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VOLUME THREE HUNDRED AND TWENTY EIGHT
INTERNATIONAL REVIEW OF
CELL AND MOLECULAR
BIOLOGY
International Review of Cell
and Molecular Biology
Series Editors
GEOFFREY H. BOURNE 1949—1988
JAMES F. DANIELLI 1949—1984
KWANG W. JEON 1967—2016
MARTIN FRIEDLANDER 1984—1992
JONATHAN JARVIK 1993—1995
LORENZO GALLUZZI 2016—
Editorial Advisory Board

KEITH BURRIDGE CARLOS LOPEZ-OTIN
AARON CIECHANOVER WALLACE MARSHALL
SANDRA DEMARIA SHIGEKAZU NAGATA
SILVIA FINNEMANN MOSHE OREN
KWANG JEON ANNE SIMONSEN
VOLUME THREE HUNDRED AND TWENTY EIGHT
INTERNATIONAL REVIEW OF
CELL AND MOLECULAR
BIOLOGY
Edited by
LORENZO GALLUZZI
Department of Radiation Oncology
Weill Cornell Medical College
New York, New York
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CONTRIBUTORS
M. Bonora
Department of Morphology, Surgery and Experimental Medicine, Section of Pathology,
Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies
(LTTA), University of Ferrara, Ferrara, Italy
F.K.-M. Chan
Department of Pathology, Immunology and Microbiology Program, University of
Massachusetts Medical School, Worcester, MA, United States
P. Codogno
Institut Necker-Enfant Malades (INEM), INSERM, Université Paris Descartes-Sorbonne
Paris Cité, Paris, France
F. Corradi
Department of Biology, University of Rome “Tor Vergata”, Rome, Italy
E. De Crignis
Department of Biochemistry, Erasmus University Medical Centre, Rotterdam, The
Netherlands
N. Dupont
V. Fossati
The New York Stem Cell Foundation Research Institute, New York, NY, United States
C. Giorgi
J.A. Glab
Department of Biochemistry, La Trobe Institute of Molecular Science, La Trobe University,
Kingsbury Drive, Melbourne, VIC, Australia
J. Huang
Department of Biological Science, Florida State University, Tallahassee, FL, United States
J.S. Lynn
ix
x Contributors
T. Mahmoudi
Department of Biochemistry, Erasmus University Medical Centre, Rotterdam, The
Netherlands
G. Manic
Regina Elena National Cancer Institute, Rome, Italy
S. Marchi
G.W. Mbogo
K. McGinnis
S. Missiroli
E. Morel
K. Moriwaki
Department of Pathology, Immunology and Microbiology Program, University of
Massachusetts Medical School, Worcester, MA, United States
A.C. Nascimbeni
S. Patergnani
P. Pinton
H. Puthalakath
Contributors xi
T. Rusielewicz
The New York Stem Cell Foundation Research Institute, New York, NY, United States
L. Schulte
A. Sistigu
Regina Elena National Cancer Institute, Rome, Italy
S. Siteni
Regina Elena National Cancer Institute; Department of Biology, University of Rome
“Roma Tre”, Rome, Italy
S. Vendramin
I. Vitale
Regina Elena National Cancer Institute; Department of Biology, University of Rome
“Tor Vergata”, Rome, Italy
M.R. Wieckowski
Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland
CHAPTER ONE
Molecular Mechanisms of
Noncanonical Autophagy
N. Dupont, A.C. Nascimbeni, E. Morel, P. Codogno*
Institut Necker-Enfant Malades (INEM), INSERM, Université Paris Descartes-Sorbonne Paris Cité,
Paris, France
*Corresponding author. E-mail address: patrice.codogno@inserm.fr
Contents
1. Introduction 2
2. The Canonical Autophagic Pathway 3
2.1 The Core ATG Machinery and the Formation of Autophagosomes 5
2.2 Maturation of Autophagosomes 6
3. Noncanonical Autophagy 7
3.1 ULK1-Independent Autophagy 7
3.2 Beclin 1-Independent and VPS34-Independent Autophagy 8
3.3 VPS34/VPS15-Independent Autophagy 13
3.4 Autophagy Independent of Ubiquitin-Like Conjugation Actors 14
4. Conclusions 16
Acknowledgments 17
References 17
Abstract
Macroautophagy is a lysosomal catabolic process that maintains the homeostasis of
eukaryotic cells, tissues, and organisms. Macroautophagy plays important physiolog-
ical roles during development and aging processes and also contributes to immune
responses. The process of macroautophagy is compromised in diseases, such as
cancer, neurodegenerative disorders, and diabetes. The autophagosome, the dou-
ble-membrane-bound organelle that sequesters cytoplasmic material to initiate
macroautophagy, is formed by the hierarchical recruitment of about 15 autophagy-
related (ATG) proteins and associated proteins, such as DFCP1, AMBRA1, the class III
phosphatidyl-inositol 3-kinase VPS34, and p150/VPS15. Evidence suggests that in
addition to the canonical pathway, noncanonical pathways that do not require the
entire repertoire of ATGs can also result in formation of autophagosomes. Here we will
discuss recent discoveries concerning the molecular regulation of these noncanonical
forms of macroautophagy and their potential roles in cellular responses to stressful
situations.
International Review of Cell and Molecular Biology, Volume 328

ISSN 1937-6448 © 2017 Elsevier Inc.
http://dx.doi.org/10.1016/bs.ircmb.2016.08.001 All rights reserved. 1
2 N. Dupont et al.
1. INTRODUCTION
Autophagy refers to catabolic processes by which eukaryotic cells

recycle their own constituents by a vacuolar transport of cargo to the
lysosomes (Boya et al., 2013). Macroautophagy (hereafter referred to as
autophagy) starts with the formation of a double-membrane-bound vacuole
named the autophagosome that unselectively or selectively sequesters
fractions of the cytoplasm. Autophagosomes deliver cargo to the lysosomes
either directly or after fusion with endosomal compartments. Autophagy
is induced in response to a variety of extracellular and intracellular stress
situations, such as nutrient shortage, hypoxia, the production of reactive
oxygen species, or the accumulation of protein aggregates. Autophagy is
necessary during development and in adult organisms maintains tissue
homeostasis and innate and adaptive immunity (Kroemer et al., 2010;
Mehrpour et al., 2010; Mizushima and Komatsu, 2011). Alterations in
autophagy are observed in pathologies, such as cancer, neurodegenerative
diseases, type II diabetes, and chronic inflammatory diseases (Choi et al.,
2013; Jiang and Mizushima, 2014).
The discovery of autophagy-related (ATG) genes in yeast was a milestone
in our understanding of the molecular aspects and the physiological functions
of autophagy (Ohsumi, 2014; Yang and Klionsky, 2010). About 40 ATG
proteins have been characterized in yeast. Eighteen ATG proteins constitute
the core machinery of the autophagosome (Mizushima et al., 2011;
Nakatogawa et al., 2009). These core ATG proteins were conserved during
the evolution and act in different modules during the assembly and the
elongation of a membrane called phagophore to form the autophagosome.
During selective autophagy, autophagy receptors recognized by the core
machinery sequester the targeted protein, organelle, protein aggregate,
bacteria, or virus into the autophagosome.
Recently, noncanonical autophagy pathways, that result in formation of
functional autophagosomes, that include only some of the core ATG
machinery have been discovered (Codogno et al., 2011). The existence of
these alternative pathways underscores the complexity of the molecular
aspects of autophagy and raises the possibility that different stimuli induce
subtle molecular changes in autophagy.
Although our understanding of noncanonical autophagy is still in its
childhood, it is important to summarize and discuss the knowledge on this
topic. Here, we discuss the differences between canonical and noncanonical
Molecular Mechanisms of Noncanonical Autophagy 3
forms of autophagy. We will not consider in this review other noncanonical

pathways involving ATG proteins, such as the recruitment of ATG to the
phagosomal membrane emanating from the plasma membrane or the for-
mation of autophagy-like structures that fuse with the plasma membrane.
Readers interested in these topics can refer to recent reviews (Bestebroer
et al., 2013; Boya et al., 2013; Munz, 2015; Subramani and Malhotra, 2013).
2. THE CANONICAL AUTOPHAGIC PATHWAY
Before discussing noncanonical autophagy, we first need to briefly

define the canonical autophagy pathway. For readers who want to have a
more complete view of the biogenesis of autophagosomes, many excellent
recent reviews on this topic can be consulted (Abada and Elazar, 2014;
Carlsson and Simonsen, 2015; Kawabata and Yoshimori, 2015; Lamb
et al., 2013; Roberts and Ktistakis, 2013; Sanchez-Wandelmer et al., 2015;
Shibutani and Yoshimori, 2014). The formation of an autophagosome can
be subdivided into different phases that involve five functional modules
(Fig. 1). Two complexes are involved in the initiation of autophagy:
the ULK1 (unc-51 like autophagy activating kinase 1) complex (ULK1 is
the mammalian homolog of yeast ATG1), phosphatidylinositol 3-kinase
complex I (which contains PIK3C3/VPS34 or class III phosphatidylinositol
3-kinase and Beclin 1, the mammalian homolog of the yeast ATG6).
Phosphatidylinositol 3-phosphate (PtdIns(3)P), which is produced by the
enzymatic activity of VPS34, recruits WD repeat domain of phosphoinosi-
tide-interacting proteins WIPI1 and WIPI2 (homologs of yeast ATG18)
to the phagophore and also recruits DFCP1 (double FYVE-domain-con-
taining protein 1) to the endoplasmic reticulum (ER) membrane, which is
the site of autophagosome formation known as the omegasome (Axe et al.,
2008). The two PtdIns(3)P-binding proteins WIPI1/2 and DFCP1 charac-
terize the third functional cassette. One of the functions of WIPI2 is to
control the transport of the multimembrane-spanning ATG9 from the pha-
gophore to a peripheral endosome/Golgi localization (Zavodszky et al.,
2013). The ATG9 protein is the fourth functional module. In yeast, the
fusion of ATG9-containing vesicles is important in the very early stages of
autophagy (Yamamoto et al., 2012). In mammalian cells, the trafficking of
ATG9 to the phagophore is also an early event that occurs soon after
autophagy induction (Orsi et al., 2012). The last functional module consists
of the two ubiquitin-like conjugation systems: ATG12–ATG5 and the
4 N. Dupont et al.
Initiation Elongation Maturation
Signaling ATG12 LC3

