Master thesis
The unconventional roles of autophagyrelated proteins in infections and immunity
By Philip Vkovski
Graduate School of Life Sciences
Master Infection and Immunity
Utrecht University
Supervisor: Dr. Jovanka Bestebroer,
Dept. Medical Microbiology, UMC Utrecht
Second reviewer: Dr. Fulvio Reggiori,
Dept. Cell Biology, UMC Utrecht
November 2012 – January 2013
The unconventional roles of autophagy-related proteins in infections and immunity
Table of contents
SUMMARY
3
1
4
INTRODUCTION
1.1
The cellular autophagy pathway
4
1.2
Autophagy in immunity and microbial adaptations
8
1.3
Scope of the thesis
2
THE UNCONVENTIONAL, NON-AUTOPHAGIC ROLES OF AUTOPHAGY PROTEINS
12
13
2.1
LC3-associated phagocytosis
13
2.2
Murine norovirus replication inhibition
16
2.3
Toxoplasma gondii’s vacuole disruption
18
2.4
Assisting bone resorption by osteoclasts
19
2.5
Regulation of STING trafficking upon dsDNA stimulation
21
3 SUBVERSION OF ALTERNATIVE PATHWAYS INVOLVING AUTOPHAGY PROTEINS BY
MICROORGANISMS
23
3.1
Coronaviruses
23
3.2
Chlamydia
26
3.3
Brucella
27
4
DISCUSSION
29
5
ACKNOWLEDGEMENTS
34
6
REFERENCES
35
2
The unconventional roles of autophagy-related proteins in infections and immunity
Summary
Autophagy is a well-known, evolutionary conserved catabolic pathway leading to the
degradation of large cytoplasmic elements by the lysosome. Autophagy-related gene (Atg)
protein
complexes
catalyze
the
formation
of
double-membrane
vesicles
called
autophagosomes. Bulk cytosol, protein aggregates and damaged organelles but also
cytoplasmic microbes such as bacteria, viruses and parasites are sequestered into doublemembrane autophagosomes and delivered to the lysosome where they are degraded. Hence,
autophagy is essentially induced upon stress situations while also providing protection
against intracellular pathogens, adding to the cellular innate immune arsenal. In turn, several
pathogens have evolved strategies to evade the autophagy pathway or even manipulate it to
sustain their intracellular life cycle.
In addition to their traditional role in autophagosome formation, recent finding have
attributed novel functions, which are distinct from the classical autophagy pathway, to
autophagy-related protein complexes. Autophagy-independent roles of Atg proteins include
the maintenance of cellular homeostasis and resistance against invading pathogens. As such,
autophagy proteins have been shown to assist and enhance the degradation of dead cells,
bacteria and parasites upon their macroendocytic engulfment. It was also demonstrated that a
subset of Atg proteins has a direct antiviral function by inhibiting Murine norovirus’
replication complex. Moreover, bone resorption by osteoclasts, innate immune regulation
upon double-stranded DNA-triggered signaling and the ER-associated degradation pathway
regulation all have in common the requirement of some of the Atg proteins in an
autophagosome formation-independent manner. On the other hand, microorganisms, such as
coronaviruses, Chlamydia or Brucella, have evolved ways to manipulate and benefit from
autophagy-independent functions of autophagy-related proteins in order to ensure the
completion of their intracellular life cycle.
These novel mechanisms that involve autophagy-related proteins independently from
their role during canonical autophagosome formation add to the functional repertoire of Atg
proteins and extend the cellular processes in which autophagy proteins are implicated.
3
Introduction
1 Introduction
1.1 The cellular autophagy pathway
Autophagy, literally “self-eating”, is a catabolic pathway, highly conserved in eukaryotes
from yeast to mammals, that sequesters large cytoplasmic elements into double-membrane
vesicles and delivers them to the lysosome for degradation. Autophagy guarantees
cellular/cytoplasmic homeostasis by disposing of long-lived proteins or protein aggregates as
well as old or damaged organelles such as mitochondria, peroxisomes and parts of the ER
(118). Autophagy was initially found to be induced in response to starvation (148). Nutrients
are therefore supplied after non-specific cytosol autodigestion and the reuse of
macromolecules resulting from this degradation. Generally, autophagy has been described as
a major stress-response process induced by nutrient and growth factor deprivation, ER and
oxidative stress as well as immune cell activation (74). Moreover, autophagy is implicated in
various human diseases (80).
There are three distinct types of autophagy: chaperone-mediated autophagy
(translocation of cytoplasmic proteins across the limiting membrane of the lysosome),
microautophagy (invagination of the lysosomal membrane engulfing part of the cytoplasm),
and macroautophagy. Macroautophagy is a rapid membrane remodeling process leading to
the sequestration of parts of the cytoplasm into so-called double-membrane autophagosomes
(138). As macroautophagy is the subject discussed in this thesis, it will hereafter be referred
to as autophagy.
Figure 1 shows the succession of events leading to the formation of autophagosomes.
Autophagosomes are generated at the phagophore assembly site (PAS), where the initial
isolation membrane elongates and expands, selectively or non-selectively engulfing the cargo
to be degraded. The exact origin of the autophagosomal membranes remains elusive, but
evidence points to multiple possible sources, including the ER or the outer mitochondrial
membranes as well as the plasma and nuclear membranes (86). Eventually, the fusion of the
ends of the membranes gives rise to the characteristic double-membrane autophagosome that
encloses the cargo and undergoes maturation (acquisition of lysosomal markers such as Rab7,
LAMP1/2, vSNARES). The outer membrane of the autophagosome then fuses with the
4
Introduction
lysosome thus generating autophagolysosomes in which the inner membrane and the
contained cargo are degraded (80).
Figure 1. Overview of the autophagy pathway and its regulation.
The bottom diagram is a schematic representation of the events leading to the formation of autophagosomes. Upon induction
of the autophagy pathway, the formation of autophagosomes is initiated at the PAS. Next, the two ends of the isolation
membrane elongate and selectively or randomly engulf the cargo. After the completion of the autophagosome, it undergoes
maturation and eventually fuses with lysosomes, generating the autophagolysosomal compartment where the cargo is
degraded. The top right box shows the molecular cascades responsible for autophagosome formation. Activation of the ULK
complex leads to its translocation to the PAS where it will recruit the other components of the core autophagy machinery.
The class III PI3K complex generates PI3P, which attracts PI3P-binding proteins that mediate the formation of the isolation
membrane. Elongation and enclosure are mediated by both the Atg12- and LC3-conjugation systems, which ultimately lead to
the lipidation of LC3 onto the inner and outer autophagosomal membrane. The remaining panels highlight stimuli inducing
(green) or suppressing (red) autophagy and several targets for cargo recognition. Reprinted from (80).
5
Introduction
The discovery of the autophagy-related genes (Atg) in yeast and its mammalian
homologues has allowed the molecular characterization of the autophagy process and the
description of the succession of events required for the completion of autophagosomes
(Figure 1, top right panel). Sixteen Atg proteins, grouped in five functional complexes,
compose the core autophagy machinery (Table 1)(118, 149).
Role of complex
Complex
ULK complex
Initiation of
autophagosome
formation
Class III PI3K
complex
Others
ATG12
conjugation
system
Elongation of
autophagosome
LC3conjugation
system
Specific proteins
General properties
ULK1/2
ATG13
FIP200
ATG101
protein kinase, phosphorylated by mTORC1
Phosphorylated by mTORC1
Scaffold for ULK1/2 and ATG13
Interacts with ATG13
VPS34
VPS15
Beclin1
ATG14
AMBRA1
UVRAG
Rubicon
ATG2
PI(3) kinase
myristoylated
BH3-only protein, interacts with and is negatively regulated by BCL2 and BCL-XL
Autophagy-specific subunit
Interacts with and activates Beclin1
A VPS38 homologue; interacts with class C VPS (HOPS) complex
Interacts with Beclin1
interacts with Atg18 in yeast
ATG9
WIPI1-4
DFCP1
VMP1
ATG12
ATG7
ATG10
ATG5
transmembrane protein
PtdIns(3)P-binding protein
PtdIns(3)P-binding ER protein
transmembrane protein
Ubiquitine-like, conjugates to ATG5
E1-like enzyme
E2-like enzyme
Conjugated by ATG12
ATG16L1
LC3 (GATE16, GABARAP)
ATG4A-D
ATG7
ATG3
Interacts with ATG5
Ubiquitine-like, conjugates to PE
LC3 carboxy-terminal hydrolase, deconjugating enzyme
E1-like enzyme
E2-like enzyme
Table 1. Key autophagy proteins and their organization in functional complexes. Adapted from (80).
In mammals, autophagy is initiated by the UNC-51-like kinase (ULK) complex, composed
of Atg13, FIP200, Atg101 and ULK1/2 (75). Mammalian target of Rapamycin complex 1
(mTORC1) negatively regulates the ULK complex by phosphorylating ULK1/2 and Atg13 (63).
Inactivation of mTORC1 leads to the translocation and activation of the ULK complex from the
cytosol to the PAS, where it recruits other elements essential for the autophagosome
formation such as class III phosphatidylinositol-3-OH kinase (PI3K) (80).
Vps34 is the only class III PI3K in eukaryotes responsible for the formation of
phosphatidylinositol 3-phosphate (PI3P) and is found in complex with Vps15, Beclin1 and
Atg14L (also known as Beclin1-associated autophagy-related key regulator) (149).
6
Introduction
The generation of PI3P at the PAS attracts PI3P-binding proteins, such as WD repeat
domain phosphoinositide-interacting proteins and double FYVE domain-containing proteins,
that mediate the formation of the isolation membrane (80). In contrast, the Vps34 complex is
involved in autophagosome maturation when UV radiation resistance-associated gene
(UVRAG) is recruited to the complex instead of Atg14L (57).
Another essential protein involved in autophagosome formation is Atg9. Atg9 is the only
transmembranous Atg protein in mammals. It cycles between nascent autophagosomes, the
trans-Golgi compartment and late endosomes. Atg9 has been named membrane shuttle as it
might be a membrane supplier. It play roles in the isolation membrane formation but its
precise functions still need to be documented (75, 85).
Elongation and enclosure of the isolation membrane are the following steps in
autophagosome formation. They are mediated by two distinct but interconnected ubiquitinlike conjugation systems. The first complex is generated by the actions of Atg7 (E1-like) and
Atg10 (E2-like) that result in the conjugation of Atg5 and Atg12, which further bind Atg16L1
to form the Atg5-Atg12/Atg16L1 complex (75). This complex is essential for autophagosome
formation and plays an additional role (E3-like enzyme) in the conjugation of the second
ubiquitin-like system and its localization (45). The latter is the conjugation of
phosphatidylethanolamine (PE) to the yeast Atg8 homologs, which are represented by the
microtubule-associated protein light-chain 3 (LC3) and γ-aminobutyric acid receptorassociated protein (GABARAP) subfamilies (75). After its synthesis, LC3 becomes processed at
the C-terminus by Atg4 and is designated as cytoplasmic non-lipidated LC3-I. LC3-I becomes
conjugated to PE at its C-terminus by Atg3 and Atg7 (E2-like enzymes) and gets incorporated
into both the inner and outer membrane of the autophagosome. The PE-lipidated form of LC3
is known as LC3-II. The GFP-tagged LC3-II (GFP-LC3) is widely used as an autophagosomal
marker, as induction of autophagy converts diffuse cytoplasmic LC3 to membrane-bound LC3
puncta (93). LC3 and its mammalian homologs are not only important for the formation of the
autophagosome but also for the recognition and selection of the cargo and the definition of
the autophagosome’s size (102, 146). In addition, LC3 is implicated in microtubule-dependent
transport of autophagosomes towards lysosomes and possesses membrane-fusion properties
as well (95, 99).
7
Introduction
The characterization of how autophagy proteins are regulated and how formation of
autophagosomes are formed (Figure 1) came with methods and agents that, besides the
knockdown, knockout or double-negative constructs of essential Atg proteins, induce or
inhibit the autophagy pathway. The most obvious way to induce autophagy is by starving
cells; this is how autophagy was identified in the first place. The insufficient nutrient
availability inhibits mTORC1, which in turn allows the activation of the entire cascade
initiated by ULK1. mTORC1, as its name suggests, is inhibited by Rapamycin, which thus also
induces the same activation effect. On the other hand, wortmannin and 3-methyladenine
inhibit autophagy by interfering with the activity of PI3K (144).
Chloroquine is a
lysosomotropic agent leading to increased endosomal and lysosomal pH, therefore blocking
the degradation of autophagy cargos by the lysosome (93). Additional ways to manipulate and
monitor autophagy have been summarized elsewhere (69, 93).
