and then fixed in 2 % (v/v)

glutardialdehyde in 200 mM HEPES buffer. For

epoxy resin embedding, cell pellets were washed

with water, fixed with 2 % (w/v) OsO4 (Electron

Microscopy Sciences), containing 1.5 % (w/v)

potassium ferricyanide, and block stained with

1.5 % (w/v) uranyl acetate (Fluka) for 30 min.

The cells were then dehydrated using a graded

ethanol series and embedded in epoxy resin

(Sigma Aldrich). 70 nm thin sections were cut on

a Leica ultramicrotome Ultracut EM UCT (Leica

Microsystems) using a diamond knife (Diatome)

and stained with 0.2 % (w/v) lead citrate (Taab)

in 0.1 N NaOH for 10 s. Sections were analyzed

with a CM100 transmission electron microscope

(FEI). The images were recorded digitally with a

Quemesa TEM CCD camera (Olympus Soft

Imaging Solutions) and iTEM software v 5.1

(Olympus Soft Imaging Solutions).

The extent of the phagosome-lysosome

fusion was categorized into arbitrarily defined

classes of small, medium and large volume

transfer. Membrane deposits which contained

less than 20 ferrofluid particles within a small

and usually a pointed membrane profile

protruding from the phagosome membrane were

defined as ‘small’. ‘Medium’ deposits were

defined as containing more than 20 ferrofluid

particles and an area of protrusion less than one

third the proportion of the latex bead. ‘Large’

deposits of ferrofluid contained more than 20

ferrofluid particles and the protrusions exceeded

one third of the latex bead area. The surface

fraction of the phagosome membrane over small

deposits of ferrofluid was analyzed from profiles

of at least 20 phagosomes on isotropic sections

per sample. Intersections of the phagosome

membrane profile with the horizontal and

vertical lines of a lined test grid were counted

and fractions were calculated from the total

counts per sample.

Analysis of in vitro fusion between latex

bead-containing phagosomes and lysosomes -

Cell-free fusion of LBPs with lysosomes was

performed as previously described (28) with

modifications: Lysosomes and LBPs were

isolated from RAW264.7 macrophages and

cytosol was prepared from RAW264.7. Where

indicated, isolated organelles were preincubated

for 10 min at 4 °C with 10 µM SaliPhe or DMSO

before addition of the remaining components of

the in vitro fusion reaction (final concentrations:

1x ATP-regenerating system, 1x salt solution,

1 mM dithiotreitol, 2 mg/ml RAW264.7

cytosol). 10 µM SaliPhe or DMSO were present

during the whole fusion reaction. After 30 min at

37 °C, the samples were set on ice for 5 min and

incubated with 0.2 mg/ml proteinase K

(Quiagen) for 15 min. Then 1.75 mg/ml

phenylmethanesulfonyl fluoride was added and

the samples filled up to 200 µl with HB (8.6 %

(w/v) sucrose, 20 mM HEPES/KOH pH 7.2,

0.5 mM EGTA). Latex bead phagosomes were

isolated from the samples placing them on top of

a 1 ml HB/25 % (w/v) sucrose cushion in a

swing out rotor (1,800 x g, 30 min). The

collected phagosomes were diluted in HB and

added to coverslips in 24-well plates followed by

centrifugation (690 x g, 15 min, 4 °C) and

fixation (3 % (v/v) formaldehyde, 2.5 % (v/v)

glutaraldehyde in HB) overnight. Remaining

aldehyde was quenched with 0.1 mg/ml NaBH4

for 30 min at RT, cells rinsed with PBS and

coverslips mounted in Mowiol (Sigma Aldrich).

Colocalization rates of latex bead-containing

phagosomes with lysosomal BSA-rhodamine

were determined from at least 600 phagosomes

per sample using fluorescence microscopy

(Axioplan, Zeiss).

Bacterial killing - Bone marrow-derived

macrophages were infected at an MOI of 5 with

E. coli DH5a or L. innocua serovar 6a (NCTC

11288) in serum-free DMEM for 15 min at

37 °C. After rinsing cells three times with PBS to

remove external bacteria, DMEM containing

10 g/ml gentamicin (Roth) was added to kill

remaining extracellular bacteria. Macrophages

were lysed at 0, 20 or 60 min after infection with

0.1 % (v/v) Triton X-100 and serial dilutions

were plated on nutrient agar plates to quantify

colony forming units the next day. Numbers of

colony forming units at 0 min were set as 100 %.

Maturation of Rhodococcus equi-

containing phagosomes - Heat-killed (15 min,

85 °C) Rhodococcus equi 103+ (31) were

surface-labeled with TAMRA-SE (5-(and 6-)

carboxytetra-methylrhodamine, succinimidyl

ester; Life Technologies) at 0.1 µg/ml for 30 min

on ice, washed and used for infection of J774E

macrophages seeded onto coverslips at an MOI

of 10. Samples were centrifuged immediately for

5 min at 15 °C and 200 x g. Medium was

replaced by fresh, pre-warmed complete medium

containing either DMSO at 0.2 % (carrier

control) or 40 nM bafilomycin A1 and

macrophages were placed at 37 °C, 10 % CO2.