ATG7
ATG5 ATG12 LC3

ATG101 ATG10 ATG3 ATG3-ATG12
ULK1
ATG13
FIP200 ATG5 ATG12
ULK -ATG16L1
LC3-PE
complex
Conjugation systems
Ambra1
Beclin-1 ATG14L WIPI-1/2
VPS34
VPS15
PIK3C3 Lysosome
complex PI3P
PI5P
PIKfyve ATG9L Phagophore

Autophagosome
vesicles Lysosome
Figure 1 ATG proteins involved in the early steps of mammalian macroautophagy.

Macroautophagy (or simply autophagy) involves the formation of double-membrane-
bound organelles called autophagosomes in which cargoes are sequestered and then
degraded after fusion with the lysosomes. Briefly, there are three important stages
in macroautophagy: initiation, which involves the nucleation step that results in
phagophore formation; elongation, which involves the closure step that results in the
autophagosome; and maturation, in which the autophagosome fuses with the
lysosome. The different modules that constitute the ATG protein core machinery
involved in the first two steps of autophagosome formation are delimited by the gray
dotted lines. Upon autophagy stimulation, the ULK complex (consisting of ULK1, FIP200,
ATG13, and ATG101 shown in yellow) activates the class III phosphatidylinositol-3-kinase
(PIK3C3) complex I (in blue), consisting of the core structure (Beclin 1, VPS15, and VPS34),
and two regulators (ATG14L and AMBRA1). This induction allows the production
of PI3P (blue lines) on the omegasome to promote the recruitment of the two
ubiquitin-like conjugation systems (in orange) through the WIPI proteins (in green).
The ATG12–ATG5–ATG16L1 complex acts as an E3-like enzyme to generate the
PE-conjugated form of LC3 (LC3-II), the well-known marker of the macroautophagic
pathway. The early stages of phagophore formation depend on vesicular transport of
the transmembrane-bound ATG9. Different noncanonical autophagy pathways also
lead to the formation of functional autophagosomes but require only certain of the
core machinery factors. Examples are ULK1- and VPS34-independent pathways. The role
of PtdIns(5)P produced by the phosphatidylinositol 5-kinase PIKfyve has also been
documented to support the autophagosome formation in a PtdIns(3)P-independent
manner. Finally, noncanonical autophagy can also require a nonclassical ubiquitin-like
conjugate (e.g., ATG12–ATG3 conjugate).
MAP1LC3 (microtubule-associated protein 1 light chain 3)–PE (phospha-

tidylethanolamine) conjugate (or LC3-II). The conjugate is the covalent link
between the C-terminus of LC3 (the mammalian homolog of yeast ATG8)
and the polar head of PE. The ubiquitin-like conjugation systems are
involved in the elongation and closure of the autophagosomal membrane
(Kabeya et al., 2000; Mizushima et al., 1998).
2.1 The Core ATG Machinery and the Formation

of Autophagosomes
Autophagy is initiated by the activation of the ULK1 complex, which
contains a serine/threonine kinase ULK1 or ULK2, ATG13, FIP200
(a 200-kDa focal adhesion kinase family interacting protein), and
ATG101. Once autophagy has been induced, this complex localizes at the
site of phagophore formation to regulate the nucleation machinery (Wirth
et al., 2013). Phagophore nucleation is highly dependent on the production
of PtdIns(3)P by VPS34. VPS34 is a part of the core phosphatidylinositol
3-kinase complex I that also contains adaptor protein VPS15, Beclin 1, and
ATG14 (Levine et al., 2015). Several proteins, such as AMBRA1 (activating
molecule in Beclin 1-regulated autophagy) or Bcl-2, can activate or inhibit
the production of PtdIns(3)P by the phosphatidylinositol 3-kinase complex I
(Wirth et al., 2013). ULK1 activates the phosphatidylinositol 3-kinase
complex I by phosphorylating Beclin 1 and AMBRA1 (Di Bartolomeo
et al., 2010; Russell et al., 2013). In turn, AMBRA1 interacts with the
E3-ligase TRAF6 (TNF receptor-associated factor 6) to induce the ubiqui-
tination of ULK1, thus increasing its stability and functional efficiency
(Nazio et al., 2013). ULK1 can also modulate the activity of other modules
of the ATG core machinery by controlling the vesicular transport of
ATG9 (Zavodszky et al., 2013) and by interacting with ATG8 homologs
via a LC3-interacting region (LIR motif) (Kraft et al., 2012).
Another component of the ULK1 complex, FIP200, interacts with
ATG16L1 (Gammoh et al., 2013; Nishimura et al., 2013), which is an
element in the ubiquitin-like cassette of the ATG core machinery. The
production of PtdIns(3)P in the phagophore membrane allows the recruit-
ment of WIPI1 and WIPI2 (Polson et al., 2010). Recently WIPI2b
[an isoform of WIPI2 functional in autophagy (Muller and Proikas-
Cezanne, 2015)] was shown to interact with ATG16L1 (Dooley et al.,
2014). These results suggest WIPI2b and ATG16L1 contribute to the expan-
sion and the closure of the vesicle in concert with the two ubiquitin-like
conjugation systems, resulting in the ATG12–ATG5–ATG16L complex,
6 N. Dupont et al.
and the formation of the PE conjugate of microtubule-associated protein

LC3. Interestingly different domains of ATG16L1 interact with FIP200 and
WIPI2b suggesting that ATG16L1 can recruit both proteins at the same time.
Autophagosome formation may occur at different membranes. During
starvation, the phagophore is nucleated at the omegasome, a subdomain of
the ER characterized by its Ω shape and by the presence of the PtdIns(3)P
binding protein DFCP1 (Axe et al., 2008). The ER-mitochondrial mem-
brane plays a pivotal role in the recruitment of ATG proteins through the
t-SNARE (target membrane—soluble N-ethylmaleimide-sensitive-factor
attachment protein receptor) protein syntaxin 17 and the ER membrane
protein VMP1 (vacuole membrane protein 1) (Hamasaki et al., 2013;
Koyama-Honda et al., 2013; Molejon et al., 2013). The plasma membrane
has also been shown to be engaged during autophagosome formation. The
t-SNARE protein, VAMP7 (vesicle-associated membrane protein 7) and its
partner v(vesicle)-SNARE (a complex of syntaxin 7, syntaxin 8, and Vti1b)
regulate the homotypic fusion of ATG16L1-positive vesicles after internal-
ization from the plasma membrane (Moreau et al., 2011). It has also been
suggested that these vesicles are precursor elements of the phagophore
(Ravikumar et al., 2010). The transmembrane protein ATG9 is also involved
in the nucleation of the phagophore membrane; it cycles between different
compartments and the phagophore (Orsi et al., 2012). The ER exit site
and the ERGIC (ER-Golgi intermediate compartment) compartment
have also been shown to play major roles in early events of autophagosome
biogenesis (Ge et al., 2013; Stadel et al., 2015). More detailed discussion on
the membrane origin in the autophagic pathway can be found in recent
reviews (Abada and Elazar, 2014; Carlsson and Simonsen, 2015; Kawabata
and Yoshimori, 2015; Lamb et al., 2013; Roberts and Ktistakis, 2013;
Sanchez-Wandelmer et al., 2015; Shibutani and Yoshimori, 2014).
2.2 Maturation of Autophagosomes