1.2 Autophagy in immunity and microbial adaptations
As mentioned in the previous section, autophagy is responsible for the random or
selective sequestration and processing of bulk cytosol and damaged/old organelles or
aggregated proteins, respectively. Moreover, autophagy has been identified as an essential
player in combating bacterial, viral and protozoan infections in a pathogen-selective, cellautonomous manner termed xenophagy. Autophagy also bridges adaptive and inflammatory
immune responses. On the other hand, the strong selective pressure provided by the host
immune response has driven microbes to evolve strategies to antagonize xenophagy. In some
cases, manipulation of autophagy by pathogens even has probacterial or proviral effects.
Pathogens are traditionally known to be cleared by innate immunity through
phagocytosis, which is a conserved process by which cells take up and digest pathogens, but
also particulate material or dead cells from the extracellular space. Specific receptors, such as
Fcγ receptors, complement receptors or scavenger receptors, first bind the particle to be
phagocytosed either directly or via molecules that have opsonized the cargo (antibodies,
complement). Subsequently, they orchestrate its internalization into a vacuole called
phagosome. Phagosomes undergo maturation and ultimately fuse with the lysosome, hence
becoming an acidic, hydrolase-containing phagolysosomes that digest their contents (40, 42,
135, 142). The latter process is similar to the degradation of autophagosomal cargo upon
8
Introduction
lysosomal fusion. Professional phagocytes, such as macrophages, neutrophils and dendritic
cells, are part of the innate immune arsenal against invading pathogens and are capable of
recognizing pathogen-associated molecular patterns via pattern recognition receptors (PRR)
(56). They subsequently elicit an innate immune response that kills the pathogen and induces
local inflammation, and provide a bridge to adaptive immune responses against the invaders.
Conserved patterns displayed by pathogens engage with Toll-like receptors (TLR) that are
located either at the surface of the plasma membrane or on internal membranes (67). TLR
signaling pathways not only lead to modulation of phagocytosis by enhancing the rate of
engulfment and fusion with the lysosomes, but also elicit an appropriate innate and adaptive
immune response.
Pathogens such as bacteria have evolved several strategies to antagonize
phagolysosomal delivery and avoid degradation. One such strategy is by blocking phagosomal
maturation and modification of this compartment that then supports replication of the
bacterium (Figure 2D, E). This is the case, for instance, for Mycobacterium tuberculosis and
Salmonella typhimurium (90). Another way employed by bacteria to survive in the host cell is
by escaping into the cytoplasm where replication can take place, as exemplified by Listeria
monocytogenes and Shigella flexneri (Figure 2B) (6, 20). Pathogens evading the
phagolysosomal degradation pathway are, however, subjects to autophagy. It has indeed
been shown that signaling through TLR activates the cellular autophagy pathway (147).
Autophagy hence provides a secondary protection level in case pathogens would escape
killing upon phagocytosis (24, 55). Thus, bacteria residing in (potentially damaged) modified
vacuoles are targeted by autophagy. This results in multi-membrane structures that
eventually deliver their content to the lysosome (Figure 2D, E) (9, 43). Cytosolic bacteria are
subjects to xenophagy as well (103, 115) (Figure 2B). They are specifically captured by the
autophagic system after being ubiquitinated or marked by other molecular tags (Figure 1,
middle right panel). These tags are linked to LC3 at the autophagosomal membranes via
adaptors such as p62, nuclear dot protein 52 or neighbor of BRCA1 gene 1 (73). In turn,
pathogenic intracellular bacteria are equipped with mechanisms to avoid recognition or the
recruitment of the autophagy machinery. Furthermore, some studies describe that in some
cases autophagy even promotes bacterial replication and that pathogenic bacteria manipulate
and exploit this compartment for their own benefit (27, 97, 126).
9
Introduction
Figure 2. Role of autophagy and autophagy proteins in the clearance of invading pathogens.
Viruses enter the host cell via the endocytic pathway and escape into the cytoplasm where they replicate. (A) Autophagy
proteins recognize viral components and eliminate them though xenophagic degradation. Phagocytosed bacteria can escape
phagosomes to avoid lysosomal delivery. (B) Cytoplasmic bacteria are targeted by the autophagy machinery for xenophagic
degradation. (C) The phagosomal membrane remnants are also discarded by autophagosomes to prevent unnecessary
immune activation. (D, E) Some bacterial species inhibit phagosomal maturation and reside in modified vacuoles, which can
be damaged (D) or intact (E). The targeting of such compartments by autophagy results in multi-membrane compartments
that are delivered to the lysosome. (F) Finally, bacteria-containing phagosomes can fuse with already-formed
autophagosomes what delivers the invader to the lysosomal compartment as well. (G) In some cases, autophagy proteins are
directly recruited to the phagosomal membrane, independently from the formation of characteristic double-membrane
autophagosomes. This leads to an enhanced phagosomal maturation and degradation of the phagocytosed cargo. In addition
to viruses and bacteria, parasites are also targets of the cellular autophagy. (H) Similarly to bacteria-containing modified
phagosomes, parasitophorous vacuoles are recognized by autophagosomes and subsequently degraded. (I) Killing of the
intracellular parasite can also be mediated by direct destruction of the pathogen-containing compartment, a process in which
autophagy proteins are importantly involved. Adapted and reprinted from (80).
Autophagy plays an evident role in antiviral defense as well. Viral detection upon virusreceptor interaction signaling, cellular stress (mostly ER) and binding to PRRs triggers
effector mechanisms leading to xenophagic degradation and antigen presentation (75)
10
Introduction
(Figure 2A). Viruses, however, have evolved strategies to impair autophagy by inhibiting key
regulators of the process. They have also been shown to manipulate this conserved pathway
for their own benefit (62). Direct roles of autophagy (but also indirect ones such as inhibition
of apoptosis or modulation of the cell metabolism) have been documented in basically every
step in the life cycle of viruses. Manipulation of autophagy to assist viral replication is the
most well-known case and has been documented for several species (27). Generally,
membranous structures of different origin are induced by RNA viruses as a niche to support
replication and prevent immune detection while providing a scaffold for the replication
machinery as well as a local concentration of essential factors necessary for the viral genome
synthesis (25, 62). Further, viruses induce and manipulate autophagy to assist their assembly,
envelopment and non-lytic release (62).
In addition to its cytoprotective and pathogen clearance activities, autophagy has been
shown to take part in a growing number of immune functions, notably in the adaptive and
inflammatory facets of immunity (26, 80). Autophagy, by its ability to bring cytosolic
components to the luminal side of the endocytic pathway, delivers pathogens residing in the
cytosol not only to the lysosomes but also to compartments containing PRR or class II major
histocompatibility complexes (MHC-II) (Figure 2F) (26). Autophagosomes have indeed been
shown to fuse with endosomes, multivesicular bodies or MHC-II-loading compartments (78,
125). Thus autophagy helps fighting against intracellular pathogens by contributing to
endogenous MHC-II peptide presentation. In addition, a role of autophagy in antigen
presentation has revealed crucial during T cell development, maturation and homeostasis (61,
100, 114). Autophagy functions in modulating the inflammasome’s activity by getting rid of
sources of danger associated molecular patterns such as, for example, reactive oxygen species
(ROS) or mitochondrial DNA that would activate the NLRP3-inflammasome (98, 154).
Moreover, pro-IL-1β or the entire activated inflammasomes can get degraded by
autophagosomes to limit inflammation (48, 127). However, autophagy can also assist
inflammatory cytokine secretion upon caspase activation and pro-IL-1β cleavage. In such a
case, IL-1β and other alarmins are unconventionally secreted outside of the cell depending on
the autophagy machinery (31). Finally, mutations in autophagy proteins have also been linked
to chronic inflammatory and autoimmune diseases (80, 138, 148).
11
Introduction
1.3 Scope of the thesis
Autophagy has long been considered uniquely as a degradative pathway, delivering
cytoplasmic components to degradative organelles. However, during the past years, literature
has emerged that offers new elements that escalate this narrow point of view. While
unconventional types of autophagy, which do not require all the components of the five
functional core complexes for autophagosome formation, have been described (101), the
recent findings that ATG proteins are implicated in pathways unrelated to autophagy has
broadened the scope of autophagy research even more (118). In contrast to conventional
routes of autophagy, several alternative processes involve some of the components of the
autophagy machinery in an autophagy-independent manner. This thesis will examine the
aspects of the unconventional use of autophagy-related proteins and has been organized in
the following way. On one hand, it will address how autophagy proteins aid in maintaining
cellular homeostasis in physiological conditions and during innate immune responses against
invading pathogens. Besides these beneficial and cytoprotective roles, it will also assess how
parts of the autophagy machinery are manipulated and hijacked by microorganisms in order
to assist viral or bacterial replication and completion of their intracellular life cycle.
12
The unconventional, non-autophagic roles of autophagy proteins
2 The unconventional, non-autophagic roles of autophagy proteins
In addition to their known role in autophagosome formation, some of the autophagy
protein complexes are involved in pathways unrelated to conventional autophagy. They play
essential roles in important pathways governing cellular homeostasis and resistance against
microbial infections. Autophagy proteins have been shown to assist macroendocytic
engulfment and degradation of dead cells as well as pathogens such as bacteria or protozoa.
Antiviral functions have also been attributed to autophagy protein complexes. Moreover,
autophagy proteins play a role in more curious cellular processes such as bone resorption by
osteoclasts, regulation of innate immune signaling and ER-associated degradation tuning.
2.1 LC3-associated phagocytosis
Autophagy is induced upon phagocytic uptake of extracellular bacteria and TLR
recognition and is responsible for the destruction of bacteria-containing vacuoles as well as
cytosolic bacteria that have escaped the phagolysosomal pathway (Figure 3 III). However,
autophagy proteins also participate in phagocytosed pathogen clearance in a mechanism
distinct from classical autophagy. LC3-associated phagocytosis (LAP) is a process by which
components of the autophagy machinery directly modify the phagosomal membrane to assist
and enhance degradation of phagocytosed elements (Figure 3 II) (124) .
Figure 3. Comparison between bacterial delivery to
the
lysosome
through
phagocytosis,
LAP
and
autophagy.
(I) Bacteria-containing phagosomes are traditionally
known to clear bacteria after fusing with early and late
endosomes
and
eventually
with
lysosomes.
(III)
Cytosolic or membrane-bound bacteria that prevent
lysosomal delivery are targeted by the autophagy system
and are degraded through xenophagy. (II) During LAP,
autophagy proteins are directly recruited to the
phagosomal
canonical
membrane
autophagy
independently
pathway
and
from
the
enhance
phagolysosomal degradation of the phagocytosed cargo.
Reprinted from (77)
13
The unconventional, non-autophagic roles of autophagy proteins
Sanjuan et al. (123) were the first to demonstrate that TLR signaling mediates the rapid
recruitment of LC3 to phagosomes. They observed that macrophages fed with latex beads
coated with TLR agonists, such as PAM3CSK4 (TLR1/2), LPS (TLR4), zymosan (TLR2), or E.
Coli recruited LC3 to the phagosomal membrane. This enhanced the degradation of the
phagocytosed cargo by a more rapid and extensive acidification of the phagosomal
compartment. Localized TLR signaling was necessary as no association of LC3 with
phagosomes was observed in TLR2 knockout macrophages having phagocytosed zymosancoated beads and in cells treated with PAM3CSK4 or LPS after having engulfed inert latex
beads. Zymosan induced the recruitment of LC3 to phagosomes in a manner dependent on
Atg5, Atg7 as well as PI3K. Beclin1 was almost instantly recruited to the phagosomal
membrane, prior to LC3. Consistently, phagosomal maturation and efficient killing of live
Saccharomyces cerevisiae was reduced in Atg7-deficient macrophages when compared to
survival of the yeast in controls (123).
In contrast to canonical autophagy, TLR-mediated LAP is not dependent on ULK1 after
engulfment of zymosan (88), nor is it affected by Rapamycin, Chloroquine or starvation
treatments. The absence of ER markers found on autophagosomes as well as double
membrane structures surrounding the engulfed material distinguish phagosomes having
recruited LC3 from conventional autophagosomes (123).
One example in which LAP was extensively studied is during Burkholderia pseudomallei
infections. B. pseudomallei are intracellular pathogenic bacteria that escape endosomes in
order to replicate in the cytoplasm after the induction of a type III secretion system (47, 134).
A first study investigating B. pseudomallei infections found a role of autophagosomes in
reducing bacterial survival in phagocytic and non-phagocytic cells (22). These conclusions
were mostly supported by the effects of autophagy-modulating drugs. Rapamycin and IFNγ
treatments, in contrast to wortmannin and 3-methyladenine, enhanced GFP-LC3
colocalization with bacteria and reduced bacterial survival in comparison to untreated
controls (22). However, a couple of years later, the same group suggested that B. pseudomallei
is subject to LAP rather than to conventional autophagy and that the previously observed
colocalization of LC3 with bacteria must result from recruitment of LC3 to the phagosomal
membrane. EM analysis revealed that although a lot of double membrane structures were
found in the cytoplasm of infected cells, these autophagosomes did not contain bacteria. B.