Cells were fixed after 2, 20, 60 and 120 min and


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prepared for immunofluorescence. Samples were

stained with anti-TfR, anti-LAMP-1, or anti-

EEA1 and corresponding secondary Alexa488-

labeled antibodies. Samples were analyzed using

a laser scanning microscope LSM510 (Zeiss) and

percentages of phagosomes colocalizing with

respective proteins were determined. Statistical analysis - All values are

expressed as the mean ± SD/SEM and analyzed

via a two-tailed, unpaired student t-test or one-

way ANOVA followed by a Tukey-Kramer test

using GraphPad Instat 3 software (*p < 0.05, **p

< 0.01).

RESULTS

V0 subunit a3-deficiency in spite of

normal pH of the lysosome - In a genetic-based

approach to analyze the role of the v-ATPase

subunit a3 in membrane fusion, we used cells

from Tcirg1oc/oc

mice, which are homozygous for

the osteosclerosis (oc/oc) mutation and naturally

lack the transmembrane a3-subunit of the v-

ATPase (24). This subunit is particularly

relevant here, because in yeast, the a-subunit has

been previously implicated in the terminal phase

of membrane fusion (11,12) and the a3 isoform

is the relevant a-isoform in macrophage

phagosomes and lysosomes (32). The a3-

deficient oc/oc mice suffer from severe

osteopetrosis and die early (24,33), so that the

preferred cell type, primary macrophages, could

not be obtained. However, we succeeded in

generating fibroblast cell lines from these mice

and we confirmed their lack of subunit a3-

expression by western blotting (Fig. 1A),

quantitative real-time PCR (Fig. 1B) and

immunostaining (Fig. 1C). The mRNA and

protein concentration of the subunit a1 was

increased in the a3-deficient cells whereas the

subunit a2 shows unaltered expression level.

Subunit a4 was only expressed at a low rate

when compared to the other three isoforms in

wild-type cells and a4-mRNA concentration was

not affected upon deletion of subunit a3 (Fig.

1A, B). Concentrations of the V0 subunits d1 and

c as well as the V1 subunit B2 remained

unchanged (Fig. 1A). Surprisingly,

fluororatiometric pH-determination using

Oregon Green 514 demonstrated no difference in

lysosome pH in a3-deficient versus wild-type

cells (Fig 1D.E) and acidification, visualized

with LysoTracker Red staining, was highly

sensitive to bafilomycin A1 treatment (Fig. 1F,G)

and hence, dependent on a functional v-ATPase

complex.

Phagosome-lysosome fusion occurs in

the absence of V0 subunit a3 - MEFs are non-

professional phagocyte fibroblast cells. To study

phagosome maturation, wild-type and oc/oc

MEFs were stably transfected with a myc-tagged

CD32-cDNA coding for the human IgG receptor

FcRII (Fig. 2A) which mediates phagocytosis of

IgG-coated particles. Cells were incubated for

15 min with IgG-coated latex beads and

maturation of the latex bead-containing

phagosomes (LBPs) was monitored over time by

determining the colocalization of the internalized

beads with the early endosome antigen 1 (EEA1)

and with late endosomal/lysosomal LAMP-2

(Fig. 2B). Immediately after uptake, most beads

colocalized with the early endosome marker

EEA1 in both genotypes (Fig. 2B,C). Co-

localization of the lysosome membrane protein

LAMP-2 with LBPs increased steadily and was

observed with almost all phagosomes at 60 min

of chase. Kinetics of EEA1 loss from and

LAMP-2 acquisition to LBPs were identical

between wild-type and a3-deficient cells,

indicating normal phagosome maturation in

either case (Fig. 2B,C).

Lysosome alkalization does not affect

maturation of phagosomes - To study if

lysosome acidification per se, rather than

absence of the v-ATPase, modulates phagosome

maturation, parallel samples were analyzed in

the presence of the v-ATPase inhibitor

bafilomycin A1, a non-covalent inhibitor of the

proton-pump, and nigericin, a fast-acting,

membrane-permeant K+/H

+-exchanger.

Concentrations were used which were sufficient

for a complete loss of lysosome acidification

(Fig. 1F,G). Neither pH modulator led to a

significant difference in the degree or timing of

colocalization of early and late endocytic marker

proteins with the latex beads (Fig. 2D). This

observation suggested a mechanism of

phagosome maturation that is independent of

luminal pH.

It is also of note that dextran (Fig. 3A)

and transferrin (data not shown) were taken up to

the same extent by both wild-type and mutant

cells. Moreover, lysosome hydrolase activities

(Fig. 3B) and processing of cathepsin-D and

cathepsin-L (Fig. 3C) also did not differ between

a3-deficient and wild-type MEF cells in steady

state. These experiments indicate that fusion

events within the endocytic pathway are not

affected by the lack of the a3 subunit.

Since we could not exclude possible

compensatory processes for a3 by the subunit a1,


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which might mask acidification-independent

functions, we further studied the consequences

of a deletion of all isoforms in professional

phagocytes, such as macrophages.

