Annexin A2 promotes phagophore assembly by

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Received 25 Jul 2014 | Accepted 13 Nov 2014 | Published 19 Jan 2015

Kateryna Morozova1, Sunandini Sidhar2, Valerio Zolla1, Cristina C. Clement1, Brian Scharf1, Zoe Verzani1,3, Antonio Diaz2, Jorge N. Larocca4, Katherine A. Hajjar3,5, Ana Maria Cuervo2 & Laura Santambrogio1,6

Plasma membrane budding of Atg-16L-positive vesicles represents a very early event in the generation of the phagophore and in the process of macroautophagy. Here we show that the membrane curvature-inducing protein annexin A2 contributes to the formation of these vesicles and their fusion to form phagophores. Ultrastructural, proteomic and FACS analyses of Atg16L-positive vesicles reveal that 30% of Atg16L-positive vesicles are also annexin A2-positive. Lipidomic analysis of annexin A2-decient mouse cells indicates that this protein plays a role in recruiting phosphatidylserine and phosphatidylinositides to Atg16L-positive vesicles. Absence of annexin A2 reduces both vesicle formation and homotypic Atg16L vesicle fusion. Ultimately, a reduction in LC3 ux and dampening of macroautophagy are observed in dendritic cells from Anxa2 / mice. Together, our analyses highlight the importance of annexin A2 in vesiculation of a population of Atg16L-positive structures from the plasma membrane, and in their homotypic fusion to form phagophore structures.

1 Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, New York 10461, USA. 2 Department of Developmental Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, New York 10461, USA. 3 Department of Cell and Developmental Biology, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, USA. 4 Department of Neurology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, New York 10461, USA. 5 Department of Pediatrics, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, USA. 6 Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, New York 10461, USA. Correspondence and requests for materials should be addressed to L.S. (email: mailto:[email protected]

Web End [email protected] ).

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Annexin A2 promotes phagophore assembly by enhancing Atg16L vesicle biogenesis and homotypic fusion

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Macroautophagy (MA) is a basic catabolic pathway that is highly conserved in eukaryotes and is used by cells for the delivery of membrane-encapsulated cellular cargos

to endosomes/lysosomes for degradation1,2. The process of encapsulation of organelles and biomolecules is initiated in the cytosol by the formation of a double-membrane structure called the phagophore3. The membrane sources that give rise to the phagophore include the endoplasmic reticulum, Golgi, mitochondria and plasma membrane413. Indeed, it is becoming increasingly apparent that the phagophore may originate differently during physiologic or stress-related conditions. Recruitment of lipids and proteins from different membrane sources increases the basal level of MA and satises the requirement to dispose of cellular structures in different locations413. In addition, different cell types may develop phagophores by preferential routes according to their specic autophagic requirements413.

For phagophores that originate from the plasma membrane, it has been proposed that Atg-16L-positive vesicles directly bud from the plasma membrane, and generate pre-phagophore structures either by direct homotypic fusion of vesicles, or after trafcking to early/recycling endosomes, by heterotypic fusion with Atg9 vesicles2,4,5. Subsequent pairing of Atg16L with the conjugated protein pair Atg12-Atg5 results in the generation of tubulovesicular double membranes, which elongate further to form the autophagosome7. Coincident with the arrival of Atg12/5 to the phagophore, Atg16L-positive compartments also acquire LC3, which gives rise to the mature phagophore. Once the elongation is complete, the double membranes seal to complete the autophagosome; Atg12, Atg5 and Atg16L are then released into the cytosol, whereas part of LC3 remains associated with the autophagosome until either its fusion with lysosomes to become an autolysosome, or its fusion with late endosomes to form an amphisome.

The machinery regulating Atg16L vesicle homotypic fusion was recently found to comprise the SNARE protein VAMP7 and its partner SNAREs (syntaxin 7, syntaxin 8 and Vti1b)7. On the other hand, heterotypic fusion of Atg16L-Atg9 vesicles relies on the SNARE protein VAMP3 (ref. 4). Both Atg16L and Atg9 vesicles have been observed in Rab11 -recycling endosomes, where the fusion events are likely to occur4,5.

Annexin A2 is a cytosolic soluble protein, present in most eukaryotic cells, that binds membrane phospholipids in presence of Ca2 . Annexin A2 has been associated with multiple intracellular functions, including vesicle budding, fusion, internalization, late endosomal biogenesis and membrane repair1418. At the plasma membrane, annexin A2 has been shown to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains, which are important for the local inward curvature of the cells lipid bilayer19,20. In ddition, annexin A2 has been shown to be pivotal for the biogenesis of the late endosomal multivesicular bodies21,22. Thus, annexin A2 enables membrane lipid segregation, membrane deformation and associated inward vesiculation2325.

A recent analysis performed in our laboratory on annexin A2 -puried vesicles26 indicated that some of these vesicles were

Atg16L . In the present study, we investigated the role of annexin A2 in Atg16L vesicle formation, and its overall participation in MA. Our data indicate that annexin A2 aids the vesiculation of Atg16L-positive vesicles at the plasma membrane by recruiting phosphatidylserine (PS) and phosphatidylinositides, and by promoting Atg16L-mediated fusion to form phagophore structures in early MA.

ResultsAnnexin A2 associates with Atg16L vesicles. In agreement with the reported biological roles of annexin A2, ultrastractural

analysis of immunogold-stained, primary mouse dendritic cells showed the presence of annexin A2 at the plasma membrane, in the cytoplasm, bound to vesicles of variable size and in late endosomal compartments (Fig. 1ac). Annexin A2 staining was also observed on phagophore-like structures present in the cytosol (Fig. 1d), on B20% of autophagosomes (Fig. 1e), and on vesicles budding from the plasma membrane (Fig. 1c), reecting results reported previously reported for Atg16L vesicles involved in phagophore formation7. Thus, we asked whether both proteins could be involved in cellular events that provide membrane for phagophore formation in the initial stages of MA.

Total cellular vesicles were prepared using ultracentrifugation of the cellular cytosol from the mouse dendritic cell line JAWS II (DC). Pelleted vesicles were stained with a monoclonal antibody to Atg16L, or an isotype control, and sorted using ow cytometry to specically separate Atg16L vesicles from the total vesicle population (Fig. 1f). Vesicle lysates were resolved using

SDSPAGE and immunoblotted for annexin A2, with total cell lysates and cytosol included as positive controls (Fig. 1g, Supplementary Fig. 1). To establish the Ca2 dependence of annexin A2 association with vesicular structures, the vesicles were left untreated or incubated with EDTA before lysis. Western blot analysis revealed the presence of annexin A2, but not annexin A5, in Atg16L-positive vesicles, and conrmed the role of Ca2 in recruitment of annexin A2 from the cytosol (Fig. 1g,

Supplementary Fig. 1). Colocalization between Atg16L and annexin A2 was further conrmed using immunouorescence (Fig. 1h).

