Golgi Inheritance Under a Block of Anterograde andRetrograde Traffic

Clement Nizak1,3, Rachid Sougrat1,2,4, FlorenceJollivet1, Alain Rambourg2, Bruno Goud1 andFranck Perez1,*

1 Institut Curie, CNRS UMR144, 26 rue d’Ulm 75248 Pariscedex 05,2Departement de Biologie Cellulaire et Moleculaire, CEA/Saclay, F-91191 Gif-sur-Yvette, France*Corresponding author: Franck Perez,franck.perez@curie.fr

In mitosis, the Golgi complex is inherited following itsdispersion, equal partitioning and reformation in eachdaughter cell. The state of Golgi membranes duringmitosis is controversial, and the role of Golgi-intersectingtraffic in Golgi inheritance is unclear. We have usedbrefeldin A (BFA) to perturb Golgi-intersecting membranetraffic at different stages of the cell cycle and followedby live cell imaging the fate of Golgi membranes in thoseconditions. We observed that addition of the drug oncells in prometaphase prevents mitotic Golgi dispersion.Under continuous treatment, Golgi fragments persistthroughout mitosis and accumulate in a Golgi-like struc-ture at the end of mitosis. This structure localizes atmicrotubule minus ends and contains all classes ofGolgi markers, but is not accessible to cargo from theendoplasmic reticulum or the plasma membrane becauseof the continuous BFA traffic block. However, it containspreaccumulated cargo, and intermixes with the reform-ing Golgi upon BFA washout. This structure also formswhen BFA is added during metaphase, when the Golgi isnot discernible by light microscopy. Together the dataindicate that independent Golgi fragments that containall classes of Golgi markers (and that can be isolated fromother organelles by blocking anterograde and retrogradeGolgi-intersecting traffic) persist throughout mitosis.

Key words: brefeldin A, Golgi apparatus, intracellularmembranes, mitosis, organelles

Received 15 January 2004, revised and accepted for pub-lication 27 January 2004

Whether the Golgi apparatus is an independent organelle

or an extension of the endoplasmic reticulum (ER) along

the secretory pathway has been a debated issue (1,2). The

Golgi is in dynamic equilibrium with the ER, and micro-

tubule disruption, for example, highlights the continuous

cycling of Golgi resident enzymes through the ER (3).

Other studies showed that the blocking of ER export by

Sar1 mutants induces accumulation of all Golgi markers in

the ER (4,5), outlining once again this recycling pathway.

The correlation between spatial distributions of the Golgi

and transitional ER in two yeast species (6) and mamma-

lian cells (7) was proposed to result from the mutual feed-

back between ER export sites and Golgi membranes.

Altogether, these arguments strongly suggest that Golgi

components are continuously cycled through the ER.

However, cytoplasts devoid of Golgi complex but still con-

taining an ER network do not form a Golgi de novo, and

secretion in these cytoplasts is blocked at the level of ER

export sites (1,8).

Studies of Golgi inheritance in mitosis reflect this contro-

versy (8–10), arguing in favor either of retrograde transport

and fusion of this organelle with the ER in prophase and

subsequent reemergence in telophase (11) or its fragmen-

tation into ER-independent partitioning units and subse-

quent reassembly from these units (8,12–15). Clearly,

determining how the Golgi apparatus disassembles and

subsequently reassembles during mitosis would allow a

deeper understanding of its function and organization

(8,16). In vitro systems (17–20) have been successful in

identifying factors that contribute to Golgi structure and

function, and the alteration of which during mitosis causes

the Golgi complex to break down (21–29).

These studies and many others support the general idea

that Golgi inheritance relies on the modulation of ER–Golgi

bidirectional transport dynamics during mitosis. Brefeldin

A has been a powerful and widely used tool to study and

perturb the ER/Golgi interface because it induces a com-

plete block of Golgi-intersecting transport pathways, Golgi

disappearance, and redistribution of Golgi resident

enzymes to the ER in interphase cells. In the presence of

this drug, Golgi matrix markers are redistributed to dis-

persed punctate structures which coincide with ER export

sites in live cells expressing fluorescent COPII chimeras

(5). It has recently been shown that these dispersed struc-

tures contain Golgi matrix markers but are devoid of Golgi

resident enzymes (the latter being relocated to the ER),

and that they persist throughout mitosis when BFA is

added to cells before they start dividing. In contrast, this

study provides biochemical evidence that these structures

are distinct from ER export sites (15), and may therefore

represent ER-independent Golgi partitioning units.

Present addresses: 3 Rockefeller University, Center for Studies inPhysics and Biology, Living Matter Laboratory, Box 34, 1230 YorkAvenue, 10021 New York, NY, USA, 4 Cell Biology andMetabolism Branch, National Institute of Child Health andHuman Development, 18 Library Drive, Building 18T/Room 101,National Institutes of Health, Bethesda, MD 20892–5430, USA.

Traffic 2004; 5: 284–299Copyright # Blackwell Munksgaard 2004

Blackwell Munksgaard doi: 10.1111/j.1600-0854.2004.00174.x

284

Figure 1: Analysis of BFA effects throughout the cell cycle. A) Unsynchronized GalNacT2-GFP HeLa cells were treated with BFA or

not (Ctrl) and imaged by time-lapse microscopy (time indicated in HH :MM). When BFA treatment was initiated in interphase, GalNacT2

was quickly relocated in the ER in 15min (00 : 15) and remained so as cells progressed through mitosis. When BFA treatment was initiated

in prometaphase, Golgi fragments did not disperse further (00 : 15), in contrast to their full dispersion in metaphase control cells (Ctrl,

01 : 15), and these fragments persisted instead throughout mitosis, aggregating into a BFA-resistant Golgi-like structure in telophase

(00 : 45). When BFA treatment was initiated in metaphase, as the Golgi is the most dispersed (at 00 : 00 the GalNacT2-GFP signal is diffuse

and only excluded from the metaphase plate), the same BFA-resistant Golgi-like structure formed in telophase, with an additional ER

background (01 : 00). When BFA treatment was initiated as the Golgi had already started to reform (late anaphase/telophase), GalNacT2

was quickly relocated in the ER (00 : 15). Bar: 10mm. B) GalNacT2-GFP HeLa cells were treated with BFA for 1 h. Telophase cells

were observed by time-lapse microscopy upon BFA washout (time indicated in HH :MM). The Golgi reformed in all cells, regardless of the

presence (lower cell) or absence (upper cell) of a Golgi-like BFA-resistant structure initially. When present, the Golgi-like structure

intermixed with the reassembling Golgi (arrows), as de novo formed GalNacT2-GFP-positive structures converged towards this site.

