3 d analysis of dictyosomes and multivesicular bodies in the green alga micrasterias denticulata by fib sem tomography

Our FIB/SEM series and 3-D reconstructions show that interphase dictyosomes of Micrasterias are not only closely associated to an ER system at their cis-side which is common in various p

3-D analysis of dictyosomes and multivesicular bodies in the green alga

Gerhard Wannera, Tillman Schäfera, Ursula Lütz-Meindlb,⇑

a

Ultrastructural Research, Faculty of Biology, Ludwig-Maximilians-University, Munich, Großhadernerstr 2-4, D-82152 Planegg-Martinsried, Germany

b

Plant Physiology Division, Cell Biology Department, University of Salzburg, Austria

a r t i c l e i n f o

Article history:

Received 21 June 2013

Received in revised form 30 September 2013

Accepted 2 October 2013

Available online 14 October 2013

Keywords:

FIB/SEM tomography

Dictyosomes

Multivesicular bodies

ER

Micrasterias denticulata

TEM

a b s t r a c t

In the present study we employ FIB/SEM tomography for analyzing 3-D architecture of dictyosomes and formation of multivesicular bodies (MVB) in high pressure frozen and cryo-substituted interphase cells of the green algal model system Micrasterias denticulata The ability of FIB/SEM of milling very thin ‘slices’ (5–10 nm), viewing the block face and of capturing cytoplasmic volumes of several hundredlm3 pro-vides new insight into the close spatial connection of the ER–Golgi machinery in an algal cell particularly

in z-direction, complementary to informations obtained by TEM serial sectioning or electron tomography Our FIB/SEM series and 3-D reconstructions show that interphase dictyosomes of Micrasterias are not only closely associated to an ER system at their cis-side which is common in various plant cells, but are surrounded by a huge ‘‘trans-ER’’ sheath leading to an almost complete enwrapping of dictyosomes by the ER This is particularly interesting as the presence of a trans-dictyosomal ER system is well known from mammalian secretory cells but not from cells of higher plants to which the alga Micrasterias is clo-sely related In contrast to findings in plant storage tissue indicating that MVBs originate from the trans-Golgi network or its derivatives our investigations show that MVBs in Micrasterias are in direct spatial contact with both, trans-Golgi cisternae and the trans-ER sheath which provides evidence that both endo-membrane compartments are involved in their formation

Ó 2013 The Authors Published by Elsevier Inc All rights reserved

1 Introduction

The plant Golgi apparatus positioned at the cross-road between

the secretory-biosynthetic and the endocytotic-vacuolar pathway

fulfills a great number of different functions ranging from

polysac-charide synthesis, protein glycosylations, and transfer, sorting of

products, regulation of vesicle trafficking up to vacuole formation

and participation in degradation processes (among numerous

oth-ers, see reviews byFaso et al (2009), Hawes and

Satiat-Jeunemai-tre (2005)) As synthesis site for most constituents of the cell wall,

it is central in plant development and plays an important role in

response to environmental impact The diverse functions of the

Golgi apparatus are reflected in the unique morphology and

struc-tural integrity of the numerous motile dictyosomes of a plant cell

(Hawes, 2005) While spatially and functionally tightly associated

to the ER from where they are supplied with proteins and lipids, the flattened closely attached cisternae of a dictyosome usually display clear cis–trans-polarity Cis-, median- and trans-cisternae

of a Golgi stack are involved in different steps of product process-ing and the tubular-reticular trans-Golgi network (TGN) acts as a sorting station that marks outgoing cargo for its destination and also holds the role of an early endosome (Hwang and Robinson, 2009; Staehelin and Moore, 1995; Viotti et al., 2010) The early endosome is the first compartment that receives cargo endocyto-sed from the plasma membrane (Otegui and Spitzer, 2008) Via different intermediate stages it may then mature to the late endo-some which is frequently named multivesicular body (MVB) MVBs are essential for membrane recycling back to the TGN, may act as constituents of the degradation pathway ending up in the vacuole (Lam et al., 2007; Otegui and Spitzer, 2008; Robinson et al., 2008; Tanchak and Fowke, 1987) or may even be involved in the forma-tion of autophagosomes (for references see below)

By means of three-dimensional high-voltage electron micros-copy early studies byMarty (1978, 1999)provided evidence that the TGN acts as starting point for generation of vesicle like provac-uoles which develop to tubular prevacprovac-uoles by microinvagination

of their membranes (see alsoBassham et al (2006)) Provacuoles, prevacuoles or prevacuolar compartments are regarded as

1047-8477/$ - see front matter Ó 2013 The Authors Published by Elsevier Inc All rights reserved.

q

This is an open-access article distributed under the terms of the Creative

Commons Attribution-NonCommercial-No Derivative Works License, which

per-mits non-commercial use, distribution, and reproduction in any medium, provided

the original author and source are credited.