Proteomic analysis of Atg16L/annexin A2 vesicles. Next, we investigated the protein composition of annexin A2 /Atg16L vesicles to further dene their role in MA. Total cellular vesicles were prepared from isolated DC cytosol. Following staining with Atg16L and annexin A2 antibodies, doubly positive vesicles were sorted (Fig. 2a). Quantitative FACS analysis indicated that 28% of the total vesicles were Atg16L. These data are consistent with the notion that, unlike other cell types, autophagy is highly active in dendritic cells, even under physiological conditions due to their role in immunosurveillance, which requires constant transport of the extra- and intracellular proteome to endo/lysosomal compartments2729. In addition, dendritic cells, as opposed to other cell types, are highly phagocytic. Thus, it is likely that many of the vesicles that give rise to phagophores derive from the plasma membrane in this cell type. Of the total Atg16L vesicles, B30%

were also annexin A2 , indicating that the combined markers dene a subpopulation of autophagic precursors (Fig. 2a).

Likewise, only about half of annexin A2 vesicles were also Atg16L (Fig. 2a), consistent with the notion that annexin A2 is implicated in other cellular functions besides autophagy. The purity of the sorted vesicles was conrmed with ultrastructural morphology (Fig. 2b).

To further analyse the origin of the vesicles, sorted material was lysed and proteins resolved with SDSPAGE. Excised bands were digested with trypsin and the retrieved peptides analysed using mass spectrometry (Fig. 2c,d, Table 1). Several proteins known to participate in vesicle formation at the plasma membrane were identied; these included clathrin, the AP-2 adaptor complex, and accessory proteins such as synaptotagmin II, an AP2 binder, and endophilin A3, a protein involved in vesicle ssion. Cytoskeletal proteins, known to be involved in vesicle rocketing and movement, were also detected; these included actin, dynactin and colin 1 (ref. 24). In addition, the SNARE protein VAMP7 and its binding partners Vti1b, Hrb and ARF 6 were also identied7. These SNAREs were previously shown to be associated with

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Atg16 vesicles (Table 1)7. VAMP3, the SNARE involved in heterotypic Atg16L-Atg9 vesicle fusion, was also detected by proteomic analysis5. Girdin and Aurora kinase A, inhibitors of LC-3 vesicle binding, were also sequenced30,31, as well as several proteins involved in phosphatidylinositol (PI) metabolism including, synaptojanin 2, an enzyme that dephosphorylates PI at positions 3, 4, 5 of the inositol ring to form PIP2. Finally, Vps13, a vacuolar sorting protein required for efcient phagocytosis, membrane bending and PIphosphate regulation, was also detected32,33 (Table 1). Together, the results clearly validate that the Atg16 -annexin A2 vesicle subpopulation originates from the plasma membrane and contains multiple molecular effectors involved in vesicle docking and fusion.

Annexin A2 generates PI- and PS-enriched Atg16L vesicles. At the plasma membrane, annexin A2 facilitates the formation of lipid microdomains enriched in the phosphoinositide PI (4,5)P2, which promotes membrane deformation and actin-mediated vesicle rocketing19,23. Conversely, annexin A2 binds PS to facilitate endosomal biogenesis or membrane repair in both endosomes and at the plasma membrane21,26. To determine whether the formation of Atg16L -annexin A2 vesicles is supported by PS and/or PI recruitment, Atg16L vesicles were sorted from the cytosol of bone marrow dendritic cells (BMDCs)

from wild-type (Anxa2 / ) and annexin A2 knockout (Anxa2 / ) mice (Fig. 3a). Total lipids were extracted from 1 105 vesicles and separated using thin layer chromatography.

500 m 200 nm

Autophagosomes **

200 nm

Annexin A2

Vesicles

Cytosol

0 +

1 10

mM EDTA

200 nm 200 nm

4% Atg16L

Atg16L

Ctr

34%

Atg16 Atg16

Annexin A2 Annexin A2

Isotype CTR

38 kDa

Annexin A2

Annexin A5

36 kDa

Total

DAPI

Figure 1 | DC phagophore precursors and autophagosome are annexin A2-positive. (ac) Immunogold staining of dendritic cells with annexin A2 antibody identies positive staining in the (a) cytosol, (a) plasma membrane, (a) multivesicular late endosomes (MVB) and (b,c) vesicular structures: nZ50 cells. (d) Immunogold labelling of phagophore structures with annexin A2 antibody; nZ50 cells. (e) Quantication of annexin A2-positive and -negative autophagosomes in primary dendritic cells; the mean and s.e. were calculated from nZ50 cells: Po0.01 (unpaired two-tailed Students t-test).

(f) FACS analysis of total DC vesicles prepared by cytosol ultracentrifugation. Vesicles were stained with an Atg16L antibody that detects B30% of positively labelled vesicles. Shown is a single representative sort (n 14 sorts). (g) Representative western blot analysis of Atg16L-positive vesicles,

FACS-sorted as in f, for annexin A2 and annexin A5. Ponceau staining is shown as loading control (n 3 blots). (h) Immunouorescence of primary

dendritic cells stained with anti-annexin A2 and anti-Atg16L antibodies; nZ75 cells. Scale bar: 10 mm.

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14 1

2 6 9

Isotype Ctr

Annexin A2

2 85

Atg 16L

Isotype Ctr

Atg 16L

100 Band 1

Band 2

Band 3

Band 4

100 Band 5

Band 6

MWAtg16L+ annexin A2+ vesicles

150 100

20%

500 nM

60 40 20

100

0 0 10 20 30 40 50 60 70 80Time (min)

Figure 2 | Proteomic characterization of Annexin A2 /Atg16 vesicles. (a) FACS analysis of annexin A2 and Atg16L vesicles (n 14 sorts).

(b) Ultrastructural analysis of annexin A2 /Atg16L-sorted vesicles. (c) Silver staining of an SDSPAGE gel loaded with lysates of annexinA2 /Atg16L-sorted vesicles. The gel was divided into six bands for mass spectrometric analysis. (d) Chromatogram of each gel band following protein extraction and trypsin digestion. Proteins identied by mass spectrometric analysis are listed in Table 1. Data presented in c,d are representative of three separate experiments.