Bar: 10mm.

Mitotic Golgi Dynamics under a BFA-transport Block

Traffic 2004; 5: 284–299 285

One essential difference between the two models for

Golgi inheritance is the role played by membrane traffic

between the Golgi and the ER. The ER-based inheritance

model relies on transport from the Golgi to the ER during

entry into mitosis and on transport from the ER towards

the reforming Golgi during mitosis exit. The fragmentation

model, on the other hand, does not require any transport

between the Golgi and another compartment during

mitosis for the Golgi to be inherited.

To address this issue, we have analyzed the sensitivity of

the Golgi complex to a BFA-induced transport block at

different stages of the cell cycle, during the dispersion

and the reformation of the Golgi apparatus in mitosis.

Figure 2: A Golgi-like BFA-resistant structure forms in mitotic cells when BFA treatment starts during mitosis. Wild-type mitotic

Hela cells were collected by shake-off from nonsynchronized cell populations and plated on coverslips for 2 h without (a) or with BFA (b).

Alternatively, cells were treated with BFA for 3 h, mitotic cells were collected by shake-off and plated for 2 h with BFA (c). Cells were then

fixed and stained for endogenous GalT. This experiment confirms that in wild-type HeLa cells a BFA-resistant structure reforms upon

mitosis exit if BFA treatment starts during mitosis, but not if the treatment starts before entry into mitosis. Bar: 10mm.

Figure 3: A 30-min BFA treatment of prometaphase/metaphase cells greatly increases the number and size of preexisting

mitotic Golgi fragments positive for both Golgi enzymes and Golgi matrix markers. Non-synchronized wild-type HeLa cells were

incubated with BFA for 30min or not incubated with BFA, fixed in MeOH and stained for Giantin and GalT. In the absence of BFA, Golgi

fragments, positive for Giantin and GalT, are visible in prometaphase, and in most metaphase cells even though the staining is essentially

diffuse for both markers in these cells. A short 30-min BFA treatment, which caused Golgi disappearance in almost all interphase cells,

increased the number and size of these fragments. Since cells are fixed 30min after initiation of BFA treatment, cells must be in prophase/

prometaphase at the time of drug addition. BFA treatment prevents the normal dispersion of prophase/prometaphase Golgi fragments

positive for Giantin and GalT.

Nizak et al.

286 Traffic 2004; 5: 284–299

Figure 4: The Golgi-like BFA-resistant

structure contains several classes of Golgi

markers. Mitotic Hela cells were collected from

nonsynchronized cell populations, immediately

incubated for 2 h in the absence (A) or presence

(B) of BFA, fixed and stained for endogenous

Giantin, GM130, p115, ERGIC53, KDELr, Rab1,

Rab6, GalT, bCOP and DNA. The BFA-resistant

structure is positive for Golgi enzymes, Golgi

matrix markers, Golgi-associated Rabs and

ER–Golgi cycling proteins, but not bCOP (B). All

these markers, including bCOP, are present on

the Golgi in control, nontreated telophase cells

(A). Bar: 10mm. (continued on following page)

Mitotic Golgi Dynamics under a BFA-transport Block

Traffic 2004; 5: 284–299 287

Figure4: Continued.

Nizak et al.

288 Traffic 2004; 5: 284–299

Results

Mitosis entry confers BFA-resistance to Golgi

membranes

While brefeldin A treatment of mitotic cells has already been

used to address the question of Golgi inheritance, those earl-

ier studies did not ask whether BFA modifies the behavior of

the Golgi in mitosis and above all did not observe whether

BFA effects depend on the stage of mitosis when it is added.

We first monitored the dynamics of the stably expressed

Golgi enzyme-based fluorescent reporter N-acetylgalactos-

aminyltransferase 2 (GalNacT2)-Green fluorescent protein

(GFP) (3) in unsynchronized HeLa cells treated with BFA at

various stages of the cell cycle (Figure1A, see Supplemental

Movie 1 at http://www.traffic.dk/video_gallery.asp). As

detailed below, our in vivo observations complete immuno-

fluorescence and biochemical results reported by others

(15,30) and our time course analysis reveals new, surprising

aspects of mitotic Golgi dynamics.

As expected, upon BFA addition, redistribution of

GalNacT2-GFP to the ER in interphase cells was complete

within 5–30min. When these cells subsequently entered

mitosis in the continuous presence of the drug, GalNacT2-

GFP remained localized in the ER throughout mitosis, as

already described for the endogenous Golgi enzyme

a-mannosidase II (15) (Figure 1A).

Similarly, if cells were treated with BFA during late ana-

phase, telophase or cytokinesis, stages at which the Golgi

had already started or finished reforming, GalNacT2-GFP

was transported back to the ER (Figure 1A). In contrast, in

cells treated in prometaphase, BFA had the exact opposite

effect. Prometaphase Golgi fragments were not redistrib-

uted to the ER; instead, BFA addition stopped the process

of progressive disappearance of the Golgi observed in

metaphase in control cells (when Golgi markers, including

GalNacT2-GFP, give a homogeneous immunofluorescence

staining throughout the cytoplasm in nontreated cells (Fig-

ure 1A, top)). Under continuous BFA treatment, GalNacT2-

GFP-positive fragments persisted in metaphase, anaphase

and accumulated in telophase into a Golgi-like structure

that was visible for several hours after cytokinesis (Figure

1A). This Golgi-like structure contains 70� 12% of the

fluorescent signal initially present in the Golgi before mitosis

(see Supplemental Movies 1 and 7 at http://www.traffic.

dk/video_gallery.asp).