Disruption of v-ATPase assembly after

conditional knockout of Atp6ap2 - We took

advantage of a recently generated conditional

knockout (cKO) mouse line in the gene for

ATP6AP2. The encoded protein is an accessory

v-ATPase subunit and also known as pro-renin

receptor. This protein is a central factor in the

assembly of the v-ATPase and its removal

results in the instability and disappearance of

complete V0 complexes (21). The generated

Atp6ap2Flox/Flox

Mx1-Cre cKO mice were treated

for one week with polyinosinic–polycytidylic

acid (pI–pC) to induce Cre expression in

interferon α-responsive cells that include

peritoneal macrophages and stem cells that we

used to induce bone marrow-derived

macrophages (BMDM). Macrophages were

analyzed 10-17 days after induction and

differentiation to BMDMs. This revealed that a

deficiency of ATP6AP2 could be indeed

obtained and that the concentrations of several

V0 subunits, including subunit a isoforms 1 – 3,

were significantly reduced in the cKO

macrophages (Fig. 4A). However, loss of

ATP6AP2 did not affect mRNA expression of

these and additionally tested subunits (Fig. 4B)

suggesting posttranscriptional events leading to

the loss of V0 subunits. Subunit a3 showed the

highest mRNA expression level of the four a-

isoforms, while a3 protein was almost

completely missing, as demonstrated by

immunoblotting and immunofluorescence

microscopy of differentiated macrophages (Fig.

4A,C). Therefore Atp6ap2 cKO is a suitable

model to analyze an almost complete absence

(up to 96%) of V0 subunits of the v-ATPase.

In these cells, the lysosome pH was only

slightly less acidic than in wild-type cells (Fig.

4D,E,F), enabling us to study an pH-independent

effect on lysosome fusion with phagosomes. It is

interesting to note that when treating cells with

bafilomycin A1, ATP6AP2-deficient

macrophages were somewhat more susceptible

to the drug than control cells (Fig. 4E) probably

also reflecting the reduced number of v-ATPase

complexes in the Atp6ap2 knockout

macrophages. As a measure of the biological

relevance of v-ATPase in phagosome

maturation, the killing capacities towards

bacteria of BMDMs from wild-type and induced

Atp6ap2 knockout mice were compared (Fig.

4G). Surprisingly, ATP6AP2-depleted

macrophages killed Escherichia coli (Gram-

negative, avirulent) and Listeria innocua (Gram-

positive, avirulent) rather more efficiently,

suggesting that the absence of the most of v-

ATPase complexes did not affect their

microbicidal functions. Compromising lysosome

pH by application of bafilomycin A1 did also not

change killing capacity of macrophages towards

Escherichia coli (data not shown), implying

bactericidal mechanisms, that are independent on

an acidic milieu. Similar to a3-deficient MEF

cells, endocytic uptake and delivery to

lysosomes of dextran and BSA (Fig. 3A) were

also not affected in Atp6ap2 cKO macrophages;

neither were maturation and activity of

lysosomal hydrolases (Fig. 3B,C).

Phagosome-lysosome fusion in

ATP6AP2-depleted macrophages - Knockout of

Atp6ap2 exclusively in cells of the myeloid

lineage, including macrophages, due to LysM-

mediated Cre deletion similarily resulted in

disappearance of the a1- and a3-V0 subunits.

Nevertheless in these cells we found unaltered

lysosome acidification and a significant

colocalization of LAMP-2 with phagocytosed

latex beads (Fig. 5), suggestive of unaltered

phagosome maturation. To analyze at an

ultrastructural level whether phagosome

maturation was affected, we performed

transmission electron microscopy (TEM)

experiments. The endocytic pathway of wild-

type and induced Atp6ap2 conditional knockout

(cKO) BMDMs was pre-labeled with 10 nm

ferroparticles and then incubated with 1.1 m

latex beads for 10 min followed by removal of

external beads and further incubation for 10 or

120 min (Fig. 6A). TEM visualization of

phagosomes (Fig. 6B) revealed three types of

nanoparticle-containing fusion profiles between

phagosomes and lysosomes, which were

categorized as small, medium and large (Fig.

6C). A significantly higher number of tight-

fitting (small) membrane profiles per bead was

observed in Atp6ap2 cKO cells compared to

wild-type cells. Medium and large profiles were

observed at a much lower frequency in both

genotypes with an even more reduced number in

the Atp6ap2 knockout cells compared to cells

with wild-type v-ATPase (Fig. 6C). We used

stereology to make an estimate for the fraction of

phagosome membrane that surrounds the small

lysosomal ferroparticle deposits. It indicates the

rate of small phagosome-lysosome fusion events

and was found to be signmificantly increased


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and observed significantly increased in

ATP6AP2-deficient macrophages at 10 min as

well as 120 min of chase (Fig. 6D). Therefore,

fusion frequency between LBPs and lysosomes

in macrophages is, if anything, increased upon

loss of a major portion of v-ATPase.

Normal phagosome-lysosome fusion

after arresting V0/V1 complexes - Our previous in

vitro fusion data (28) and the above latex bead

experiments demonstrated that neither

bafilomycin A1 nor nigericin inhibited

phagosome-lysosome fusion. Similarly,

bafilomycin A1 treatment, sufficient to increase

lysosome pH (Fig. 7A), did not alter maturation

of phagosomes containing heat-killed bacteria

(31) within macrophages (Fig. 7B). This

suggested that vesicle acidification per se is not a

prerequisite for phagosome maturation.

Recently, salicylihalamide, a v-ATPase inhibitor,

was described to lock V0 and V1 sectors of v-

ATPases together (34). Because it has been

postulated (11,35) that the V0 subunit catalyzes

membrane fusion only when being free of V1, we

investigated the V0 - V1 locking effect in vitro.