Table 1 | Proteomic analysis of Annexin A2/Atg16L vesicles.

Atg16L/Annexin A2 vesicles Function Reference

Actin Vesicle trafckingMicrotubule-actin cross-linking factor 1 Vesicle trafckingTwinlin Modulator of Actin polymerizationWAS/WASL interacting protein (WIPF1) Modulator of Actin polymerizationCoronin-1 Autophagosome formation 42

Cortactin Autophagosomelysosomal fusion 43

Dynactin Autophagosome movement 44

Colin 1 Modulator of Actin polymerizationAP-2 (beta and mu subunits) Atg16L interaction 7

Clathrin Atg16L interaction 7

Synaptotagmin II EndocytosisSynaptojanin 2 PI(4,5)P2 hydrolysisEndophilin A3 Endocytosis; vesicular transportAnnexin A2 Lipid segregation (PS and PIs)

Protein S100A11 Annexin A2-binding partnerASAP2 ARF6 regulatorVesicle-fusing ATPase Vesicle trafckingARF6 PIP2 formation via PIP5K; PLD Inhibition 6

Girdin Autophagy regulator 30

VAMP 7 SNARE 7

VAMP 3 SNARE 5

VtiB SNARE 7

Ubiquitin Binding to Atg16L 45

Galectin 3 EndocytosisAurora A kinase Autophagy regulators 31

Vps 13c Endocytosis; Regulation PI phosphatesHrb VAMP 7 adaptor 7

MA, macroautophagy; PI, phosphatidylinositol; PS, phosphatidylserine. Proteins previously reported to be associated with MA are referenced.

Puried lipids were used as controls (Fig. 3b). No differences were observed between Anxa2 / and Anxa2 / vesicles with respect to cholesterol and cholesterol esters, the most abundant vesicular lipids (Fig. 3b). In addition, no apparent differences were observed in the abundance of PE, the lipid required for LC3

recruitment to the phagophore, between vesicles from the two genotypes. On the other hand, a quantitative decrease in recovered PS was observed in Anxa2 / versus Anxa2 / vesicles. Of the three analysed phosphatides (PI, PIP2 and PIP3), only trace amounts of PI were detected in Anxa2 / , but not

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Anxa2+/+

Anxa2/

Anxa2

Ctr +/+ / Ctr +/+

+/+

1% 38%

29%

Cholesterol and esters

Anxa2

PIP PIP3

Ctr (FITC)

Atg16L (FITC)

100

Atg16++/A2++ m/z 863.70 36:1 PI

Atg16++/A2++ m/z 885.60 18:0/20:4 PI

18:0 LPI-H20

18:0 LPI-H

PI-signature

Atg16++/A2 m/z 863.70

Atg16++/A2 m/z 885.60

Atg16++/A2 m/z 782.00

845.76

18:0 FA-H

283.28 18:0 LPA-H20

581.43

419.28 599.49 803.89

759.90

297.13

522.28

747.78

803.78

257.34 451.26

0 0 200 400 600 800

841.85

867.85

841.93

867.85

100

0 400 600 800

200 400 600 800

Atg16++/A2 m/z 812.00

18:0 FA-H/

22:6 FA-CO2-H

283.33

18:O FA-H/

22:6 FA-CO2-H

180:O LPI-H20-H

38:4 PI-H2O-H

581.26

18:0 LPA-H2O-H

20:4 FA-H

419.25

303.25

18:0 LPI-H

20:4 LPIH20-H

823.85

813.91

695.64

601.55

20:4 LPA-H20-H

439.26

599.34

601.51

876.93

339.43 507.51

589.64

200 400 600 800

0 200 400 600 800

100

Atg16++/A2++ m/z 782.00 36:4 PS fragment

With neutral loss of: 36:4 PS-CO2-H

763.68

36:4 PS-H2O-H

PS signature

763.64

349.35

20:4 FA-H 20:4 LPA-H2O-H

16:0 FA-H

737.75

H2N

623.51

721.62

677.70

303.35 255.38

439.36

695.60

449.41 511.20

797.83

Atg16++/A2++ m/z 812.00 38:3 PS fragment

With neutral loss of:

18:0 LPA-H20-H

20:3 FA-H 18:0 LPA-H

18:0 FA-H

18:2 FA-H 38:3 PS-H20

724.57

38:3 PS-PA-H

793.66

100

PS signature

283.35

279.36 304.35

H2N

419.35

437.31

793.71

767.78

751.64

707.67

283.36

382.33

0 200 400 600 800

Figure 3 | Atg16L vesicles from Anxa2 / mice are decient in PI and PS. (a) FACS analysis of Atg16L vesicles in primary dendritic cells from Anxa2/ and Anxa2 / mice. (b) Thin layer chromatography of cholesterol, PE and PS extracted from Atg16L vesicles sorted from dendritic cells of Anxa2/ and Anxa2 / mice. (c) Thin layer chromatography of PI, PIP2 and PIP3 extracted from Atg16L vesicles sorted from Anxa2/ and

Anxa2 / dendritic cells. Data presented in a,b are representative of four separate experiments. (d) Mass spectrometry of lipids extracted from Atg16L vesicles sorted from Anxa2 / and Anxa2 / dendritic cells; data are representative of four separate experiments.

Anxa2 / , vesicles (Fig. 3c); this result is not surprising considering the low sensitivity of thin layer chromatography and the very low abundance of each of these signalling lipids in membrane structures.

To further analyse the lipid content of Anxa2 / and Anxa2 / vesicles, we performed mass spectrometry (MS/MS)

analysis on puried vesicle-derived lipids (Fig. 3d, Supplementary Data 1). MS/MS revealed that both PA and PE were present in vesicle lipids from both sources, whereas neither PS nor PI was detected in Anxa2 / vesicles (Supplementary Data 1, Fig. 3).

Using MS, we were able to detect neither PIP2 nor PIP3, both of

which are known to be present in trace amounts. Thus, even though it is possible that both lipids are present in the vesicles, they are probably found in very low concentrations, below our level of experimental detection. Together, these data support the requirement for annexin A2 in plasma membrane vesiculation of Atg16L vesicles, and its role in forming PIPS-enriched microdomains to sustain this process.