⇑ Corresponding author Address: Cell Biology Department, Plant Physiology

Division, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.

E-mail addresses: wanner@lrz.uni-muenchen.de (G Wanner), tillman.schaefer@

gmx.de (T Schäfer), ursula.meindl@sbg.ac.at (U Lütz-Meindl).

convergence point for either the endocytotic pathway or

autoph-agy Provacuoles may sequester portions of cytoplasm by forming

digitate extensions thus developing to early double-membrane

autophagosomes lateron (Marty, 1978, 1999) Although the

obser-vations of Marty were based on chemically fixed tissue in which

artificial membrane alterations cannot be excluded, the results

are intriguing and deserve verification by means of a more reliable

structure preservation technique More recent publications on high

pressure frozen Nicotiana BY-2 cells (Tse et al., 2004) and

Arabidop-sis root tips (Scheuring et al., 2011) identified MVBs as prevacuolar

compartments or provacuoles arising from one particular domain

of the TGN and able to fuse with the vacuole in a non-vesicular

way This process requires both TGN integrity and V-ATPase

activ-ity (Scheuring et al., 2011) In protein storage tissue of Arabidopsis

embryos it has been found that two different populations of TGN

derived vesicles containing either the storage protein or the

processing enzymes, fuse into MVBs functioning as pre-vacuolar

compartments (Otegui et al., 2006) Whereas numerous

partici-pants in these different degradation pathways have been

identi-fied, the structural transformation from the TGN into the MVB is

still obscure This process however is crucial for both

understand-ing the endocytotic and the degenerative pathway

High pressure freeze fixation (HPF) for best structural

preserva-tion (Staehelin et al., 1990) combined with 3-D analysis such as

electron tomography has been proved to be an excellent tool for

getting insight into the development of dictyosomal or ER derived

structures and their functions at high resolution (Donohoe et al.,

2006; Hayashi-Nishino et al., 2009; Kang and Staehelin, 2008;

Kang et al., 2011; Knott et al., 2008; Mogelsvang et al., 2004;

Ylä-Anttila et al., 2009) Although the benefit of this technique

for detailed structural analysis is undoubted, its limitations arise

from the maximum thickness of the sections (400 nm; see

Dono-hoe et al (2006)), from the maximum tilt angle of about 70°

caus-ing a ‘‘misscaus-ing wedge’’ and from the relatively small volume (max

25lm3) that can be calculated for the 3-D reconstructions The

new technique of focused ion beam milling and viewing by field

emission scanning electron microscopy (FIB/SEM) overcomes these

problems and provides additional structural information by its

ability of sectioning very thin slices (5–10 nm) parallel to the block

face and by covering volumes of several hundreds oflm3(Knott

et al., 2008; Schroeder-Reiter and Wanner, 2009; Schroeder-Reiter

et al., 2009, 2012) The resolution of FIB/SEM tomography does not

yet reach the resolution of TEM in x/y but is close to it as clearly

demonstrated in a recent publication (Villinger et al., 2012) In

re-spect to analysis of dictyosomal derived membranes an additional

advantage of this method is provided by the fact that several

dict-yosomes of a cell can be captured at the same time

In the present study we employed this technique for analyzing

the 3-D architecture of interphase dictyosomes of the algal model

system Micrasterias denticulata (Meindl, 1993) and to obtain insight

into structural connections between MVBs and endomembrane

systems This alga is very well suited for such investigations as it possesses large dictyosomes with a constant average number of

