December 4th, 2001D. Tyteca - PhD Thesis 1
Azithromycin, a pharmacological agent whichselectively inhibits some pathways of endocytosis:
characterization, interests and mechanism ofaction.
Donatienne Tyteca, Pharm.Thesis submitted for the dregree of Doctor in Pharmaceutical Sciences (PhD)
Promotor: Prof. M.P. Mingeot-LeclercqCopromotors: Profs P.J. Courtoy & P.M. Tulkens
Brussels, December 4th, 2001
December 4th, 2001D. Tyteca - PhD Thesis 2
➥➥➥➥ mammalian cells take up extracellular material by a variety of mechanisms collectively termed endocytosis
➥➥➥➥ implications of endocytosis in physiology:●●●● uptake of extracellular nutrients,
●●●● cellular cholesterol homeostasis,●●●● regulation of hormonal response,●●●● maintenance of cell polarity,●●●● antigen presentation,●●●● ....
➥➥➥➥ implications of endocytosis in pathology: ●●●● atherosclerosis, ●●●● entry of pathogens and toxins, ●●●● neurogenerative diseases (Alzheimer, prion), ●●●● ....
(Mukherjee et al, 1997)
Endocytosis
December 4th, 2001D. Tyteca - PhD Thesis 3
pinocytosis phagocytosis
lateendosome
lysosome
earlyphagosome
latephagosome
zipper
trigger
sortingendosome
non-coated pit
recyclingcompartment
coated pit
Pathways of endocytosis studied in this thesis
December 4th, 2001D. Tyteca - PhD Thesis 4
membrane organization in domains
membrane fluidity
membraneasymmetry
Membrane lipids are implicated at various stagesof the endocytic process
December 4th, 2001D. Tyteca - PhD Thesis 5
budding coat proteins:clathrin, APsCOPs
cytoskeleton adhesion to acurved particle
membranetension andasymmetry
fission dynamin
H+
H+ H+
H+acidificationvacuolarH+-ATPase
dockingRabPI 3-kinase
fusionSNARE/SNAPmembrane
transportactinmicrotubules
Molecular machineries of endocytic pathways
December 4th, 2001D. Tyteca - PhD Thesis 6
Conditions, mutations and agents have been extensively usedto dissect cellular mechanisms of endocytosis:
➥➥➥➥ conditions:●●●● K+ depletion (Cupers et al, 1994)●●●● incubation in hypertonic medium (Cupers et al, 1994; 1997)●●●● cytosol acidification (Sandvig et al, 1987)
➥➥➥➥ mutants:●●●● clathrin, adaptor proteins and associated proteins●●●● COP●●●● dynamin●●●● Rabs●●●● .... (for a review, see Dautry-Varsat, 2001)
What is the place of pharmacological inhibitorsto dissect the endocytic apparatus ?
December 4th, 2001D. Tyteca - PhD Thesis 7
budding cytoskeleton adhesion to acurved particle
membranetension andasymmetry
transportactinmicrotubules
fission dynamin
dockingRabPI 3-kinase
fusionSNARE/SNAPmembraneH+
H+H+
H+acidificationH+-ATPase
➥➥➥➥ agents:
wortmannin
nocodazole
cytochalasins
phospholipidosis
gentamicin
coat proteins:clathrin, APCOP
chlorpromazine benzyl alcohol
bafilomycins chloroquine
December 4th, 2001D. Tyteca - PhD Thesis 8
limitations:
➥➥➥➥ almost all are unspecific and show pleiotropic effects
➥➥➥➥ none inhibit the earliest steps of clathrin-independent pinocytosis
and azithromycin ?