Annexin A2 promotes homotypic fusion of Atg16L vesicles. Although annexin A2 is not a fusogenic protein per se, it has been

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30 Single Single Double

Fusion

Double

Fusion

Average # events

**** ****

0 - 2 Ca2+(m)

100 nm

100 nm 100 nm

Single

Double Double

Fusion

Figure 4 | Annexin A2 is required for homotypic Atg16L vesicle fusion. (ad) Analysis of Atg16L /annexin A2 vesicles fusion events at different Ca2 concentrations; Atg16L /annexin A2 vesicles were sorted with Alexa-488 or Texas-Red-conjugated antibody to obtain mixed colour-positive vesicles. In vitro fusion was analysed at different Ca2 concentrations. Single vesicles refer to structures as depicted in b; double vesicles refer to structures as depicted in c and multiple fusion refers to structures as depicted in d; the mean and s.e. were calculated from n4300 events. *Po0.05; **Po0.01; ****Po0.0001. (e) Ultrastructural analysis of phagophore-like structures derived from the in vitro fusion of Atg16L/annexin A2 vesicles.

(f) Quantication of fusion events among Atg16L vesicles sorted from Anxa2/ and Anxa2 / DCs; the mean and s.e. were calculated from n4300 events. **Po0.01; ***Po0.001. (g,h) Quantication of fusion events among Atg-16L vesicles sorted from Anxa2 / and Anxa2 / DCs, plus or minus reconstitution with annexin A2; the mean and s.e. were calculated from n4300 events. *Po0.05; ****Po0.0001 (all unpaired two-tailed Students t-test).

Figure 5 | Reduced autophagosome biogenesis and autophagic ux in DC from Anxa2 / mice. (a) Immunouorescence for LC3 in dendritic cells of Anxa2/ and Anxa2 / mice. Nuclei are highlighted with DAPI. Insert shows higher magnication. Scale bars: 10 and 5 mm (insert). (b) Quantication of the number of puncta per cell, and average size of LC3-positive puncta, in micrographs such as shown in a; the mean and s.e. were calculated from n475 cells; *Po0.05. (c) Electron microscopy of the same cells. Arrows mark autophagosomes (blue) and autolysosomes (green). Scale bar: 10 mm.

(d) Immunoblot for LC3 in Anxa2 / and Anxa2 / dendritic cells untreated (none) or incubated with lysosomal protease inhibitors (PI) for the indicated times. Actin is shown as loading control. (eg) Quantication of steady-state levels of LC3-II (e), increase in LC3-II levels upon lysosomal proteolysis blockage (f) and increase in LC3-II at the two times of PI treatment (g) calculated from immunoblots shown in d. n 3. (h) Direct uorescence

microscopy in Anxa2/ and Anxa2 / dendritic cells transiently transfected with the tandem reporter mCherry-GFP-LC3, and maintained in serum-supplemented or serum-depleted medium for 6 h. Inserts show higher magnication images. Scale bars: 10 and 5 mm (insert). (i) Quantication of the number of total autophagic vacuoles (AV; mCherry ). Autophagosome (APG, mCherry and GFP ) and autolysosomes (APGL, mCherry

and GFP ) in images such as in h; the mean and s.e. were calculated from n475 cells; *Po0.05. (j) Quantication of the percentage of autophagosomes

and autolysosomes; *Po0.05. (km) Rates of degradation of long-lived proteins in Anxa2 / and Anxa2 / dendritic cells maintained in the presence or absence of serum for 24 h. Total rates of protein degradation (k), and degradation sensitive to lysosomal inhibition (combination of ammonium chloride and leupeptin (N/L)) or to inhibition of macroautophagy (3-methyl adenine (3MA)), in cells maintained in the presence (l) or absence(m) of serum. n 6; *Po0.05 (all unpaired two-tailed Students t-test).

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** **** *

20 200

100

Average # events

***

Anxa2: +/+

100

Average # events

****

- 10 100

Annexin A2 (pg)

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associated with Ca2 -mediated membrane fusion events. In association with its binding partners (S100A10 and S100A11), annexin A2 is known to bridge disparate membranes and promote fusion17,20. We, therefore, evaluated the potential role for

annexin A2 in Atg16L-mediated phagophore formation and elongation using a comparative uorescence-based fusion assay. Atg-16L vesicles were isolated from Anxa2 / BMDC using

Atg16 antibody and FACS sorting. The vesicles were labelled with

+/+

Number of puncta per cell

+/+

LC3-DAP1

Average size

of puncta

n >50 cells * P<0.01

10 m

Annexin A2:

+/+

Hours:

LC3-II levels 4

+/+

-Actin

LC-3

LC3-II levels

(folds+/+)

0.5

LC3-II flux

LC3-II synthesis

AV APGL

APG

1.2

+/+

LC3-II PI/none

(folds +/+)

# Vesicles per cell

0.6

LC3-II 42h

(folds +/+)

0.6

0 +/+

0 +/+ /

+/+

Serum +

Serum:

100

% APG

% APGL

+/+

AV (% total AVs)

+/+

Serum

Serum:

Serum +

Serum

+/+

Total long-lived protein

degradation

24 h proteolysis (%)

% Of total protein degradation

+/+

20 *

0 Serum +

Serum

3MA

0 N/L 0

N/L

3MA

Sensitive

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either red or green uorochromes, incubated for 30 min in fusion buffer, and equilibrated in a range of Ca2 concentrations. As expected, fusogenic events were quantitatively proportional to the

Ca2 concentration (Fig. 4ad). In addition, visualization of fusion products by EM conrmed the presence of elongated, double-membrane structures that possessed ultrastructural characteristics of an emerging phagophore (Fig. 4e).

Next, Atg16L vesicles isolated from Anxa2 / and Anxa2 / BMDC were subjected to in vitro fusion at 20 mm

Ca2 . In immunouorescence studies, we noted an equal presence of single and double vesiculate structures, as well as elongated fusion products, in Anxa2/ samples, whereas the majority of Anxa2 / samples contained mainly single vesicles with a dramatically reduced number of fusion structures (Fig. 4f).

Fusion events could be partially restored in Anxa2 / samples upon addition of recombinant annexin A2 (Fig. 4g,h). Together, the data indicated an additional role of annexin A2 in homotypic fusion of Atg16L vesicles7.