11 cisternae throughout the cell cycle and their vesicular products during different developmental stages are well defined (Eder and Lütz-Meindl, 2008; Eder et al., 2008; Lütz-Meindl and Brosch-Salo-mon, 2000; Oertel et al., 2004) Moreover, numerous studies influ-encing the secretion pathway have provided information on the regulation of the secretory machinery (Lehner et al., 2009; Salomon and Meindl, 1996) Recently evidence has been obtained that Micrasterias is capable of performing autophagy and programmed cell death upon induction by abiotic stressors such as oxidative stress, high salinity or cadmium (Affenzeller et al., 2009a,b; Ando-sch et al., 2012) Coincidently with the occurrence of autophagy, environmental stress evokes severe structural alterations at dictyo-somes (Affenzeller et al., 2009a,b; Darehshouri et al., 2008; Volland

et al., 2011) Particularly cadmium induces a dose and time depen-dent complete disintegration of dictyosomes combined with an increase in the number of MVBs (Andosch et al., 2012) Although, like in other plant and animal systems, the ER contributes to auto-phagosome formation in Micrasterias there are several indications that MVBs may functions as sources for stress induced degenera-tion or even autophagosome formadegenera-tion as well (see above) Infor-mation on the structural origin of MVBs as provided by FIB/SEM tomography will therefore be important as a basis to understand stress induced degradation processes The present study shows that MVBs are frequently found in structural contact with both trans-Golgi cisternae and a ‘‘trans-ER’’ As Micrasterias is a member of Streptophyta belonging to the closest relatives of higher plants (Wodniok et al., 2011) we expect that the results obtained may

be of general interest for plant cells and may also provide insight into evolutionary aspects of the first steps of degradation

2 Materials and methods All chemicals were purchased from Roth (Karlsruhe, Germany)

or Sigma–Aldrich (Vienna, Austria) unless stated differently 2.1 Cell cultures

The unicellular freshwater green alga M denticulata was cultivated

in a liquid Desmidiacean medium (Schlösser, 1982) in Erlenmeyer flaks at constant temperature of 20 °C and a light/dark regime of 14/10 h Cells were subcultured every 4–5 weeks To obtain defined interphase stages for FIB/SEM analyses, developmental stages were collected and were allowed to grow in nutrient solution for 48 h 2.2 High pressure freeze fixation for TEM and FIB/SEM tomography Interphase cells of M denticulata were high-pressure frozen and cryo-substituted following the protocol of Aichinger and Fig.1 (a), and (b) Different 3-D views of the trans-side of a dictyosome of M denticulata reconstructed from FIB/SEM series Cisternal rims are lacerated, outermost-trans-cisterna are shorter than others and undulated Secretory vesicles (grey) are connected to edges of trans-outermost-trans-cisternae From red to green: cis- to trans-outermost-trans-cisternae.

Lütz-Meindl (2005), Meindl et al (1992) In brief the cells were

wrapped in cotton fibers to which they stick due to their surface

mucilage, and frozen in a Leica EMPACT HPF device (Leica

Micro-systems, Austria) Freeze substitution was performed in a Leica

EM AFS in 2% OsO4and 0.05% uranyl acetate in anhydrous acetone

Cells were embedded in epoxy resin (Agar low viscosity resin; Agar

Scientific, Essex, UK) and sectioned on a Leica UC7 ultramicrotome

for TEM 60–80 nm sections were mounted on formvar coated

cop-per grids and investigated in a LEO 912 AB Omega TEM (Zeiss,

Oberkochen, Germany) at 80 kV by using zero-loss energy filtering

(Lütz-Meindl and Aichinger, 2004) Sections of all samples were

viewed in TEM prior to analysis by FIB/SEM tomography

‘‘slice and view’’ technique using a Zeiss Auriga 60 dual beam workstation (Carl Zeiss Microscopy, Oberkochen, Germany) For slicing with the focused ion beam, the conditions were as follows:

500 pA milling current of the Ga-emitter; with each step 7–10 nm

of the epoxy resin was removed with the FIB SEM images were re-corded with an aperture of 60lm in the high current mode at 1.5 kV of the In-lens EsB detector with the EsB grid set to

1400 V Line averaging 4 was performed The image pixel size was 5.3  5.3 nm in x/y The dimensions of the recorded images were 2048  1536 pixel According to the Nyquist criterion the resolution lies thus in the range of 12 nm The time to acquire one frame was 90s The slice and view process was repeated 500–1500 times to obtain the different datasets The contrast of the images was inversed, so that they appeared like a conventional bright field TEM image

2.4 Data processing and 3-D reconstruction Image stacking and alignment was performed with the open source software ImageJ (http://rsbweb.nih.gov/ij/index.html) Segmentation was performed using Amira Software (VSG; Visualization Sciences Group)