The advantage of this drug is that both the

reduced availability of free V0 subunit and the

effect of increased lysosome pH can be studied

in one sample. We observed a clearly increased

amount of assembled complexes in presence of

the equipotent salicylihalamide analog

saliphenylhalamide A (Fig. 8A-C) as well as loss

of LysoTracker Red signals caused by an

increased lysosome pH (Fig. 8D). Yet, there was

no effect on phagosome-lysosome fusion in vitro

(Fig. 8E). Saliphenylhalamide A also did not

change the degree of fusion of BSA-rhodamine-

labeled lysosomes with latex bead-containing

phagosomes in macrophages (Fig. 8F), again

questioning a central role of v-ATPase

participation in fusion.

DISCUSSION

Does proton-pumping v-ATPase regulate

and possibly even catalyze phagosome-

endosome and phagosome-lysosome fusion and

how does it participate in pathogen killing?

Recent work by other groups suggested that the

V0 sector, the membrane integral parts of v-

ATPases, acts downstream of SNARE complex

formation and contributes to lipid mixing and

membrane fusion (11,35,36). V0 sectors in only

one of the two fusion partners were reported to

be sufficient to support fusion (35). The fusion

of inter-membrane fusion complexes appeared to

require calcium release but was independent of

the proton pumping activity of the v-ATPase

(12,37). We have chosen pharmacological and

genetic approaches to analyze the contribution of

the v-ATPase complex to fusion between

phagosomes and endo(lyso)somes which contain

large quantities of v-ATPase. In summary,

surprisingly, neither of the above v-ATPase

manipulations yielded a significant change in the

endocytic or phagocytic uptake, in lysosome

acidification, or in phagosome-lysosome fusion.

Our data suggest that neither v-ATPase in

general nor the a3 subunit in particular, have an

apparent direct role as a promoter of phagosome-

lysosome fusion in our systems.

The role of the v-ATPase complex and

specifically the membrane bound V0 sector as a

fusion-enhancing factor has been a matter of

extensive investigations (13,38). With respect to

the phagocytic pathway, this role has only been

studied by looking at apoptotic body removal in

zebrafish brain (16) where the a1-subunit of v-

ATPase -despite an unaltered lysosome pH- was

critical for fusion. This suggests that different

phagocytic cargos with different degrees of

danger signals, e.g. apoptotic blebs versus IgG-

opsonized latex béad phagosomes or bacteria,

may feed into different maturation pathways.

Also, the use of different organisms and tissues,

zebrafish brain versus mouse macrophage and of

different strategies for gene inactivation,

morpholino addition versus conditional knock

out, may have caused this. Another suggestive

correlation between v-ATPase possession and

phagosome maturation comes from phagosomes

containing Mycobacterium tuberculosis. These

phagosomes almost completely lack v-ATPase

and they are arrested at an early stage along the

endocytic/phagocytic continuum (39). This

correlation suggested that the lack of the proton

pump may not only be indicative for altered

phagosome trafficking of this pathogen-

containing phagosome, but may actually cause it.

Our approach to study proton-pumping-

independent contributions of v-ATPases to

membrane fusion was the generation of cell lines

disturbed in v-ATPase complex assembly which

contain fewer V0 complexes (62-96% reduction

varying between different subunits), likely due to

increased protein degradation. Further, we have

used cells with a knockout of the a3 subunit.

Both types of experiments challenged the

cellular functions of the transmembrane V0

sector (7). Of the four possible isoforms of the v-

ATPase a-subunit, a3 is arguably the most


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important one on macrophage lysosomes and

may be relevant for phagosome acidification

(32). It was suggested that the a3 subunit is

likely the most prominent in J774 and

RAW264.7 macrophage cell lines (40,41), in

which it is localized to phagosomes and needed

for bactericidal function (32). a3-knockout

mouse embryonic fibroblasts have normal

endocytic functions and unaltered activities of

lysosomal hydrolases at a normal lysosome pH.

The complete lack of a3 did also not change the

kinetics of phagosome maturation, strongly

suggesting that fusion of late

endosomes/lysosomes with phagosomes occurs

to the same extent in the absence of this a-

subunit of the v-ATPase. It may well be that at

least a portion of the a3 subunit is replaced by

the a1-subunit of v-ATPase as suggested by the

increased expression of this subunit in the a3-

deficient MEF cells. Expression levels of other

subunit a-isoforms were unaltered or very low in

protein and mRNA levels making a

compensatory role of these subunits unlikely. A

limited functional compensation for subunit a3

by subunit a1 has been discussed in osteoclasts, a

cell population with predominant expression of

subunit a3 and only low levels of subunit a1

(42). Knockout of a3 did not suffice to

completely block the osteoclast´s v-ATPase

function (43).

Our studies using bone marrow-derived

macrophages from mice deleted in Atp6ap2, a

gene essential for vacuolar ATPase assembly,

revealed that deletion resulted in a significant

drop in the concentrations of all v-ATPase V0

subunits. Similar to the situation in

cardiomyocytes and kidney (21,44,45) the

presence of ATP6AP2 appears to be a

prerequisite for v-ATPase assembly in the

endoplasmic reticulum. Taken the pivotal role of

the v-ATPase complex into account it is not

surprising that a complete deficiency of v-

ATPase may lead to early cell death (46). We

expected that the pronounced loss of the v-

ATPase levels would cause both a strong

reduction in lysosome acidification and in

membrane fusion between late endocytic

organelles. Surprisingly, the ability to acidify

these compartments was only slightly, if at all,

affected in the absence of functional v-ATPase.