Annexin A2 is required to sustain autophagic ux. To analyse the consequences of annexin A2 depletion on MA, we rst stained Anxa2 / and Anxa2 / dendritic cells for endogenous LC3 (Fig. 5a,b). We found a signicant reduction in the number of LC3 vesicles in Anxa2 / cells, but no change in

the average size of the LC3 puncta (Fig. 5b). Electron micro

scopy also conrmed the lower abundance of autophagic vacuoles in Anxa2 / cells, with no apparent change in the morphology and size of completed vesicles (Fig. 5c). These ndings support the hypothesis that annexin A2 is required for the early steps of autophagosome biogenesis (nucleation); however, it is not a determinant of nal autophagosome size, in which Atg-16L fusogenicity is less important.

The reduced content of autophagic vacuoles in Anxa2 / dendritic cells could reect either slower formation or accelerated lysosomal degradation of the formed vesicles. Direct analysis of autophagic ux, assessed by measuring LC3 degradation (Fig. 5dg), and maturation of autophagic compartments, highlighted by the tandem reporter mCherry-GFP-LC3 (Fig. 5hj), suggests that depletion of annexin A2 leads to reduced autophagic ux. Immunoblot analysis revealed lower steady-state levels of LC3-II (Fig. 5d,e, Supplementary Fig. 1), and reduced accumulation of LC3-II upon treatment with inhibitors of lysosomal proteolysis in Anxa2 / versus Anxa2 / cells (Fig. 5d,f). Analysis of autophagosome biogenesis, assessed as the increase in LC3-II at two times during proteolysis inhibition, suggests that the reduced autophagic ux in Anxa2 / cells is due mainly to reduced autophagosome formation (Fig. 5i,j). We also used the tandem reporter mCherry-GFP-LC3, which allows for a more dynamic measurement of both autophagosome formation and maturation; acidication of autophagosomes upon lysosomal fusion quenches their green uorescent protein (GFP) uorescence, whereas mCherry uorescence persists, allowing for identication of autolysosomes as red-only puncta. Direct uorescence conrmed that Anxa2 / cells maintained in serum-containing medium had a reduced number of both autophagosomes and autolysosomes (Fig. 5h,j). In agreement with these morphological data, Anxa2 / cells maintained under these conditions also showed a signicant decrease in the total degradation rates of long-lived proteins (Fig. 5k) that was even more pronounced when we analysed the fraction of proteins degraded in the lysosomal compartment, as evidence by their sensitivity to inhibition of lysosomal proteolysis, or those whose degradation was directly dependent on active autophagy, as evidenced by their sensitivity to the inhibition of autophagosome biogenesis by the PI3K inhibitor 3-methyladenine (Fig. 5l).

Interestingly, the reduced autophagosome content in Anxa2 / cells was no longer observed upon induction of autophagy by starvation (Fig. 5h,i). These data suggest that the contribution of the plasma membrane, and consequently the involvement of annexin A2 in early precursor fusion, is more important for basal, quality-control autophagy than for induced autophagy.

Lastly, analysis of the compartments highlighted by the tandem LC3 reporter revealed that, in addition to the reduced number of autophagic vacuoles in the Anxa2 / cells, there was a higher proportion of autophagosomes than autolysosomes in these cells (Fig. 5h,j). The slower maturation of autophagic compartments in cells defective in annexin A2 was also conrmed by electron microscopy analysis (Fig. 5c). In contrast to the advanced degradation of cargo in most of autophagic vacuoles in Anxa2 / cells (compatible with post-lysosomal fusion compartments), cargo was still clearly distinguishable inside autophagic vacuoles (pre-lysosomal fusion) in the Anxa2 / cells. In fact, this defective maturation was still observed upon serum removal, despite the fact that autophagosome biogenesis was partially restored under these conditions (Fig. 5i,j). In agreement with these ndings, the fraction of long-lived proteins degraded by MA (sensitive to 3MA) upon serum removal was still signicantly lower in Anxa2 / cells (Fig. 5m). Interestingly, despite reduced autophagic degradation in Anxa2 / cells during serum deprivation, total degradation rates of long-lived protein were comparable in both cell groups under these conditions (Fig. 5k). This sustained protein degradation is likely due to compensatory upregulation of other autophagic pathways, because the fraction of lysosomal degradation persistent upon inhibition of MA was elevated in these cells upon serum removal (Fig. 5m). It is possible that the differences in lipid composition (PS and PI) in autophagosomes formed in the absence of annexin A2 determine their lower fusogenic capability with respect to endosomal and lysosomal compartments.

Overall, our ndings reveal that annexin A2 is required for the homotypic fusion of Atg16L vesicles in the formation of pre

autophagosome structures, and suggest that proper recruitment of phospholipids to the forming autophagosome membrane is required for the later maturation of the autophagic compartments.

DiscussionDuring the last few years it has become apparent that the phagophore may arise from multiple membranes, including the ERGolgi, mitochondria and plasma membrane1,4,7,9,10,710,13. It is possible that in different cell types, the phagophore originates preferentially from different subcellular locations, or that, even within the same cell, the site of origin can change according to the nature of the stressor that induces MA. The plasma membrane origin of the phagophore relies on Atg16L-positive vesicles, which bud inwardly and give rise to phagophores through homotypic fusion or heterotypic fusion with Atg9 vesicles after trafcking to the recycling endosomes4,5,7. SNARE proteins involved in homotypic fusion have been identied as VAMP7, and its partners, ARF6 and Vtib, whereas the SNARE that controls heterotypic Atg16 Atg9 fusion is VAMP3 (ref. 4,7).

Formation of the phagophore from the Golgi membrane requires generation of PI(3)P, from PI, through the action of the class III phosphoinositide kinase (PI3K-III) Vps34 (ref. 1). In addition, a PI3K-III-independent mechanism, which generates PI(3-4)P2 and relies on PI3K-II, has also been described34. Alternative sources of PI(3)P formation are dephosphorylation of PI(3-4)P2 and PI(3-5)P2 by PI4 or PI5 phosphatases, respectively35,36. At the plasma membrane, on the other hand, Atg16L interacts with the clathrin heavy chain and ARF6, and

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promotes autophagosome formation through activation of PIP5 kinase, thus generating PI(4-5)P2. Both ARF6 knockdown and impaired generation of PI(4-5)P2 decrease formation of pre-phagophore structures6,7.

Annexin A2 is a ubiquitously expressed, soluble protein that has been implicated in multiple biological processes related to membrane trafcking and fusion17. Our analyses of the early events involved in MA identied two additional functions for annexin A2. First, annexin A2 participates in the generation of PIPS-enriched Atg16L-positive vesicles. It was previously shown in liposome-based assays that annexin A2 promotes the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into liposomes, and also promotes membrane indentations at sites rich in PI(4,5)P2, leading to inward membrane budding and vesiculation19,20,23. These functions can be performed by the full-length protein, as well as by the C-terminal protein core domain. Annexin A2 has also been shown to recruit PS at both the plasma and endosomal membrane, and it has been proposed that annexin A2 has a scaffolding function in the biogenesis of membrane structures. In particular, by organizing lipid microdomains, annexin A2 regulates membrane curvature, providing a driving force for budding37.