3 Results Dictyosomes of Micrasterias interphase cells are mostly accu-mulated in the cell center around the nucleus or along the edges

of the large chloroplast which covers each of the two semicells FIB/SEM slicing and 3-D analysis of the block-face images shows that the Golgi stacks consist of flat unfenestrated cisternae with lacerated rims (Fig 1a,b) Interconnections between the cisternae are frequently visible at the images series The outermost trans-cis-terna is frequently shorter and more involute and differs in its structure from the others On FIB/SEM series (Fig 2) and TEM micrographs (Fig 3a,b) it appears chain-like constricted resulting

in an undulated surface in the 3-D reconstruction (Fig 1b) During interphase the main products of dictyosomes in Micrasterias are large mucilage vesicles which origin from the edges of the outer-most trans-cisternae and are also found accumulated around the dictyosomes (Fig 4,Suppl animation) Additionally smaller secre-tory vesicles are present These findings confirm earlier results ob-tained from serial sectioning of HP frozen or chemically fixed Micrasterias cells and TEM (Meindl et al., 1992) The trans-most cisterna is frequently separated from the dictyosome and appears circular (Fig 2) Depending on how it is oriented it may be assigned

to a TGN Like in other plant cells (Faso et al., 2009; Moreau et al.,

2007) almost all dictyosomes are in close spatial relationship to an

ER cisterna at their cis-side (Figs.2,3b,5,6,Suppl animation) and transitorial vesicles between these two compartments of the endo-membrane system are visible For the first time FIB-serial section-ing and 3-D reconstruction shows that the dictyosomes in Micrasterias are almost entirely engulfed by a huge ER sheath which is not limited to the cis-Golgi side but also envelopes the trans-Golgi area to a large extent (Figs 5 and 6,Suppl animation) Frequently the membranes of the trans-cisternae are in close

Fig.2 FIB/SEM series of two dictyosomes of an interphase Micrasterias cell

Trans-most Golgi cisternae are shorter and involute respectively circular (arrows) ER

cisternae present at both cis- and trans-side of the right dictyosome Framed area

indicates vesicular contact between a trans-Golgi cisterna and the trans-ER Small

secretory vesicles and large mucilage vesicles are visible around the left

dictyo-some 10 nm slices, every second image shown.

contact with the ER membranes either directly or via vesicular

con-nections (Fig 6) This finding yields a new picture of the close

spa-tial connection of the ER-Golgi machinery which became only

possible by the capability of FIB/SEM tomography of providing

3-D analysis of large volumes particularly in z-direction Although

single ER cisternae have been observed in vicinity to the

dictyoso-mal trans-cisternae in TEM as well (Fig 3b), the presence of such a

huge enwrapping ‘‘trans-ER’’ sheath would have been never

deduc-ible from 2-D TEM micrographs The fact that several dictyosomes

of a cell can be serial sliced and visualized by FIB/SEM at a time,

en-ables comparison of different Golgi-ER associations thus yielding

additional information on the generality of these observations

MVBs in Micrasterias are mostly found in close association to

dictyosomes both in interphase cells (Figs 7 and 8) and during cell

growth (not shown) As in higher plant cells (Lam et al., 2007;

Ote-gui and Spitzer, 2008; OteOte-gui et al., 2006; Scheuring et al., 2011;

Wang et al., 2011) they appear always spherical and contain

intra-luminal vesicles with a diameter of about 50 nm in Micrasterias

(Fig 3c) Their maximum diameter averages 400 nm Several MVBs may be found in close vicinity of one dictyosome (Fig 3d) All pre-vious studies on MVB maturation mostly done in protein storage tissue, have indicated that they originate from either the TGN or from dictyosomal derived vesicles that fuse to form a MVB (see ref-erences above) Our study employing FIB/SEM analysis provides evidence that MVB formation in Micrasterias is not only a function

of the trans-Golgi region but involves a ‘‘trans-ER’’ as well Most of the MVBs investigated in Micrasterias were in spatial contact to both, an ER and a trans-dictyosomal cisterna Examples of these contacts are visible in the series ofFigs 7 and 8 Occasionally direct contacts existed exclusively between MVB and ER although the MVB was located in close vicinity to trans-Golgi cisternae (Fig 9a,b,Suppl animation) The contact between the membranes

of the ER and the MVB appears to be a direct one: membranes of ER and MVBs are frequently attached to each other (Fig 3e,f) Connec-tions to dictyosomal derived tubular structures indicating that MVBs originate from the TGN as observed in Arabidopsis root tips

