12

STRUCTURAL ASPECTS OF AUTOPHAGY

Per O. Seglen, Trond Olav Berg, Henrietta Blankson, Monica Fengsrud, Ingunn Holen, and Per Eivind Stmmhaug

Department of Tissue Culture Institute for Cancer Research The Norwegian Radium Hospital Montebello N-031O Oslo, Norway

SUMMARY

As a first step towards isolation of autophagic sequestering membranes (phagopho­res), we have purified autophagosomes from rat hepatocytes. Lysosomes were selectively destroyed by osmotic rupture, achieved by incubation of hepatocyte homogenates with the cathepsin C substrate glycyl-phenylalanyl-naphthylamide (GPN). Mitochondria and perox­isomes were removed by Nycodenz gradient centrifugation, and cytosol, micro somes and other organelles by rate sedimentation through metrizamide cushions. The purified auto­phagosomes were bordered by dual or mUltiple concentric membranes, suggesting that autophagic sequestration might be performed either by single autophagic cisternae or by cisternal stacks.

Okadaic acid, a protein phosphatase inhibitor, disrupted the hepatocytic cytok­eratin network and inhibited autophagy completely in intact hepatocytes, perhaps suggesting that autophagy might be dependent on intact intermediate filaments. Vin­blastine and cytochalasin D, which specifically disrupted microtubules and microfila­ments, respectively, had relatively little (25-30%) inhibitory effect on autophagic sequestration.

In a cryo-ultrastructural study, the various autophagic-lysosomal vacuoles were immunogold-labelled, using the cytosolic enzyme superoxide dismutase as an auto­phagic marker, Lgp 120 as a lysosomal membrane marker, and bovine serum albumin as an endocytic marker. Vinblastine (50 f..1M) was found to inhibit both autophagic and endocytic flux into the 1ysosomes, with a consequent reduction in lysosomal size. Asparagine (20 mM) caused swelling of the lysosomes, probably as a result of the ammonia formation that could be observed at this high asparagine concentration. Autophagosomes and amphisomes (autophagic-endocytic, prelysosomal vacuoles) ac­cumulated in asparagine-treated cells, reflecting an inhibition of autophagic flux that might be a consequence of lysosomal dysfunction.

Intracellular Protein Calaho/isl1l, Edited by Koichi Suzuki and Judith Bond Plenum Press, New York, 1996 103

104 P. O. Seglen et al.

INTRODUCTION

Autophagy is a major mechanism for the degradation of protein as well as of other intracellular macromolecules and organelles, yet its structural basis is poorly understood. In rat hepatocytes, autophagy is initiated when the levels of certain amino acids become low (Poso et al., 1982; Seglen et al., 1980), possibly resulting in the detachment of these amino acids from regulatory cell surface receptors (Kadowaki et al., 1992). The resulting signal activates, by an unknown mechanism, sequestering organelles called phagophores, which have the morphological appearance of electron-dense membrane sheets containing single or multiple (stacked) collapsed membrane cisternae (Seglen, 1987), probably derived from the endoplasmic reticulum (Dunn, 1994). The phagophores envelop whole regions of cytoplasm in a nonselective manner (Kopitz et al., 1990), except in the case of autophagy-dependent regression following peroxisome proliferation, where some organelle specificity can be demonstrated (Luiken et al., 1992). The sequestration of cytoplasm by the phagophore results in the formation of a sealed vacuole, an autophagosome, where the phagophore now forms the wall around a section of completely normal-appearing cytoplasm. It is not known how the extension, curving and vacuole sealing performed by the phagophore membranes is directed, but an involvement of the cytoskeleton has been indicated (Holen et al., 1992; Aplin et aI. , 1992). Eventually, the autophagosome delivers its contents to a lysosome, probably by fusion, and the contents are degraded by the lysosomal enzymes. There are biochemical data suggesting that at least some autophagosomes may fuse with acidic endosomes before they reach the lysosomes, forming "amphisomes" (Gordon and Seglen, 1988; Stmrnhaug and Seglen, 1993; Dunn, 1994).

The first step in the autophagic-lysosomal pathaway, i.e., the sequestration of cytoplasm by the phagophore, can be measured as the transfer, in intact cells, of a cytosolic marker from the cytosol to sedimentable autophagic vacuoles (autophagosomes, am­phi somes and lysosomes). The marker can be an electroinjected, inert small molecule, e.g., [3H]raffinose (Seglen et al., 1986), or an endogenous, cytosolic enzyme, e.g., lacatate dehydrogenase (LDH), which accumulates if its lysosomal degradation is prevented by leupeptin (Kopitz et al., 1990).