14
The unconventional, non-autophagic roles of autophagy proteins
pseudomallei were either surrounded by single membranes or were found free in the cytosol.
Autophagy of cytosolic or bacteria-containing phagosomes was hence excluded as no double
or multiple membranes, respectively, were observed around bacteria (Figure 3) (38).
A majority of B. pseudomallei escapes endosomes and is resistant to canonical autophagy
by a process that still needs to be described. On the other hand, bacteria that do not escape
are mostly found in phagosomes positive for both LC3 and the lysosomal marker LAMP1.
Consistently, B. pseudomallei type III secretion system mutants bopA and bipD are unable to
escape phagosomes and show increased colocalization with LC3 as well as LAMP1 when
compared to wild type bacteria, therefore suggesting an enhanced routing towards the
lysosome (38). Surprisingly, heat-killed B. pseudomallei display diminished recruitment of LC3
to phagosomes, indicating that live bacteria and signaling routes other than TLR activation
might be required for LAP to be induced (38).
The most recent findings in the field show that, in agreement with previous studies (22,
123), LC3-associated phagocytosis of B. pseudomallei is dependent on Beclin1 and is enhanced
by starvation and Rapamycin treatments and confirm that LAP is necessary for bacterial
retention in phagosomes and efficient killing. However, in contrast to starvation conditions
and untreated controls that depend on Beclin1, Beclin1 knockdown does not affect LAP in
Rapamycin treatments. This independency raises further questions with respect to
recruitment of different pools of LC3, Beclin1-independent autophagy and LAP as well as
alternative PI3P formation at the phagosomal membrane (82).
LC3-associated phagocytosis is not only employed as a cellular mechanism to combat
pathogen infections. Modifications of endocytic vesicles can also assist the removal of
apoptotic, necrotic or entotic cells. Cells that have undergone programmed cell death must be
cleared from the extracellular space in order to avoid unnecessary inflammation and
autoimmunity. Specific receptors on phagocytes recognize phosphatidylserine (PtdSer),
which is normally absent on the surface of healthy cells but exposed at the membrane after
apoptosis or necrosis (116). Engagement of PtdSer with TIM4 has been shown to be
responsible for the engulfment of apoptotic or necrotic bodies and mediates, by a yet
unidentified pathway, the rapid recruitment of LC3 to single membrane phagosomes
containing the dead cell bodies that further mature into phagolysosomes (88).
15
The unconventional, non-autophagic roles of autophagy proteins
Entosis is a novel form of nonapoptotic cell death by which an epithelial cell engulfs
another live cell that is then destroyed by lysosomal enzymes upon fusion of the entotic
vacuole with the lysosome (106). Similarly to apoptotic and necrotic cell disposal, the limiting
membranes of endosomes containing entotic cells rapidly and transiently recruit LC3 for
phagolysosomal delivery as evidenced by the localization of LAMP1 and cathepsins on and in
the vacuole, respectively (35).
Both entosis and clearance of apoptotic and necrotic cells are dependent on Atg5, Atg7
and LC3-II as well as on the presence of PI3K (VPS34) and Beclin1 on the limiting membrane
of the compartment. In contrast to classical autophagy, the preinitiation complex containing
ULK1 or FIP200 is however not required. LAP enhances lysosomal fusion and degradation of
dead and entotic cells. It further reduces secretion of inflammatory cytokines such as IL-1β
and IL-6 upon phagocytosis of apoptotic and necrotic corpses while promoting the secretion
of anti-inflammatory factors IL-10 and TGFβ (35, 88).
Additionally,
gene-silencing experiments performed
in vivo
demonstrate
the
engagement of LAP in removal of apoptotic corpses during the embryonic development of
Caenorhabditis elegans. It was indeed shown that the VPS34 complex component BEC-1 was
required to simultaneously recruit the LC3 homologue LGG-1 and RAB5, a protein known for
its role in phagosome maturation and degradation of its cargo, to the phagosomal membrane
containing the apoptotic bodies (35, 81, 88).
The studies mentioned in this section have gone some way towards enhancing our
understanding of LAP. They all share the feature that LC3 and other autophagy proteins, once
recruited to the single macroendocytic membrane in a process distinct from canonical
autophagy, regulate the fusion of non-autophagic compartments with the lysosome. However,
how LAP is initiated in these different situations still needs to be clarified. Two extensive
reviews summarize the current knowledge in this young field of research (36, 77).
2.2 Murine norovirus replication inhibition
In addition to their known antiviral function in autophagosome formation (Figure 2A),
autophagy protein complexes have been found to play autophagy-independent roles by
inhibiting critical steps in the viral life cycle, as exemplified by the case of Murine norovirus
(MNV) infections.
16
The unconventional, non-autophagic roles of autophagy proteins
MNV is a non-enveloped single-stranded positive-sense RNA virus used as a model to
study human gastroenteritis caused by this viral family (143). IFNγ plays an essential role in
antiviral defense, especially in the absence of type I interferon IFN/β, which can play a
compensatory role (52, 122). Hwang et al. (52) demonstrated in a recent study that the Atg5Atg12/Atg16L1 complex, known to locate at the autophagosomal membrane where it
mediates elongation and closure of the autophagosome by assisting LC3 lipidation, was
necessary for IFNγ-mediated antiviral activity against MNV. Experiments showed that Atg5 is
required for MNV control in IFNγ treated macrophages (Figure 4); the absence of this
essential autophagy protein having neither effect on viral replication nor on IFNγ-induced
transcriptional changes and JAK-STAT signaling cascade. However, modulation of canonical
autophagy by a multitude of treatments does not affect MNV replication or IFNγ antiviral
activity, indicating that Atg5 is an autophagy-independent key factor for the antiviral activity
triggered by IFNγ (52).
Figure 4. Atg5-dependent control of MNV replication upon IFNγ stimulation of macrophages.
Western blot analysis of MNV polymerase (A) and capsid (B) protein expression in control (Atg5flox/flox) and Atg5-deficient
(Atg5flox/flox +LysMcre) macrophages after IFNγ treatment. Reprinted from (52).
Expression of Atg5 mutants that are unable to conjugate with Atg12 or bind to Atg16L1
cannot complement the lack of resistance against MNV infections in Atg5-deficient
macrophages. In addition, Atg16L1- and Atg7-deficient cells fail to control the virus upon IFNγ
treatments. Consistently, while Atg7 has a dual role in both assisting Atg5-Atg12 conjugation
and lipidation of LC3, the latter facet didn’t play a role in combatting MNV, as confirmed by
dominant negative constructs of Atg4B that block LC3 lipidation. Hence, these experiments
indicate the implication of the entire Atg5-Atg12/Atg16L1 complex in the antiviral defense
(Figure 5) (52).
17
The unconventional, non-autophagic roles of autophagy proteins
Atg16L1 localizes at replicative membranous
structures induced upon infection and associates
with the MNV polymerase. Further, Atg5 was shown
to be responsible for the inhibition of viral
polymerase expression (but not immediate viral
protein synthesis from incoming genome), synthesis
of positive and anti-sense viral genome as well as
structural protein synthesis (Figure 4). In a nutshell,
the function of Atg5-Atg12/Atg16L1 complex,
independently
from
its
role
in
canonical
autophagosomes formation, can be attributed to
inhibiting the viral replication complex formation in
consequence
to
macrophage
IFNγ-mediated
responses (52).
Figure 5. The Atg5-Atg12/Atg16L1 complex and
Atg7,
independently
from
their
role
in
autophagosome formation, are required for the
IFNγ-dependent inhibition of the formation of
MNV replication complex. Reprinted from (52).
2.3 Toxoplasma gondii’s vacuole disruption
Autophagy proteins are involved in autophagy-independent defense mechanisms
against intracellular protozoan parasites as well, as observed in Toxoplasma gondii infections
(Figure 2I).
T. gondii persists in non-activated macrophages by inhibiting, upon entry, the fusion of
the modified parasitophorous vacuole, in which it replicates, with the lysosome (129).
However, activated macrophages can limit replication, kill and clear the pathogen by two
independent ways (151), either by activation of autophagy in macrophages by CD40-CD40L
signaling (Figure 2 H) (5, 111) or by IFNγ/LPS-induced disruption and stripping of the
parasitophorous vacuole membrane followed by clearance of the damaged parasite and
membranous debris (Figure 2I) (83). The latter involves IIGP1, a p47 GTPase (87), and Atg5
that plays an autophagy-independent role in this process (152).
Zhao and colleagues (152) have shown that Atg5-deficient macrophages are unable to
clear T. gondii infections even when previously treated with IFNγ/LPS. Wild type IFNγ/LPSactivated cells show extensive vesiculation and stripping of the parasite-containing vacuolar
membranes followed by autophagosomal detection of free damaged parasites in the cytosol
18
The unconventional, non-autophagic roles of autophagy proteins
(Figure 6C, D). In contrast, vacuoles in Atg5-knockout macrophages remain intact (Figure 6A,
B). It further appeared that Atg5 deficiency hinders the rapid recruitment of IIGP1 to the
parasitophorous vacuole membrane. IIGP1 was instead severed in intracellular vesicles thus
raising speculation about a lack of trafficking to the T. gondii vacuole (152).
Figure 6. Atg5-dependent killing of T. gondii (Tg) residing in
parasitophorous vacuoles (PV).
Electro micrographs showing the lack of damage to the vacuole
containing
T.
gondii
in
Atg5-deficient
IFNγ/LPS-treated
macrophages (A, B) compared to control cells (C, D). Reprinted
from (152)
The cellular localization of LC3 in Atg5 knockouts and their control cells was regrettably
not investigated in this study. However, an earlier research reports an accumulation of LC3 at
disrupted parasitophorous vacuoles in vicinity of intense IIGP1 signals in infected IFNstimulated astrocytes (87). Furthermore, no neighboring membrane structures resembling
autophagosomes were detected by EM analysis, hence leaving intriguing unanswered
questions concerning the function of Atg5 and LC3 in immunity against T. gondii.
2.4 Assisting bone resorption by osteoclasts
LAP is not the only case in which cellular membranes are modified by LC3. This
phenomenon has also been encountered in osteoclasts, specialized multinucleated cells of
myeloid origin responsible for bone resorption, where LC3 decorates distinct areas of the
plasma membrane.
Osteoclasts play an essential role in bone tissue homeostasis (Figure 7). There is a fine
balance between bone formation and resorption in order to maintain skeletal integrity
(subject to growth and mechanical constraints), mineral homeostasis (calcium and
phosphate) and the bone marrow environment (11). The bone-apposed membrane of
19
The unconventional, non-autophagic roles of autophagy proteins
osteoclasts is sealed by an actin ring, the formation of which is regulated by microtubules and
small GTPases, such as Cdc42, delimitating an extracellular space referred to as resorptive
organelle (29, 58, 59). This membrane develops into a ruffled border that increases the
surface through which ions (mainly H+ and Cl-) are pumped into the resorptive organelle in
order to degrade the mineral matrix. Lysosomal enzymes, such as the cathepsin K collagenase,
digest the organic components of the bone; they are delivered through directional fusion of
secretory lysosomes with the ruffled border (21, 133).
Figure 7. Osteoclasts and bone resorption.
Osteoclasts are multinucleated cells responsible for bone
resorption. Their bone-apposed membrane, which is sealed by
an actin ring, is called the ruffled border and contains channels
and pumps through which ions are transported to the
extracellular space. Lysosomal enzymes are delivered to the
resorptive organelle through directional fusion of secretory
lysosomes and vesicles. Reprinted from (37).
In relation to autophagy, maturation of osteoclasts is associated with an increased
synthesis of LC3-II, Atg4B and Atg5, none of which, however, is correlated with enhanced
autophagic fluxes (17). Formation of the ruffled border, fusion of secretory lysosomes at the
ruffled border and bone resorption activity are dependent on the Atg5-Atg12 conjugates, Atg7
and Atg4B. This indicates an essential role of LC3-II, which localizes at the bone apposed
membrane in association with the actin ring but is not present on secretory lysosomes (28). In
addition, LC3 associates with microtubules in proximity of the actin ring and its knockdown
by siRNA affects the formation of the actin belt as well as, similarly to the other autophagy
proteins mentioned above, cathepsin K secretion and consequently the bone resorption
activity of osteoclasts (17).