Through proteomic analysis, we have discovered that a subset of annexin A2 vesicles are also Atg16L . Further proteomic assessment of vesicle content has identied several proteins previously known to be associated with Atg16L phagophore precursors, thus establishing the plasma membrane as the site of vesicular origin. Lipidomic analyses have demonstrated, furthermore, that annexin A2 is required for PS recruitment in Atg16L vesicles from the plasma membrane. Although we could not directly demonstrate the presence of PI (4,5)P2 in the Atg16L vesicles, it is clear that annexin A2 is required for enrichment of vesicles with the PI (4,5)P2 precursor, PI.

The importance of PI and PS enrichment in Atg16L vesiculation is highlighted by the 30% reduction in the proportion of Atg 16L vesicles in Anxa2 / dendritic cell cytosol.

Phospholipid enrichment and associated vesiculation are generated by the ability of annexin A2 to bind PS and PI in a Ca2 -dependent manner, and also by the ability of annexin A2 to bind and organize cytoskeleton proteins for inward budding.

Indeed, annexin A2 can bind the barbed ends of F-actin laments and generate vesicle rocketing23. Annexin A2-mediated vesiculation is likely aided by other proteins, such as WASP, which induces actin assembly at the surface of endomembranes, because the WASP-binding partner WIPF1 was identied in our proteomic analysis. Together, our data indicate that annexin A2 generates PS and PI microdomains, and promotes Atg16L vesiculation. It remains to be established whether the remaining

Atg16L vesicles originate from other subcellular compartments in an annexin A2-independent manner.

A second annexin A2 function, reported here, is to facilitate homotypic Atg16L vesicle fusion, which is required for phagophore formation and elongation. Our in vitro assay demonstrates that vesicles lacking annexin A2 fuse less, and the autophagosomes that do form display further fusion defects when they encounter endosomes and lysosomes. Importantly, this decreased fusion capacity translates in vivo into a decrease in dendritic cell autophagic ux as determined by a reduction in LC3 processing.

In Atg16L vesicle fusion events, annexin A2 likely serves to bridge disparate vesicles. This role is well characterized for the heterotetrameric complex formed by the association of two molecules of annexin A2 and two of an S100 protein, either S100A10 or S100A11. During the bridging event, each molecule of annexin A2 engages vesicular PS, or other anionic phospholipid, electrostatically, whereas S100A10/11 proteins serve to

stabilize the annexin A2 dimer. This function is mediated by the core domain of annexin A2 and requires Ca2 (ref. 17).

It is important to note that our model was developed in primary dendritic cells. Compared with other cell types, dendritic cells have the highest rate of plasma membrane turnover, due to their incessant endocytic activity, which is tightly connected to their role in immunosurveillance. Thus, in this cell type it is possible that the contribution of plasma membrane to the origin of phagophore precursors is greater than in other cell types. Indeed, among the total vesicle population prepared from dendritic cell cytosol, a large proportion was Atg16L , indicating a cellular commitment to autophagic processes.

In conclusion, our data support the concept that annexin A2 plays a central role in the formation and fusion of Atg16L vesicles by orchestrating recruitment of phosphoinositides and PS to vesicular membranes, and by coordinating vesicular budding and homotypic fusion. Our data elucidate a key early mechanistic step in MA.

Methods

Mice and bone marrow cultures. Female, Anxa2 / and Anxa2 / mice on the C57Bl6 background, 812 weeks old, were maintained in animal facilities at

Weill Cornell Medical College and Albert Einstein College of Medicine. Animal killing and bone marrow harvesting were carried out according to protocols approved by the Animal Institute Committee of both institutions. To obtain BMDCs, the bone marrow was harvested from the femurs and cells were cultured for 7 days in granulocytemonocyte colony-stimulating factor (10 ng ml 1) in complete DMEM26.

Immunogold labelling of annexin A2. BMDCs were xed in 3.7% paraformaldehyde 0.1% glutaraldehyde in dPBS for 20 min followed by permeabilization in 0.1% Triton X100 for 15 min at 25 C. Cells were extensively washed with dPBS and incubated in blocking buffer (1% FBS, 1% BSA in dPBS) for 45 min. BMDCs were then incubated overnight with 4 mg ml l goat anti-mouse annexin A2 antibody (clone A-15, Santa Cruz Biotechnology) at 4 C, and subsequentlywith 0.8 mg ml 1 of ultrasmall gold-conjugated rabbit anti-goat IgG (Electron

Microscopy Sciences, Hateld, PA, USA). Silver enhancement of ultrasmall conjugates was performed using the Aurion R-Gent SE-EM kit (Electron Microscopy Sciences). The cells were briey post-xed in 1.0% aqueous osmium tetroxide and processed for transmission electron microscopy (TEM).

Transmission electron microscopy. Primary BMDCs were xed in 2.5% glutaraldehyde, 2% paraformaldehyde in sodium cacodylate buffer 0.1 M, pH 7.4 for 3 h at 4 C. Samples were post-xed in 1.0% aqueous osmium tetroxide (pH 7.4) for 1 h at 4 C and dehydrated in a series of water/acetone mixtures progressing to 100% acetone. Cells were inltrated in sequentially increasing concentrations of Embed 812-Araldite (Electron Microscopy Sciences), and embedded in BEEM capsules. Ultrathin sections were stained with uranyl acetate followed by lead citrate, and viewed with a Jeol JEM-1200EX transmission electron microscope (Jeol Ltd., Akishima, Japan) at 80 kV.

Immunouorescence. BMDCs were cultured on 22 40-mm glass coverslips

coated with poly-L-lysine in 60-mm Petri dishes. Samples were xed in 4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature, permeabilized with PBS and 0.5% saponin for 10 min, and blocked in 1% BSA in PBS for 30 min. BMDCs were then incubated with annexin A2 1:100 (Clone A-15, Santa Cruz Biotechnology) and Atg16L1 1:200 (Clone AB1, Sigma or Clone 1F12, MBL), in 1% BSA in PBST overnight at 4 C. Samples were incubated with secondary antibodies (goat anti-Alexa-488 and rabbit anti-Alexa 568) for 1 h at room temperature. Samples were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen), and imaged using an Olympus IX70 inverted microscope equipped with theI.P. Lab Spectrum imaging software (Scanalytics Inc., Fairfax, VA, USA).