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INCUBATION TIME (min)

Figure 1. Effects of okadaic acid, vinblastine and cytochalasin D on autophagic sequestration of LDH.

Structural Aspects of Autophagy 105

Figure 2. Effect of okadaic acid on the organization of cytokeratin (CK8) intennediate filaments.

Role of the Cytoskeleton in Autophagic Sequestration

By incubating hepatocytes with inhibitors of the various cytoskeletal elements, it should be possible to see whether autophagic sequestration requires the structural integrity of any of these elements.

Hepatocytes were incubated at 37°C for the length of time indicated, in the presence of 0.3mM leupeptin, with no further additions (0); with 30 nM okadaic acid (.); I 0 ~M vinblastine (VB), or 1 O~m cytochalasin D ("'). The net amount of cytosolic LDH sequestered at each time point was measured and expressed as per cent of the total cell-associated LDH. Each point is the mean ± S.E. of three independent experiments.

As shown in Fig. 1, neither vinblastine, at a concentration demonstrated by immu­nostaining to specifically fragment hepatocytic microtubules, nor cyctochalasin D, at a concentration found to specifically fragment actin microfilaments, had much effect on autophagic sequestration. Neither microtubules nor microfilaments would, therefore, seem to be required for autophagic sequestration in hepatocytes. On the other hand, okadaic acid, which disrupted the cytokeratin network (Fig. 2) without affecting microtubules or micro­filaments, inhibited autophagic sequestration nearly completely (Fig. I).

Hepatocytes were incubated for Ih at 37°C with (A), no addition (control), or (B), okadaic acid, 30 nM. After incubation, the cells were sedimented onto glass slides (Cy­tospin), fixed in 100% methanol and processed for indirect immunofluorescence staining of cytokeratin intermediate filaments, using a monoclonal antibody against CK8.

Several protein kinase inhibitors, including the flavonoid naringin (Gordon et aI., 1995) and KN-62, thought to specifically inhibit the Ca2+/calmodulin-dependent protein kinase II (Holen et at., 1992), offered parallel protection of autophagy and cytokeratin against okadaic acid, suggesting that the same protein kinase may control both processes. Although a causal relationship remains to be proven, it would seem reasonable to assume that ifthere is a cytoskeletal involvement in hepatocytic autophagy, the most likely candidate would be the cytokeratin intermediate filaments. Other cell types may have other cytoskeletal requirements: in a fibroblastic kidney cell line (NRK), autophagosome formation was suppressed by cytochalasins, suggesting a need for intact microfilaments (Aplin et at., 1992). It should be noted that the overall autophagic-lysosomal degradation is strongly suppressed by microtubule inhibitors, but the microtubular requirement apparently relates to the delivery

106 P. O. SegJen et al.

e'

••

e e

Figure 3. Immunogold labelling of a lysosome.

of autophagocytosed material to lysosomes rather than to the initial autophagic sequestration (H0yvik et al., 1986).

Effects of Autophagic Flux Inhibitors (Vinblastine, Asparagine) on the Distribution of Autophagic Vacuoles, Characterized by Cryoelectron Microscopy and Gold-Immunostaining

Microtubule inhibitors like vinblastine inhibit both autophagic and endocytic flux in hepatocytes, resulting in the accumulation of autophagosomes as well as of endosomes. Asparagine, on the other hand, has been found to interrupt autophagic flux in an apparently selective manner, causing an accumulation of undegraded, autophagocytosed material in autophagosomes (Seglen, 1987) as well as in the prelysosomal autophagic vacuoles called amphisomes (because they are also able to receive endocytosed material) (Gordon and Seglen, 1988; H0yvik et al., 1991). Since the autophagic vacuoles accumulating during different types of inhibitor treatment have been poorly characterized, an ultrastructural study was undertaken, using gold-immunostaining of hepatocyte cryosections to identify the various vacuole categories.

The cytosolic enzyme superoxide dismutase (SOD) is relatively degradation-resis­tant; autophagocytosed SOD can, therefore, be detected in lysosomes as well as in earlier autophagic vacuoles, making it a perfect marker for autophagic vacuoles in general (Rabouille et al. , 1993). As a lysosomal marker, the lysosomal membrane glycoprotein Lgp120 was chosen (Dunn, 1990), whereas endocytosed bovine serum albumin (BSA)-gold was used as an endocytic marker (Fig. 3).