Fig.3 TEM micrographs of ultrathin sections of high-pressure frozen and freeze substituted interphase cells of M denticulata (a) Dictyosomes with adjacent ER cisterna at cis- and trans-side Trans-most cisterna is chain-like constricted Large mucilage vesicles (MV) are in contact with rims of trans-cisternae Two MVBs (arrows) are located in close proximity to the trans-Golgi side and to a trans-ER cisterna (b) MVB (arrow) is in contact with vacuolar membrane translucent vesicles (arrow head) are in contact with trans-dictyosomal cisterna and MVB ER = endoplasmic reticulum (c) Representative MVB of Micrasterias with intraluminal vesicles (d) Trans-side of dictyosome (D) with stack of mucilage vesicles (MV) Three MVBs (arrows) are in close proximity to the dictyosome and to a trans-ER cisterna ER = endoplasmic reticulum (e) Dictyosome and parts of the enwrapping ER Contact between MVB (arrow) and part of a trans-ER cisterna visible MV = mucilage vesicle; ER = endoplasmic reticulum; P = Plastid (f) Part of a dictyosome and MVB attached to an ER cisterna ER = endoplasmic reticulum (g,h) MVBs in spatial contact with small electron-dense vesicle (arrow) and with larger translucent vesicle (arrow head) (i,j) MVBs attached to mucilage vesicles (MV) (j) Intraluminal vesicle of MVB discharged into upper mucilage vesicle (framed area).

M = mitochondrion.

(Scheuring et al., 2011) were never found in Micrasterias Two

dif-ferent vesicle populations were frequently seen in contact with

MVBs: small electron-dense vesicles with a diameter of approx

50 nm corresponding to the intraluminal vesicles of the MVBs both

in size and in electron density and larger, electron translucent

ves-icles with an average diameter of 130 nm (Fig 3g,h) Electron

translucent vesicles were frequently found to be in contact to the

rims of trans-cisternae suggesting that they originate from the

dictyosomes (Fig 3b) whereas the origin of the small vesicles is

unclear Contacts between the MVBs and the dictyosomal cisternae

may occur either via these vesicles, or directly between the

dicty-osomal membrane and the membrane of the MVB (Fig 7) Most

contacts between MVBs and dictyosomes were observed with the

outermost trans-cisterna or with one of the other trans-cisternae,

only rarely with medium cisternae MVBs with no visible contact

to either the ER or a dictyosome were also rarely found by FIB/

SEM tomography However, it cannot be excluded that the

corresponding endomembrane system in these cases was located

in a larger distance above or below the MVB and was simply not

included in the respective series

In addition to their structural connections to trans-Golgi

cister-nae and the ER, MVBs were observed to be in contact with each

other (Figs 5 and 9a,b) and with vacuoles (Fig 3b) Occasionally

MVBs were found tightly attached to membranes of large mucilage

vesicles (Fig 3i) and their transluminal vesicles seemed to be

ization of a huge ER sheath at the trans-side of the dictyosomes which together with the common cis-Golgi associated ER system known from different plant cells (among others see Hawes and Satiat-Jeunemaitre (2005), Moreau et al (2007)) almost entirely enwraps the dictyosomes in Micrasterias Neither TEM serial sectioning nor electron tomography would be able to provide information on the magnitude of this ER envelope Due to the relatively thick sections (60–70 nm) information is lost in TEM and only parts of this ‘‘trans-ER system’’ are depicted On the other hand, electron tomography is limited to a relatively small mic volume and would not allow calculating such large cytoplas-mic areas Additionally our FIB/SEM series indicate that this

‘‘trans-ER’’ sheath may also be involved in MVB formation which has been so far attributed solely to trans-Golgi compartments and their derivatives in higher plant cells (e.g (Scheuring et al., 2011; Tse et al., 2004; Wang et al., 2011))

The presence of a ‘‘trans-ER’’ system is well known from mam-malian secretory cells where it represents a specialized ER cisterna

in close association with the trans-most Golgi cisterna (reviewed

byMogelsvang et al (2004)) In higher plant cells indications for

a ‘‘trans-ER’’ possibly involved in secretion in concert with the dictyosomes, have been obtained from early thick-sectioning techniques in mung bean cells revealing direct connections between the ER and trans-dictyosome cisternae (Harris and