Cdc42 is located near the actin ring as well, where it colocalizes with LC3. Its activity and
location are both regulated by Atg5 and LC3 (17). Similar results have been obtained for Rab7,
another small GTPase involved, among others, in the generation of the ruffled border (28,
150). While DeSelm et al. (28) suggest that LC3-II decorated membranes are targets for fusion
with lysosomes, as reminiscent to autophagosomal and LC3-associated phagosome fusion
20
The unconventional, non-autophagic roles of autophagy proteins
with lysosomes, Chung and coworkers (17) provide evidence that LC3 has a role in the actin
ring formation by specifically regulating Cdc42 and providing a link between microtubules,
which are known to be essential for the initiation of the actin ring formation, and Cdc42 (30).
Hence LC3 has a role, unrelated to autophagy, in the formation of the actin belt and
formation of the ruffled membrane thus enabling secretory lysosomes to release their bonedegrading components after fusion at the bone apposed plasmalemma in osteoclasts.
2.5 Regulation of STING trafficking upon dsDNA stimulation
Some autophagy proteins have been shown to intervene in signal transduction
pathways, notably in the activation of Stimulator of interferon genes (STING) upon dsDNA
detection. However, instead of acting as immune effectors, here, autophagy proteins execute a
regulatory function.
Cytoplasmic nucleic acids are recognized by TLR (TLR 3, 7, 8, 9) or RIG-I-like receptors
that subsequently mediate innate inflammatory immune responses. Concerning the detection
of double-stranded DNA (from bacterial or DNA virus origin), AIM2 signaling through the
inflammasome complex and activation of Caspase-1 is responsible for the production of
proinflammatory cytokines IL-1β and IL-18 (50). On the other hand, STING has been
identified for initiating responses leading to IFN gene transcription (13). A lot about the
localization, trafficking, signaling pathways and recognized nucleic acid targets of STING is
still unknown and necessitates further investigation. Nevertheless, it has been shown that
upon dsDNA recognition, STING translocates from the ER, where it originally resides, to the
Golgi apparatus before associating with TBK1 and p62 in cytoplasmic concentrated foci (121).
No localization of STING on endosomes, lysosomes and peroxisomes has been observed.
Phosphorylation of IRF3 as well as STAT6 leads to their translocation into the nucleus and
activation of IFN gene transcription (13).
Colocalization of Atg proteins with STING was examined upon stimulation of MEF cells
with dsDNA. LC3 and Atg9a colocalized with STING whereas ULK1, Atg5 and Atg14L did not.
Atg7 and Atg16L1 knockout cells supported normal activation of membrane trafficking of
STING upon dsDNA stimulation and secreted IFNβ levels similar to controls. Atg9a is the only
transmembrane Atg protein and is supposed to provide membranes for autophagosome
formation by shuttling between the Golgi, endosomes and autophagosomes. Atg9a-knockout
cells are deficient in autophagy. However, in response to dsDNA stimulation, cytoplasmic
21
The unconventional, non-autophagic roles of autophagy proteins
TBK1-associated STING puncta were largely increased in Atg9a-deficient cells, as were IRF3
phosphorylation and IFNβ, IL-6 and CXCL10 production (121). This aberrant activation of
downstream signaling indicates that a minor subset of Atg proteins, independently from their
implication in autophagosome formation, plays a role in the regulation of innate signaling
upon detection of dsDNA. The exact mechanisms of such a modulation, however, still need to
be elucidated.
22
Subversion of alternative pathways involving autophagy proteins by microorganisms
3 Subversion of alternative pathways involving autophagy proteins by
microorganisms
Pathogens are known to usurp essential cellular pathways during their intracellular life
cycle. Whereas subversion of classical autophagosomes has been extensively described, the
recently-published experiments discussed in this section point out cases in which microbes
co-opt, for their own benefit, part of the autophagy machinery in a manner that is unrelated to
canonical autophagy.
3.1 Coronaviruses
Coronaviruses, such as Severe Acute Respiratory Syndrome (SARS)-CoV or the model
virus Mouse Hepatitis Virus (MHV), have been shown to hijack vesicles derived from the ER in
order to form their replicative structures. Autophagy-related proteins are essential to this
process whereas the latter does not depend on the induction of conventional autophagy
pathways.
Coronaviruses are enveloped viruses containing a large single-stranded, positive-sense
RNA genome. They belong the order of the Nidovirales and to the family of the Coronaviridae
(89). After entry, MHV and SARS-CoV induce the formation of double membrane vesicles
(DMV) to which they target their replication and transcription complex (RTC) (39). The exact
source of DMVs membranes still remains elusive although several lines of evidence support
an ER origin. Nsp3-4, which are components of the RTC, are N-glycosylated and ectopic
expression of nsp4 is localized to the ER (46, 66, 104, 105). In addition, inhibition of the ER to
Golgi transport reduces the amount of DMVs (140). Ultrastructural studies show that DMVs
and convoluted membranes are interconnected in a reticular network continuous with the ER
while some ER markers were found on DMVs as well (71, 130, 139).
A possible role of autophagy in the generation of DMV was proposed, as their double
lipid bilayers morphology resembles that observed for autophagosomes (23). However,
conflicting results on the recruitment of LC3 to DMV membranes and the requirement of Atg5
for virus replication subsequently appeard (112, 113, 130, 153). An element of answer was
brought by a series of experiments showing that MHV hijacks LC3-I-coated ER-derived
vesicles known to be involved in the regulation of ER-associated degradation (ERAD) (120).
23
Subversion of alternative pathways involving autophagy proteins by microorganisms
Hence, MHV manipulates a subset of autophagy-related proteins for DMV formation and viral
replication without depending on canonical autophagosomes.
ER-associated degradation (ERAD) is a cellular pathway responsible for the degradation
of folding-defective polypeptides generated in the ER (91). Upon recognition of their modified
sugar chains by ERAD lectins, misfolded polypeptides are extracted from the calnexinchaperone system, retrotranslocated into the cytosol, i.e. via the E3 ubiquitin ligasecontaining HRD1 dislocon, and targeted for proteasomal degradation (Figure 8) (2, 91, 136).
ER degradation-enhancing alpha-mannosidase-like 1 (EDEM1) and osteosarcoma
amplified 9 (OS-9) are stress-inducible regulators of protein disposal by ERAD (2, 94). Unlike
the vast majority of ER resident chaperones, EDEM and OS-9 are short lived and rapidly
degraded by the lysosome (Figure 8). This mechanism is independent of coat protein complex
II but dependent on Atg5, thus suggesting a role for autophagy. EDEM and OS-9 were found to
locate in small ER-derived, LC3-I-coated vesicles named EDEMosomes and do not colocalize
with classical ER markers. These vesicles are distinct from autophagosome populations and
induction of autophagy by starvation reduces EDEM disposal (possibly by reducing LC3
availability), suggesting an alternative pathway to conventional autophagy (14).
Figure 8. Schematic representation of the ERAD tuning pathway.
EDEM1 and OS9 are short-lived regulators of protein disposal by ERAD that are constitutively removed from the ER and
degraded by the lysosome in a process known as ERAD tuning. In the absence of misfolded proteins, EDEM1 and OS9 bind
SEL1L that otherwise that otherwise stabilize the HRD1 dislocon and engage ERAD. SEL1L recruits LC3-I, which assists the
sequestration of EDEM1 and OS9 in vesicles called EDEMosomes. These vesicles are distinct from traditional
autophagosomes and activation of the autophagy pathway is not required for their generation. Coronaviruses have been
shown to manipulate the ERAD tuning pathway and manipulate EDEMosomes in order to form their replicative DMVs while
preventing the maturation of EDEMosomes and the eventual fusion with the lysosome (red arrows). Adapted from (119).
24
Subversion of alternative pathways involving autophagy proteins by microorganisms
Constitutive removal of the EDEM and OS-9 regulators from the ER and their lysosomal
degradation at steady state has been termed ERAD tuning. It guarantees low intralumenal
EDEM and OS-9 concentrations in physiological conditions, what avoids premature
degradation of nascent polypeptides and interference with ongoing folding programs. SEL1L
is a type I membrane protein and is a component of the HRD1 dislocon (Figure 8). EDEM1 and
OS-9 selectively associate with SEL1L in the lumen of the ER, while LC3-I binds on the
cytosolic side, assisting the formation of EDEMosomes (7, 19). The presence of misfolded
proteins in the ER lumen outcompetes the binding of EDEM1 and OS-9 to SEL1 and inhibits
recruitment of LC3. This stabilizes the SEL1-HRD1 complex and thus engages the ERAD
pathway while increasing EDEM1 and OS-9 intralumenal concentrations (7, 16, 54). Hence,
SEL1L is referred to as ERAD tuning receptor.
Coronaviruses have been shown to usurp the EDEM1 and OS-9 degradation pathway for
DMV generation (Figure 8) (120). Although depletion of Atg5 has a major impact on MHV
infections (153), DMV formation and MHV replication is independent of Atg7, indicating that
MHV infections do not require conventional autophagy to complete their life cycle. Similarly
to EDEMosomes, DMV are non-covalently associated with LC3-I and are distinct from LC3-IIpositive autophagosome populations. Knockdown of LC3A, LC3B or SEL1L impairs EDEM1
and OS-9 degradation as well as MHV replication. DMV membranes lack conventional ER
markers but contain EDEM1 and OS-9 during the whole time course of infection, even if the
silencing of the latter has no influence on the outcome of the infection.
These experiments show significant similarities between DMVs and EDEMosomes and
consistently support the hypothesis that MHV hijacks the cellular ERAD tuning pathway in
order to replicate within double-membrane-associated structures (7, 120). This process
happens without an intact autophagy machinery and requires LC3 in a role distinct from its
well-known one in autophagosome formation.
Almost identically to MHV, Equine arteritis virus (EAV) has been shown to usurp the
ERAD-tuning pathway and autophagy proteins that function distinctively from their role in
canonical autophagy (96). EAV also belongs to the order of the Nidovirales and shows
similarities with MHV as to its replication strategy (70, 109). Additionally, the completion of
EAV’s intracellular cycle is independent of Atg7 but dependent on LC3-I, which colocalizes
with viral RNA intermediates and EDEM1 on DMVs (96). The striking alikeness between these
25
Subversion of alternative pathways involving autophagy proteins by microorganisms
two viruses arises the question whether this mechanism is common to other viral families in
the same order. The identification of the viral factors that are necessary for the establishment
of the replicative structures and their interaction with host factors awaits clarification.
Furthermore, how DMV originate from single membranes and their destiny still remain
elusive. These questions will certainly be subjected to future investigations.
3.2 Chlamydia
Similarly to Coronaviruses, the non-lipidated form of LC3 (LC3-I) associates with
bacterial inclusions induced by Chlamydia trachomatis and is essential for the progression of
the bacterium into its replicative stage. Here, LC3 is suggested to act as a microtubuleassociated protein instead of its function in autophagosome formation.
C. trachomatis is a species of obligate intracellular pathogenic bacterium that relies on
its host for nutrient supply. It exhibits an alternate biphasic developmental cycle in which
infectious elementary bodies that are metabolically inert are responsible for the
dissemination of the infection (1). Upon uptake and internalization into the host cell, it
switches towards the metabolically active form that is no longer infectious and develop in a
membrane-bound compartment, modified by the bacterium, termed inclusion (34).
Chlamydia-containing inclusions escape the endocytic route before being acidified and travel
to the perinuclear region near the Golgi apparatus. There they interact with the exocytic
pathway where the bacterium can replicate (49, 145). Eventually, chlamydia exits the host cell
through lysis or extrusion (53). While one study (107) revealed an ambiguous interplay
between Chlamydia and the autophagy system, a recent publication brought to light an
autophagy-independent process involving autophagy components necessary for chlamydial
trafficking to its replication site and intracellular development (4).
Early findings hypothesized a potential interaction of chlamydia with the autophagic
pathway, as chlamydial growth in the occlusions was hindered after inhibition of autophagy
by an excess of a panel of single amino acids as well as with 3-methyladenine (3). These
results were discredited by probable adverse effects of these amino acid and 3-methyladenine
treatments that might be responsible for the chlamydia growth inhibition (12, 92). Still,
localization of endogenous MAP-LC3 as well as the ER markers SERCA2 and Calreticulin at the
periphery of the occlusions could suggest the involvement of autophagy. However, chlamydial
26
Subversion of alternative pathways involving autophagy proteins by microorganisms
inclusions did not accumulate the autophagosomal stain MDC, indicating that the inclusions
do not fuse with autophagosomes (3).
Pachikara et al. (107) were the first to suggest that chlamydia had a more subtle
interplay with the autophagy pathway as neither induction of autophagy by starvation or
Rapamycin nor inhibition upon Atg5 depletion in MEF cells had effects on chlamydial growth.