This can possibly be explained by the presence

of some v-ATPase holo-complexes, which may

also be a prerequisite for the survival of the

knockdown cells. The expression levels of all a-

isoforms, except for the isoform a4, which is not

expressed in macrophages, were significantly

lower in Atp6ap2 knockout than in wild-type

macrophages. It is likely that remaining v-

ATPase complexes are sufficient to fulfill their

proton-pumping function, as acidification was

still bafilomycin A1-sensitive. However, less

bafilomycin A1 is needed to increase the pH in

Atp6ap2 knockout cells suggesting a lower

number of functional v-ATPase complexes.

The presence of a normally low

lysosome pH allowed us to monitor the fusion

capacity of lysosomes with phagosomes without

the need to consider abnormal acidification as a

reason for any of the phenotypes we would

observe. After all, disturbances of trafficking

events in the endocytic pathway caused by pH

neutralization, have been described (5). Despite

the significant drop in the number of v-ATPase

complexes in Atp6ap2 knockout cells, all tested

functions of endocytic and phagocytic

compartments were unaffected and phagosome-

lysosome fusion, as well as killing of bacteria

was even somewhat pronounced. We expected

that the large decrease in complex concentration

would affect the number of fusion events.

However, no effect on fusion was observed. Hence, the residual levels of v-ATPase

complexes may still contribute to membrane

fusion.

The removal of the bulky V0

transmembrane protein complex (47,48) from

membranes might decrease steric hindrance for

membrane fusion catalyzed by other factors such

as the SNARE complexes, thereby increasing

fusion rates. In this context it is interesting to

note that the size of protrusion of the yeast V0/V1

complex from the membrane has been estimated

to be 415 nm (49). The assumption that this huge

complex might impair the contact between

membranes also finds support in our electron

microscopy-based observation of more fusion

events in the Atp6ap2-deleted macrophages as

compared to the wild-type cells.

This interpretation is in contrast to

studies in yeast and mammalian cells, which

implicated a direct V0 sector involvement in

fusion, that is independent of the intraluminal pH

(11-13,16,37). However, some of this work is

disputed as a recent study on homotypic vacuole

fusion in yeast argues strongly that acidification

rather than the presence of the v-ATPase is a

major driving factor of membrane fusion (17).

Contrary to these findings, in our

experiments, acidification also did not seem to

be a driving force, as neither nigericin nor


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bafilomycin A1 treatment significantly altered

the kinetics of LAMP-2 acquisition by

phagosomes containing latex beads or heat-killed

bacteria. These obervations suggest efficient

phagosome-lysosome fusion under conditions of

increased lysosome pH. Also, locking many of

the V0 - V1 sectors together by

saliphenylhalamide A did not affect the extent of

phagosome-lysosome fusion, although it could

have been expected that this locking would

reduce the supply of free V0 and place extra

bulky protein complexes between the two

membranes destined to fuse (35).

Taken together, our data with cells either

mostly lacking the v-ATPase complex or

completely lacking its a3 subunit did not reveal

an absolute necessity of the v-ATPase in fusion.

Strongly supportive of these findings is the fact,

that reconstituted fusion with purified

components of yeast vacuoles proceeded in the

complete absence of a v-ATPase complex (50).

The same was true for reconstituted homotypic

early endosome fusion (51). Another piece of

data pointing to a non-mandatory role for v-

ATPases in membrane fusion is that

compartments which naturally lack a v-ATPase

complex, such as postmitotic nuclear vesicles

(52), can still fuse. Although the above

arguments and data do not unequivocally

exclude a regulatory role of v-ATPases in fusion

(53), they argue that the presence of the v-

ATPase is not obligatory and that acidification-

independent roles of v-ATPases in fusion may be

rather the exception than the rule.


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Acknowledgements: We would like to thank Kristin Schröder, Christine Desel, Sönke Rudnik and

Janna Schneppenheim for technical support. We are also very grateful to John Lucocq for his help

with the stereology.

FOOTNOTES

Funding: This work was supported by the Deutsche Forschungsgemeinschaft (GRK1459 to P.S. and

SPP1580 to A.H., G.G. and P.S). We thank the Electron microscopy laboratory at the Department of

Bioscience, University of Oslo, Norway.

Author contribution statement: GG, AH and PS designed experiments; SK, CH, UR, CK, KB

performed experiments; SK, CH, UR, KB analyzed data; AH and PS wrote the manuscript; AI, MS,

BL, JB provided reagents and material and commented on the manuscript.

Abbreviations: ATP6AP2, ATPase, H+-transporting lysosomal accessory protein 2; cKO, conditional

knockout; CtsD, cathepsin D; CtsL, cathepsin L; DIC, differential interference contrast; EEA1, early

endosome antigen 1; LAMP-1, lysosomal-associated membrane protein 1; LAMP-2, lysosomal-

associated membrane protein 2; LBPs, latex bead-containing phagosomes; MOI, multiplicity of

infection; SaliPhe, saliphenylhalamide A; TfR, transferrin receptor

FIGURE LEGENDS

Figure 1: The V0 subunit a3 is dispensable for lysosome acidification.