Autophagosome maturation and final fusion with the lysosome occurs in the
vicinity of the centrosome and depends on several lysosomal membrane
proteins, such as the small GTPase Rab7 (Ras-related protein 7) and
the transmembrane lysosome-associated membrane protein 2 (LAMP2)
(Eskelinen, 2005). These fusion events are also dependent on SNAREs.
VAMP3 contributes to the fusion of multivesicular bodies with autophago-
somes to form amphisomes (Fader et al., 2009). Recently, syntaxin 17
has been shown to be targeted by autophagosomes to control fusion with
endosomes/lysosomes (Itakura et al., 2012). Interestingly ATG14L binds
syntaxin 17 and stabilizes the syntaxin 17-SNAP29 (synaptosomal-associated

protein 29) complex (Diao et al., 2015). This interaction primes the inter-
action with VAMP8 to control autophagosome–endosome fusion.
In contrast to its role in autophagosome biogenesis, the role of ATG14L
in autophagosome maturation requires its homooligomerization (Diao
et al., 2015). Syntaxin 17 and ATG14L are involved in the early and late
stages of autophagosome formation (Hamasaki et al., 2013). This is only
one of the examples in which the same actors have been demonstrated to
be involved in different steps of the autophagic pathway. The phosphory-
lation of LC3 at position Thr50 by the Hippo kinase STK3/STK4 (serine/
threonine-protein kinase 3/serine/threonine-protein kinase 4) (Wilkinson
et al., 2015) and the activity of the ER calcium pump CaP60A/SERCA are
also critical for the fusion of autophagosomes with lysosomes (Mauvezin
et al., 2015). The multivalent adaptor protein PLEKHM1 (pleckstrin
homology domain-containing family M member 1), which interacts with
Rab7, LC3, and the HOPS (homotypic fusion and protein sorting) com-
plex, is a central hub for both the maturation of autophagosomes and
the progression of endocytosis (McEwan et al., 2015). The reformation
of lysosomes through a process called autophagosome-lysosome reforma-
tion (ALR) is fundamental to lysosomal identity (Yu et al., 2010).
Phosphatidylinositol 3-phopshate and VPS34, which are necessary in the
early stage of autophagy, are important regulators of ALR with the protein
UVRAG as a partner (Munson et al., 2015).
3. NONCANONICAL AUTOPHAGY
Noncanonical autophagy does not require all of the ATG proteins to

build-up the double-membrane-bound autophagosome. There is evidence
for several types of noncanonical autophagy; each will be discussed in the
following sections.
3.1 ULK1-Independent Autophagy

A form of autophagy that bypasses the canonical ULK1 initiation step has
been reported to occur in response to ammonia or to glucose deprivation
(Cheong et al., 2011). Interestingly, Gammoh et al. (2013) identified a
FIP200-binding domain (FBD) in ATG16L1 adjacent but distinct from
the WIPI2b binding site (Dooley et al., 2014). This domain is required for
amino acid starvation-induced autophagy (ULK-dependent autophagy) but
8 N. Dupont et al.
is not required for glucose deprivation-induced autophagy (which is ULK

independent). These results suggest that ATG16L1 can integrate upstream
signals independently of the ULK1 complex. Probably other ATG16L1
domains in addition to the FBD domain are also involved in the signaling
because ATG16L2, an ATG16L1 homolog in mammalian cells, does not
have an FBD domain and is unable to support autophagy initiation
(Gammoh et al., 2013).
The C-terminal WD40 repeats of ATG16L1 are not required for amino
acid starvation-dependent autophagy. However, this region is required for
selective forms of autophagy (Lassen et al., 2014). Recently Stork and cow-
orkers reported that a ULK1/2 binding-deficient ATG13 partially restores
amino acid-induced and serum starvation-induced autophagy in ATG
13-deficient cells (Alers et al., 2011). These results suggest that ULK1-
independent autophagy can occur in the absence of amino acids either
mediated by a low-affinity interaction between ULK1 and the truncated
form of ATG13 or by an ULK1-dependent but ULK1-ATG13-independent
event. Although amino acid-dependent autophagy is impaired in ULK1/2-
deficient cells (Cheong et al., 2011; McAlpine et al., 2013), some LC3-II
formation is present in the absence of amino acids (McAlpine et al., 2013).
Overall these data challenge the view of the absolute requirement of the
ULK1/2 complex to initiate autophagy.
3.2 Beclin 1-Independent and VPS34-Independent Autophagy

Beclin 1 and VPS34 can form complexes with different partners. These
complexes promote the induction of autophagy (in concert with ATG14)
or the maturation of autophagosomes (in concert with UVRAG and
Rubicon) (Funderburk et al., 2010; Wirth et al., 2013). However, the
Beclin 1–VPS34 complex is not obligatory under all circumstances (Codogno
et al., 2011; Proikas-Cezanne and Codogno, 2011). Noncanonical Beclin
1-independent autophagy occurs after cells have been treated with proapop-
totic compounds (Table 1).
The first evidence for Beclin 1-independent autophagy was shown in
the context of neuronal cell death induced by the neurotoxin 1-methyl-
4-phenylpyridinium (Zhu et al., 2007). Treatment of human breast cancer
cells with the polyphenol resveratrol also induces Beclin 1-independent
autophagy (Scarlatti et al., 2008). Furthermore, Z18, a compound
that targets the BH3 binding groove of Bcl-XL and Bcl-2, causes Beclin
1-independent autophagosome formation in HeLa cells (Tian et al., 2010),
and other proapoptotic compounds, such as staurosporine, MK801, and
Molecular Mechanisms of Noncanonical Autophagy
Table 1 Inducers of noncanonical autophagy.
Methods used and
Inducers ATGs bypassed inhibitorsa Cell types References
Ammonia (metabolic ULK1, ULK2 DKO Ulk1/2 (WB LC3) Mouse embryonic fibroblasts Cheong et al.
intermediate) (2011)
Glucose deprivation ULK1, ULK2 DKO Ulk1/2 (WB LC3, Mouse embryonic fibroblasts Cheong et al.
IFLC3) (2011)
Carbonyl cyanide FIP200, KO Fip200, KD ATG13, Mouse embryonic fibroblasts, Chen et al.
m-chlorophenylhydrazone ATG13, ATG14, Beclin 1 epithelial cells (HeLa), (2013)
(CCCP) ATG14, (WB LC3, IFLC3) glioblastoma cells (U251)
Beclin 1
Glucose deprivationb PIK3C3 WM (WB LC3, IF LC3) Mouse embryonic fibroblasts McAlpine et al.
(2013)
Unsaturated fatty acids Beclin 1, KD, WM, 3-MA (WB Epithelial cells (HeLa) and Niso-Santano
VPS34 LC3, IF LC3) human breast cancer cells et al. (2015)
(U2OS)
Glucose deprivation VPS34 KD, KO, WM (WB LC3, Mouse embryonic fibroblasts, Vicinanza et al.
IF LC3, EM) epithelial cells (HeLa) (2015)
Resveratrol Beclin 1, KD, 3-MA, WM Human breast cancer cells Scarlatti et al.
VPS34 (WB LC3, IF LC3) (U2OS, MCF7) (2008);
Mauthe et al.
(2011)
1-Methyl-4-phenylpyridinium Beclin 1 KD, 3-MA (WB LC3, Neurons (SH-SY5Y) Zhu et al. (2007)
(MPP) IF LC3)
(Continued )
9
10
Table 1 Inducers of noncanonical autophagy.—cont'd.
Methods used and
Inducers ATGs bypassed inhibitorsa Cell types References
Z18 Beclin 1, KD, 3-MA (WB LC3, Human epithelial cells (HeLa) Tian et al. (2010)
VPS34 IF LC3)
Staurosporin Beclin 1 KD (WB LC3, IF LC3) Primary cortical neurons Grishchuk et al.
(2011)
MK801 Beclin 1 KD (WB LC3, IF LC3) Primary cortical neurons Grishchuk et al.
(2011)
Gossypol Beclin 1 KD (WB LC3, IF LC3) Human epithelial cells (HeLa Gao et al. (2010)
cells and not MCF7)
Arsenic trioxide Beclin 1 KD (WB LC3, IF LC3) Ovarian cells (HEY cells) Smith et al.
(2010)
Etoposide Beclin 1 KD (WB LC3, IF LC3) Primary cortical neurons Grishchuk et al.
(2011)
α-Hemolysin Beclin 1 KD, 3-MA, WM Chinese hamster ovary Mestre et al.
(IF LC3) fibroblast (CHO) (2010)
Recombinant capsid Beclin 1 KD (WB LC3, EM) Murine macrophage cells Liao et al. (2013)
protein VP1 (RAW264.7)
1,3-Dibutyl-2-thiooxo- Beclin 1 KD, 3-MA (WB LC3) Colorectal carcinoma cells Wong et al.
imidazolidine-4,5-dione (C1) (HCT116) (2010)
3-Methylcyclopentylidene-[4- Beclin 1 KD (WB LC3, IF LC3) Human lung carcinoma cells Ragazzoni et al.
(40 -chlorophenyl)thiazol-2-yl] (H1299) (2013)
hydrazone (CPTH6)
N. Dupont et al.
Molecular Mechanisms of Noncanonical Autophagy
Epoxomicin, lactacystin, Beclin 1 KD, 3-MA, WM (WB Human hepatocarcinoma cells Liu et al. (2013)
bortezomib, MG132 LC3, IF LC3) (HepG2)
Raclopride ATG7 KD ATG7 (WB LC3, Cardiac myocytes Yan et al. (2013)
WB p62)
Etoposide, staurosporine AG5, ATG7, KO Atg5, Atg7; KD Mouse embryonic fibroblasts, Nishida et al.
ATG9, ATG9, ATG12, erythroid cells (2009); Honda
ATG12, ATG16 (EM, et al. (2014)
ATG16 proteolysis)
Amino acid and growth factor ATG9 KO, KD (WB LC3, IF Mouse embryonic fibroblasts, Orsi et al. (2012)
deprivation LC3) human embryonic kidney
cells (HEK)
a
DKO, Double knockout; EM, electron microscopy; IF, immunofluorescence (or fluorescence); KD, knockdown; KO, knockout; 3-MA, 3-methyladenine; WB, Western
blot; WM, wortmannin.
b
These authors demonstrated a partial inhibition of autophagy in ULK1/2 DKO MEFs upon glucose starvation.
11
12 N. Dupont et al.
etoposide, induce Beclin 1-independent autophagy in primary cortical neu-