This hypothesis was recently confirmed by precise characterization of the recruitment of LC3
to the inclusion membrane (4). Recruitment of LC3-I was demonstrated by antibodies against
intrinsic endogenous LC3 that associated with both autophagosomes and inclusions, whereas
GFP-LC3 only localized at autophagosomal membranes. Of all the human orthologues of LC3,
only GABARAPL1, which is known to associate with microtubules and promote tubulin
polymerization, was recruited to the inclusions. Additionally, LC3 and its interaction partners
microtubule-associated protein 1A and 1B (MAP1) colocalized at bacterial inclusions situated
in proximity of the nucleus and in association with the Golgi at later time points post infection.
This was dependent on early but not late bacterial protein synthesis. In contrast, no
recruitment of LC3 and MAP1 was observed to the dispersed inclusions in the cytoplasm
shortly after infection, thus demonstrating that bacterial effectors actively modulate host cell
processes. LC3 and MAP1 associated with microtubules in the cytoplasm and in the vicinity of
inclusions in a Nocodazole-, a microtubule-disrupting drug, sensitive manner. Finally, siRNA
knockdown of LC3 or MAP1 decreased bacterial progeny as well as inclusion size and
infectivity.
In sum, the data presented by Al-Younes et al. (4) show that LC3-I is recruited to the
chlamydial inclusion and acts in an autophagy-independent way while closely associating
with MAP1A-B and the microtubule network thus allowing transport to the Golgi for
replication.
3.3 Brucella
Brucella abortus is another example of intracellular bacterium that is able to invade
several cell types in order to replicate and resides in autophagy-like compartments for
intracellular survival. After phagocytic uptake and internalization of the bacterium, B. abortus
resides in a modified compartment referred to as the Brucella-containing vacuole (BCV). The
vacuole undergoes limited fusion with endosomes and the lysosome but avoids the
degradative pathway (110, 132). Acidification of the vacuole is necessary for the induction of
27
Subversion of alternative pathways involving autophagy proteins by microorganisms
B. abortus’s VirB operon encoding for a type IV secretion system that secretes bacterial
effectors into the cytoplasm of the host cell and mediates BCV transport and translocation to
the ER (10, 15, 18). There, B. abortus replicates in an ER-derived replicative BCV (rBCV) (15,
110). Replicating B. abortus is contained in a single membrane vesicle that is positive for ER
markers and coated with ribosomes and, in this stage, does not require proteins involved in
the cellular autophagic pathway. Nevertheless, conversion of a rBCV into a vacuole with
autophagic properties termed autophagic BCV (aBCV) has been shown to be essential for
completion of the intracellular life cycle of B. abortus and its cell-to-cell spread (131).
In contrast to aBCV, rBCVs are LAMP1-negative and calreticulin-positive. On the other
hand, aBCVs are LAMP1- and Rab7-positive and calreticulin-negative. They are acidic and
enwrapped by double-membrane crescents or multiple membranes. aBCV formation depends
on autophagy initiation factors Beclin1, ULK1 PI3K. However, depletion of Atg5, Atg7,
Atg16L1, Atg4B and LC3B, known as elongation factors, has no effect. None of these
treatments affected rBCV formation or B. abortus replication (131).
During conventional autophagy, Beclin1 associates either with Atg14L for initiation of
autophagosome formation or UVRAG and Rubicon for maturation of autophagosomes (which
also involves Rab9). Depletion of Atg14L in cells infected with B. abortus had profound effects
on recruitment of Beclin1-VPS34-VPS15 complex to the ER and aBCV formation (131),
whereas absence of UVRAG and Rab9 displayed a similar phenotype as control conditions.
These results emphasize the role of autophagy proteins involved in initiation but not
maturation of aBCV formation.
Cells infected with B. abortus give rise to secondary infections. Similarly, secondary
infection foci could also be observed when Atg5-, Atg7- or LC3B-depleted cells were infected;
however, a significant reduction was observed for Beclin1 and ULK1 knockdowns (131). Thus,
only Beclin1 and ULK1 of the examined autophagy proteins have a role in the completion of
the intracellular life cycle of B. abortus and cell-to-cell spread.
Taken together, these experiments demonstrate that autophagy initiation but not
maturation factors are required in post-ER replication stages of B. abortus and are crucial for
terminating B. abortus’s intracellular cycle.
28
Discussion
4 Discussion
Besides its traditional roles in cytosol degradation during bulk autophagy and selective
organelle removal, autophagy also manifests in immunological functions through its ability to
specifically sequester and clear intracellular pathogens and regulate immune mediators.
However, recent discoveries have added to and have extended this new paradigm. This thesis
has specifically shed light on novel functions of autophagy proteins in which they act in
processes that are independent of the classical autophagosomal pathway. It exposed
situations where subsets of autophagy-related proteins assist the innate immune arsenal
during infections by driving or enhancing the degradation of intracellular pathogens such as
bacteria or protozoan parasites, and inhibiting viral replication. It was examined how
autophagy proteins additionally intervene in maintaining cellular and physiological
homeostasis, for example during dsDNA-triggered responses, bone degradation by
osteoclasts, clearance of apoptotic and necrotic bodies as well as regulation of the ERAD
pathway. In the second part, instances where microbes exploit the autophagy-independent
function of autophagy-related proteins during membrane dynamics or vesicle trafficking
processes were discussed. These findings opened the door to an enthralling new field of
research.
LC3 undoubtedly has a central role in autophagy, but it was long considered as being
solely an autophagosome-associated component involved in autophagosome formation.
However, what has to be kept in mind is that LC3 was initially discovered to be a microtubuleassociated protein. LC3 was first identified as a 18kDa protein that co-precipitates with LC1
and LC2, both known to be part of the MAP protein complex (76). Further studies
characterized the LC3 light-chain subunit, which interacts with both MAP1A/LC2 and
MAP1B/LC1 complexes (84). LC3 associates with microtubules and has been shown to have a
stabilizing effect on them (32, 44). In mammalian cells, even if highly debated, microtubules
do not seem to be required in autophagosome formation and elongation, although they may
assist and enhance the efficiency of this process (33, 64). On the other hand, it is commonly
accepted that microtubules guide the newly formed autophagosomes, which are generated in
the cytoplasm periphery, towards the perinuclear region. There are located the microtubuleorganizing center, as well as the ER, the Golgi apparatus and, most importantly for autophagy,
29
Discussion
the lysosomes. The microtubule minus-end-directed trafficking of autophagosomes facilitates
the fusion of autophagosomes with endosomes or lysosomes (33, 60). This directional
vesicular transport towards the perinuclear region happens via direct or indirect binding of
LC3 to the molecular motor dynein (60, 68, 117). Altogether, the close association of LC3 and
microtubules during autophagy is reminiscent with instances in which autophagy-related
proteins play autophagy-independent roles.
It is tempting to speculate that LC3 determines the destiny of autophagosomes and
other compartments that are decorated by it. Indeed, LC3 has two spatially distinct domains
that might exert very different functions in vesicle trafficking (72). The amino-terminus of
LC3 is responsible for the attachment to microtubules via the dynein motor and the transport
towards the minus-end of microtubules in both canonical autophagy and non-canonical
processes involving autophagy proteins. In addition to LAP compartments, which will be
discussed in more details below, it has been shown in a study published almost ten years ago
that Chlamydia-containing vacuoles are trafficked towards the perinuclear region as well
(41). This happened in a manner dependent on dynein but not on the p50 subunit of the
dynactin complex, which is known to link cargos to the dynein motor protein. Furthermore, it
has been recently demonstrated that Chlamydia’s directional transport is dependent on the Nterminal domain of LC3-I and MAPs, both of which are bound to the inclusions (4). Perhaps
that LC3-I is part of the answer of how Chlamydia is linked to microtubules and transported
close to the Golgi, where it could get access to nutrients, in order to replicate. Curiously, MHVinduced DMVs are also decorated by LC3-I and might as well travel to the perinuclear region,
as later stages in the viral life cycle are known to interact with the ER-Golgi intermediate
compartment and the Golgi compartment before the virions are released by exocytosis
through the secretory pathway (89, 120).
On the other hand, the carboxy-terminus of LC3, where the lipidation takes places, is
known to interact with other Atg proteins and is responsible for the targeting of LC3 to the
autophagosomal membrane. In addition, it may selectively link the trafficked cargo (137). A
plausible hypothesis could be that this domain regulates the interaction with additional
factors such as GTPases (or adaptor proteins), to which LC3 has repeatedly been found to be
associated with. Such an interaction could determine the target membrane to which the
autophagosome or other LC3-decorated compartment would fuse. By way of illustration, the
Cdc42 GTPase is closely associated to and regulated by LC3 during the actin ring and ruffled
30
Discussion
border formation in osteoclasts (17). Another example is the association observed between
Atg5 (thus most probably also LC3 (87)) and the p47 GTPase IIGP1 during T.gondii infections
(152). Most importantly, Rab7, which is a GTPase as well, is a factor well known to associate
with maturating autophagosomes and endosome before they fuse with lysosomes. A recent
study has characterized an adaptor protein capable of both binding LC3 and Rab7 (108).
Additionally, C. trachomatis and MHV, which have both been shown to recruit LC3-I to their
modified cellular membranes instead of LC3-II, do not fuse with lysosomes although, similarly
to autophagosomes and LAP, they are both trafficked to the perinuclear region (4, 120).
Taken together, the broad functional repertoire of LC3 that extends beyond its roles in
autophagosome formation might be the reason why it is often encountered in situations
where autophagy-related proteins are involved in autophagy-independent cellular processes.
The challenge will be to pinpoint which and how different factors are recruited to the diverse
membranous vesicles and to determine the interactions leading to the specific outcome of the
studied compartment. Further investigation into LC3 and its homologues is required to
determine their redundant or specific functions in these diverse processes as well.
With regards to LAP, recent investigation points out a multitude of cases in which LC3
participates in the regulation of the fusion of non-autophagosomal and lysosomal
compartments in a manner distinct to canonical autophagy. Independently from the initial
breakthrough in the field by Sanjuan and al. (123), a proteomic analysis of phagosomal
membranes consistently established a link between phagocytosis and proteins involved in
cellular autophagy, which associate with phagosomes (128). The study of B. pseudomallei
remarkably illustrates this purpose. Although initially thought to be targeted by conventional
autophagy, only EM analysis demonstrated the lipidation of LC3 onto single-membrane
phagosomes that contain the engulfed bacterium (38). Nowadays EM seems to be the only
reliable tool to distinguish LAP from autophagy. Traditional clustering of LC3 and detection of
GFP-LC3 puncta cannot, by itself, be interpreted as activation of only the autophagy pathway
as LC3 puncta are also encountered on phagosomes. Therefore, numerous cases of LAP may
have been overlooked in the past.
Furthermore, the conservation of LAP during evolution (and therefore its importance)
might have been underestimated until now. A vast diversity of phagocytosed material is
subjected to LAP, notably dead yeast (123), apoptotic and necrotic cells (88), entotic cells (35)
31
Discussion
as well as different species of bacteria (38, 65, 79, 123). Vesicles have also been shown to
recruit LC3 to their membranes during macropinocytosis and such events, additionally to
entosis, are classified as LAP in this thesis (35). LAP has been detected, in vitro, in various cell
lines such as RAW macrophage-like cells (38, 79, 123), primary bone marrow-derived
macrophages (88), mouse embryonic fibroblasts (65) and MCF10 epithelial cells (35) as well
as in vivo in C. elegans (81). Moreover, there is evidence that LAP possibly takes over when
autophagy of bacteria-containing vesicle is disrupted, as observed during Salmonella
infections (65) or even happens simultaneously with autophagy as observed for Listeria (8).
Taken together, these pieces of evidence might testify to an essential, evolutionary conserved
cellular mechanism that considerably improves the clearance of engulfed material, therefore
maintaining cellular homeostasis and providing protection against invading pathogens.
The signaling pathways leading to the initiation of LAP still remain to be precisely
determined. It has been suggested that the autophagy complex Atg5-Atg12-Atg16L1 directly
lipidates LC3 on the phagosomal membrane in a rapid and transient manner, as Atg12
localizes at phagosomes containing IgG-coated beads, similarly to those containing beads
coated with TLR agonists (51). Signaling through TLR of Fcγ receptors might drive the
assembly of the NOX2 NADPH oxidase on the phagosomal membrane (51). In turn,
recruitment of LC3 lipidation machinery may depend on the localized generation of ROS
(141). It seems, however, that signaling through receptors might not be sufficient, as
exemplified by the diminished recruitment of LC3 to phagosomes in cells having taken up
heat-killed B. pseudomallei (38). Hence, other bacterial factors may play an essential role in
this process. Similar findings for C. trachomatis, although in a different context, show that
early bacterial protein synthesis is essential for LC3-I and MAP recruitment to the bacterial
inclusion and add to this hypothesis (4). For the engulfment of dead cells, which evidently do
not interact with TLR or Fcγ receptors, no pathway has yet been identified except for the
requirement of TIM4 (88). Again, the challenge for the future will be to identify the cellular
receptor and foreign body ligand interactions as well as the factors necessary for the initiation
of LAP.