Murine embryonic fibroblasts (MEFs) generated from mice carrying the Tcirg1oc

allele were analyzed

for v-ATPase expression and their ability to acidify lysosomes. (A) Immunoblot analysis of v-ATPase

subunit expression in whole cell lysates from wild-type (Tcirg1+/+

) and a3-/-

(Tcirg1oc/oc

) MEFs. β-

actin staining was used to control for equal protein load. (B) Expression of the four subunit a isoforms

was analyzed by qRT-PCR in both genotypes. Shown are mean relative mRNA levels normalized to

the most stable house-keeping genes ± standard error from three independent experiments (** P <

0.01, unpaired, two-tailed Student´s t test). (C) Lysosomal localization of V0 subunit a3. Wild-type

and mutant cells were fixed and co-immunostained with anti-V0 a3 and anti-LAMP-2 antibodies.

Colocalization between v-ATPase subunit a3 and LAMP-2 was quantified to be 78 % (Pearson´s

correlation coefficient/PCC = 0.74) in wild-type cells and 15 % (PCC = 0.09) in a3-deficient cells. (D

and E) Ratiometric quantification of lysosome pH in wild-type and a3-/-

MEFs. Late endocytic

compartments of MEFs were pulse-chase labeled with dextran-Oregon Green 514 and cells subjected

to live cell fluorescence imaging. (D) Intensity ratios 440/488 as readouts for lysosome pH were

calibrated in situ with nigericin at defined pH. Additional control samples were pre-incubated for

15 min with bafilomycin A1 (B, 200 nM) or nigericin (N, 10 µg/ml, pH > 6.5 in both cases). (E) The

values for lysosome pH are comparable between a3-deficient and wild-type cells. Data are presented

as means ± standard errors from three independent experiments. (F and G) LysoTracker Red staining

of MEF cells. Wild-type and a3-deficient MEFs were incubated with LysoTracker Red for 20 min and

fixed for fluorescence imaging. Where indicated, bafilomycin A1 (0 - 500 nM) or nigericin (0 - 20

µg/ml) was added for 15 min before LysoTracker Red staining as a control for a lysosome pH

equilibrated with the cytosol and extracellular buffer, respectively. Representative fluorescence images

are shown in F. (G) LysoTracker Red signal intensities were measured and plotted against the

concentrations of bafilomycin A1 or nigericin to create dose-response curves. Shown are mean

LysoTracker Red signal intensities relative to carrier treated controls cells ± standard deviation of

three independent experiments.

Scale bars = 10 µm.


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Figure 2: Loss of V0 subunit a3 does not influence phagosome-lysosome fusion.

(A) Total cell lysates of wild-type and a3-deficient MEFs, stably transfected with human Fc-γ

receptor II-myc (FcγRII-myc), were probed with the indicated antibodies in western blot analysis.

GAPDH was used to control for equal protein load. (B) Analysis of latex bead phagocytosis in FcγRII-

myc expressing MEFs. Cells were pulsed with human IgG (hIgG)-opsonized latex beads for 15 min

and maturation of latex bead-containing phagosomes monitored for chase periods from 0 – 60 min.

External latex beads were labeled with an anti-hIgG antibody and samples fixed for immunostaining.

Colocalization rates between internalized latex beads (no hIgG-signal) and EEA1 or LAMP-2 were

determined by laser-scanning confocal microscopy (LSCM) after the indicated chase periods. (C)

Representative fluorescence images are shown for experiments conducted in B including magnified

regions of interest (ROI). Arrows point to areas with colocalization (scale bars = 10 µm). (D)

Influence of manipulation of luminal pH on phagosome maturation. Experiments in B were conducted

in the presence of the v-ATPase inhibitor bafilomycin A1 (200 nM), the ionophore

nigericin (10 µg/ml) or carrier control (DMSO) with the treatment starting either 15 min before

(bafilomycin A1, DMSO) or concurrent with latex beads incubation (nigericin). Percentages of

phagosomes colocalizing with LAMP-2 and EEA-1 were determined by LSCM. A minimum of 100

phagosomes was analyzed per condition. Data are presented as means ± standard errors from 2-3

independent experiments.

Figure 3: Endocytic maturation and degradation in lysosomes is normal a3- in and ATP6AP2-

deficient cells Lysosome functions were analyzed in wild-type and a3

-/- MEFs and wild-type and Atp6ap2 cKO

macrophages. (A) Visualization of vesicle maturation upon macropinocytosis and degradation within

lysosomes. Cells were incubated either in the presence of dextran-Texas Red (0.5 mg/ml) for 30 min

plus 2 h chase period or overnight in the presence DQ-BSA (0.1 mg/ml). Samples were fixed and

immunolabeled with anti-V0 a3 and anti-LAMP-2 antibodies. Distribution of the fluorescent marker

proteins and their signal intensities were similar in the analyzed genotypes of both cell types. Shown

are representative immunofluorescence images (scale bars = 10 µm). (B) Total β-hexosaminidase

activity was comparable between a3-deficient and wild-type MEFs and between Atp6ap2 cKO and

wild-type macrophages, respectively. Shown are means ± standard errors from 7-9 independent

experiments. (C) Intracellular proteolytic processing of cathepsins D and L was analyzed by western

blotting in the cell types mentioned in A-B. No impairment of enzyme maturation is visible. Arrows

point to proform (P), intermediate (I) and mature (M) forms of the respective cathepsin. Lysates from

cathepsin D (CtsD-/-

) and cathepsin L (CtsL-/-

) knockout MEFs were used to control for antigen

specifity of the antibodies (* unspecific signal).