rons (Grishchuk et al., 2011). These studies suggest that it might become
feasible to use prodeath compounds that induce noncanonical autophagy for
cancer therapy when the functions of canonical autophagy proteins are
compromised.
Beclin 1-independent autophagy has also been observed in settings
unrelated to cell death, such as during differentiation (Arsov et al., 2011),
bacterial toxin uptake (Mestre et al., 2010), and viral infection (Berryman
et al., 2012). Interestingly cis-unsaturated fatty acids (oleate and arachido-
nate) trigger autophagy that is independent of Beclin 1 and VPS34, whereas
saturated fatty acids (palmitate and stearate) cause autophagy that is depen-
dent on these two factors (Niso-Santano et al., 2015). The existence of
Beclin 1-independent autophagy induced by unsaturated fatty acids has been
confirmed in mice, in the nematode Caenorhabditis elegans, and in the yeast
Saccharomyces cerevisiae. Whether the stimulation of noncanonical autophagy
by cis-unsaturated fatty acids underlies the beneficial effects of these nutrients
on obesity, atherosclerosis, and neurodegenerative disease is an intriguing
possibility.
BAG3 (Bcl-2 associated athanogene 3), a cochaperone of HSP70
(70-kDa heat shock protein) family, is engaged in Beclin 1-independent
autophagy in response to proteasome inhibition (Liu et al., 2013) and in
estrogen receptor-mediated autophagy in breast cancer cells (Felzen et al.,
2015). A role of BAG3 in the regulation of LC3 translation has been recently
reported (Rodriguez et al., 2016). How this is related to the role of BAG3 in
noncanonical autophagy remains to be elucidated. It may be that BAG3 is a
bona-fide signature for Beclin 1-independent autophagy (Liu et al., 2013).
Interestingly, Beclin 1-independent autophagy is not synonymous with
pathways that exclude the VPS34–WIPI–ATG5-LC3 route. Exposure of
human tumor cells to arsenic trioxide (Smith et al., 2010) or gossypol
promotes autophagy that is VPS34-dependent, but Beclin 1-independent.
The autophagy activated by these inducers involves WIPI-1 under some
circumstances (Gao et al., 2010). Recently, resveratrol was found to pro-
mote WIPI-1-dependent LC3 lipidation in the absence of induced
phagophore formation, indicating that different membrane sites may be
used during noncanonical autophagosome formation (Mauthe et al.,
2011). In fact, WIPI-1 specifically localizes to both the plasma membrane
and the ER in response to the induction of autophagy, indicating that
the WIPI–ATG5–LC3 pathway can function at several different membrane
sites.
Table 2 In vivo detection of noncanonical autophagy by the use of Atg-knockout mouse

models.
KO In vivo autophagosome
model formation References
Vps15 Muscle Nemazanyy et al. (2013)
Vps34 Mature sensory neurons Zhou et al. (2010)
Vps34 T lymphocytes McLeod et al. (2011)
Vps34 T lymphocytes Willinger and Flavell (2012)
Vps34 Heart and liver Jaber et al. (2012)
Vps34 T lymphocytes Parekh et al. (2013)
Atg5 Brain, liver, heart, Nishida et al. (2009); Honda et al.
erythrocytes in embryos (2014); Ma et al. (2015)
3.3 VPS34/VPS15-Independent Autophagy

Recent studies suggest that autophagy can be independent of VPS34 and
p150/VPS15 (Table 2). In Vps34/ sensory neurons, autophagosomes and
LC3-II production are observed (Zhou et al., 2010). In the same mouse
model, autophagy was observed in T lymphocytes (McLeod et al., 2011).
However, in other genetic mouse models in which Vps34 is ablated, autop-
hagy is absent or only minimally observed (Jaber et al., 2012; Willinger and
Flavell, 2012). A recent report shows that VPS15-deficient mouse skeletal
muscle is capable of forming LC3-positive autophagosomes (Nemazanyy
et al., 2013). It is possible that PtdIns(3)P is required in these situations. This
would suggest that this lipid is produced either by the degradation of PtdIns
(3,4)P2 or PtdIns(3,4,5)P3 or by the activity of another phosphatidylinositol
3-kinase (Dall’Armi et al., 2013).
In fact, class II phosphatidylinositol 3-kinase (PIK3C2) has been
shown to contribute to the pool of PtdIns(3)P involved in autophagosome
biogenesis (Devereaux et al., 2013). The formation of PtdIns(3)P by
PIK3C2 would explain why autophagy is sometimes insensitive to classical
PIK3 inhibitors, such as 3-methyladenine and wortmannin (Rubinsztein
et al., 2012).
In autophagy induced by glucose starvation, PtdIns(3)P is not required
to initiate autophagy (McAlpine et al., 2013; Vicinanza et al., 2015).
Recently, the role of PtdIns(5)P produced by the phosphatidylinositol
5-kinase PIKfyve in support of autophagosome formation in the absence
of PtdIns(3)P has been documented (Vicinanza et al., 2015). Whether
PtdIns(5)P-dependent autophagy requires Beclin 1 is not known. It
14 N. Dupont et al.
remains to be investigated whether DFCP1, similarly to WIPI2b, can bind

to PtdIns(3)P and PtdIns(5)P. The fact that PtdIns(5)P makes up a minor
fraction of the cellular phosphoinositide suggests that autophagy induction
is dependent on subtle molecular and cellular conditions.
3.4 Autophagy Independent of Ubiquitin-Like

Conjugation Actors
Although the formation of LC3-II is not observed in ATG5/ cells,
transition electron microscopy reveals the presence of autophagosomes
and amphisomes (preautolysosomal vacuoles that are formed by
autophagosome–endosome fusion) in ATG5/ cells treated with etoposide
over a prolonged period of time (Nishida et al., 2009). Based on the unusual
lamination of the membrane forming the phagophores and autophagosomes
in this context, the trans-Golgi network seems to be the membrane source
for this form of alternative autophagy, which is initiated by ULK1 and
VPS34 complexes in a manner similar to canonical autophagy (Codogno
et al., 2011). In contrast to the canonical pathway, the elongation of the initial
autophagosomal membrane does not require ATG9 or the ATG proteins of
the ubiquitin-like conjugation system (ATG7, ATG5, and LC3). Instead, the
monomeric GTPase Rab9, which is involved in vesicular trafficking
between the trans-Golgi network and late endosomes, is required to elongate
the initial autophagosomal membrane through fusion events with Rab9-
positive vesicles. The resulting noncanonical, double-membrane autopha-
gosome can mature and fuse with the lysosomal compartment to deliver
cargo for degradation.
The form of autophagy that is independent of the ubiquitin-like
conjugation systems has been observed in various cell types and embryonic
tissues (Codogno et al., 2011) and plays a role in the removal of mito-
chondria during erythrocyte maturation and mitophagy in vivo (Nishida
et al., 2009).
However, in erythroblasts, mitochondria are also degraded by autop-
hagy in a manner dependent on ATG5 and ATG7 (Mortensen et al., 2011).
These results suggest that both canonical autophagy and noncanonical
autophagy may contribute to the elimination of mitochondria during
erythroblast differentiation. Interestingly, ULK1-dependent and ATG5/
ATG7-independent autophagy is the major pathway that eliminates mito-
chondria from fetal erythroblasts (Honda et al., 2014). ATG5-independent
autophagy also functions in mitochondrial clearance during induced
pluripotent stem cell (iPSC) reprogramming (Ma et al., 2015). Since

this metabolic reprogramming is similar to that observed during tumori-
genesis, noncanonical autophagy may be involved in pathophysiological
situations.
Recently, a form of autophagy that is independent of ATG3 and ATG7
has been reported in the programmed reduction of cell size during intes-
tinal cell death in Drosophila (Chang et al., 2013). This noncanonical
autophagy involves ATG8 and Uba1 (Ubiquitin-like modifier-activating
enzyme 1), the E1 enzyme that catalyzes the first step in ubiquitination.
Uba1 is not a substitute for the E1 activity of ATG7 in the conjugation
of ATG8 to PE (ATG3 functions as the E2-conjugating enzyme in the
formation of ATG8-PE). As Chang et al. propose, Uba1 could function at
a different stage of the autophagy pathway (Chang et al., 2013). The
mechanism by which ATG8 is recruited to the autophagosomal membrane
remains to be elucidated.
Evidence indicates that noncanonical forms of autophagy exist in differ-
ent phyla, suggesting that noncanonical forms of autophagy did not in fact
emerge recently in the course of evolution. Interestingly, the intracellular
bacterium Brucellaabortus requires a vacuole with autophagic features during
replication (Starr et al., 2012). The formation of this compartment is depen-
dent on ULK1 and components of the Beclin 1 complex (including
ATG14L1 and a VPS34 activity) but is independent of ATG4B, ATG5,
ATG7, and LC3B, suggesting that ATG5-independent autophagy or autop-
hagic-like processes can be subverted by microorganisms.
Noncanonical autophagy may also involve nonclassical ubiquitin-like
conjugates. Recently ATG3, the E2-like enzyme involved in LC3 lipida-
tion during autophagy, has been shown to be an ATG12 conjugation
target (Radoshevich et al., 2010). The same group reported that the
ATG12–ATG3 conjugate interacts with the ESCRT (endosomal sorting
complex required for transport)-associated protein Alix to control Alix-
dependent functions, such as multivesicular body distribution, exosome
biogenesis and virus budding, and facilitation of basal autophagy, but
not starvation-induced autophagy (Murrow et al., 2015). In basal autop-
hagy, the ATG12–ATG3 conjugate contributes to the maturation of
autophagosomes. This role is distinct from the role of ATG3 and ATG12
during autophagosome formation; however, the mechanism by which the
ATG12–ATG3 conjugate contributes to basal autophagy remains to be
elucidated.
16 N. Dupont et al.
4. CONCLUSIONS
Noncanonical autophagy pathways and structures have the same func-

