The Golgin Tether Giantin Regulates the Secretory Pathway by Controlling Stack Organization within Golgi Apparatus.
ABSTRACT Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications.
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ABSTRACT: Blood samples were harvested from the antecubital vein of 20 fasting patients with acute cerebral infarction at 1, 7 and 15 days after onset to prepare blood platelet suspension. Fasting antecubital vein blood was collected from an additional 20 normal adults as controls. Under transmission tron microscope, platelet Golgi tubules and vesicles became significantly thickened, enlarged, and irregular after acute cerebral infarction. Alpha granules in platelets significantly reduced in number, especially 1 day after cerebral infarction. Under immunoelectron microscopy, a few alpha granules aggregated around Golgi tubules and vesicles after infarction. These results suggested that platelet Golgi apparatus displayed significant morphological changes, which were possibly associated with enhanced synthetic and secretory functions of activated platelets after acute cerebral infarction. This study used Golgi apparatus blocking agent Brefeldin A to block Golgi apparatus in an aim to study the effects of Golgi apparatus on CD40L expression on the surface of activated platelets. Flow cytometry revealed that CD40L expression on activated platelet surfaces decreased significantly when Golgi apparatus was blocked, which indicated that Golgi apparatus participated in the synthesis and transport of CD40L to the platelet surface.Neural Regeneration Research 08/2013; 8(23):2134-43. · 0.23 Impact Factor
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ABSTRACT: This review summarizes the data describing the role of cellular microtubules in transportation of membrane vesicles - transport containers for secreted proteins or lipids. Most events of early vesicular transport in animal cells (from the endoplasmic reticulum to the Golgi apparatus and in the opposite recycling direction) are mediated by microtubules and microtubule motor proteins. Data on the role of dynein and kinesin in early vesicle transport remain controversial, probably because of the differentiated role of these proteins in the movements of vesicles or membrane tubules with various cargos and at different stages of secretion and retrograde transport. Microtubules and dynein motor protein are essential for maintaining a compact structure of the Golgi apparatus; moreover, there is a set of proteins that are essential for Golgi compactness. Dispersion of ribbon-like Golgi often occurs under physiological conditions in interphase cells. Golgi is localized in the leading part of crawling cultured fibroblasts, which also depends on microtubules and dynein. The Golgi apparatus creates its own system of microtubules by attracting γ-tubulin and some microtubule-associated proteins to membranes. Molecular mechanisms of binding microtubule-associated and motor proteins to membranes are very diverse, suggesting the possibility of regulation of Golgi interaction with microtubules during cell differentiation. To illustrate some statements, we present our own data showing that the cluster of vesicles induced by expression of constitutively active GTPase Sar1a[H79G] in cells is dispersed throughout the cell after microtubule disruption. Movement of vesicles in cells containing the intermediate compartment protein ERGIC53/LMANI was inhibited by inhibiting dynein. Inhibiting protein kinase LOSK/SLK prevented orientation of Golgi to the leading part of crawling cells, but the activity of dynein was not inhibited according to data on the movement of ERGIC53/LMANI-marked vesicles.Biochemistry (Moscow) 09/2014; 79(9):879-93. · 1.35 Impact Factor
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ABSTRACT: Figure optionsDownload full-size imageDownload high-quality image (295 K)Download as PowerPoint slide01/2014;
The Golgin Tether Giantin Regulates the Secretory
Pathway by Controlling Stack Organization within Golgi
Mayuko Koreishi1, Thomas J. Gniadek2, Sidney Yu3, Junko Masuda4, Yasuko Honjo5, Ayano Satoh1*
1The Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan, 2Department of Pathology, Johns Hopkins University School of
Medicine, Baltimore, Maryland, United States of America, 3School of Biomedical Sciences and Epithelial Cell Biology Research Center, The Chinese University of Hong
Kong, Shatin, N.T., Hong Kong SAR, People’s Republic of China, 4Mucosal Immunity Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 5The Research Core for Interdisciplinary Sciences (RCIS), Okayama University,
Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest
golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment
protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus.
Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks.
Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment
than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an
increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known
to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells
resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial
organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications.
Citation: Koreishi M, Gniadek TJ, Yu S, Masuda J, Honjo Y, et al. (2013) The Golgin Tether Giantin Regulates the Secretory Pathway by Controlling Stack
Organization within Golgi Apparatus. PLoS ONE 8(3): e59821. doi:10.1371/journal.pone.0059821
Editor: Catherine L. Jackson, Institut Jacque Monod, Centre National de la Recherche Scientifique, France
Received July 5, 2010; Accepted February 21, 2013; Published March 21, 2013
Copyright: ? 2013 Koreishi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding sources The Kurata Memorial Hitachi Science and Technology Foundation, the special Coordination Fund for Promoting Science and
Technology, and JSPS KAKENHI Grant Number23570167 of MEXT (Ministry of Education, Sport, Culture, Science and Technology in Japan), Hayashi Memorial
Foundation for Female Natural Scientists, Ryobi Teien Memorial Foundation, the Naito Foundation, the Uehara Memorial Foundation, and the National Institutes
of Health (AG030101 and GM060919, USA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The majority of intracellular membrane traffic involves the
formation, transport, and selective fusion of membrane-bound
vesicles. To bud from a donor membrane, vesicles generally
require coat proteins that induce membrane curvature. Well-
studied coat protein complexes include coatomer protein I (COPI)
within the Golgi apparatus, coatomer protein II (COPII) at
endoplasmic reticulum exit-sites, and clathrin at the trans-Golgi
and cell surface membrane [1,2,3].