December 4th, 2001D. Tyteca - PhD Thesis 9
O
H3C
OH
OH
OH
CH3
H3CH2C O
O
CH3
CH3
H3C
O
O
OHO
(H3C)2N
CH3
OH
1
3'
N
CH3
CH3
9
H3C
H3C
H3CO
9a
Azithromycin (AZ),a dicationic amphiphile
December 4th, 2001D. Tyteca - PhD Thesis 10
➥➥➥➥ spectrum of activity ●●●● Gram + ●●●● some Gram -
➥➥➥➥ therapeutic use ●●●● upper and lower respiratory tract infections ●●●● skin infections ●●●● sexually transmitted diseases ●●●● Mycobacterium avium complex in AIDS patients
➥➥➥➥ pharmacokinetic properties in vivo ●●●● exceptionally high and rapid accumulation in tissues, and slow release (Foulds et al, 1990) ●●●● consequences ➥➥➥➥ low serum concentrations ➥➥➥➥ decrease of the length of treatment ➥➥➥➥ toxicity ??? Pharmacological properties of AZ
Pharmacological properties of AZ
December 4th, 2001D. Tyteca - PhD Thesis 11
➥➥➥➥ accumulates in lysosomes of fibroblasts and macrophages (Carlier et al, 1994)
➥➥➥➥ acidotropic sequestration (de Duve et al, 1974)
H+
B B B
BH+ BH+ BH+
H+H+
Extracellular fluid
Extralysosomal space
Lysosomes
Cellular pharmacokinetic properties of AZ
December 4th, 2001D. Tyteca - PhD Thesis 12
➥➥➥➥ induces a lysosomal phospholipidosis in fibroblasts (Van Bambeke et al, 1996)
➥➥➥➥ inhibits lysosomal phospholipase A1 (Montenez et al, 1996)
Cellular toxicological properties of AZ
December 4th, 2001D. Tyteca - PhD Thesis 13
➥➥➥➥ binds to negatively-charged bilayers at acidic pH (Montenez et al, 1996)
➥➥➥➥ perturbs the fusion of lysosomes with horseradish peroxidase (HRP)-containing endosomes (unpublished observation of Van Bambeke)
December 4th, 2001D. Tyteca - PhD Thesis 14
●●●● Selection of experimental system Rat foetal fibroblasts
➥➥➥➥ avidly accumulate azithromycin ➥➥➥➥ develop lysosomal phospholipidosis ➥➥➥➥ extensively characterized system
●●●● Experimental test Fluid-phase endocytosis
Azithromycin, a lysosomotropic antibiotic, impairs fluid-phaseendocytosis in cultured fibroblastsD. Tyteca, P. Van Der Smissen, F. Van Bambeke, K. Leys, P.M. Tulkens, P.J.Courtoy & M.-P. Mingeot-LeclercqEur. J. Cell Biol. 80: 466-478 (2001)
➀➀➀➀ Could AZ affect earlier steps of the endocytic apparatus ?
December 4th, 2001D. Tyteca - PhD Thesis 15
HRP
(a)
(b)
lateendosome
lysosome
earlyphagosome
latephagosome
zipper
trigger
sortingendosome
non-coated pit
recyclingcompartment
coated pit
(Cupers etal, 1994)
Fluid-phase endocytosis➥➥➥➥ horseradish peroxidase (HRP)
December 4th, 2001D. Tyteca - PhD Thesis 16
●●●● General experimental protocol
confluent cells
pretreatment with AZ (from 0 to 3 days)
incubation with the endocytic tracer (from 0 to 4 h)
washing, recovering and sonication
assays:➥➥➥➥ endocytic tracer➥➥➥➥ proteins➥➥➥➥ AZ➥➥➥➥ phospholipids
December 4th, 2001D. Tyteca - PhD Thesis 17
control cells
cells pretreatedwith AZ
AZ slows down fluid-phase endocytosis
December 4th, 2001D. Tyteca - PhD Thesis 18
Inhibition of fluid-phase endocytosis correlates with AZcontent
but is independent of phospholipidosis
December 4th, 2001D. Tyteca - PhD Thesis 19
CT
3 h AZ
3 days AZ
5 min HRP 2 h HRP
AZ causes a major reduction of the number of endosomes and lysosomes andimpairs accessibility of HRP to swollen and overloaded endosomes/lysosomes
December 4th, 2001D. Tyteca - PhD Thesis 20
Azithromycin inhibits clathrin-independent pinocytosis and slowsdown sequestration of ligand-receptor complexes into endocytic andrecycling vesicles of J774 macrophagesD. Tyteca, P. Van Der Smissen, M. Mettlen, F. Van Bambeke, P.M. Tulkens, M.-P. Mingeot-Leclercq & P.J. CourtoySubmitted for publication
●●●● Selection of experimental system J774 mouse macrophages
➥➥➥➥ homogeneous cell line ➥➥➥➥ high endocytic activity ➥➥➥➥ well-characterized system for pinocytosis and phagocytosis
●●●● Experimental tests ➥➥➥➥ fluid-phase endocytosis ➥➥➥➥ bulk-membrane endocytosis ➥➥➥➥ receptor-mediated endocytosis ➥➥➥➥ phagocytosis
➁➁➁➁ Is AZ specific to fluid-phase endocytosis ?