If cells were treated with BFA at metaphase, when the

Golgi was no longer discernible (with only rare fragments

present), we observed that a pericentriolar structure, posi-

tive for GalNacT2-GFP, reformed during the following ana-

phase/telophase stages (Figure 1A). The proportion of

resisting membranes was then lower: 50� 15% of the

signal initially present in the Golgi before mitosis was

found in the Golgi-like structure in telophase, and some

ER staining was clearly visible (see Supplemental Movies

1 and 7 at http://www.traffic.dk/video_gallery.asp).

With washout of the drug, Golgi reformation occurred in all

cells (Figure 1B and Supplemental Movie 2 at http://

www.traffic.dk/video_gallery.asp), regardless of the pre-

sence (lower cell) or not (upper cell) of a BFA-resistant

Golgi-like structure before washout. When present, the

BFA-resistant Golgi-like structure was intermixed with, or

directly contributed to, Golgi reassembly upon BFA wash-

out (Figure 1B, lower cell, arrows).

BFA-resistant membranes in telophase/cytokinesis cells

often aligned along microtubules, dynamically accumu-

lated near the centrosomes and extremities of the mid-

body and exchanged continuously between these two

microtubule minus end-associated membrane pools in

each daughter cell (see Supplemental Movie 5 at http://

www.traffic.dk/video_gallery.asp). This dynamic associ-

ation with microtubule minus ends is similar to the behavior

of the Golgi apparatus in control, nontreated telophase

cells (data not shown).

In separate experiments, we confirmed and quantified this

BFA-resistance of prometaphase–metaphase Golgi by

immunofluorescence of endogenous proteins in wild-type

HeLa cells. To avoid any secondary effect due to cell

synchronization, we treated HeLa cells with BFA and fol-

lowed the fate of the Golgi apparatus in mitotic cells

obtained by the shake-off method on unsynchronized cell

populations (Figure 2). When cells were incubated with

BFA in interphase (3 h before shake-off), mitosis pro-

ceeded normally and Golgi enzymes were still found in

the ER in telophase/cytokinesis (Figure 2c). However, in

85� 5% of cells treated with BFA immediately after the

shake-off, and thus for most of those cells in prometa-

phase/metaphase, we observed that a BFA-resistant

Golgi-like structure containing Golgi enzymes reformed

upon mitosis exit. This Golgi-like structure was more com-

pact and discontinuous than the Golgi apparatus in control,

nontreated telophase cells (Figure 2a), and persisted dur-

ing the late stages of mitosis (Figure 2b) in the continuous

presence of BFA. Protection from mitosis-induced Golgi

disappearance was already clear during the metaphase of

treated cells since both the number and the size of Golgi

remnants were increased (Figure 3).

Composition and ultrastructure of the BFA-resistant

Golgi-like structure

In contrast to the proposal that only Golgi matrix structures

serve as partitioning units (15), the BFA-resistant structure

we observed is positive for Golgi enzymes. We character-

ized further its composition by staining for other classes of

endogenous Golgi-associated proteins, and all the ones

tested were present on the Golgi-like structure (Figure 4

and data not shown): Golgi enzymes (GalNacT2-GFP, endo-

genous b1,4-Galactosyltransferase (GalT) and a-mannosidase

II), Golgi matrix proteins (GM130, Giantin, p115), Rabs

(Rab1, Rab6), ER/Golgi cycling proteins (KDEL receptor

(KDELr), ERGIC53). ERGIC53 is less enriched than Giantin

Mitotic Golgi Dynamics under a BFA-transport Block

Traffic 2004; 5: 284–299 289

in the BFA-resistant structure, as it is in the Golgi of control,

nontreated telophase cells (Figure 4). BFA-resistance is not

due to a general resistance to BFA of mitotic cells since

COPI was fully released in the cytosol and absent from

Golgi-like membranes, even though it is normally Golgi-

associated in untreated cells at the same stage of the cell

cycle (Figure 4). Moreover, endosomes are known to

become tubular in the presence of BFA, and this tubulation

process was observed in cells containing BFA-resistant

Golgi-like structures (see below).

We then analyzed treated cells by electron microscopy.

We observed the frequent appearance of clusters of

vesiculo-tubular membranes in cells treated by BFA during

prometaphase/metaphase. The location of some of these

clusters, in the vicinity of the centrosome (Figure 5a) or of

Figure 5: The BFA-resistant Golgi-like structure is an accumulation of juxtaposed ‘frozen’ mitotic Golgi clusters. Mitotic Hela

cells, either wild-type or stably expressing ManII-HRP, were collected from nonsynchronized cell populations, immediately incubated for

2 h with or without BFA until they reached telophase/cytokinesis. Cells were then processed for electron microscopy (see Materials and

Methods). ManII-HRP HeLa cells were additionally incubated with DAB/H2O2 to specifically stain Golgi structures. a) Tubulovesicular

clusters, reminiscent of mitotic Golgi clusters, were found in a pericentriolar area (ctr: centrosome) in wild type HeLa cells treated with

BFA from metaphase to telophase, but not in cells treated from interphase to telophase (not shown) or nontreated cells (in which Golgi

stacks have reformed, not shown). b) In ManII-HRP HeLa cells, these clusters were weakly stained by DAB/H2O2, indicating that they are

of Golgi origin. c) These structures (arrow, enlarged in the inset) were often found in close proximity to the midbody (mb), in agreement

with our observation by light microscopy. d) In the same cell area, nontreated telophase cells were found to reform normal Golgi stacks (g)

positive for ManII-HRP. a) bar¼ 0.35mm, b) bar¼0.28mm, c) bar¼0.5 mm, d) bar¼0.34mm.

Nizak et al.

290 Traffic 2004; 5: 284–299

chromosomes (not shown) suggested that these were the

structures positive for Golgi markers observed at the light

microscopy level. To confirm the identity of these mem-

branes, we used a HeLa cell line that we recently selected

and that in the Golgi apparatus stably expresses horse-

radish peroxidase fused to the transmembrane segment

of the medial Golgi enzyme a-mannosidase II (ManII-HRP).