Opar-ka, 1983) However, in the recent literature referring preferentially

to cryo-fixed samples or to studies in living cells, reports of the presence of a trans-Golgi-ER association are missing (see reviews

byFaso et al (2009), Hawes and Satiat-Jeunemaitre (2005)) From

an evolutionary point of view it is interesting that this

trans-Golgi-ER association appears in an alga such as Micrasterias which is closely related to higher plants (Wodniok et al., 2011) Moreover, the FIB/SEM series show that direct structural contacts between the ‘‘trans-ER’’ and the trans-most Golgi cisterna – as postulated

by early studies in mammalian and plant cells (Hand and Oliver, 1977; Harris and Oparka, 1983; Novikoff et al., 1971) – do in fact occur in Micrasterias

The presence of structural connections of MVBs not only to trans-Golgi cisternae and their derivatives but also to a ‘‘trans-ER’’ may point towards a particular way of degradation According

to the early GERL hypothesis ofNovikoff et al (1971)postulating that newly synthesized lysosomal enzymes are directly delivered from the ER to the trans-most Golgi cisterna in neurons, the ER

in Micrasterias may supply MVBs with lytic enzymes whereas the cargo proteins are secreted through the Golgi apparatus In this way the MVBs would become lytic compartments and starting points for degradation as postulated in early TEM studies (Tanchak and Fowke, 1987) In fact an increase in the number of MVBs has been observed in Micrasterias upon exposure to environmental stress such as high salinity or cadmium exposure (Affenzeller

et al., 2009a; Andosch et al., 2012), indicating their involvement

in stress induced degradation Golgi-derived vesicles resembling the ‘‘dense vesicles’’ carrying storage proteins in cotyledons or embryos of legumes (Hinz et al., 1999; Otegui and Spitzer, 2008; Robinson et al., 1998) and electron-dense small vesicles with the same size as the interluminal vesicles have been found to be in contact with MVBs in Micrasterias and are interpreted to contribute

Fig.4 3-D reconstruction of interphase dictyosome of Micrasterias with

enwrap-ping ER system (blue) and different vesicles populations Small secretory vesicles

are shown in green, mucilage vesicles in grey Dictyosomal cis-side in red,

trans-side in green.

Fig.5 3-D reconstruction of dictyosome and ER envelope (blue) at cis- and

trans-side of the dictyosome Two MVBs (yellow) are visible in proximity of the

dictyosomal trans-cisternae.

to their formation Our data on frequent, close structural contacts

between different compartments suggest that discharge of MVBs

occurs both into vacuoles or lytic compartments but also into

mucilage vesicles Mucilage vesicles are secreted throughout the cell cycle of Micrasterias (Oertel et al., 2004) and were recently found to represent vehicles for heavy metal detoxification (Volland

Fig.6 FIB/SEM series of dictyosome in contact to a cis- and a ER cisterna Trans-ER covers the side of the dictyosome almost entirely At least two of the trans-cisternae (framed areas) are in contact with the trans-ER either directly or via vesicular connections The trans-most cisterna is vesicular and disconnected from Golgi stack appearing as TGN Large mucilage vesicles (MV) are visible in close vicinity to dictyosome 10 nm slices, every 10th image shown.

et al., 2011) Heavy metals are sequestered in mucilage vesicles

and removed from the cell by mucilage secretion In the same

way degrading proteins inside MVBs may be disposed via the

secretion pathway Cytochemical and immuno-TEM studies including cells after stress exposure will be required to verify this hypothesis

Fig.7 FIB/SEM series of MVB in contact with trans-most dictyosomal cisterna via

small vesicles (arrow) Maturating and ripe mucilage vesicle in contact to each

other and close to trans-Golgi cisternae 10 nm slices, every 4th image shown.

Fig.8 FIB/SEM series of MVB in direct spatial contact to both a trans-dictyosomal cisterna and a trans-ER cisterna (arrow) The MVB also contacts one of the maturating mucilage vesicles.

We gratefully acknowledge the excellent technical assistance

by Silvia Dobler and Ancuela Andosch and the valuable suggestions

and support by Max Scheungrab during the manual segmentation

for the 3-D reconstructions The study was financially supported by

the Austrian Science Fund project 21035-B16 to U.L-M

Appendix A Supplementary data

Supplementary data associated with this article can be found, in

the online version, athttp://dx.doi.org/10.1016/j.jsb.2013.10.003

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