Figure 4: Killing of bacteria within macrophages is increased by a reduced v-ATPase

concentration.

Conditional knockout of the v-ATPase accessory protein 2 (ATP6AP2) was induced in

Atp6ap2Flox/Flox

Mx1-Cretg/+

mice by pI-pC treatment and mice sacrificed 10 days post induction for

isolation of peritoneal (A-F) or generation of bone marrow-derived macrophages (BMDMs, G). Cells

derived from Atp6ap2Flox/Flox

Mx1-Cre+/+

mice served as wild-type controls. (A) Western blot analysis

of v-ATPase expression. Cells were lysed and subjected to immunoblotting against v-ATPase

subunits. Anti-ATP6AP2 antibodies to the full length (fl), N-terminal (NTF) and C-terminal (CTF)

fragments of ATP6AP2 were applied to determine Atp6ap2 knockout efficiency. β-actin was used to

control for equal protein load. (B) mRNA expression levels of different v-ATPase subunits as

determined by qRT-PCR. Values are presented in relation to the most stable house-keeping genes as

means ± standard errors from three (knockout) to four (wild-type) macrophage preparations. (C)

Immunofluorescence analysis of localization of V0 subunit a3. Macrophages were fixed and labeled

with anti-V0 a3 and anti-LAMP-2 antibodies. Colocalization between a3 and LAMP-2 was measured

to be 72 % (PCC = 0.78) in wild-type and 24 % (PCC = 0.27) in Atp6ap2 knockout cells. (D and E)

Primary macrophages were analyzed for lysosome acidification by LysoTracker Red staining. (D)

Representative fluorescence images of macrophages either untreated or pre-treated with bafilomycin

A1 (100 nM) for 15 min. (E) A dose-response curve was generated by measuring LysoTracker Red


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intensity after 15 min pretreatment with various concentrations of bafilomycin A1 (0 – 200 nM).

Shown are mean intensities in relation to DMSO treated samples ± standard deviation from 2

independent experiments. (F) Lysosome pH in Atp6ap2 cKO cells was determined using

ratiofluorometric dextran-Oregon Green 514 measurements. Treatment for 1 h with bafilomycin A1

(200 nM) yielded a pH > 6.5 (not shown). Data are presented as means ± standard errors from four

(wild-type) to six (knockout) macrophage preparations. * P < 0.05 according to unpaired two-tailed

Student´s t test. (G) Killing of bacteria within BMDMs. Macrophages were infected with either

Escherichia coli or Listeria innocua (MOI = 5) (both non-pathogenic bacteria) and colony forming

units were determined after the indicated infection periods by macrophage lysis and propagation on

nutrient agar. Data are presented as per cent of bacterial colonies recovered from agar plates relative to

the numbers recovered from zero-time samples. Shown are means ± standard errors from 2-5

independent experiments. * P < 0.05 according to unpaired two-tailed Student´s t test between

genotypes. Scale bars = 10 µm, insets show DIC image.

Figure 5: Knockout of Atp6ap2 in LysM-Cre transgenic mice. Peritoneal macrophages harvested from Atp6ap2

Flox/Flox LysM-Cre

tg/+ (Atp6ap2 LysM-ko) and

Atp6ap2Flox/Flox

LysM-Cre+/+

(control) mice were analyzed for v-ATPase expression and activity. (A)

Expression of ATP6AP2 full length (fl) and C-terminal fragment (CTF) as well as expression of

designated v-ATPase subunits was analyzed by immunoblotting of cell lysates from macrophages.

β-actin was used to control for equal protein load. (B) Quantification of lysosome pH with ratiometric

dextran-Oregon Green 514 measurements. Data are presented as means ± standard errors from five

macrophage preparations for each genotype. (C) Uptake and intracellular delivery of latex beads.

Atp6ap2 LysM-ko and control cells were incubated with murine IgG (mIgG)-opsonized latex beads for

15 min and incubated for further 60 min after washout of bead excess. Before fixation of the cells for

immunofluorescence analysis, external latex beads were labeled with an anti-mIgG antibody (1 min).

Lysosomal structures are visualized with an anti-LAMP-2 antibody. An anti-V0 a3 staining was

performed to control for Atp6ap2 knockout. Scale bars = 10 µm.

Figure 6: Knockout of Atp6ap2 enhances fusion between phagosomes and lysosomes.

Fusion between phagosomes and lysosomes was analyzed in BMDMs from Atp6ap2 knockout

(Atp6ap2Flox/Flox

Mx1-Cretg/+

) and control mice by electron microscopy. (A) Scheme showing

experimental procedure, lysosomes are colored in red, phagosomes in orange: BMDMs were

incubated with ferrous nanoparticles (10 nm, in black) and cultured in the absence of nanoparticles for

further 3 h to ensure lysosomal localization of the tracer. Pre-labeled macrophages were then fed with

latex beads for 10 min at 37 °C. Phagosome maturation was stopped 10 min or 2 h later and samples

were processed for electron microscopy. (B) Representative micrographs show fusion profiles between

latex bead-containing phagosomes (LBPs) and nanoparticle-labeled lysosomes (Ly). Scale bars =

500 nm. Nanoparticle deposits within the phagosome (indicated by arrows) were considered to reflect

a recent fusion event. (C) Fusion events as depicted in B were then categorized according to their size

and their average number per bead was compared between wild-type and Atp6ap2 knockout cells after

2 h of chase. Atp6ap2 knockout macrophages show an increase in the total number of fusion events,

reflected by an increase in the number of small deposits as the major population. (D) The fraction of

phagosome membrane surrounding the small deposits as a measure of the fusion rate was quantified

by sterology and compared between both genotypes for at least 20 phagosomes per sample after 10

min and 2 h chase. Data in C and D represent means ± standard errors from 4 (wild-type) to 7

(knockout) macrophage preparations. * P < 0.05, ** P < 0.01 according to unpaired two-tailed

Student´s t test.