Electron microscopy observation of interphase cells

stained with diaminobenzidine (DAB) confirmed the correct

localization of the fusion protein (Jollivet et al., in prepara-

tion). Similarly, ManII-HRP is found in the reforming Golgi

structure during mitosis exit (Figure 5d). The tubulovesicular

clusters are also positive for ManII-HRP, confirming their

Golgi origin (Figure 5b,c). Note that, in contrast to control

cells (Figure 5d), no stacked structures could be observed in

cells containing BFA-resistant structures. These tubulo-

vesicular structures are very similar to the mitotic Golgi

clusters observed by others (13,20,31–33). Note also that

the DAB precipitate is not homogeneously distributed over

the different vesicles and tubules of a given cluster: it is

possible that, even though all classes of markers were

found in BFA-resistant structures by fluorescent micro-

scopy, each particular vesicle or tubule is enriched in a

particular subset of Golgi markers, for instance according

to their cis–trans distribution in interphase (34).

The BFA-resistant Golgi-like structure is not

connected with the ER and is distinct from ER exit

sites

In interphase cells treated with BFA, the Golgi matrix

markers GM130 and GRASP65-GFP, and the recycling

protein p58-GFP (ERGIC53’s rat ortholog fused to GFP)

colocalize with fluorescent COPII chimeras at ER exit

sites, and Golgi enzymes are completely redistributed

in the ER (5). Therefore, the resistant Golgi-like

structures observed could represent fragments of the ER

or ER exit sites. We therefore stained for Sec31, a compon-

ent of the COPII coat, which revealed that these struc-

tures are clearly distinct from ER export sites (Figure 6A).

This result is consistent with the partial localization of

ERGIC53 to the BFA-resistant Golgi-like structure: the

abundance of a given marker in the Golgi-like structure

correlates with its abundance in the Golgi of control,

nontreated cells.

We next tested by Fluorescence Loss In Photobleaching

(FLIP) whether the BFA-resistant Golgi-like structures that

form in anaphase/telophase were directly connected to the

underlying ER (Figure 6B and Supplemental Movie 3 at

http://www.traffic.dk/video_gallery.asp). In all telophase

cells, whether treated with BFA or not, a portion of the

ER of one of the daughter cells was repetitively photo-

bleached for 15min and the decrease of fluorescence

was quantified. In BFA-treated cells that did not show

any resistant Golgi-like structure because they had already

reached telophase when the treatment started (Figure 6B,

middle panel), a 90% decrease in total GFP fluorescence

was observed. This experiment shows that, as expected,

when the GalNacT2-GFP pool is entirely relocated in the

ER because of BFA, it is highly sensitive to FLIP. Note that

in the second daughter cell, no photobleaching was

observed, presumably because the ER/Golgi systems of

the two daughter cells are independent at this late stage of

mitosis, which provided us with an internal control for

photobleaching. The same FLIP experiment was carried

out in cells containing a BFA-resistant Golgi-like structure

and in control, nontreated cells (Figure 6B, lower panel,

and upper panel, respectively). No noticeable intensity

change in the Golgi-like or control Golgi pattern was

observed when the ER fluorescence decreased by 90%

(Figure 6B, postbleach). Therefore, the BFA-resistant

Golgi-like structures that reform upon mitosis exit are

independent of the ER, at least on a time-scale of 15min,

and consequently not directly connected to it, like Golgi

membranes in control, nontreated cells.

The BFA-resistant Golgi-like structure is built from

preexisting Golgi remnants

We then questioned the origin of these BFA-resistant

membranes, and whether they reemerged from the ER.

To address this issue we tested whether these Golgi-like

structures were accessible to cargo transported from the

ER or from the plasma membrane. BFA blocks Golgi-

intersecting membrane transport pathways in interphase

cells, and we confirmed that it still does even when the

BFA-resistant Golgi-like structure forms from mitotic Golgi

fragments.

Secretion is known to resume at telophase (35), and we

observed that tsO45VSVG was normally secreted at the end

of mitosis in control cells (Figure 7a–c). When BFA was

added in prometaphase/metaphase, tsO45VSVG was not

present in the Golgi-like structure but remained in the ER,

indicating that its transport from the ER to the BFA-

resistant Golgi-like structure and the plasma membrane

was blocked (Figures 7d–f).

We followed a second cargo of the ER to Golgi pathway

using Fluorescence Recovery After Photobleaching (FRAP)

experiments on GalNacT2-GFP. We observed that while

GalNacT2-GFP is efficiently transported from the ER to the

Golgi during the telophase/cytokinesis of control cells, it is

no longer transported in the presence of BFA. The Golgi

area in control cells or the Golgi-like structure in BFA-

treated cells was slowly photobleached for 2min to ensure

that GalNacT2-GFP fluorescence recovery would not be

dominated by rapid lateral diffusion in Golgi or Golgi-like

membranes. Fluorescence recovery typical of ER–Golgi

transport (method originally described in Zaal et al. (11))

was observed only in control cells and not in the presence

of BFA, indicating that GalNacT2-GFP did not accumulate

to the Golgi-like structure from an ER pool in BFA-treated

cells, and confirming that anterograde transport was

blocked (Figure 7B and Supplemental Movie 4 at http://

Mitotic Golgi Dynamics under a BFA-transport Block

Traffic 2004; 5: 284–299 291

www.traffic.dk/video_gallery.asp). Altogether, this strongly

suggested that the Golgi-like structure that reappeared in

BFA-treated metaphase cells did not reemerge from the ER.

We also asked whether this BFA-resistant structure could

arise from downstream compartments by retrograde

transport. We observed that Shiga toxin B subunit (STxB)

retrograde transport (36,37) from the plasma membrane to

the reforming Golgi and the ER functions during telophase/

cytokinesis (Figure 8a–c). We used StxB as a marker to

test whether BFA-resistant Golgi-like membranes were

accessible to retrograde cargo. In the presence of BFA,

Figure 6: The Golgi-like BFA-resistant structure is distinct from ER exit sites and is not connected to the ER. A) Mitotic Hela cells

were collected from nonsynchronized cell populations, immediately incubated for 2h with BFA, fixed and stained for endogenous Giantin and

Sec31. The Golgi-like structure does not correspond to COPII-positive ER exit sites. Bar: 10mm. B) Mitotic GalNacT2-GFP HeLa cells were

collected from nonsynchronized cell populations, BFA was then immediately added or not, and cells were allowed to reach telophase. As

illustrated in Figure 1, if BFA was added during anaphase/telophase, GalNacT2-GFP was relocated in the ER (middle panel). In these condi-

tions, a 15-min FLIP of the ER pool of GalNacT2-GFP within the designated region of interest (ROI) led to a 95% reduction in total cell

fluorescence (the second daughter cell is not photobleached and provides an internal control). In contrast, the BFA-resistant Golgi-like

structure observed in cells treated during metaphase (bottom panel) and the Golgi in control, nontreated cells (upper panel) were not

bleached upon photobleaching of the ER by the same 15-min FLIP procedure. Therefore, the Golgi-like structure is not directly connected to

the ER, like the normal Golgi in control, nontreated cells. Bar: 10mm.