The scope of research in the autophagy field has considerably expanded during recent
years and advances have been made in understanding the generation, regulation and cellular
implication of autophagosomes and autophagosome-like compartments. However, new
32
Discussion
questions have arisen from recent discoveries and need to be answered. Firstly, basic issues in
the canonical autophagy pathway need to be solved, notably the source of autophagosomal
membranes and their conversion from initial single membranes into double-membrane
autophagosomes. Furthermore, Nishida et al. (101) described an alternative autophagy
pathway independent of Atg5 and Atg7, although these proteins were previously thought to
be indispensably required for the formation of autophagosomes. Next to alternative
autophagy, a subset of autophagy-related proteins was found to be implicated in novel
functions that are independent from classical autophagy, further expanding the repertoire of
cellular functions in which autophagy proteins participate. We are just beginning to
appreciate the impact that autophagy-related proteins have on a vast variety of cellular
processes and the repercussion that such pathways have when they are dysfunctional or
deregulated.
33
Acknowledgments
5 Acknowledgements
I would like to address a major thanks to Jovanka Bestebroer for her supervision, her
very helpful advice and corrections, and for the many interesting discussions we had.
I also would like to thank Fulvio Reggiori for introducing me to the field of autophagy
and for all the inspiring conversations.
34
References
6 References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Abdelrahman, Y., and R. Belland. 2005. The chlamydial
developmental cycle. FEMS microbiology reviews 29:949959.
Aebi, M., R. Bernasconi, S. Clerc, and M. Molinari. 2010.
N-glycan structures: recognition and processing in the ER.
Trends in biochemical sciences 35:74-82.
Al-Younes, H., V. Brinkmann, and T. Meyer. 2004.
Interaction of Chlamydia trachomatis serovar L2 with the
host autophagic pathway. Infection and immunity 72:47514762.
Al-Younes, H. M., M. A. Al-Zeer, K. Hany, G. Joscha, K.
Alexander, M. Nikolaus, B. Volker, R. B. Peter, and F. M.
Thomas. 2011. Autophagy-independent function of MAPLC3 during intracellular propagation of Chlamydia
trachomatis. Autophagy 7.
Andrade, R., M. Wessendarp, M.-J. Gubbels, B. Striepen,
and C. Subauste. 2006. CD40 induces macrophage antiToxoplasma gondii activity by triggering autophagydependent fusion of pathogen-containing vacuoles and
lysosomes. The Journal of clinical investigation 116:23662377.
Bernardini, M., J. Mounier, H. d'Hauteville, M. CoquisRondon, and P. Sansonetti. 1989. Identification of icsA, a
plasmid locus of Shigella flexneri that governs bacterial
intra- and intercellular spread through interaction with Factin. Proceedings of the National Academy of Sciences of
the United States of America 86:3867-3871.
Bernasconi, R., C. Galli, J. Noack, S. Bianchi, C. de Haan, F.
Reggiori, and M. Molinari. 2012. Role of the SEL1L:LC3-I
complex as an ERAD tuning receptor in the mammalian ER.
Molecular cell 46:809-819.
Birmingham, C., V. Canadien, N. Kaniuk, B. Steinberg, D.
Higgins, and J. Brumell. 2008. Listeriolysin O allows
Listeria monocytogenes replication in macrophage
vacuoles. Nature 451:350-354.
Birmingham, C., A. Smith, M. Bakowski, T. Yoshimori,
and J. Brumell. 2006. Autophagy controls Salmonella
infection in response to damage to the Salmonellacontaining vacuole. The Journal of biological chemistry
281:11374-11383.
Boschiroli, M., S. Ouahrani-Bettache, V. Foulongne, S.
Michaux-Charachon, G. Bourg, A. Allardet-Servent, C.
Cazevieille, J. Liautard, M. Ramuz, and D. O'Callaghan.
2002. The Brucella suis virB operon is induced
intracellularly in macrophages. Proceedings of the National
Academy of Sciences of the United States of America
99:1544-1549.
Boyce, B., E. Rosenberg, A. de Papp, and L. T. Duong.
2012. The osteoclast, bone remodelling and treatment of
metabolic bone disease. European journal of clinical
investigation 42:1332-1341.
Braun, P., H. Al-Younes, J. Gussmann, J. Klein, E.
Schneider, and T. Meyer. 2008. Competitive inhibition of
amino acid uptake suppresses chlamydial growth:
involvement of the chlamydial amino acid transporter BrnQ.
Journal of bacteriology 190:1822-1830.
Burdette, D., and R. Vance. 2013. STING and the innate
immune response to nucleic acids in the cytosol. Nature
immunology 14:19-26.
Calì, T., C. Galli, S. Olivari, and M. Molinari. 2008.
Segregation and rapid turnover of EDEM1 by an autophagylike mechanism modulates standard ERAD and folding
activities.
Biochemical
and
biophysical
research
communications 371:405-410.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
Celli, J., C. de Chastellier, D.-M. Franchini, J. PizarroCerda, E. Moreno, and J.-P. Gorvel. 2003. Brucella evades
macrophage killing via VirB-dependent sustained
interactions with the endoplasmic reticulum. The Journal of
experimental medicine 198:545-556.
Christianson, J., T. Shaler, R. Tyler, and R. Kopito. 2008.
OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the
Hrd1-SEL1L ubiquitin ligase complex for ERAD. Nature cell
biology 10:272-282.
Chung, Y.-H., S.-Y. Yoon, B. Choi, D. Sohn, K.-H. Yoon, W.J. Kim, D.-H. Kim, and E.-J. Chang. 2012. Microtubuleassociated protein light chain 3 regulates Cdc42-dependent
actin ring formation in osteoclast. The international journal
of biochemistry & cell biology 44:989-997.
Comerci, D., M. Martinez-Lorenzo, R. Sieira, J. Gorvel,
and R. Ugalde. 2001. Essential role of the VirB machinery
in the maturation of the Brucella abortus-containing
vacuole. Cellular microbiology 3:159-168.
Cormier, J., T. Tamura, J. Sunryd, and D. Hebert. 2009.
EDEM1 recognition and delivery of misfolded proteins to
the SEL1L-containing ERAD complex. Molecular cell
34:627-633.
Cossart, P. 2011. Illuminating the landscape of hostpathogen interactions with the bacterium Listeria
monocytogenes. Proceedings of the National Academy of
Sciences of the United States of America 108:19484-19491.
Coxon, F., and A. Taylor. 2008. Vesicular trafficking in
osteoclasts. Seminars in cell & developmental biology
19:424-433.
Cullinane, M. a., L. Gong, X. Li, N. Lazar-Adler, T. Tra, E.
Wolvetang, M. Prescott, J. Boyce, R. Devenish, and B.
Adler. 2008. Stimulation of autophagy suppresses the
intracellular survival of Burkholderia pseudomallei in
mammalian cell lines. Autophagy 4:744-753.
de Haan, C., and F. Reggiori. 2008. Are nidoviruses
hijacking the autophagy machinery? Autophagy 4:276-279.
Delgado, M. n., R. Elmaoued, A. Davis, G. Kyei, and V.
Deretic. 2008. Toll-like receptors control autophagy. The
EMBO journal 27:1110-1121.
den Boon, J., and P. Ahlquist. 2010. Organelle-like
membrane compartmentalization of positive-strand RNA
virus replication factories. Annual review of microbiology
64:241-256.
Deretic, V. 2012. Autophagy: an emerging immunological
paradigm. Journal of immunology (Baltimore, Md. : 1950)
189:15-20.
Deretic, V., and B. Levine. 2009. Autophagy, immunity, and
microbial adaptations. Cell host & microbe 5:527-549.
DeSelm, C., B. Miller, W. Zou, W. Beatty, E. van Meel, Y.
Takahata, J. Klumperman, S. Tooze, S. Teitelbaum, and
H. Virgin. 2011. Autophagy proteins regulate the secretory
component of osteoclastic bone resorption. Developmental
cell 21:966-974.
Destaing, O., F. d. r. Saltel, J.-C. Géminard, P. Jurdic,
and F. d. r. Bard. 2003. Podosomes display actin turnover
and dynamic self-organization in osteoclasts expressing
actin-green fluorescent protein. Molecular biology of the
cell 14:407-416.
Destaing, O., F. d. r. Saltel, B. Gilquin, A. Chabadel, S.
Khochbin, S. p. Ory, and P. Jurdic. 2005. A novel RhomDia2-HDAC6 pathway controls podosome patterning
through microtubule acetylation in osteoclasts. Journal of
cell science 118:2901-2911.
35
References
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
Dupont, N., S. Jiang, M. Pilli, W. Ornatowski, D.
Bhattacharya, and V. Deretic. 2011. Autophagy-based
unconventional secretory pathway for extracellular delivery
of IL-1β. The EMBO journal 30:4701-4711.
Faller, E., T. Villeneuve, and D. Brown. 2009. MAP1a
associated light chain 3 increases microtubule stability by
suppressing microtubule dynamics. Molecular and cellular
neurosciences 41:85-93.
Fass, E., E. Shvets, I. Degani, K. Hirschberg, and Z. Elazar.
2006. Microtubules support production of starvationinduced autophagosomes but not their targeting and fusion
with lysosomes. The Journal of biological chemistry
281:36303-36316.
Fields, K., and T. Hackstadt. 2002. The chlamydial
inclusion: escape from the endocytic pathway. Annual
review of cell and developmental biology 18:221-245.
Florey, O., S. Kim, C. Sandoval, C. Haynes, and M.
Overholtzer. 2011. Autophagy machinery mediates
macroendocytic processing and entotic cell death by
targeting single membranes. Nature cell biology 13:13351343.
Florey, O., and M. Overholtzer. 2012. Autophagy proteins
in macroendocytic engulfment. Trends in cell biology
22:374-380.
Goltzman, D. 2002. Discoveries, drugs and skeletal
disorders. Nature reviews. Drug discovery 1:784-796.
Gong, L., M. Cullinane, P. Treerat, G. Ramm, M. Prescott,
B. Adler, J. Boyce, and R. Devenish. 2011. The
Burkholderia pseudomallei type III secretion system and
BopA are required for evasion of LC3-associated
phagocytosis. PloS one 6.
Gosert, R., A. Kanjanahaluethai, D. Egger, K. Bienz, and S.
Baker. 2002. RNA replication of mouse hepatitis virus takes
place at double-membrane vesicles. Journal of virology
76:3697-3708.
Greenberg, S., and S. Grinstein. 2002. Phagocytosis and
innate immunity. Current opinion in immunology 14:136145.
Grieshaber, S., N. Grieshaber, and T. Hackstadt. 2003.
Chlamydia trachomatis uses host cell dynein to traffic to the
microtubule-organizing center in a p50 dynamitinindependent process. Journal of cell science 116:37933802.
Grimsley, C., and K. Ravichandran. 2003. Cues for
apoptotic cell engulfment: eat-me, don't eat-me and comeget-me signals. Trends in cell biology 13:648-656.
Gutierrez, M., S. Master, S. Singh, G. Taylor, M. Colombo,
and V. Deretic. 2004. Autophagy is a defense mechanism
inhibiting BCG and Mycobacterium tuberculosis survival in
infected macrophages. Cell 119:753-766.
Halpain, S., and L. Dehmelt. 2006. The MAP1 family of
microtubule-associated proteins. Genome biology 7:224.
Hanada, T., N. Noda, Y. Satomi, Y. Ichimura, Y. Fujioka, T.
Takao, F. Inagaki, and Y. Ohsumi. 2007. The Atg12-Atg5
conjugate has a novel E3-like activity for protein lipidation
in autophagy. The Journal of biological chemistry
282:37298-37302.
Harcourt, B., D. Jukneliene, A. Kanjanahaluethai, J.
Bechill, K. Severson, C. Smith, P. Rota, and S. Baker.
2004. Identification of severe acute respiratory syndrome
coronavirus replicase products and characterization of
papain-like protease activity. Journal of virology 78:1360013612.
Harley, V., D. Dance, G. Tovey, M. McCrossan, and B.
Drasar. 1998. An ultrastructural study of the phagocytosis
of Burkholderia pseudomallei. Microbios 94:35-45.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
Harris, J., M. Hartman, C. Roche, S. Zeng, A. O'Shea, F.
Sharp, E. Lambe, E. Creagh, D. Golenbock, J. Tschopp, H.
Kornfeld, K. Fitzgerald, and E. Lavelle. 2011. Autophagy
controls IL-1beta secretion by targeting pro-IL-1beta for
degradation. The Journal of biological chemistry 286:95879597.
Heinzen, R., D. Howe, L. Mallavia, D. Rockey, and T.