Negative staining of vesicles. Before and after fusion, FACS-sorted vesicles(10 ml) were spotted on glow discharged, formvar-coated 300-mesh copper grids. Samples were left on grids for 2 min before blotting of excess liquid. Following blotting, grids were immediately stained with 1% phosphotungstic acid (PTA) and viewed with a Jeol JEM-1200EX transmission electron microscope at 80 kV.

Vesicle preparation and FACS analysis. Vesicles were prepared from either JAWS II (ATCC) or Anxa2 / and Anxa2 / dendritic cells. After cell disruption by cavitation in 0.25 M sucrose in DPBS, and homogenization for 10 times with a Dounce homogenizer, the post-nuclear supernatant fraction was centrifuged

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at 2,000 g for 15 min. The supernatant was then centrifuged at 100,000 g for 1 h

to eliminate residual fragments of the plasma membrane, ER, organelles and Golgi. The fraction of interest, which contained mostly vesicles, was harvested from the supernatant by pelleting at 300,000 g for 1 h.

Vesicles were labelled with primary anti-annexin A2 (Clone A-15, Santa Cruz Biotechnology) and anti-Atg16L (Clone AB1, Sigma or Clone 1F12, MBL) antibodies, followed by the secondary goat anti-Alexa-488 and rabbit anti-Texas red-conjugated (Jackson ImmunoResearch) in staining buffer containing 220 mM KCl, 5 mM NaCl, 5 mM NaH2PO4, 0.5 mM MgCl2, pH 6.0. Antibody-stained vesicles were sorted on a Becton Dickinson FACS-Aria high-speed cell-sorting ow cytometer. Data acquisition and analysis were performed using the BD FACS Diva software.

Proteomic analysis. FACS-sorted annexin A2 /Atg16L vesicles were lysed in 1% NP40 50 mM Tris/HCl and 150 mM NaCl for 1 h on ice. Protein concentration was determined by the Bradford method (Bio-Rad Laboratories). Proteins (15 mg)

were resolved on a 10% SDSPAGE, and protein bands visualized with the Silver Stain Kit for Mass Spectrometry (Pierce). Each lane was cut into slices, which were destained, and washed with 50 mM ammonium bicarbonate (NH4HCO3) and acetonitrile. Samples were then trypsin-digested, and peptides subjected to nano-LC-MS/MS sequencing on a LTQ-Orbitrap Velos HR mass spectrometer. MGF les were generated from the raw data les and searched against the SwissProt Mus musculus database using Mascot (version 2.1.04).

Western blot analysis of Atg16L vesicles. Atg16L FACS-sorted vesicles were lysed in 1% NP40, 50 mM Tris/HCl and 150 mM NaCl lysis buffer. Protein concentration was determined by the Bradford method (Bio-Rad Laboratories). Total proteins (20 mg) were resolved on 415% gradient SDSPAGE minigels (Bio-Rad).

Total cell lysate and BMDC cytosol were run as positive controls. After transfer to nitrocellulose membranes and blocking in PBS with 0.1% Tween 20 and 5% nonfat milk, the membranes were incubated with anti-mouse annexin A2 (1:4,000, Clone C-10 Santa Cruz Biotechnology) or anti-mouse annexin A5 (1:200, Clone H-3, Santa Cruz Biotechnology), followed by horse radish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch). Proteins were visualized by chemiluminescence (Supersignal, WB kit, Thermo Scientic).

Vesicle fusion. Total cytosolic vesicles were prepared from Anxa2 / and Anxa2 / BMDC, as well as JAWS II cells by cavitation and ultracentrifugation, as described above. Vesicles were stained with Atg16L primary antibody; half of each vesicle preparation was stained subsequently with Alexa-488-conjugated mouse anti-rabbit IgG (Jackson ImmunoResearch), and the other half with TexasRed-conjugated goat anti-rabbit IgG (Invitrogen). In some experiments, Atg16L green and red FACS-sorted vesicles were incubated with increasing concentrations of Ca2 (0, 2, 20 and 200 mM) for 40 min at 37 C in 220 mM KCl, 5 mM NaCl, 5 mM NaH2PO4, 0.5 mM MgCl2, at pH 6.0. In other experiments, recombinant annexin A2 was added in the fusion buffer of Anxa2 / vesicles. In all experiments, single vesicles, double vesicles and fusion structures were enumerated in both green and red channels under an Olympus IX70 inverted microscope equipped with I.P. Lab Spectrum imaging software (Scanalytics Inc). A minimum of 300 vesicles and fusion events were counted in each experiments. For electron microscopy analyses, 10 ml of each sample was applied on Formvar-coated grid, and negatively stained with 1% PTA.

Analysis of autophagic ux. Autophagic ux was determined by immunoblot analysis of cells treated with or without with 20 mM NH4Cl and 100 mM leupeptin to block lysosomal proteolysis1. The difference in LC3 levels between cells treated with or without the inhibitors was used to calculate autophagic ux, whereas the difference in LC3 levels at two times during the inhibition of proteolysis was used as an index of autophagosome formation. Autophagic ux was also measured upon transfection of the cells with the tandem reporter mCherry-GFP-LC3 (Addgene38). Cells were imaged 24 h after transfection and the number of mCherry-positive vesicles (autophagic vacuoles), mCherry and GFP-positive vesicles (autophagosomes), and mCherry-only-positive vesicles (autolysosomes) was calculated after thresholding, using the particle measure tool of the Image J software (NIH).

Extraction of lipids and phosphoinositides. Extraction of lipids from the ATG16L /Anxa2 / and from the ATG16L /Anxa2 / autophagic vesicles was performed using a modication of the protocol developed by Honeyman39.

Briey, an acidic HCl solution was used to improve recovery of phosphoinositides, which tend to bind strongly to proteins during the standard extraction with chloroform/methanol solvent. Lyophilized vesicles were solubilized in 100 ml of

H2O, to which 375 ml of chloroform/methanol/12N HCl (2/4/0.1, v/v) was added. After thorough mixing, 125 ml of chloroform was added, and the solution vortexed for 30 s followed by the addition of another 125 ml of H2O. After 10 min of centrifugation at 2,000 g, the lower chloroform layer was removed and

transferred to a glass tube for evaporation in a vacuum centrifuge. The lipid lm was rapidly redissolved in 50 ml of 1:1:0.3 chloroform/methanol/water, and

subjected to further analysis by thin layer chromatography (TLC) and nanoLC MS/MS.