Nizak et al.

292 Traffic 2004; 5: 284–299

Figure 7: TheBFA-resistantGolgi-like structure forms in the absenceof anterograde transport towards theGolgi.A) The anterograde

transport of tsO45VSVGwasmonitored in asynchronous populations of HeLa cells transfectedwith tsO45VSVG-GFP. The cells shown herewere

fixed for 180min after the temperature shift (t¼ 0min) and stained for endogenousGalT (a, d, red c, f) andDNA (DAPI, blue c, f). tsO45VSVG-GFP

was entirely distributed in the ER at t¼ 0min in all cells after the 39.5 �C temperature block, and therefore efficiently accumulated in the ER

before the temperature shift (not shown). BFAwas then added or not and cells were simultaneously shifted at 37 �C (t¼0min), which allowed

for both normal cell division and tsO45VSVG-GFP transport. At t¼120min, tsO45VSVG-GFP was present in the Golgi area and already visible at

the plasma membrane in interphase cells as well as cytokinesis cells in the absence of BFA (not shown), and at t¼180min it was mostly

distributed at the plasma membrane (a–c), in particular at the level of filipodia (highlighted by arrows). The anterograde transport of VSVG was

thus normal in nontreated cells duringmitosis exit. In the presence of BFA, tsO45VSVG-GFP remained accumulated in the ER from t¼0min to

t¼ 180min in all cells, including those that had formed the Golgi-like BFA-resistant structure upon mitosis exit (d–f). Therefore the Golgi-like

structure forms in the absence of anterograde transport. Bar: 10mm. B) The Golgi-like structure or the Golgi area was photobleached in one of

the two daughter cells in cells treated with BFA in prometaphase/metaphase (lower panel) or nontreated cells (upper panel), respectively. The

2-min photobleaching procedure ensured that recovery would not be due to lateral diffusion within connected membranes. The fluorescence

recovery was then monitored for 30min. No recovery was observed in BFA-treated cells. Normal recovery, typical of ER–Golgi anterograde

transport, was observed in control cells. Bar: 10mm.

Mitotic Golgi Dynamics under a BFA-transport Block

Traffic 2004; 5: 284–299 293

STxB accumulated in endosomes (tubularized due to the

presence of the drug) and reached neither the Golgi-like

structure nor the ER (Figure 8d–f).

This shows that Golgi-intersecting pathways are blocked

by BFA in these cells as in interphase cells (36), and that

BFA is fully active in these pathways even if added in

prometaphase/metaphase. Altogether, our data indicate

that the BFA-resistant Golgi-like structure is isolated both

from the ER and from the plasma membrane/endosomes

system, and that the BFA-resistant Golgi-like structure

forms and persists independently of these compartments.

This reinforced our hypothesis that these Golgi fragments

were already present in metaphase, in a BFA-resistant

state, and that addition of BFA caused their accumulation

at microtubule minus ends. This would imply that all the

Golgi markers present in the Golgi-like structure in the

presence of BFA were not transported from another com-

partment but rather preexisted in prometaphase/meta-

phase Golgi fragments, as suggested by the FRAP

experiment (Figure 7B). Should this be the case, if cargo

were accumulated in the Golgi before mitosis, they would

still be present in the BFA-resistant Golgi-like structure.

Cells were thus allowed to accumulate STxB into the

Golgi before mitosis, and then treated with BFA in pro-

metaphase/metaphase. In these conditions, STxB was this

time efficiently recovered in the resistant Golgi-like struc-

ture (Figure 9). This indicates that preaccumulated cargo

are still present in the BFA-resistant Golgi-like structure but

new cargo cannot access this structure. Therefore, it

is likely that all the markers present within the Golgi

apparatus before mitosis are also present in prometaphase

and metaphase, at the time of BFA addition, in mitotic

Golgi fragments. Upon BFA treatment, these fragments

become isolated from other compartments and accumu-

late in telophase into a BFA-resistant Golgi-like structure in

the absence of anterograde or retrograde transport

towards this structure.

Discussion

In vivo imaging reveals a short time window at prometa-

phase/metaphase for the BFA effect on mitotic Golgi.

Figure 8: The BFA-resistant Golgi-like structure forms in the absence of retrograde transport towards the Golgi. Mitotic HeLa

cells were collected and immediately incubated for 2 h (insets, 1 h) with Cy3-STxB (red) in the absence (a–c) or presence (d–f) of BFA. Cells

were fixed and stained for endogenous GalT (green) and DNA (blue). STxB retrograde transport functions at the end of mitosis in untreated

cells (a–c). BFA treatment blocks STxB in tubularized endosomes (d–f), showing that new retrograde cargo cannot access the BFA-

resistant Golgi-like structures. Bar: 10mm.

Nizak et al.

294 Traffic 2004; 5: 284–299

Previous studies have reported a role for COPI-dependent

and independent pathways in mitotic Golgi breakdown

(8,17,38,39). More generally, the role of membrane traffic

(8), its arrest and subsequent resumption (14,40), and the

tuning of the anterograde/retrograde balance (9), have

been proposed to account for mitotic Golgi disassembly

and subsequent reassembly. Our use of BFA on nonsyn-

chronized cells together with in vivo experiments revealed

two key phenomena:

1 BFA addition prevents the fragmented prometaphase

Golgi from disappearing. These resistant fragments sub-

sequently persist throughout mitosis in the continuous

presence of BFA, are inherited by daughter cells, and

accumulate into a Golgi-like structure that contributes to

Golgi reassembly upon BFA washout. This sharp transi-

tion from an interphase BFA-sensitive Golgi to a mitotic

BFA-resistant one is probably due to sudden changes in

the regulation of Golgi dynamics.