tion as canonical autophagy: these pathways specifically or nonspecifically
sequester a portion of the cytoplasm and compartmentalize pathogens. In
many cases, material sequestered by noncanonical autophagy is ultimately
degraded in the lysosomal compartment. Noncanonical autophagy, which
requires only a subset of ATG proteins, has been observed in various settings.
It is not yet clear how some ATG modules can be bypassed to form a
functional autophagosome. Recent evidence notably suggests that PtdIns
(3)P can be produced in the absence of Beclin 1 and VPS34 by the activity
of PIK3C2 (Devereaux et al., 2013). This provides a rational for the fact
that the PtdIns(3)P effector WIPI1/2 is necessary for initiation of LC3
lipidation in noncanonical autophagy in many settings (Codogno et al.,
2011). Alternatively, other phosphoinositides, such as PtdIns(5)P, can
initiate autophagy.
Evidence indicates that components of ubiquitin-like systems can recruit
effectors that bypass the conventional route for membrane elongation and
sealing. Since the LC3 family is large in mammals, a careful investigation of
the roles of different members of the LC3/GABARAP family must be
done when a bypass of LC3 is suspected. A growing body of evidence shows
that GABARAP family members can substitute for LC3B.
One important question is whether noncanonical forms of autophagy
have specific functions in cell physiology or pathological situations. From the
literature available on Beclin 1-independent autophagy, it is difficult to
identify a single function corresponding to this form of noncanonical autop-
hagy. For example, Beclin 1-independent autophagy has been reported in
contexts, such as survival, death, and proliferation, and immune cell devel-
opment (Codogno et al., 2011; Proikas-Cezanne and Codogno, 2011).
Beclin 1-independent autophagy is the most commonly reported nonca-
nonical form of autophagy. Beclin 1 is a key autophagy protein with a large
interactome (Behrends et al., 2010; He and Levine, 2010). It is involved in
both the formation and maturation of autophagosomes, depending on the
interacting partners (Funderburk et al., 2010; Wirth et al., 2013). Moreover,
Beclin 1 is targeted by many viruses to block autophagy at different stages.
Beclin 1-independent autophagy could, therefore, be an evolutionary adap-
tation that prevents the blockade of autophagy by an invading virus, which
would otherwise compromise cell survival.
Another intriguing question involves the relationship between nonca-