December 4th, 2001D. Tyteca - PhD Thesis 21
HRPLY (a)
(b)
lateendosome
lysosome
earlyphagosome
latephagosome
zipper
trigger
sortingendosome
non-coated pit
recyclingcompartment
coated pit
(Cupers etal, 1994;Swanson etal, 1987)
Fluid-phase endocytosis ➥➥➥➥ HRP and lucifer yellow (LY)
December 4th, 2001D. Tyteca - PhD Thesis 22
AZ
HRP
LY
AZ inhibits fluid-phase endocytosis and this inhibition isreversible
December 4th, 2001D. Tyteca - PhD Thesis 23
lateendosome
lysosome
earlyphagosome
latephagosome
zipper
trigger
sortingendosome
non-coated pit
recyclingcompartment
coated pitaggregates
ofN-Rh-PE
Bulk-membrane endocytosis➥➥➥➥ N-rhodamine-phosphatidylethanolamine (N-Rh-PE)
(a)
(Kok et al, 1990)
December 4th, 2001D. Tyteca - PhD Thesis 25
Receptor-mediated endocytosis➥➥➥➥ 125I-labelled transferrin
(a)
TfTfRY
lateendosome
lysosome
earlyphagosome
latephagosome
zipper
trigger
sortingendosome
non-coated pit
recyclingcompartment
coated pit
YY
YYY
Y
YY YY
Y
Y
(b)
(c)
YY
YY
YY
YY
(Cupers et al, 1994)
December 4th, 2001D. Tyteca - PhD Thesis 26
AZ strongly decreases the surface-pool of transferrinreceptors
December 4th, 2001D. Tyteca - PhD Thesis 27
(b)
(c)
AZ delays sequestration of ligand/receptor in endocytic pits andrecycling vesicles
December 4th, 2001D. Tyteca - PhD Thesis 28
PAPFc?R
Receptor-mediated endocytosis➥➥➥➥ PAP immune complexes
Y
lateendosome
lysosome
earlyphagosome
latephagosome
zipper
trigger
sortingendosome
non-coated pit
recyclingcompartment
coated pit
(a)
Y
YY
YY YY Y
(b)
YYY
YY
Y
Y
Y
Y YY
Y
Y
(Mellman et al,1984; Kiss andRohlich, 1984;
1987)
December 4th, 2001D. Tyteca - PhD Thesis 29
(a) (b)
AZ marginally decreases the surface-pool of Fc? receptors anddelays sequestration of ligand/receptor complexes into endocytic pits
December 4th, 2001D. Tyteca - PhD Thesis 30
lateendosome
lysosome
earlyphagosome
latephagosome
zipper
trigger
sortingendosome
non-coated pit
recyclingcompartment
coated pit
(a)
Phagocytosis➥➥➥➥ latex beads of 1 and 0.1 µm
(Pratten andLloyd, 1986)
beads
December 4th, 2001D. Tyteca - PhD Thesis 32
HRP(fluid-phase endocytosis)
PAP(receptor-mediated endocytosis)
latex beads (phagocytosis)
CT 3 h AZ
AZ impairs accessibility of HRP and PAP, but not of latex beads,to swollen endosomes/lysosomes
December 4th, 2001D. Tyteca - PhD Thesis 33
Latex beads move within the structures vacuolated by AZ,demonstrating their presence in these structures
December 4th, 2001D. Tyteca - PhD Thesis 34
Does AZ perturb endocytosis by:
●●●● a general toxic effect ? NO
●●●● phospholipidosis ? NO
●●●● AZ accumulation ? YES
Interpretation
December 4th, 2001D. Tyteca - PhD Thesis 35
➥➥➥➥ pH neutralization of endosome/lysosome ?
➥➥➥➥ swelling of endosome/lysosome ?