Hackstadt. 1996. Developmentally regulated synthesis of
an unusually small, basic peptide by Coxiella burnetii.
Molecular microbiology 22:9-19.
Hornung, V., A. Ablasser, M. Charrel-Dennis, F.
Bauernfeind, G. Horvath, D. Caffrey, E. Latz, and K.
Fitzgerald. 2009. AIM2 recognizes cytosolic dsDNA and
forms a caspase-1-activating inflammasome with ASC.
Nature 458:514-518.
Huang, J., V. Canadien, G. Lam, B. Steinberg, M. Dinauer,
M. Magalhaes, M. Glogauer, S. Grinstein, and J. Brumell.
2009. Activation of antibacterial autophagy by NADPH
oxidases. Proceedings of the National Academy of Sciences
of the United States of America 106:6226-6231.
Hwang, S., N. Maloney, M. Bruinsma, G. Goel, E. Duan, L.
Zhang, B. Shrestha, M. Diamond, A. Dani, S. Sosnovtsev,
K. Green, C. Lopez-Otin, R. Xavier, L. Thackray, and H.
Virgin. 2012. Nondegradative role of Atg5-Atg12/ Atg16L1
autophagy protein complex in antiviral activity of interferon
gamma. Cell host & microbe 11:397-409.
Hybiske, K., and R. Stephens. 2007. Mechanisms of host
cell exit by the intracellular bacterium Chlamydia.
Proceedings of the National Academy of Sciences of the
United States of America 104:11430-11435.
Iida, Y., T. Fujimori, K. Okawa, K. Nagata, I. Wada, and N.
Hosokawa. 2011. SEL1L protein critically determines the
stability of the HRD1-SEL1L endoplasmic reticulumassociated degradation (ERAD) complex to optimize the
degradation kinetics of ERAD substrates. The Journal of
biological chemistry 286:16929-16939.
Into, T., M. Inomata, E. Takayama, and T. Takigawa.
2012. Autophagy in regulation of Toll-like receptor
signaling. Cellular signalling 24:1150-1162.
Ishii, K., S. Koyama, A. Nakagawa, C. Coban, and S. Akira.
2008. Host innate immune receptors and beyond: making
sense of microbial infections. Cell host & microbe 3:352363.
Itakura, E., C. Kishi, K. Inoue, and N. Mizushima. 2008.
Beclin 1 forms two distinct phosphatidylinositol 3-kinase
complexes with mammalian Atg14 and UVRAG. Molecular
biology of the cell 19:5360-5372.
Ito, Y., S. Teitelbaum, W. Zou, Y. Zheng, J. Johnson, J.
Chappel, F. Ross, and H. Zhao. 2010. Cdc42 regulates bone
modeling and remodeling in mice by modulating RANKL/MCSF signaling and osteoclast polarization. The Journal of
clinical investigation 120:1981-1993.
Itzstein, C., F. Coxon, and M. Rogers. 2011. The regulation
of osteoclast function and bone resorption by small
GTPases. Small GTPases 2:117-130.
Jahreiss, L., F. Menzies, and D. Rubinsztein. 2008. The
itinerary of autophagosomes: from peripheral formation to
kiss-and-run fusion with lysosomes. Traffic (Copenhagen,
Denmark) 9:574-587.
Jia, W., and Y.-W. He. 2011. Temporal regulation of
intracellular organelle homeostasis in T lymphocytes by
autophagy. Journal of immunology (Baltimore, Md. : 1950)
186:5313-5322.
Jordan, T., and G. Randall. 2012. Manipulation or
capitulation: virus interactions with autophagy. Microbes
and infection / Institut Pasteur 14:126-139.
Jung, C., C. Jun, S.-H. Ro, Y.-M. Kim, N. Otto, J. Cao, M.
Kundu, and D.-H. Kim. 2009. ULK-Atg13-FIP200
complexes mediate mTOR signaling to the autophagy
machinery. Molecular biology of the cell 20:1992-2003.
36
References
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
K√ ∂ chl, R., X. Hu, E. Chan, and S. Tooze. 2006.
Microtubules facilitate autophagosome formation and
fusion of autophagosomes with endosomes. Traffic
(Copenhagen, Denmark) 7:129-145.
Kageyama, S., H. Omori, T. Saitoh, T. Sone, J.-L. Guan, S.
Akira, F. Imamoto, T. Noda, and T. Yoshimori. 2011. The
LC3 recruitment mechanism is separate from Atg9L1dependent membrane formation in the autophagic response
against Salmonella. Molecular biology of the cell 22:22902300.
Kanjanahaluethai, A., Z. Chen, D. Jukneliene, and S.
Baker. 2007. Membrane topology of murine coronavirus
replicase nonstructural protein 3. Virology 361:391-401.
Kawai, T., and S. Akira. 2010. The role of patternrecognition receptors in innate immunity: update on Tolllike receptors. Nature immunology 11:373-384.
Kimura, S., T. Noda, and T. Yoshimori. 2008. Dyneindependent movement of autophagosomes mediates
efficient encounters with lysosomes. Cell structure and
function 33:109-122.
Klionsky, D., F. Abdalla, H. Abeliovich, R. Abraham, and
A. Acevedo-Arozena. 2012. Guidelines for the use and
interpretation of assays for monitoring autophagy.
Autophagy 8:445-544.
Knoops, K., M. Bàrcena, R. Limpens, A. Koster, A.
Mommaas, and E. Snijder. 2012. Ultrastructural
characterization of arterivirus replication structures:
reshaping the endoplasmic reticulum to accommodate viral
RNA synthesis. Journal of virology 86:2474-2487.
Knoops, K., C. Swett-Tapia, S. van den Worm, A. Te
Velthuis, A. Koster, A. Mommaas, E. Snijder, and M.
Kikkert. 2010. Integrity of the early secretory pathway
promotes, but is not required for, severe acute respiratory
syndrome coronavirus RNA synthesis and virus-induced
remodeling of endoplasmic reticulum membranes. Journal
of virology 84:833-846.
Kouno, T., M. Mizuguchi, I. Tanida, T. Ueno, T.
Kanematsu, Y. Mori, H. Shinoda, M. Hirata, E. Kominami,
and K. Kawano. 2005. Solution structure of microtubuleassociated protein light chain 3 and identification of its
functional subdomains. The Journal of biological chemistry
280:24610-24617.
Kraft, C., M. Peter, and K. Hofmann. 2010. Selective
autophagy: ubiquitin-mediated recognition and beyond.
Nature cell biology 12:836-841.
Kroemer, G., G. Mari √ ± o, and B. Levine. 2010.
Autophagy and the integrated stress response. Molecular
cell 40:280-293.
Kuballa, P., W. Nolte, A. Castoreno, and R. Xavier. 2012.
Autophagy and the immune system. Annual review of
immunology 30:611-646.
Kuznetsov, S., and V. Gelfand. 1987. 18 kDa microtubuleassociated protein: identification as a new light chain (LC-3)
of microtubule-associated protein 1 (MAP-1). FEBS letters
212:145-148.
Lai, S., and R. Devenish. 2012. LC3-Associated
Phagocytosis (LAP): Connections with Host Autophagy.
Cells.
Lee, H., L. Mattei, B. Steinberg, P. Alberts, Y. Lee, A.
Chervonsky, N. Mizushima, S. Grinstein, and A. Iwasaki.
2010. In vivo requirement for Atg5 in antigen presentation
by dendritic cells. Immunity 32:227-239.
Lerena, M. a., and M. a. Colombo. 2011. Mycobacterium
marinum induces a marked LC3 recruitment to its
containing phagosome that depends on a functional ESX-1
secretion system. Cellular microbiology 13:814-835.
Levine, B., N. Mizushima, and H. Virgin. 2011. Autophagy
in immunity and inflammation. Nature 469:323-335.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
Li, W., W. Zou, Y. Yang, Y. Chai, B. Chen, S. Cheng, D. Tian,
X. Wang, R. Vale, and G. Ou. 2012. Autophagy genes
function sequentially to promote apoptotic cell corpse
degradation in the engulfing cell. The Journal of cell biology
197:27-35.
Li, X., M. Prescott, B. Adler, J. Boyce, and R. Devenish.
2012. Beclin 1 is required for starvation-enhanced, but not
rapamycin-enhanced, LC3-associated phagocytosis of
Burkholderia pseudomallei in RAW 264.7 cells. Infection
and immunity.
Ling, Y., M. Shaw, C. Ayala, I. Coppens, G. Taylor, D.
Ferguson, and G. Yap. 2006. Vacuolar and plasma
membrane stripping and autophagic elimination of
Toxoplasma gondii in primed effector macrophages. The
Journal of experimental medicine 203:2063-2071.
Mann, S., and J. Hammarback. 1994. Molecular
characterization of light chain 3. A microtubule binding
subunit of MAP1A and MAP1B. The Journal of biological
chemistry 269:11492-11497.
Mari, M., J. Griffith, E. Rieter, L. Krishnappa, D. Klionsky,
and F. Reggiori. 2010. An Atg9-containing compartment
that functions in the early steps of autophagosome
biogenesis. The Journal of cell biology 190:1005-1022.
Mari, M., S. Tooze, and F. Reggiori. 2011. The puzzling
origin of the autophagosomal membrane. F1000 biology
reports 3:25.
Martens, S., I. Parvanova, J. Zerrahn, G. Griffiths, G.
Schell, G. Reichmann, and J. Howard. 2005. Disruption of
Toxoplasma gondii parasitophorous vacuoles by the mouse
p47-resistance GTPases. PLoS pathogens 1.
Martinez, J., J. Almendinger, A. Oberst, R. Ness, C. Dillon,
P. Fitzgerald, M. Hengartner, and D. Green. 2011.
Microtubule-associated protein 1 light chain 3 alpha (LC3)associated phagocytosis is required for the efficient
clearance of dead cells. Proceedings of the National
Academy of Sciences of the United States of America
108:17396-17401.
Masters, P. 2006. The molecular biology of coronaviruses.
Advances in virus research 66:193-292.
Méresse, S., O. Steele-Mortimer, E. Moreno, M.
Desjardins, B. Finlay, and J. Gorvel. 1999. Controlling the
maturation of pathogen-containing vacuoles: a matter of life
and death. Nature cell biology 1:8.
Meusser, B., C. Hirsch, E. Jarosch, and T. Sommer. 2005.
ERAD: the long road to destruction. Nature cell biology
7:766-772.
Mizushima, N. 2004. Methods for monitoring autophagy.
The international journal of biochemistry & cell biology
36:2491-2502.
Mizushima, N., T. Yoshimori, and B. Levine. 2010.
Methods in mammalian autophagy research. Cell 140:313326.
Molinari, M., V. Calanca, C. Galli, P. Lucca, and P.
Paganetti. 2003. Role of EDEM in the release of misfolded
glycoproteins from the calnexin cycle. Science (New York,
N.Y.) 299:1397-1400.
Monastyrska, I., E. Rieter, D. Klionsky, and F. Reggiori.
2009. Multiple roles of the cytoskeleton in autophagy.
Biological reviews of the Cambridge Philosophical Society
84:431-448.
Monastyrska, I., M. Ulasli, P. Rottier, J.-L. Guan, F.
Reggiori, and C. de Haan. 2012. An autophagyindependent role for LC3 in equine arteritis virus
replication. Autophagy 9.
Mostowy, S., and P. Cossart. 2012. Bacterial autophagy:
restriction or promotion of bacterial replication? Trends in
cell biology 22:283-291.
37
References
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
Nakahira, K., J. Haspel, V. Rathinam, S.-J. Lee, T. Dolinay,
H. Lam, J. Englert, M. Rabinovitch, M. Cernadas, H. Kim,
K. Fitzgerald, S. Ryter, and A. Choi. 2011. Autophagy
proteins regulate innate immune responses by inhibiting
the release of mitochondrial DNA mediated by the NALP3
inflammasome. Nature immunology 12:222-230.
Nakatogawa, H., Y. Ichimura, and Y. Ohsumi. 2007. Atg8,
a ubiquitin-like protein required for autophagosome
formation, mediates membrane tethering and hemifusion.
Cell 130:165-178.
Nedjic, J., M. Aichinger, J. Emmerich, N. Mizushima, and
L. Klein. 2008. Autophagy in thymic epithelium shapes the
T-cell repertoire and is essential for tolerance. Nature
455:396-400.
Nishida, Y., S. Arakawa, K. Fujitani, H. Yamaguchi, T.
Mizuta, T. Kanaseki, M. Komatsu, K. Otsu, Y. Tsujimoto,
and S. Shimizu. 2009. Discovery of Atg5/Atg7-independent
alternative macroautophagy. Nature 461:654-658.
Noda, N., Y. Ohsumi, and F. Inagaki. 2010. Atg8-family
interacting motif crucial for selective autophagy. FEBS
letters 584:1379-1385.