2 A BFA-resistant Golgi-like structure, positive for Golgi

enzymes and for most of the Golgi markers, also

forms upon mitosis exit when BFA is added in meta-

phase, a stage where no, or very few, Golgi structures

are visible. We show that these structures did not

reemerge from the ER (or from the plasma membrane/

endosomes).

Although BFA has already been used to study Golgi inherit-

ance, the phenomena we describe have not been

observed before, probably because BFA was added before

mitosis or because a dynamic analysis of nonsynchronized

cells had not been undertaken. For example, Seemann

et al. (15) treated premitotic cells with BFA and observed

that when cells subsequently divide, Golgi enzymes

remain in the ER and GM130 in dispersed punctate struc-

tures throughout mitotis (15), which corresponds to our

observations in the case of cells treated before they enter

mitosis (Figure 2C). These dispersed structures may cor-

respond to ER exit sites (5) or, as proposed by Seemann

et al. (15), to ER-independent partitioning units, the com-

position of which would, however, require further charac-

terization. It is worth noting that the duration of mitosis is

dependent on cell type. We observed that the BFA-

resistance prometaphase/metaphase time window is much

shorter in cells that divide faster, like the normal rat kidney

cells used by Seemann et al. (15). Consequently, it is very

unlikely that one would be able to find the BFA-resistant

structure in populations of fast dividing cells without fol-

lowing the Golgi in live cells or adding BFA immediately

after a shake-off of unsynchronized cells, which could

explain why the BFA-resistant Golgi-like structure was

not observed. Interestingly, the Golgi inheritance contro-

versy may result in part from cell type-dependent mitotic

kinetics.

Another study provided biochemical data showing that

mitotic Golgi and ER membranes of nocodazole-arrested

mitotic-like HeLa cells can be separated in the presence of

BFA only if BFA is added during nocodazole arrest and not

prior to it (30). However, the use of nocodazole in that

study complicates the analysis of the results because

BFA effects depend on the presence of functional micro-

tubules (41). Moreover, mitosis is blocked in an artificial

metaphase-like state, at the spindle assembly checkpoint,

and does not complete under these conditions. Herewe show,

in agreement with Lee et al. (31) but using nocodazole-free

conditions, that BFA is indeed unable to induce ER/Golgi fusion

in prometaphase/metaphase. However, we also show that

BFA inhibits the normal disappearance of the Golgi observed

in mitosis and probably its fusion with the ER, hence exagger-

ating the ER–Golgi independence.

A speculative mechanism for the mitotic BFA effect

Our in vivo real time observations show that Golgi rem-

nants remain BFA-resistant during anaphase/telophase

Figure 9: The BFA-resistant Golgi-like structure contains preaccumulated cargo. HeLa cells were first incubated with Cy3-STxB for

1 h, then washed, and mitotic cells collected by shake-off. STxB-loaded mitotic cells were then incubated for 2 h with BFA. Cells were

fixed and stained for endogenous GalT (green) and DNA (blue). Pre-accumulated STxB is present in the Golgi-like structure. Bar: 10mm.

Mitotic Golgi Dynamics under a BFA-transport Block

Traffic 2004; 5: 284–299 295

and can still be observed after the completion of cytokin-

esis if the drug is added in prometaphase/metaphase in

unsynchronized cells. Our findings are reminiscent of the

effect of compounds affecting the activity of G proteins,

namely GTPgS and AlF4, in a mitotic Golgi disassembly/

reassembly in vitro system (18). The latter inhibit Golgi

reassembly if already present during disassembly but not

if present during reassembly only. In other words, Golgi

membranes ‘remember’ at which point the treatment

starts. This ‘memory effect’ of drug treatment implies

that mitosis modifies Golgi membranes and drives

them into a unique state that renders them insensitive to

subsequent treatment with these GTPase-perturbing

compounds. Because BFA has more than one target (42),

it is difficult to hypothesize a precise mechanism for the

BFA-insensitivity of prometaphase Golgi fragments and

the formation of this BFA-resistant structure in telophase.

Unpublished data from our lab (A. Roux, Bruno Goud)

suggest that numerous proteins are recruited onto the

Golgi upon BFA treatment. SDS-PAGE analysis of rat liver

Golgi membranes incubated with rat liver cytosol in the

presence of BFA revealed that about 20 unknown proteins

are recruited onto Golgi membranes in these conditions,

implying that many additional candidate proteins have to

be considered. For example, a complete understanding of

this process should take into account the BFA-induced

ADP-ribosylation of CtBP/BARS-50 and GAPDH (43) and

their effect on Golgi membrane dynamics.

Although Golgi membranes ‘remember’ that they were

treated by BFA during prometaphase/metaphase, the

resistant Golgi-like structures regain part of their inter-

phasic identity upon mitosis exit. The minus end-directed

movement of BFA-resistant Golgi-like membranes sug-

gests that the dynein machinery is already active at this

stage (44,45), and that its binding to membranes and

microtubule-based movement are normally restored in

the presence of BFA. This is surprising since the spec-

trin/ankyrin meshwork necessary for dynein/dynactin

membrane attachment is highly sensitive to BFA (46).

This may indicate that part of the Golgi reassembly

process still occurs in the presence of BFA at mitosis

exit. Completion of normal Golgi maturation is, however,

inhibited by BFA. For instance, our electron microscopy

observations show that the Golgi-like structure is an accu-

mulation of juxtaposed Golgi fragments and no stacked

structure could be observed under these conditions. The

incomplete maturation into a normally structured Golgi

indicates that BFA-sensitive factors, possibly COPI, as

well as BARS50 and GAPDH, play a role in the process

of Golgi membrane fusion into cisternae and their stacking,

which could be directly tested in in vitro systems.

The ‘memory effect’ of mitotic BFA treatment we report

implies that the initial state of Golgi membranes during

BFA treatment is crucial. When treatment starts during

prometaphase or metaphase, either factors necessary for

BFA action are depleted from the Golgi initially (or inactiv-

ated, see (47)) and cannot associate with membranes in

the presence of BFA, or factors protecting the Golgi from

BFA action cannot dissociate from those membranes in

the presence of BFA, or both. Many speculative molecular

schemes can be drawn up to explain the memory effect.