Hepatocytes were incubated for 2h at 37°C, cryosectioned and double-immunola­beled for 19p120 (20-nm gold; large arrowhead) and SOD (lO-nm gold; small arrowhead). 5-nm BSA-gold (arrow) was taken up by endocytosis. Bar 200 nm.

Although Lgp 120 has been reported to be present in prelysosomal endocytic vacuoles in certain cell types (Green et al., 1987), we found no Lgp120 labelling in hepatocytic endosomes (BSA+/SOD-). Virtually all Lgpl20-positive vacuoles (96%) were SOD+, i.e., all hepatocytic lysosomes would appear to be "auto"lysosomes, engaging in autophagy. Most of them (76%) had also received BSA within 2h, indicating that they were "am­phi"lysosomes, participating in endocytosis as well.

Structural Aspects of Autophagy 107

Treatment with vinblastine strongly suppressed the labelling of autophagic vacuoles with BSA. Small, BSAnegative lysosomes (LGP+/BSA-) and autophagosomes (LGP-/BSA) accumulated, consistent with inhibition of both endocytic and autophagic flux (Table I).

Asparagine, added at a high concentration (20 mM), induced swelling of BSA positive, but not of BSA negative autophagic vacuoles, perhaps suggesting that swelling requires endocytic fluid delivery. Autophagosomes (LGP-/BSA-), amphisomes (LGP­IBSA+) and BSA negative Iysosomes (LGP+/BSA-) accumulated (Table I), indicating that swelling of the BSA positive vacuoles might impair their ability to fuse with vacuoles of other categories. Altogether, the results suggest that the marker proteins used are well suited for the study of autophagic vacuole kinetics.

ISOLATION OF AUTOPHAGOSOMES

Since the inhibition of autophagic processing by vinblastine caused a considerable accumulation of autophagosomes (Table I), vinblastine-treated hepatocytes were used in an attempt to isolate these organelles in a purified form . As a biochemical marker for auto­phagosomes, autophagically sequestered LDH was used. In the absence of leupeptin, any enzyme reaching the lysosomes will be degraded, causing LDH to accumulate in prelysoso­mal autophagic vacuoles only (i.e. , in the presence of vinblastine, in autophagosomes).

Hepatocytes incubated with vinblastine (50flM) for 2h were electropermeabilized and homogenized, and large-granule fractions were prepared and fractionated further on (A) sucrose or (B) Nycodenz density gradients. Gradient fractions were analyzed for LDH (0), acid phosphatase (.) and cytochrome C oxidase (t.) as markers of autophagosomes, lysosomes and mitochondria, respectively. Enzyme activities per fraction are given as per cent of the respective total cellular acitivities.

In a density gradient study. autophagosomes were found to be well separated from mitochondria in Nycodenz gradients, whereas they coincided with Iysosomes in gradients of either sucrose or Nycodenz (Fig. 4). A number of methods were tried in order to achieve separation of autophagosomes from lysosomes; eventually, a procedure for the selective

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Tab

le 1

. E

ffec

ts o

f vi

nbla

stin

e an

d a

spar

agin

e o

n a

utop

hagi

c va

cuol

e n

um

ber

and

siz

e (v

olum

e) i

n ra

t he

pato

cyte

s

prof

ile,

mm

3 /cm

3 )

Vac

uole

num

ber

(no.

of v

acuo

le p

rofi

les/

cell

pro

file

) V

acuo

le s

ize

(vol

. fr

acti

on/v

ac.)

Con

trol

V

inbl

asti

ne

Asp

arag

ine

Con

trol

V

inbl

asti

ne

Asp

arag

ine

Asp

arag

ineA

utop

hagi

c va

cuol

es (

21)

(SO

D+)

(2

0)

(10)

(2

1)

(20)

(1

0)

(21)

Aut

opha

goso

mes

(L

GP

'/BS

A-)

3.

3 ±

0.5

16.7

± I

. 1<

5.4

± 0.

8"

1.8

± 0.

6 2.

0 ±

0.4

2.2

± 0.

6 A

mph

isom

es

(LG

P-/B

SA +)

3.

2 ±

0.5

1.0

± O

.4C

5.

4 ±

0.5"

2.

5 ±

0.9

2.2

± 1.

3 4.

3 ±

0.8

"Aut

o"ly

soso

mes

(L

GP+

/BSA

-)

4.0

± 0.