TLC lipid analysis. Lipid extracts from Atg16L /Anxa2 / and Atg16L / Anxa2 / vesicles were spotted on silica-coated plates, and developed in a closed jar with chloroform:methanol:water (65:25:1), supplemented with 12%

phosphoric acid (H3PO4) to enable identication of major phospholipids and cholesterol. After the run, the TLC plate was air-dried and developed with iodine vapours. The dark-brown spots corresponding to the lipids of interest were identied using the corresponding lipid standards (PS phosphatidylserine,

PE phosphatidylethanolamine, PC phosphatidylcholine, cholesterol and the

specic phophoinositides (PI phosphatidyl inositol, PIP phosphatidyl

inositol monophosphate, PIP2 phosphatidyl inositol diphosphate and

PIP3 phosphatidyl inositol triphosphate).

MS analysis of lipid/phospholipid extracts. Lipid extract samples were diluted 1/10 in 1:1:0.3 (v/v/v) CHCl3:MeOH:H2O containing 25 mM piperidine40. Each sample (10 ml) was loaded into a 2-mm static nanospray emitter (PicoTips, New Objective). Using the static nanospray source at 1.01.4 kV, the sample typically lasted as a stable spray for 30 min. A high-resolution mass spectrometer (Orbitrap Velos, Thermo Scientic) was used in negative ionization mode for targeted and untargeted tandem MS (MS/MS), using a 1.5 m/z isolation width, and either collision-induced disassociation, higher collision disassociation or both at energies of 0 and 55%. Data collected at 0% provided the precursor ion m/z values obtained from the Orbitrap or from the Ion Trap. The untargeted approach utilized the Ion Map feature of the Orbitrap where prole MS/MS data (502,000 m/z) were collected from precursor ions from 200 to 2,000 m/z, at 2 m/z step size. Data were collected with the Xcalibur package (ThermoFinnigan).

Lipidomics. The MS Analysis Tool provided by the Resources at the LIPID MAPS website (http://www.lipidmaps.org/resources/resources.html

Web End =http://www.lipidmaps.org/resources/resources.html) was used to identify lipid species from MS/MS fragmentation proles (Supplementary Data 1). Phospholipids (PI and PS) showing exact matches of their fragment ions with our experimental MS/MS data are shown in Fig. 3. The phospholipids are presented with the class abbreviation preceded by the xx:y, where xx is the total carbon atoms in the fatty acid chains and y is the number of double bonds.

Analysis of intracellular protein degradation rates. Degradation of long-lived proteins in cultured cells was measured following 48-h labelling of cells with [3H] leucine (2 mCi ml l) at 37 C and pulse-chase experiments41. Cells were then extensively washed and placed in chase medium containing excess unlabelled leucine. Aliquots of the medium taken at different times were subjected to acid precipitation, and then ltered through a 0.22-mm to separate the lter-retained fraction (proteins) from the ow-through fraction (small peptides and amino acids). Proteolysis was calculated as the amount of acid-precipitable radioactivity (protein) transformed to acid soluble (amino acids) radioactivity during the incubation. MA-dependent degradation was inhibited using 10 mM 3-methyladenine, and lysosome-dependent degradation was inhibited using a mixture of 20 mM ammonium chloride and 100 mm leupeptine (N/L)41.

Statistical methods. All numerical results are reported as means.e.m. Statistical signicance of the difference between experimental groups was analysed using the unpaired two-tailed Students t-test.

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Acknowledgements

This work was supported by the National Institutes of Health Grant AG031782 to A.M.C., and L.S. and HL042493 to KAH. We acknowledge Edward Nieves and the Laboratory for Macromolecular Analysis and Proteomic at Albert Einstein College of Medicine (NIH grant 1S10RR019352) and MS-Bioworks (Ann Arbor, MI) for the mass spectrometry services. We gratefully acknowledge the expert technical assistance of Dena Almeida. V.Z. is supported by the PhD programme in Genetics and Cell Biology at the University of Tuscia, Department of Ecology and Biology (DEB), Viterbo, Italy.

Author contributions

L.S., A.M.C., J.L. and K.A.H. designed the experiments; K.M., S.S., V.Z., C.C.C., B.S., Z.V., A.D. and J.L. performed the experiments; K.M., L.S., K.A.H. and A.M.C. wrote the paper.

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How to cite this article: Morozova, K. et al. Annexin A2 promotes phagophore assembly by enhancing Atg16 vesicle biogenesis and homotypic fusion. Nat. Commun. 6:5856 doi: 10.1038/ncomms6856 (2015).

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NATURE COMMUNICATIONS | 6:5856 | DOI: 10.1038/ncomms6856 | http://www.nature.com/naturecommunications

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Plasma membrane budding of Atg-16L-positive vesicles represents a very early event in the generation of the phagophore and in the process of macroautophagy. Here we show that the membrane curvature-inducing protein annexin A2 contributes to the formation of these vesicles and their fusion to form phagophores. Ultrastructural, proteomic and FACS analyses of Atg16L-positive vesicles reveal that 30% of Atg16L-positive vesicles are also annexin A2-positive. Lipidomic analysis of annexin A2-deficient mouse cells indicates that this protein plays a role in recruiting phosphatidylserine and phosphatidylinositides to Atg16L-positive vesicles. Absence of annexin A2 reduces both vesicle formation and homotypic Atg16L vesicle fusion. Ultimately, a reduction in LC3 flux and dampening of macroautophagy are observed in dendritic cells from Anxa2^-/- mice. Together, our analyses highlight the importance of annexin A2 in vesiculation of a population of Atg16L-positive structures from the plasma membrane, and in their homotypic fusion to form phagophore structures.

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Title

Annexin A2 promotes phagophore assembly by enhancing Atg16L+ vesicle biogenesis and homotypic fusion

Author

Morozova, Kateryna; Sidhar, Sunandini; Zolla, Valerio; Clement, Cristina C; Scharf, Brian; Verzani, Zoe; Diaz, Antonio; Larocca, Jorge N; Hajjar, Katherine A; Cuervo, Ana Maria; Santambrogio, Laura

Pages

5856

Publication year

2015

Publication date

Jan 2015

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms6856

ProQuest document ID

1646391417

Annexin A2 promotes phagophore assembly by enhancing Atg16L+ vesicle biogenesis and homotypic fusion

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