We believe that there is a molecule X, a target of BFA, that

is modified (say, ribosylated) by BFA addition and is also

modified in mitosis (say, successively phosphorylated in

prophase and dephosphorylated in anaphase). Clearly, if

the BFA-induced modification occurs in prophase, it can

interfere with the successive action of prophase kinases

and anaphase phosphatases on X if, for example, the

BFA-induced modification prevents recognition of X by

anaphase phosphatases as a substrate (hence the mem-

ory effect). Again, the large spectrum of candidate kinases

and phosphatases already involved in mitotic Golgi

dynamics precludes a detailed mechanistic explanation of

the memory effect at the present time.

A recently published article could give at least part of the

answer to this mechanistic question (48). ADP-ribosylation

factor 1 (Arf1), a major target of BFA in interphase, is

naturally inactivated and released from membranes early

in mitosis to allow for Golgi dispersion. BFA might interfere

with the Arf1 activity regulation machinery precisely

in early mitosis in a way that prevents complete Golgi

dispersion (although we observed that COPI, effector of

Arf1, remains cytosolic, Figure 4), with a memory effect

linked to the successive inactivation and reactivation of

Arf1.

Persistence of an independent Golgi throughout

mitosis which is transiently BFA-resistant in

prometaphase and metaphase

The resistance of prometaphase Golgi membrane to BFA

treatment was unexpected since BFA induces a rapid

retrograde transport of Golgi membranes to the ER within

minutes of treatment in interphase cells, and since,

according to the ER-based inheritance model, Golgi disap-

pearance may occur as a consequence of an accelerated

retrograde transport of Golgi markers to the ER. In add-

ition, the structures we observed by electron microscopy

are reminiscent of tubulovesicular mitotic Golgi clusters

described before (13,20,31–33). This suggests that BFA

addition during prometaphase or metaphase somehow

freezes Golgi remnants in their mitotic state even after

mitosis completion. That Golgi remnants are present

even during metaphase, when Golgi markers appear com-

pletely diffuse at the light microscope level, is further

suggested by the observed reappearance of Golgi-like

structures after BFA treatment of metaphase cells. We

show that no transport occurs between these Golgi-like

structures and the ER, thus implying that they do not

reemerge from the ER but rather preexisted and converge

towards microtubule minus ends upon mitosis exit. This is

further suggested by the fact that although these struc-

tures are not accessible to new cargo, preaccumulated

Nizak et al.

296 Traffic 2004; 5: 284–299

cargo efficiently label them. We would thus propose that,

although they cannot be individually observed by light

microscopy (even though they are visible in the spindle

area, from which the ER is excluded, see (12)) mitotic

Golgi remnants, positive for all Golgi markers tested here,

and for preaccumulated cargo, exist throughout mitosis.

We thus propose the following model, synthesizing the

two current extreme but nonexclusive views of mitotic

Golgi inheritance, to explain our results (11–13,15,49).

The Golgi starts disassembling in prophase and generates

fragments containing all classes of Golgi markers and

preaccumulated cargo. TheseprometaphaseGolgi fragments

are in a unique state in the sense that BFA treatment

would not induce their fusion with the ER but would

block their normal mitotic dispersion. A continuous BFA-

induced block of Golgi-intersecting traffic would isolate

them from other compartments throughout mitosis in

this ‘frozen’ state but not prevent them from aggregating

towards microtubule minus ends in telophase. These Golgi

fragments containing Golgi enzymes and many other mark-

ers cannot form when Golgi membranes are redistributed

to the ER by BFA treatment prior to mitosis (15), explaining

the absence of the Golgi-like structure under these condi-

tions (Figures 1A, 2).

From prometaphase to metaphase, Golgi fragments are

further dispersed in the form of mitotic Golgi clusters and

free vesicles (12,31,32). It is also likely that at the same

time some of these Golgi remnants disappear by progres-

sive retrograde transport and fusion with the ER (11).

Blocking ER–Golgi transport with BFA in prometaphase

results in the persistence of very large and numerous

fragments, and we observed that more ER staining, sensi-

tive to FLIP, is observed when BFA is added in metaphase

as compared to prometaphase. It is possible that these

very numerous and large mitotic Golgi fragments result

from the inhibition of free vesicle shedding (12) by BFA

treatment, a process for which we cannot provide a

molecular explanation for the time being. In any case, our

results indicate that some fragments still remain in meta-

phase in HeLa cells, and that BFA addition at this stage

freezes them in this state. In the absence of drug

treatment, these metaphase fragments then coalesce

and contribute to Golgi reassembly in anaphase/telophase

(see Supplemental Movie 6 at http://www.traffic.dk/

video_gallery.asp), probably parallel with massive export

of Golgi markers from the ER (11).

In conclusion, our study unravels a new temporal view of

the evolution of Golgi membranes in mitosis. A sharp

transition in prometaphase drives Golgi membranes into

a unique state in which they become BFA-insensitive, and

the presence of BFA locks them in this state, in extreme

isolation from the ER. This sudden transition could be the

outcome of a Golgi-associated control of mitosis entry

(50,51), even though we did not observe a cell cycle

block in cells that do not fully disperse their Golgi because

of BFA (maybe the checkpoint is already passed at the

time of drug addition). Then, as cells progress towards

metaphase, the Golgi is progressively dispersed further,

but Golgi remnants remain that bear the same BFA-

resistanceasprometaphaseGolgi fragments. Theseprometa-

phase and metaphase Golgi fragments can be artificially

isolated from other compartments by blocking Golgi-

intersecting anterograde and retrograde traffic. Therefore, a

subset of Golgi membranes naturally persists throughout

mitosis that contains all classes of Golgi markers and

aggregates upon mitosis exit to contribute to Golgi reas-

sembly.