nonical autophagy and selective forms of autophagy, such as mitophagy,
which specifically sequesters mitochondria (Youle and Narendra, 2011).
ATG5- and ATG7-independent autophagy is known to be involved in the
clearance of mitochondria during erythroid maturation (Nishida et al.,
2009), and determining the roles of the different ATG proteins in the various
forms of selective autophagy may provide a way to identify the molecular
signatures of autophagy that lie beyond the selective recognition of cargo
(Weidberg et al., 2011).
Another important question is whether both noncanonical autophagy
and canonical autophagy can be activated in a coordinated manner in the
same cell in response to a stressful situation. Being able to distinguish
between noncanonical autophagic pathways and the canonical autophagic
pathway by means of specific markers would make a significant contribution
to the molecular understanding of how autophagy is regulated and functions.
ACKNOWLEDGMENTS
A.C.N. and N.D. are supported by fellowships from the “Association pour la Recherche sur le
Cancer” (ARC). Studies in Patrice Codogno’s laboratory are supported by institutional
funding from INSERM (Institut National de la Santé et de la Recherche Médicale),
CNRS (Centre National de la Recherche Scientifique), University Paris-Descartes, and
grants from ANR (French National Research Agency), and INCa (French National
Cancer Institute). Conflict of interest: The authors declare that they have no conflict of
interest.
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“Well, then, if she has, she’ll see my papa and mama, and tell
them about me, and oh, Mam’selle, won’t they be glad to hear from
me?”
“I hope she will tell them how dearly I love you, and what you are
to me,” murmured Mam’selle, pressing her cheeks to the bright little
head resting against her shoulder.
“Look up there, Mam’selle Diane, do you see those two beautiful
stars so near together? I always think they are mama and papa,
watching me. Now I know mama is there, too, and will never come
back again; and see, near those there is another very soft and bright,
perhaps that is your mama shining there with them.”
“Perhaps it is, my dear—yes, perhaps it is,” and Mam’selle Diane
raised her faded eyes toward the sky, with new hope and strength in
their calm depths.
About that time Paichoux began a most laborious correspondence
with a fashionable jeweler in New York, which resulted in some very
valuable information concerning a watch with a diamond monogram.
CHAPTER XXX
AT MRS. LANIER’S
I T was a few days before the following Christmas, and Mrs. Lanier,
who had just returned from Washington, was sitting alone one
evening in her own pretty little parlor, when a servant handed her a
card.
“Arthur Maynard,” she read. “Let him come up at once”; and as the
servant left the room she added to herself: “Dear boy! I’m so glad
he’s come for Christmas.”
In a moment a handsome young fellow was in the room, shaking
hands in the most cordial way.
“You see I’m home, as usual, for the holidays, Mrs. Lanier,” he
said, showing a row of very white teeth when he laughed.
“Yes, you always do come for Christmas and Mardi-gras, don’t
you? You’re such a boy still, Arthur,” and Mrs. Lanier looked at him
as if she approved of his boyishness. “Sit down and let us have a
long chat. The children have gone to the theater with Mr. Lanier. I
was too tired to go with them. You know we reached home only this
morning.”
“No. I didn’t know that or I wouldn’t have come. You don’t want to
be bothered with me when you’re so tired,” said Arthur, rising.
“Nonsense, Arthur; sit down. You always cheer me up. You’re so
full of life and spirits, I’m really glad to see you.”
While Mrs. Lanier was speaking, the young fellow’s bright, clear
eyes were traveling about the room, and glancing at everything,
pictures, bric-à-brac, and flowers. Suddenly he uttered an
exclamation, and, springing up, seized a photograph in a velvet
frame that stood on a cabinet near him.
It represented a family group, father, mother, and child; and for a
moment he seemed too surprised to speak. Then he asked, in a very
excited tone, “Mrs. Lanier, where did you get this—and who is the
lady?”
“She is a friend of mine,” said Mrs. Lanier, much surprised. “Why
do you ask—have you ever seen her?”
“Yes, yes; and I have a copy of this picture. It is such a strange
story; but first, before I say a word, please tell me who she is, and all
about her.”
“Why, Arthur, you seem greatly interested,” returned Mrs. Lanier,
with a smile. “The lady is my dear friend, Jane Chetwynd. We were
classmates at boarding-school in New York; her father is the rich Mr.
Chetwynd. You have heard of him, haven’t you?”
“Yes, indeed; but please go on.”
“Do you want all the history?”
“Everything, please. I’ve a serious reason for wanting to know all
about the originals of this photograph.”
“Well, the gentleman is Jane’s husband, Mr. Churchill, an
Englishman, and the little girl is ‘Lady Jane,’ their only child. There’s
quite a romance connected with Jane’s history, and I’m just now
floundering in a sea of darkness in regard to that same Jane
Chetwynd.”
“If you please, go on, and perhaps I can help you out,” urged the
young man, eagerly and abruptly.
“Well, as it’s a subject I’m greatly interested in, I don’t mind telling
you the whole story. Jane Chetwynd was the only daughter; her
mother died when she was a child. Jane was her father’s idol; he
had great plans for her, and when she was only eighteen he hoped
she would marry one of the rich Bindervilles. Jane, however, married
a young Englishman who was in her father’s employ. The young man
was handsome, as you can see by his picture, well born, and well
educated; but he was unknown and poor. To Richard Chetwynd that
was unpardonable, and therefore he disowned Jane—cut her off
entirely, refused to see her, or even to allow her name to be
mentioned.
“A cousin of Mr. Churchill, who lived in England, owned a fine
ranch in Texas, and there the young couple went to pass their
honeymoon. They were delighted with the ranch, and decided to
make it a permanent home.
“Their little girl was born there, and was named for her mother. On
account of some dainty little ways, and to avoid confusion, her father
called her Lady Jane.
“In her frequent letters to me, my friend spoke of her as a
remarkable child, and of course she was the idol of her parents. In
spite of the trouble with her father, Jane never regretted her choice,
and even her isolated life had many charms for her. She was of a
quiet, domestic disposition, and loved the country. Indeed, I know
her life there was one of idyllic happiness. When the child was three
years old Jane sent me that picture; then about two more years
passed, during which time I heard from her frequently, and after that
suddenly the correspondence stopped. I was in Europe for a year,
and when I returned I set to work to find out the cause. Many letters
were returned from San Antonio, the nearest post-office; but finally
we succeeded in communicating with the overseer on the ranch,
who informed us that Mr. Churchill had died suddenly of a prevalent
fever, the summer before,—more than two years ago now,—and that
Mrs. Churchill with her little girl had left the ranch directly after her
husband’s death to return to New York, since which time he had
received no news of her; and the overseer also expressed surprise
in his letter at her long silence, as he said she had left many valuable
things that were to be sent to her when and where she should direct,
after she reached New York; he had since received no instructions,
and the property was still lying there.
“Then I wrote directly to New York to a friend who was very
intimate at one time with the Chetwynds, for some information about
Jane; but she could tell me nothing more than the newspapers told
me, that Richard Chetwynd had gone abroad, to remain some years.
Of Jane I could not hear a word.
“Sometimes I think she may have followed her father to Europe,
and that they are reconciled and living there together. But why does
she not write to me—to the friend whom she always loved so dearly?
“Then there is another thing that has worried me no little, although
in itself it is a trifle. When we were at school together I had a little
birthday gift made at Tiffany’s for Jane, a silver jewel-box, engraved
with pansies and forget-me-nots, and a lot of school-girl nonsense. I
made the design myself, and the design for the monogram also.
About a year ago I found that very box for sale at Madame
Hortense’s, on Canal Street. When I asked Hortense where she got
it, she told me that it was left with her to sell by a woman who lived
down town on Good Children Street, and she gave me the name and
address; but when I went there a day or two afterwards the woman
had gone,—left mysteriously in the night, and none of the neighbors
could tell me where she went. Of course the woman’s sudden
disappearance made me feel that there was something wrong about
her, and I can’t help thinking that she got the little box dishonestly. It
may have been stolen, either in Texas or in New York, and finally
drifted here for sale. I got possession of it at once, very thankful that
such a precious relic of my girlhood should have accidentally fallen
into my hands; but every time I look at it I feel that it is a key which
might unlock a mystery if only I knew how to use it.”
All the while Mrs. Lanier was speaking, Arthur Maynard followed
every word with bright, questioning eyes and eager, intense interest.
Sometimes he seemed about to interrupt her; then he closed his lips
firmly and continued to listen.
Mrs. Lanier was looking at him inquiringly, and when he waited as
if to hear more she said: “I have told you all. Now what have you to
tell me?”
“Something quite as strange as anything you have told me,”
replied Arthur Maynard, with an enigmatical air. “You must not think
you’re the only one with a mystery worthy the skill of a Parisian
detective. If I had any such talent I might make myself famous, with
your clues and my clues together.”
“What in the world do you mean, Arthur? What do you know?—for
pity’s sake, tell me! You can’t think how Jane Chetwynd’s long
silence distresses me.”
“Fool that I was!” cried the young fellow, jumping up and pacing
the room with a half-tragic air. “If I hadn’t been an idiot—a simpleton
—a gosling—if I’d had a spark of sense, I could have brought that
same Jane Chetwynd, and the adorable little Lady Jane, straight to
your door. Instead of that, I let them get off the train at Gretna alone
when it was nearly dark, and—Heaven only knows what has
happened to them!”
“Arthur Maynard, what do you mean?” asked Mrs. Lanier, rising to
her feet, pale and trembling. “When—where—where is she now—
where is Jane Chetwynd?”
“I wish I knew. I’m as wretched and anxious as you are, Mrs.
Lanier, and what has happened to-day has quite upset me; but I
must tell you my story, as you have told yours.”
And then, while Mrs. Lanier listened with clasped hands and intent
gaze, Arthur Maynard told of the meeting with Lady Jane and her
mother on the train, of the gift of “Tony,” the blue heron, and of the
separation at Gretna.
“Oh, Arthur, why—why didn’t you go with them and bring them to
me? She was a stranger, and she didn’t know the way, and your
being our friend and all.”
“My dear Mrs. Lanier, she never mentioned your name or number.
How could I guess you were the friend to whom she was going? and
I didn’t want to seem presuming.”
“But where did she go? She never came here!”
“Wait till I tell you the rest, and then we will discuss that. I stood on
the platform until the train started, and watched them walking toward
the ferry, the mother very feebly, and the child skipping along with
the little basket, delighted with her new possession; then I went back
to my seat, angry enough at myself because I wasn’t with them,
when what should I see on the floor, under their seat, but a book
they had left. I have it now, and I’ll bring it to you to-morrow; inside of
the book was a photograph—a duplicate of this, and on the fly-leaf
was written ‘Jane Chetwynd.’”
“I thought so! I knew it was Jane!” exclaimed Mrs. Lanier excitedly.
“But she never came here. Where could she have gone?”
“That’s the mystery. She may have changed her mind and gone to
a hotel, or something may have happened to her. I don’t know. I
don’t like to think of it! However, the next day I advertised the book,
and advertised it for a week; but it was never claimed, and from that
day to this I’ve never been able to discover either the mother or the
child.”
“How strange, how very strange!” said Mrs. Lanier, greatly
troubled. “Why should she have changed her mind so suddenly? If
she started to come to me, why didn’t she come?”
“The only reasonable solution to the problem is that she changed
her mind and went on to New York by the night-train. She evidently
did not go to a hotel, for I have looked over all the hotel registers of
that time, and her name does not appear on any of them. So far
there is nothing very mysterious; she might have taken the night-
train.”
“Oh, Arthur, she probably did. Why do you say she might have?”
“Because you see I have a sequel to my story. You had a sequel to
yours, a sequel of a box. Mine is a sequel of a bird—the blue heron I
gave the little Lady Jane. I bought that same blue heron from a bird-
fancier on Charter Street this very morning.”
“How can you be sure that it is the same bird, Arthur? How can
you be sure?”
“Because it was marked in a peculiar way. It had three distinct
black crosses on one wing. I knew the rogue as soon as I saw him,
although he has grown twice the size, and—would you believe it?—
he has the same leather band on his leg that I sewed on more than
two years ago.”
“And you found out where the fancier bought him?” asked Mrs.
Lanier breathlessly.
“Of course I asked, the first thing, and all the information I could
get from the merchant was that he bought him from an Italian a few
days before, who was very anxious to sell him. When I called the
bird by his name, Tony, he recognized it instantly. So you see that he
has always been called by that name.”
“The child must have lost him, or he must have been stolen. Then
the box, the jewel-box here too. Good heavens! Arthur, what can it
mean?”
“It means that Mrs. Churchill never left New Orleans,” said Arthur
decidedly.
“My dear Arthur, you alarm me!” cried Mrs. Lanier; “there is
something dreadful behind all this. Go on, and tell me everything you
know.”
“Well, after I bought the bird, and while I was writing my address
for the man to send him home, a funny little old Frenchman came in,
and suddenly pounced on Tony, and began to jabber in the most
absurd way. I thought he was crazy at first; but after a while I made
him understand that the heron belonged to me; and when I had
calmed him down somewhat I gathered from his remarks that this
identical blue heron had been the property of ’one leetle lady’ who
formerly lived on Good Children Street.”
“Good Children Street,” interrupted Mrs. Lanier; “what a
remarkable coincidence!”
“That the bird had been lost, and that he had searched
everywhere to find it for the ‘leetle lady.’ Then I asked him for a
description of the ‘leetle lady.’ And, as I live, Mrs. Lanier, he
described that child to the life,”—and Arthur Maynard pointed to the
photograph as he spoke.
“Oh, Arthur, can it be that Jane Chetwynd is dead? What else can
it mean? Where is the child? I must see her. Will you go with me to
Good Children Street early to-morrow?”
“Certainly, Mrs. Lanier. But she is not there; the old man told me a
long story of a Madame Jozain, who ran away with the child.”
“Madame Jozain!” cried Mrs. Lanier excitedly—“the same woman
who had the jewel-box.”
“Evidently the same, and we are on her track—or we should be, if
she were alive; but unfortunately she’s dead. The little Frenchman
says so, and the child is now in Margaret’s Orphans’ Home.”
“Oh, I see it all now! It is as clear as day to me!” cried Mrs. Lanier,
springing from her chair and walking excitedly back and forth. “It is
all explained—the mysterious attraction I felt for that child from the
first. Her eyes, her voice, her smile are Jane Chetwynd’s. Arthur,
would you know her again if you saw her?”
“Certainly; she hasn’t grown out of my recollection in two years,
though of course she may not resemble the photograph so much.
You see it is four or five years since that was taken; but she can’t
have changed in two years so that I won’t know her, and I’m very
sure also that she’ll remember me.”
“Well, come to-morrow at eleven, and I think I can have her here.
The lovely child in Margaret’s Home, in whom I have felt such an
interest, must be the one. Her name is Jane. I will write to Margaret
at once to bring her here to-morrow morning, and, Arthur, if you can
identify her she is Jane Chetwynd’s child without a doubt;—but Jane
—poor Jane! What has happened to her? It is a mystery, and I shall
never rest until it is explained.”
“And perhaps you will hate me for my stupidity,” replied Arthur,
looking very much cast down, as he shook hands and said good-
night.
“No, no, my dear boy. You were not in the least to blame, and
perhaps your generosity in giving Lady Jane the blue heron may be
the means of restoring her to her friends.”
Thinking the matter over from Mrs. Lanier’s point of view, Arthur
went away somewhat comforted, but still very anxious about the
developments the next day might bring forth.
CHAPTER XXXI
LADY JANE COMES TO HER OWN
T HE next morning, when Margaret brought little Jane, Mrs. Lanier