5 16

.5 ±

1.2

c 8.

3 ±

0.8c

2.

5 ±

0.7

0.9

± 0.

2"

1.7

± 0.

6 "A

mph

i"ly

soso

mes

(L

GP+

/BSA

+)

10.1

± 1

.2

1.0

± 0.

3c

10.7

± 1

.2

3.1±

0.7

1.4

± 0.

9 5.

1 ±

0.9"

Isol

ated

rat

hep

atoc

ytes

wer

e in

cuba

ted

for

2h a

t 37

°C w

ith g

old-

conj

ugat

ed b

ovin

e se

rum

alb

umin

(B

SA)

and

vinb

last

ine

(50

/-1M

) or

asp

arag

ine

(20

mM

) as

in

dica

ted.

T

he c

ells

wer

e th

en c

ryos

ecti

oned

and

im

mun

osta

ined

with

spe

cifi

c an

tibo

dy a

nd p

rote

in A

-gol

d.

Aut

opha

gic

vacu

oles

wer

e id

enti

fied

in e

lect

ron

mic

rogr

aphs

by

posi

tive

gol

d-im

mun

osta

inin

g fo

r su

pero

xide

dis

mut

ase

(SO

D+)

, Iy

soso

mes

by

posi

tive

im

mun

ogol

d st

aini

ng f

or l

ysos

omal

mem

bran

e gl

ycop

rote

in I

gp 1

20 (

LG

P+),

and

endo

cyti

c in

put b

y th

e pr

esen

ce o

f 2h

endo

cyto

sed

albu

min

-gol

d (B

SA +)

. E

ach

valu

e is

the

mea

n ±

S.E

. of t

he n

umbe

r o

f cel

l pr

ofil

es g

iven

abo

ve e

ach

colu

mn.

"p<

O.0

5; b

p<O

.Ol;

cp<O

.OO

l fo

r si

gnif

ican

ce v

s. c

ontr

ol a

ccor

ding

to t

he t

-tes

t.

-<:> ClC

~

$' ~ '" = ~ ~

Structural Aspects of Autophagy 109

5 A B 0.5 W LDH C/J-C/J c(C/J ...J P\CTR 0.4 z...J ...J 4

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W CJW

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0

1.06 1.10 1.14 1.18 1.06 1.10 1.14 1.18

FRACTION DENSITY Ig/ml)

Figure 5. Effect ofGPN on the density distributions oflysosomes and autophagosomes in isotonic Nycodenz density gradients.

destruction of Iysosomes was developed (Berg et ai., 1994). By incubating electrodisrupted hepatocytes with the cathepsin C substrate glycyl-L-phenylalanine-2-naphthylamide (GPN), a selective osmotic rupture of the Iysosomes could be achieved (Fig. 5),

Hepatocytes, prepared from a rat that had been injected with 25/lg [125I]-TC-AOM 18h prior to cell isolation, were incubated for 2h at 37°C with vinblastine (50/lM), then electrodisrupted. The disruptates were incubated either for 5 min at 37°C without additions (0) or for 5 min at 37°C with 0,5 mM GPN (.), Cytosol-free cell corpses were then isolated and homogenized, and large-granule fractions from the corpses were fractionized further on isotonic Nycodenz gradients. [125I]-TC-AOM radioactivity (A) and LDH (B) were meas­ured in the fractions, and expressed as per cent (per fraction) of the corresponding total amounts in the disruptate.

Figure 6. Purified autophagosomes from rat liver. Bar 400 nm.

110 P. O. Seglen et al.

Figure 7. Emptied autophagosomes. The autophagosome walls are seen to consist of many concentric membranes. Bar 200 nOm.

In combination with Nycodenz cushions (to remove mitochondria) and several rate sedimentation steps (to remove cytosol, nuclei and microsomes), relatively pure preparations of autophagosomes could be obtained (Fig. 6).

Preliminary attempts have been made to empty the autophagosomes and recover their limiting membranes (the phagophores). The results indicate that the walls of auto­phagosomes are built up of from two to numerous concentric unit membranes (Fig. 7), suggesting that either a single cisterna or a stack of flattened cisternae may fold up to form a multilayered bag. Hopefully, analysis of the molecular composition of the phagophore membranes may eventually provide some clues as to how this "bagging" process is executed.

ACKNOWLEDGEMENTS

This work has been generously supported by The Research Council of Norway and by the Norwegian Cancer Society.

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