Figure 7: Lack of an effect of bafilomycin A1 on maturation of phagosomes containing heat-

killed bacteria. (A) Effect of bafilomycin A1 on lysosome pH in J774E macrophages. Cells were treated for 40 min

with 10 nM bafilomycin A1 and then subjected to LysoTracker Red staining. Representative

fluorescence images are shown (DIC channel in insets). Scale bars = 10 µm. (B) Influence of increased

lysosome pH after bafilomycin A1 treatment on maturation of Rhodococcus equi-containing

phagosomes. J774E macrophages were fed with heat-killed Rhodococcus equi 103+ and further


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incubated in the presence of 40 nM bafilomycin A1 or 0.2 % (v/v) DMSO (carrier control). Cells

were fixed after the indicated incubation periods and prepared for immunofluorescence analysis by

staining for EEA1, LAMP-1, or transferrin receptor (TfR). At least 100 phagosomes were analyzed for

each condition. Data represent colocalization rates between phagosomes and the respective marker

protein as means ± standard deviation from 2 - 5 independent experiments.

Figure 8: Increased formation of V0 / V1 complexes does not affect phagosome-lysosome fusion. Influence of the v-ATPase inhibitor saliphenylhalamide A (SaliPhe) on in vitro and in cellulo fusion

between phagosomes and lysosomes was analyzed in RAW264.7 macrophages. (A - C) Effect of

SaliPhe on v-ATPase complex formation. (A) Subcellular fractionation of RAW264.7 cells after 2 h

treatment with 10 µM SaliPhe or DMSO, as indicated. T= total lysate, S1 = low speed

supernatant/perinuclear supernatant, P1 = low speed pellet/cells and nuclei, S2 = high speed

supernatant/cytosolic proteins, P2 =high speed pellet/membrane bound proteins. SaliPhe treatment

shifts v-ATPase V1 subunits B und E1 from the cytosolic to the membrane-bound fraction. LAMP-1

and V0c localization were not influenced by SaliPhe. Shown are representative immunoblots from 2

independent experiments (* unspecific signal). (B) SaliPhe increases V0-V1 interaction.

Immunoprecipitates (anti-V1B2) were prepared from cells treated as in (A) and analyzed for co-

precipitation of V0 subunits. LAMP-1 was included as negative control for interaction. Representative

blots from 2 independent experiments are shown. (C) Lysosomes from RAW264.7 cells were co-

labeled with BSA-rhodamine and nanomagnets and segregated via magnetic sorting. Isolated

organelles were then incubated for 60 min under in vitro fusion conditions and processed for western

blotting. Where denoted, isolated lysosomes were pretreated for 10 min with SaliPhe (10 µM) and the

treatment continued throughout the experiment. Staining with the indicated antibodies shows more V1

bound to lysosomes when SaliPhe was present during the incubation. (D) Lysosome pH is increased

by SaliPhe treatment. RAW264.7 cells were treated with 200 nM bafilomycin A1 or 10 µM SaliPhe

for 100 min before LysoTracker Red staining. Shown are representative fluorescence images (insets:

DIC channel). Scale bar = 10 µm. (E) In vitro fusion experiments were carried out with lysosomes,

treated as described in C and latex bead-containing phagosomes (LBPs) isolated from macrophages.

Both organelle types were either pretreated for 10 min with SaliPhe or left untreated. In vitro fusion

was then allowed to proceed for 60 min at 37 °C in the presence or absence of SaliPhe or carrier

(DMSO). Incubation at 4 °C served as a negative control. (F) Influence of SaliPhe on in cellulo fusion

of BSA-rhodamine-labeled lysosomes with LBPs. RAW264.7 macrophages were cultivated in

presence of BSA-rhodamine overnight and the label chased into lysosomes for 2 h while incubating

with SaliPhe or DMSO. Macrophages were then allowed to ingest latex beads for 10 min and

incubated for further 0, 20 or 80 min with addition of SaliPhe/DMSO. Cells were lysed and

phagosomes isolated to analyze the frequency of colocalization between BSA-rhodamine and LBPs.

Data are presented as means ± standard errors from 3 independent experiments.


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Figure 1


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Figure 8


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Brabander, Albert Haas and Paul SaftigBargen, Gareth Griffiths, Atsuhiro Ichihara, Beth S. Lee, Michael Schwake, Jef De

Sandra Kissing, Christina Hermsen, Urska Repnik, Cecilie Kåsi Nesset, Kristine vonVacuolar ATPase in Phagosome-Lysosome Fusion

published online April 22, 2015J. Biol. Chem.

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