Materials and Methods

Cell culture and mitotic shake-off

Wild-type or stably expressing GalNacT2-GFP HeLa cells

(3) were cultured as described (36). Mitotic cells were

obtained by shake-off in the absence of any synchroniza-

tion protocol and plated on coverslips. Cells were then

either fixed in MeOH (�20 �C, 4min) or 3% paraformalde-

hyde in phosphate-buffered saline at different time-points

after shake-off or directly observed by conventional video

microscopy or using a Leica SP2 confocal system (Wetzlar,

Germany). This method yielded 85� 5% prometaphase/

metaphase cells and 15� 5% anaphase/telophase cells

immediately after the shake-off.

tsO45VSVG-GFP transport

HeLa cells were transfected with a tsO45VSVG-GFP plas-

mid, incubated at 39.5 �C for 10 h, and then at 37 �C in the

presence or absence of BFA. The usual change to 32 �Ccould not be done because mitosis completion was

impaired at this low temperature, and we observed thattsO45VSVG–GFP transport occurred normally at 37 �C in the

absence of BFA.

Antibodies and reagents

Monoclonal antibodies were obtained from G. Warren

(p115), H.-P. Hauri (ERGIC53), Transduction laboratories

(GM130) (BD, Franklin Lakes, NJ), Calbiochem (Giantin)

(San Diego, CA) and Sigma (bCOP, mAD antibody)

(St. Louis, MO). Polyclonals were obtained from E.G. Berger

(GalT), B.L. Tang (Sec31), H.-P. Hauri (KDELr), M. Rojo

(p23) and K. Moremen (ManII). Secondary antibodies

were from Molecular probes (Alexa 488) (Eugene, OR)

and Jackson Immunoresearch (Cy3) (Westgrove, PA).

DAPI was from Sigma, BFA (used at 5mg/mL for treat-

ment) was from Epicentre (Madison, WI). Purified STxB

was obtained from L. Johannes, labeled with Cy5 (labeling

kit fromAmersham, Freiburg, Germany) and used at 5mg/mL.

Electron microscopy

MannII-HRP HeLa cells were fixed in 4% paraformalde-

hyde and 0.5% glutaraldehyde for 4 h at room temperature

and incubated 40min with DAB (0.25mg/mL, Sigma) and

Mitotic Golgi Dynamics under a BFA-transport Block

Traffic 2004; 5: 284–299 297

0.003% H2O2 (Sigma) in phosphate buffered solution at

pH7.4. Cells were then incubated in 2.5% glutaraldehyde

buffered in sodium cacodylate (0.1M) at pH7.4 and placed

at 4 �C for at least 24 h. Cells were washed by several

changes of 0.1 M sodium cacodylate buffer and postfixed

1 h at room temperature in reduced osmium (1 : 1 mixture

of 2% aqueous osmium tetroxide and 3% aqueous potas-

sium ferrocyanide, according to the Karnovsky procedure).

After postfixation, the cells were scrapped with a rubber

policeman, embedded in Agar (2%), dehydrated in ethanol

and processed for Epon embedding. Thin (80 nm) sections

were cut on a Reichter-E ultramicrotome (Vienna, Austria)

and collected on copper or gold grid and stained with lead

citrate (2min). Alternatively, when DAB/H2O2 staining was

not used, sections were stained with a technique derived

from a thiocarbohydrazide-silver proteinate method:

sections placed on gold grids were floated for 15min at

room temperature on a 0.5% aqueous solution of thiocarbo-

hydrazide, rinsed with water, stained 20min on a 1%

aqueous silver proteinate and carefully rinsed in distilled

water. Sections were then examined with a CM 12

Philips electron microscope at 80kV (Eindhoven, the

Netherlands).

Imaging of fixed and live cells

Time-lapse and immunofluorescence data were acquired

on a Leica DMRA microscope using Princeton Charge-

Coupled Device cameras (Princeton Instruments, Mon-

mouth Junction, NJ). For time-lapse data, six optical sections

were acquired every 5min through fluorescent FITC filter

and Phase contrast set-ups with a 63� objective and a

0.5� lens (to enlarge the field). For immunofluorescence

data, a 100� objective was used instead. Photobleaching

analysis was performed on a Leica SP2 confocal micro-

scope with a 40� objective (the field depth of which is

much larger than the cell thickness) and a 488nm laser.

For FLIP experiments, stacks of four optical sections were

acquired every 1min at low laser intensity, and eight high

intensity scans restricted to the outlined regions were

done between these acquisitions. For FRAP experiments,

stacks of four optical sections were acquired every 1min

(prebleach¼ 2 stacks, postbleach¼ 30 stacks), before and

after six high intensity scans with photobleaching

restricted to the outlined regions (with 20s between

scans, to allow for relaxation due to lateral diffusion within

membranes). In all types of live cell experiments, cells that

were observed progressed through mitosis normally even

in the case of FRAP and FLIP (see supplemental movies).

Images were processed with Metamorph (Universal

Imaging, Downingtown, PA), Adobe Image Ready and

Adobe Photoshop (San Jose, CA).

Online Supplemental Movies (http://www.traffic.dk/

video_gallery.asp)

TIFF image stacks of time-lapse acquisitions were

assembled on an iMac-G4 into Quicktime movies (Apple,

Cupertino, CA) for online publication with Adobe Image

Ready and Image J (available at http://rsb.info.nih.gov/ij/).

The movies correspond to Figure 1A, B, Figures 7B and 8B

and three supplemental in vivo experiments.

Acknowledgments

We thank E.G. Berger, B. L. Tang, G.Warren, H.-P. Hauri, M. Rojo and

K.Moremen for the kind gift of antibodies, J.White for the GalNacT2-GFP

HeLa cell line and tsO45VSVG-GFP plasmid, L. Johannes for purified STxB,

N. Altan-Bonnet and J. Lippincott-Schwartz for communicating results prior

to publication and many stimulating discussions, L. Ovtracht for expert

technical assistance, and we are indebted to J.-B. Sibarita, V. Fraisier and

J.Salamero for managing the imaging facility at the Institut Curie, on which

this work has essentially relied. We also thank D.Cassel and M.van Overbeek

for helpful comments on the manuscript. This work was supported

by the Institut Curie, the Centre National de la Recherche Scientifique, and

a grant from the Association pour la Recherche sur le Cancer (ARC 5747).

C.N. was the recipient of a 4th year of PhD grant from the Association

pour la Recherche sur le Cancer, and R.S. was supported by LVMH (Paris,

France).

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