➥➥➥➥ membrane interaction ?
AZ accumulation ...
December 4th, 2001D. Tyteca - PhD Thesis 36
Azithromycin, a macrolide antibiotic that impairs endocytictrafficking, directly interacts with biomembranes and perturbs theirorganization and fluidityD. Tyteca, A. Schanck, Y. F. Dufrêne, M. Deleu, P.J. Courtoy, P.M. Tulkens &M.-P. Mingeot-Leclercq(to be submitted)
●●●● Selection of experimental system ➥➥➥➥ liposomes ➥➥➥➥ Langmuir-Blodgett monolayers ➥➥➥➥ J774 mouse macrophages
●●●● Experimental tests➥➥➥➥ interaction with membranes ➥➥➥➥ membrane organization in domains➥➥➥➥ insertion of membrane probes in the plasma membrane➥➥➥➥ membrane fluidity
➂➂➂➂ How does AZ inhibit pinocytosis ?
December 4th, 2001D. Tyteca - PhD Thesis 37
interaction of AZ with membranes➥➥➥➥ 31P nuclear magnetic resonance (NMR)
100 50 0 -50 -100ppm
s ’////////
s ’⊥⊥⊥⊥
= s ’//////// - s ’⊥⊥⊥⊥ ’s
s ’i
100 50 0 -50 -100ppm
s ’⊥⊥⊥⊥
= s ’i -’s eff. s ’⊥⊥⊥⊥
December 4th, 2001D. Tyteca - PhD Thesis 38
pH 5.4
pH 7.0
pH 6.0
AZ interacts with lipids of liposomes made ofcholesterol: PC: SM: PI in a pH-independent fashion
December 4th, 2001D. Tyteca - PhD Thesis 39
A B
C D
AZ interacts with lipids and perturbs the organization ofDPPC: cholesterol Langmuir-Blodgett monolayers
membrane organisation in domains➥➥➥➥ atomic force microscopy (AFM)
December 4th, 2001D. Tyteca - PhD Thesis 40
insertion of membrane probes in the plasma membrane of J774
➥➥➥➥ monomers; external leaflet of the PM
➥➥➥➥ monomers; deep in the PM
➥➥➥➥ aggregates
AZ reduces incorporation in the plasma membrane ofthree membrane tracers
December 4th, 2001D. Tyteca - PhD Thesis 41
2 p3 - p
r =TMA-DPH
plasma membrane fluidity of J774➥➥➥➥ fluorescence anisotropy of TMA-DPH
December 4th, 2001D. Tyteca - PhD Thesis 43
lateendosome
lysosome
earlyphagosome
latephagosome
zipper
trigger
sortingendosome
non-coated pit
recyclingcompartment
coated pitHRPLY
Fluid-phase endocytosis
Rh-PE
Bulk-membrane endocytosis
TfPAP
Y
YY Y
YY YY
Y
YYY YYYY
Y
Receptor-mediated endocytosis
beads
Phagocytosis
General conclusion
December 4th, 2001D. Tyteca - PhD Thesis 44
AZ decreases membrane fluidity(J774 plasma membrane)
AZ perturbs membrane organization in domains(monolayers)
AZ decreasesincorporation of:
N-Rh-PE,C6-NBD-SMTMA-DPH
(J774 plasmamembrane)
AZ interacts withphospholipidheadgroups(liposomes)
December 4th, 2001D. Tyteca - PhD Thesis 45
➥➥➥➥ Mechanism of AZ action:
●●●● role of membrane properties
➥➥➥➥ membrane fluidity, tension, transverse asymmetry and
composition
➥➥➥➥ receptor mobility in the plasma membrane and
incorporation into coated pits
●●●● role of vacuolation
●●●● role of pH neutralization
Short-term perspectives
December 4th, 2001D. Tyteca - PhD Thesis 46
Usefulness of AZ
●●●● inhibition of selective endocytic modes/pathways/steps
study of a series of ligands, e.g. cholera toxin internalization
●●●● differential modulation by drug concentration
Practical application: mechanism and function
●●●● clathrin-independent endocytosis
●●●● recycling pathway
●●●● fusogenicity between endosomes and lysosomes
➥➥➥➥
➥➥➥➥
Long-term perspectives
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