sent for them to come to her room, and there she heard the
strange story that Paichoux had told Margaret.
Putting together one thing and another, the incidents seemed to
form a chain of which there was only one link missing, and that was
an explanation of the mystery surrounding the fate of the young
mother. What had become of her? And how had Madame Jozain got
possession of the child, as well as of the property?
“It is work for a skilful detective,” said Mrs. Lanier, when Margaret
had told her of Paichoux’s plan.
And Margaret replied that, with the aid of a little money, the snarl
could soon be unraveled.
“The money will be forthcoming,” returned Mrs. Lanier. “It shall be
my sacred duty to begin an investigation as soon as the child’s
identity is established. Mr. Lanier will interest himself with me, and
every possible effort shall be made to get at the bottom of the
mystery. Meanwhile, my good Margaret, you must leave little Jane
with me. Jane Chetwynd’s child must not be dependent on charity.”
To this Margaret readily agreed, and then Lady Jane was called
from the nursery, where she had been with Mrs. Lanier’s little girls
during this long serious conversation.
The child came in dressed in her homely orphan’s garb, with all
her beautiful hair braided and hanging stiffly down her back; but she
was lovely in spite of her unlovely attire, her sweet little face was
dimpled with smiles, and her wide eyes were full of pleasant
expectation.
“Come here, my dear,” said Mrs. Lanier, holding out her hands.
“Now tell me, which name do you like best, Lady Jane, or simply
Jane?”
She hesitated a moment and looked wistfully at Margaret, while a
slight shadow passed over her face. “I like Lady Jane; but Mother
Margaret likes Jane best.”
Then Mrs. Lanier opened a drawer and took out a photograph in a
velvet frame. “My dear,” she said, holding it before her, “who are
these?”
In an instant the child’s face changed; every vestige of color fled
from it, as she fixed her eyes on the picture with a look of eager
affection and pitiful surprise. “It’s papa and mama!” she exclaimed
passionately. “It’s my dear, dear mama!” Then, with a cry of distress,
she threw herself into Margaret’s arms and sobbed bitterly.
“This is proof enough for me,” said Mrs. Lanier, as she laid the
picture away; “the recognition was instantaneous and complete. She
is Jane Chetwynd’s child. Margaret, leave her to me; I will love her
and comfort her.”
An hour after Mrs. Lanier was sitting in her library, writing hastily
and excitedly, when the doorbell rang, and, just as she was
addressing a letter to “Richard Chetwynd,” Arthur Maynard entered.
The boy looked quite pale and anxious, as he glanced at Mrs.
Lanier’s flushed, excited face.
“Don’t ask me any questions; just wait a moment,” she said, with a
reassuring smile.
Presently there was a sound of children’s voices on the stairs, and
three little girls entered the room quietly and demurely. They were
dressed exactly alike in dainty white frocks and broad sashes; two
were pale and dark; they were Ethel and May Lanier; and one was
fair and rosy, with wonderful golden hair hanging in burnished,
waving masses below her waist, while the thick fringe across her
forehead, although it looked a little refractory, as though it had just
been cut, gave her a charmingly infantile and picturesque
appearance.
The moment the little Laniers saw Arthur Maynard they ran to him
talking, and laughing gaily, while Lady Jane,—for it was she, quite
metamorphosed through the skill of Mrs. Lanier’s French maid, and
one of Ethel’s dainty suits,—remained standing shyly in the center of
the room.
Mrs. Lanier was watching her sweet little face with its puzzled,
anxious expression. She held her hands tightly clasped, and her soft
brows were slightly contracted, while she looked at the merry group
with large, serious eyes. Presently a winsome smile broke over her
face, and going slowly forward she said softly: “If you please, aren’t
you the boy who gave me the blue heron?”
Arthur Maynard was quite beside himself with delight. Holding out
both hands, he drew her to him, and putting his arm about her
caressingly he said gaily: “Yes, Lady Jane, I’m the very boy. And so
you remember me? I thought you’d forgotten me long ago.”
“Oh, no, no, I hadn’t; but,” with a little, tremulous smile, “you—you
didn’t know me, did you?”
“Yes, you darling, I did; I was only waiting to see if you really
remembered me.”
“Oh, but you didn’t know I saw you once before.”
“No, indeed. When and where was it?” asked Arthur eagerly.
“It was a long while ago. It was Mardi-gras, and I was lost; but you
couldn’t see me, because I had on a domino,” replied Lady Jane,
with dancing eyes and roguish little smile. “I called you, and you
heard me, because you looked around; but you couldn’t see me.”
“Well, I declare! Now I remember! Of course, I couldn’t guess that
the little pink crumpled thing was Lady Jane. Why didn’t you call me
again?”
“Oh,” with a little sigh. “I thought maybe you didn’t remember me.”
“As if I could ever forget; but where is Tony? have you given him
away?” and he looked into her eyes with a smile.
“No, I didn’t give him away. I love him too much to give him to any
one; but he’s lost. He broke his string, while I was out singing, and
Tante Pauline was too lame to catch him, and I searched and looked
everywhere for him, and then I couldn’t sing any more—and—” and
here she paused, flushing deeply while the tears gathered on her
lashes.
“She’s just the same adorable little creature,” whispered Arthur to
Mrs. Lanier, while he stroked her hair softly. Then he bent over her
and asked her very earnestly and gravely:
“Do you remember that day on the cars, Lady Jane, when I gave
you Tony?”
“Why, yes,—or I wouldn’t know you,” she replied ingenuously.
“Well, your mama was with you then. Where is she now?”
“Oh,” with a very sad sigh, “I don’t know; she’s gone away. I
thought she’d come back, and I waited and waited; but now I don’t
look any more. I think she’s with papa, and isn’t coming back.”
“When did she go? My darling, try to remember about your
mama,” urged Mrs. Lanier gently.
“It was so long ago, I can’t tell when it was,” she said dejectedly. “I
was ill, and when I got well Tante Pauline said she had gone.”
“Was it in Good Children Street that she went?”
“No. It was before. It was away across the river, because Tante
Pauline, and Mr. Raste, and I, and Tony in his basket, all came in a
big boat.”
“You see Jane Chetwynd never left Gretna,” said Mrs. Lanier in an
awe-struck voice.
“Where is Tante Pauline now?” continued Arthur.
“I don’t know. I ran away, and I haven’t seen her for ever so long.”
“Why did you run away from her? Didn’t you love her?”
“No, no! Please don’t ask me,—please don’t,” and suddenly she
covered her little flushed, troubled face with both hands and began
to cry silently.
“We mustn’t question her any more, Arthur,” said Mrs. Lanier
softly, as she soothed the child. “Her little heart has been probed to
the very depths. She is a noble little soul, and she won’t utter a
complaint against that wretched woman.”
“Never mind, my darling; forget all about Tante Pauline. You will
never see her again, and no one shall make you unhappy. You are
my child now, and you shall stay with me always, and to-morrow we
are going to buy Christmas presents for all your friends in Good
Children Street.”
“And I”—whispered Arthur, pressing his cheek close against her
golden head—“I have a Christmas present for you; so wipe away
your tears, and prepare to be very happy.”
“I have just written to her grandfather,” said Mrs. Lanier, after they
had sent her away to the children, all smiles and dimples again. “I
see by the papers that he has returned from Europe. There’s not the
least doubt that she is Jane’s child, and, if he has any heart, he’ll
come and investigate this mystery. I don’t dare to do anything until I
hear from him.”
“That will be very soon; he will probably be here in a day or two,
for he is on his way now.”
“Arthur, what do you mean? How has he heard?”
“Oh, Lady Jane has a great many friends who are deeply
interested in her. Paichoux, the dairyman, has been in
correspondence with the millionaire, and I have been interviewing
Paichoux. The little Frenchman put me on Paichoux’s track. It seems
that Paichoux got Mrs. Churchill’s watch from Madame Jozain’s son,
and Paichoux was inspired to write to the jeweler in New York,
whose name and the number of the watch were on the inside of the
case, to find out for whom that especial watch was made. After some
delay a letter came from Mr. Richard Chetwynd himself, telling
Paichoux that the watch was made for his daughter Jane Chetwynd.
The jeweler had forwarded Paichoux’s letter to Mr. Chetwynd, who
was in Paris, and the millionaire has hastened home to investigate,
which is a favorable omen for Lady Jane.”
The next day, the day before Christmas, and just one year from
the time when Lady Jane sat on the church steps eating the bread
and apple supplied her by a charitable impulse, she was making
almost a royal progress in Mrs. Lanier’s carriage, as lovely in her rich
dress as a little fairy, and every bit as much admired as Pepsie had
predicted she would be when, in the future, she should ride in a blue
chariot drawn by eight white horses. Mrs. Lanier’s generosity allowed
her to remember every one with suitable gifts, and her visit to Good
Children Street was something to be long remembered. Mrs. Lanier
almost blushed with shame and regret when she found herself once
more in the presence of Diana d’Hautreve, to think that for all these
years she had forgotten one who was once a queen in society both
by right of birth and wealth. “It is unpardonable in me,” she said to
herself when she saw the gentle, lonely woman hold the child to her
heart so fondly. “It is unpardonable to forget and neglect one so
entirely worthy of the best, simply because she is poor. However,
now that I have discovered her through Lady Jane, I will try to make
up for the indifference of years, by every attention that I can show
her.”
While these thoughts were passing through Mrs. Lanier’s mind,
Lady Jane was unfolding before Mam’selle Diane’s dazzled eyes a
rich mourning silk. “You must have it made right away,” she
whispered, pressing her rosy cheek to her friend’s, “for Mrs. Lanier
says you will visit your friends again, and I want you to wear my
Christmas present the first time you go out.”
Then Pepsie was made happy with a beautiful wheeled chair for
the street, which was so arranged with numerous springs that she
could be lifted over rough places without hurting her poor back, and
Madelon was the recipient of a beautiful warm cloak, and Tite’s love
of finery was fully gratified by a gay hat “wid fedders on it.” Little Gex
was fitted out with a supply of useful articles, and the Paichoux, one
and all, were remembered with gifts suitable for each; while the
orphans’ Christmas tree was loaded with presents from Lady Jane,
who only the year before had clung to the railings, cold and hungry,
and peeped in at the glittering display which was being prepared for
other little orphans not half as friendless and needy as she was.
And the homely, kind face of Margaret fairly shone with happiness,
as she watched her little favorite dispensing her pretty gifts.
And there was one hour of that happy Christmas eve that Lady
Jane never forgot. It was when Margaret took her into the chapel and
bade her kneel before the statue of our Saviour, who was once a
little child, and thank him devoutly for all the good things that had
come to her. Then, when she rose from her knees, the sister who
had taught her music played a sweet Ave Maria on the organ, and
the child’s angelic voice rose upward in a rapturous song of praise
and adoration; while Margaret knelt, with bowed head and clasped
hands, patient, humble, resigned, but yet sorrowful at losing the lamb
she had taken to her heart and cherished so tenderly.
CHAPTER XXXII
A MERRY CHRISTMAS
I T was Christmas evening, and Mrs. Lanier’s beautiful house was