Ogawa, M., T. Yoshimori, T. Suzuki, H. Sagara, N.
Mizushima, and C. Sasakawa. 2005. Escape of intracellular
Shigella from autophagy. Science (New York, N.Y.) 307:727731.
Oostra, M., M. Hagemeijer, M. van Gent, C. Bekker, E. te
Lintelo, P. Rottier, and C. de Haan. 2008. Topology and
membrane anchoring of the coronavirus replication
complex: not all hydrophobic domains of nsp3 and nsp6 are
membrane spanning. Journal of virology 82:12392-12405.
Oostra, M., E. te Lintelo, M. Deijs, M. Verheije, P. Rottier,
and C. de Haan. 2007. Localization and membrane topology
of coronavirus nonstructural protein 4: involvement of the
early secretory pathway in replication. Journal of virology
81:12323-12336.
Overholtzer, M., A. Mailleux, G. Mouneimne, G.
Normand, S. Schnitt, R. King, E. Cibas, and J. Brugge.
2007. A nonapoptotic cell death process, entosis, that occurs
by cell-in-cell invasion. Cell 131:966-979.
Pachikara, N., H. Zhang, Z. Pan, S. Jin, and H. Fan. 2009.
Productive Chlamydia trachomatis lymphogranuloma
venereum 434 infection in cells with augmented or
inactivated autophagic activities. FEMS microbiology letters
292:240-249.
Pankiv, S., and T. Johansen. 2010. FYCO1: Linking
autophagosomes to microtubule plus end-directing
molecular motors. Autophagy 6.
Pedersen, K., Y. van der Meer, N. Roos, and E. Snijder.
1999. Open reading frame 1a-encoded subunits of the
arterivirus replicase induce endoplasmic reticulum-derived
double-membrane vesicles which carry the viral replication
complex. Journal of virology 73:2016-2026.
Pizarro-Cerdà, J., S. Méresse, R. Parton, G. van der Goot,
A. Sola-Landa, I. Lopez-Goni, E. Moreno, and J. Gorvel.
1998. Brucella abortus transits through the autophagic
pathway and replicates in the endoplasmic reticulum of
nonprofessional phagocytes. Infection and immunity
66:5711-5724.
Portillo, J.-A. C., G. Okenka, E. Reed, A. Subauste, J. Van
Grol, K. Gentil, M. Komatsu, K. Tanaka, G. Landreth, B.
Levine, and C. Subauste. 2010. The CD40-autophagy
pathway is needed for host protection despite IFN-Γdependent immunity and CD40 induces autophagy via
control of P21 levels. PloS one 5.
Prentice, E., W. Jerome, T. Yoshimori, N. Mizushima, and
M. Denison. 2004. Coronavirus replication complex
formation utilizes components of cellular autophagy. The
Journal of biological chemistry 279:10136-10141.
Prentice, E., J. McAuliffe, X. Lu, K. Subbarao, and M.
Denison. 2004. Identification and characterization of
severe acute respiratory syndrome coronavirus replicase
proteins. Journal of virology 78:9977-9986.
114. Pua, H., I. Dzhagalov, M. Chuck, N. Mizushima, and Y.-W.
He. 2007. A critical role for the autophagy gene Atg5 in T
cell survival and proliferation. The Journal of experimental
medicine 204:25-31.
115. Py, B. n. d., M. Lipinski, and J. Yuan. 2007. Autophagy
limits Listeria monocytogenes intracellular growth in the
early phase of primary infection. Autophagy 3:117-125.
116. Ravichandran, K. 2010. Find-me and eat-me signals in
apoptotic cell clearance: progress and conundrums. The
Journal of experimental medicine 207:1807-1817.
117. Ravikumar, B., A. Acevedo-Arozena, S. Imarisio, Z.
Berger, C. Vacher, C. O'Kane, S. Brown, and D.
Rubinsztein. 2005. Dynein mutations impair autophagic
clearance of aggregate-prone proteins. Nature genetics
37:771-776.
118. Reggiori, F. 2012. Autophagy: New Questions from Recent
Answers. ISRN Molecular Biology 2012.
119. Reggiori, F., C. de Haan, and M. Molinari. 2011.
Unconventional use of LC3 by coronaviruses through the
alleged subversion of the ERAD tuning pathway. Viruses
3:1610-1623.
120. Reggiori, F., I. Monastyrska, M. Verheije, T. Calì, M.
Ulasli, S. Bianchi, R. Bernasconi, C. de Haan, and M.
Molinari. 2010. Coronaviruses Hijack the LC3-I-positive
EDEMosomes, ER-derived vesicles exporting short-lived
ERAD regulators, for replication. Cell host & microbe 7:500508.
121. Saitoh, T., N. Fujita, T. Hayashi, K. Takahara, T. Satoh, H.
Lee, K. Matsunaga, S. Kageyama, H. Omori, T. Noda, N.
Yamamoto, T. Kawai, K. Ishii, O. Takeuchi, T. Yoshimori,
and S. Akira. 2009. Atg9a controls dsDNA-driven dynamic
translocation of STING and the innate immune response.
Proceedings of the National Academy of Sciences of the
United States of America 106:20842-20846.
122. Samuel, C. 2001. Antiviral actions of interferons. Clinical
microbiology reviews 14:778.
123. Sanjuan, M., C. Dillon, S. Tait, S. Moshiach, F. Dorsey, S.
Connell, M. Komatsu, K. Tanaka, J. Cleveland, S. Withoff,
and D. Green. 2007. Toll-like receptor signalling in
macrophages links the autophagy pathway to phagocytosis.
Nature 450:1253-1257.
124. Sanjuan, M., S. Milasta, and D. Green. 2009. Toll-like
receptor signaling in the lysosomal pathways.
Immunological reviews 227:203-220.
125. Schmid, D., M. Pypaert, and C. Münz. 2007. Antigenloading compartments for major histocompatibility
complex class II molecules continuously receive input from
autophagosomes. Immunity 26:79-92.
126. Shahnazari, S., and J. Brumell. 2011. Mechanisms and
consequences of bacterial targeting by the autophagy
pathway. Current opinion in microbiology 14:68-75.
127. Shi, C.-S., K. Shenderov, N.-N. Huang, J. Kabat, M. AbuAsab, K. Fitzgerald, A. Sher, and J. Kehrl. 2012. Activation
of autophagy by inflammatory signals limits IL-1β
production by targeting ubiquitinated inflammasomes for
destruction. Nature immunology 13:255-263.
128. Shui, W., L. Sheu, J. Liu, B. Smart, C. Petzold, T.-Y. Hsieh,
A. Pitcher, J. Keasling, and C. Bertozzi. 2008. Membrane
proteomics of phagosomes suggests a connection to
autophagy. Proceedings of the National Academy of
Sciences of the United States of America 105:16952-16957.
129. Sibley, L. 2003. Toxoplasma gondii: perfecting an
intracellular life style. Traffic (Copenhagen, Denmark)
4:581-586.
130. Snijder, E., Y. van der Meer, J. Zevenhoven-Dobbe, J.
Onderwater, J. van der Meulen, H. Koerten, and A.
Mommaas. 2006. Ultrastructure and origin of membrane
vesicles associated with the severe acute respiratory
syndrome coronavirus replication complex. Journal of
virology 80:5927-5940.
38
References
131. Starr, T., R. Child, T. Wehrly, B. Hansen, S. Hwang, C. L√
≥pez-Otin, H. Virgin, and J. Celli. 2012. Selective
subversion of autophagy complexes facilitates completion
of the Brucella intracellular cycle. Cell host & microbe
11:33-45.
132. Starr, T., T. Ng, T. Wehrly, L. Knodler, and J. Celli. 2008.
Brucella intracellular replication requires trafficking
through the late endosomal/lysosomal compartment.
Traffic (Copenhagen, Denmark) 9:678-694.
133. Stenbeck, G. 2002. Formation and function of the ruffled
border in osteoclasts. Seminars in cell & developmental
biology 13:285-292.
134. Stevens, M., M. Wood, L. Taylor, P. Monaghan, P. Hawes,
P. Jones, T. Wallis, and E. Galyov. 2002. An Inv/Mxi-Spalike type III protein secretion system in Burkholderia
pseudomallei modulates intracellular behaviour of the
pathogen. Molecular microbiology 46:649-659.
135. Stuart, L., and R. Ezekowitz. 2008. Phagocytosis and
comparative innate immunity: learning on the fly. Nature
reviews. Immunology 8:131-141.
136. Tamura, T., J. Sunryd, and D. Hebert. 2010. Sorting things
out through endoplasmic reticulum quality control.
Molecular membrane biology 27:412-427.
137. Tanida, I., T. Ueno, and E. Kominami. 2004. Human light
chain 3/MAP1LC3B is cleaved at its carboxyl-terminal
Met121 to expose Gly120 for lipidation and targeting to
autophagosomal membranes. The Journal of biological
chemistry 279:47704-47710.
138. Todde, V., M. Veenhuis, and I. van der Klei. 2009.
Autophagy: principles and significance in health and
disease. Biochimica et biophysica acta 1792:3-13.
139. Ulasli, M., M. Verheije, C. de Haan, and F. Reggiori. 2010.
Qualitative and quantitative ultrastructural analysis of the
membrane rearrangements induced by coronavirus.
Cellular microbiology 12:844-861.
140. Verheije, M., M. Raaben, M. Mari, E. Te Lintelo, F.
Reggiori, F. van Kuppeveld, P. Rottier, and C. de Haan.
2008. Mouse hepatitis coronavirus RNA replication depends
on GBF1-mediated ARF1 activation. PLoS pathogens 4.
141. Vernon, P., and D. Tang. 2012. Eat-Me: Autophagy,
Phagocytosis, and Reactive Oxygen Species Signaling.
Antioxidants & redox signaling.
142. Vieira, O., R. Botelho, and S. Grinstein. 2002. Phagosome
maturation: aging gracefully. The Biochemical journal
366:689-704.
143. Wobus, C., L. Thackray, and H. Virgin. 2006. Murine
norovirus: a model system to study norovirus biology and
pathogenesis. Journal of virology 80:5104-5112.
144. Wu, Y.-T., H.-L. Tan, G. Shui, C. Bauvy, Q. Huang, M.
Wenk, C.-N. Ong, P. Codogno, and H.-M. Shen. 2010. Dual
role of 3-methyladenine in modulation of autophagy via
different temporal patterns of inhibition on class I and III
phosphoinositide 3-kinase. The Journal of biological
chemistry 285:10850-10861.
145. Wyrick, P. 2000. Intracellular survival by Chlamydia.
Cellular microbiology 2:275-282.
146. Xie, Z., U. Nair, and D. Klionsky. 2008. Atg8 controls
phagophore expansion during autophagosome formation.
Molecular biology of the cell 19:3290-3298.
147. Xu, Y., C. Jagannath, X.-D. Liu, A. Sharafkhaneh, K.
Kolodziejska, and N. Eissa. 2007. Toll-like receptor 4 is a
sensor for autophagy associated with innate immunity.
Immunity 27:135-144.
148. Yang, Z., and D. Klionsky. 2010. Eaten alive: a history of
macroautophagy. Nature cell biology 12:814-822.
149. Yang, Z., and D. Klionsky. 2010. Mammalian autophagy:
core molecular machinery and signaling regulation. Current
opinion in cell biology 22:124-131.
150. Zhao, H., T. Laitala-Leinonen, V. Parikka, and H. V√§√
§n√§nen. 2001. Downregulation of small GTPase Rab7
impairs osteoclast polarization and bone resorption. The
Journal of biological chemistry 276:39295-39302.
151. Zhao, Y., D. Wilson, S. Matthews, and G. Yap. 2007. Rapid
elimination of Toxoplasma gondii by gamma interferonprimed mouse macrophages is independent of CD40
signaling. Infection and immunity 75:4799-4803.
152. Zhao, Z., B. Fux, M. Goodwin, I. Dunay, D. Strong, B.
Miller, K. Cadwell, M. Delgado, M. Ponpuak, K. Green, R.
Schmidt, N. Mizushima, V. Deretic, L. Sibley, and H.
Virgin. 2008. Autophagosome-independent essential
function for the autophagy protein Atg5 in cellular
immunity to intracellular pathogens. Cell host & microbe
4:458-469.
153. Zhao, Z., L. Thackray, B. Miller, T. Lynn, M. Becker, E.
Ward, N. Mizushima, M. Denison, and H. Virgin. 2007.
Coronavirus replication does not require the autophagy
gene ATG5. Autophagy 3:581-585.
154. Zhou, R., A. Yazdi, P. Menu, and J. r. Tschopp. 2011. A role
for mitochondria in NLRP3 inflammasome activation.
Nature 469:221-225.
39