bright with lights and flowers, and merry with music and laughter.
There were, besides the little Laniers and Lady Jane, a dozen
children or more, who had been invited to see the wonderful
Christmas-tree, which Mr. and Mrs. Lanier and Arthur Maynard had
spent a good part of the day in decorating. It stood at one end of the
drawing-room, and its broad branches were fairly bending beneath
the treasures heaped upon them. It glowed and sparkled with the
light of a hundred wax candles, reflected over and over by
innumerable brilliant objects until it seemed like Moses’s burning
bush, all fire and flame; and amid this radiant mass of color and light
were the most beautiful gifts for every member of the family, as well
as for the happy little visitors. But the object which attracted the most
curiosity and interest was a large basket standing at the foot of the
tree.
“Whom is that basket for, papa?” asked Ethel Lanier of her father,
who was unfastening and distributing the presents.
“We shall see presently, my dear,” replied Mr. Lanier, glancing at
Lady Jane, who stood, a radiant little figure, beside Arthur Maynard,
watching every movement with sparkling eyes and dimpling smiles.
At last, with a great deal of difficulty, the basket was untied, and
Mr. Lanier read in a loud, distinct voice from a card attached to it,
“For Lady Jane Churchill. With Arthur Maynard’s love and good
wishes.”
“There, I thought it was for Lady Jane,” cried Ethel delightedly. “I
know it’s something lovely.”
Mr. Lanier, with no little ceremony, handed the basket to Arthur,
who took it and gave it to Lady Jane with a low bow.
“I hope you will like my present,” he said, smiling brightly, while he
helped the wondering child untie the strings that fastened the cover.
Her little face was a study of mingled curiosity and expectancy,
and her eyes sparkled with eagerness as she bent over the basket.
“OH, OH! IT’S TONY!” CRIED LADY JANE
“It’s so large. What can it be? Oh, oh! It’s Tony!” she cried, as the
cover was lifted, and the bird hopped gravely out and stood on one
leg, winking and blinking in the dazzling light. “It’s Tony! dear, dear
Tony!” and in an instant she was on her knees hugging and kissing
the bird passionately.
“I told you I would find him for you,” whispered Arthur, bending
over her, almost as happy as she.
“And you knew him by the three little crosses, didn’t you? Oh,
you’re so good, and I thank you so much,” she said, lifting her lovely,
grateful eyes to the boy’s face. She was smiling, but a tear glistened
on her lashes.
“What a darling she is!” said Mrs. Lanier fondly. “Isn’t it pretty to
see her with the bird? Really it is an exquisite picture.”
She was like an anxious mother over a child that had just been
restored to her. “You know me, Tony, don’t you? and you’re glad to
see me?” she asked, over and over, while she stroked his feathers
and caressed him in the tenderest way.
“Do you think he remembers you, Lady Jane?” asked Mr. Lanier,
who was watching her with a smile of amusement.
“Oh yes, I know he does; Tony couldn’t forget me. I’m sure he’ll
come to me if I call him.”
“Please try him. Oh, do try him!” cried Ethel and May.
Mr. Lanier took the bird and placed him behind a chair at the
extreme end of the room, where he stood gravely blinking and
nodding, but the moment he heard Lady Jane’s little chirp, and
“Tony, Tony,” he ran fluttering to her and nestled close against her.
Every one was pleased with this exhibition of the bird’s
intelligence, and the children were quite wild over the new
acquisition. The other presents were forgotten for the moment, and
they could do nothing but watch every movement with admiration
and wonder.
To Lady Jane the recovery of her lost treasure was the crowning
point of happiness, and she consented reluctantly to leave him alone
in the conservatory, where he was to spend the night, and where he
looked very comfortable, as well as picturesque, standing on one leg
under a large palm.
“Doesn’t she dance like a little fairy!” said Arthur admiringly to Mrs.
Lanier, as they stood, a little later, watching the children dancing.
“Yes, she is very graceful and altogether charming,” replied Mrs.
Lanier. “It is delightful to see her so happy after all she has suffered.”
“I don’t imagine she will care half as much for her rich grandfather
as she does for Tony,” returned Arthur. “You see she’s acquainted
with Tony, and she isn’t acquainted with her grandfather. I hope he’ll
be decent to her,” he added anxiously.
“It is almost time for him to be here,” said Mrs. Lanier, glancing at
the clock. “Mr. Lanier will meet him at the station and bring him here,
if he will accept our hospitality. I’ll confess I’m filled with
consternation. He used to be such a stern, cold man; he never even
softened to Jane’s young friends; he was polite and kind, but never
genial, and I dare say he has quite forgotten me. It’s a trial for me to
meet him with this awful mystery hanging over Jane’s last days. Oh,
I hope he will take kindly to the child! He idolized her mother before
she thwarted his plans, and now I should think his remorse would be
terrible, and that he would do everything to atone for his
unkindness.”
“I have faith in Lady Jane,” laughed Arthur. “It must be a hard heart
to withstand her winning ways. I’ll wager before a week that the old
millionaire will be her devoted slave.”
Just at that moment a servant entered, and handed Mrs. Lanier a
card. “It is Mr. Chetwynd,” she said to Arthur. “They have come; he is
in the library, and Mr. Lanier asks me to bring the child.”
A few moments later, Mrs. Lanier led Lady Jane into the room
where Richard Chetwynd waited to receive her. He was a tall, pale
man, with deep, piercing eyes, and firmly closed lips, which gave
character to a face that did not lack kindliness of expression. As she
advanced a little constrainedly, holding the child by the hand, he
came forward to meet her with an air of friendly interest.
“Perhaps you have forgotten me, Mrs. Lanier,” he said, cordially
extending his hand, “but I remember you, although it is some time
ago that you used to dine with my daughter in Gramercy Park.”
“Oh, no, I have not forgotten you, Mr. Chetwynd; but I hardly
expected you to recall me among all Jane’s young friends.”
“I do. I do perfectly,” he replied, with his eyes fixed on Lady Jane,
who clung to Mrs. Lanier and looked at the tall, grave stranger with
timid scrutiny.
Then he held out his hand to the child. “And this is Jane
Chetwynd’s daughter. There is no doubt of it. She is the image of her
mother,” he said in a low, restrained voice. “I was not prepared to

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International Review of Cell and

Molecular Biology Volume 328 Lorenzo

International Review of Cell and Molecular Biology

International Review of Cell and Molecular Biology

International Review of Cell and Molecular Biology

International Review of Cell and Molecular Biology 1st

International Review of Cell and Molecular Biology 1st

International Review of Cell and Molecular Biology 1st

International Review of Cell and Molecular Biology

International Review of Cell and Molecular Biology 1st

Editorial Advisory Board

Publisher: Zoe Kruze

International Review of Cell and Molecular Biology, Volume 328

Autophagy refers to catabolic processes by which eukaryotic cells

forms of autophagy. We will not consider in this review other noncanonical

2. THE CANONICAL AUTOPHAGIC PATHWAY

Before discussing noncanonical autophagy, we first need to briefly

Initiation Elongation Maturation

Signaling ATG12 LC3

ATG5 ATG12 LC3

PIKfyve ATG9L Phagophore

Figure 1 ATG proteins involved in the early steps of mammalian macroautophagy.

MAP1LC3 (microtubule-associated protein 1 light chain 3)–PE (phospha-

2.1 The Core ATG Machinery and the Formation

and the formation of the PE conjugate of microtubule-associated protein

2.2 Maturation of Autophagosomes

syntaxin 17 and stabilizes the syntaxin 17-SNAP29 (synaptosomal-associated

Noncanonical autophagy does not require all of the ATG proteins to

3.1 ULK1-Independent Autophagy

is not required for glucose deprivation-induced autophagy (which is ULK

3.2 Beclin 1-Independent and VPS34-Independent Autophagy

etoposide, induce Beclin 1-independent autophagy in primary cortical neu-

Table 2 In vivo detection of noncanonical autophagy by the use of Atg-knockout mouse

3.3 VPS34/VPS15-Independent Autophagy

remains to be investigated whether DFCP1, similarly to WIPI2b, can bind

3.4 Autophagy Independent of Ubiquitin-Like

pluripotent stem cell (iPSC) reprogramming (Ma et al., 2015). Since

Noncanonical autophagy pathways and structures have the same func-

Another intriguing question involves the relationship between nonca-

T HE next morning, when Margaret brought little Jane, Mrs. Lanier

I T was Christmas evening, and Mrs. Lanier’s beautiful house was

“OH, OH! IT’S TONY!” CRIED LADY JANE

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