For accurate vesicular transport, vesicles must dock and fuse
with their proper target membrane, which involves coordinated
and specific protein-protein interactions. For example, the
targeting of COPI vesicles is considered to be a multi-layered
process that requires Rab\Arl GTPases, tethers, and soluble N-
ethylmaleimide-sensitive factor attachment protein receptors
(SNAREs) . Tethers include coiled-coil proteins of the golgin
family and multi-protein complexes, such as TRAPP and COG
[5,6,7]. Once a vesicle is tethered to its target membrane, vesicle
docking and membrane fusion are mediated by SNAREs and
accessory proteins . SNARE proteins contain evolutionarily
conserved 60–70 amino acid SNARE motifs arranged in heptad
repeats that confer specificity to vesicle membrane (v-SNAREs)
and target membrane (t-SNARE) interactions. The asymmetric
distribution of v-SNAREs and t-SNAREs among intracellular
membrane compartments allows for specific vesicle and target
membrane fusion events. However, the earliest stage of SNARE
pairing between membranes cannot occur at distances of more
than 25 nm, suggesting that tethers play an important role in the
initial contact between vesicles and their target membrane. Along
these lines, we have previously shown that golgin tethers play a
role in specifying vesicle fusion sites within the Golgi apparatus .
Golgins are a family of coiled-coil proteins that are anchored
either directly, via a membrane spanning domain, or indirectly,
through interactions with other golgins or Rab/Arl GTPases .
Specific golgins are found at different locations within the Golgi
apparatus, where they organize Golgi and cisternal stacking and
tether COPI vesicles to the Golgi membranes .
The ‘‘cis-golgin tether’’ is one of the most well-characterized
golgin tether complexes. It is composed of the COPI vesicle-
associated golgin giantin linked to Golgi membrane-associated
GM130 via p115. GM130 is in turn linked to GRASP65 via a
PDZ-like domain. GRASP65 is anchored to the Golgi membrane
through N-terminal myristoylation as well as through binding to
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other Golgi proteins . Together, these proteins appear to
mediate vesicle tethering at the cis-Golgi membrane.
We have also identified a new golgin tether consisting of the
COPI vesicle-associated protein golgin-84 and the Golgi mem-
brane-associated protein CASP. It appears that COPI vesicles
tethered by the golgin-84/CASP are involved in Golgi enzyme
transport, whereas COPI vesicles tethered by the cis-golgin tether
giantin/GM130 are involved in cargo transport . Interestingly,
COPI vesicles utilizing golgin-84/CASP tethers lack anterograde
cargo and p24 family proteins, which are putative cargo receptors
within COPI vesicles. In contrast, COPI vesicles utilizing the
giantin/GM130 tethering complex are enriched for anterograde
cargo and the p24 family of cargo receptor proteins . Taken
together, these results suggest that these two different golgin tether
complexes may define functionally distinct sub-populations of
COPI vesicles. Conceptually, we propose that giantin and golgin-
84 should be considered vesicle-associated tethers (v-ATs), similar
Giantin is the largest identified golgin protein . Previous
studies have demonstrated that giantin consists of a transmem-
brane domain with either a short or no luminal domain at its C
terminus, depending on the species . Giantin binds directly to
the C terminus of p115 [12,13] to form part of the cis-golgin
tether. Although the contribution of Rab1 to cis-golgin tether
formation is currently unclear, giantin has been shown to bind
directly to Rab1 [14,15]. We have proposed a model wherein
giantin and GM130 facilitate p115-mediated recruitment of rab1
to Golgi membranes, since giantin and GM130 stimulate p115
binding to Rab1 . In addition to members of the cis-golgin
tether, giantin has several other binding partners, including Rab6
and GCP60 [15,16]. Given its large size, giantin could potentially
interact simultaneously with its various binding partners.
Giantin Localized to COPI Vesicles and Golgi Buds and
To gain additional insight into the function of giantin, we
analyzed the localization of giantin by immunoelectron microsco-
py (immune-EM) using anti-giantin pAb. As shown in Figure 1A
and quantitated in Figure 1B, approximately 45% of giantin was
localized to vesicles, Golgi buds, and Golgi cisternal rims. This is
in agreement with previously reported biochemical and electron
microscopy data, which show that giantin is found in COPI
vesicles . In addition, giantin was found in all compartments in
the Golgi cisternae, unlike GM130, whose localization is highly
restricted to cis-Golgi cisternae [17,18,19,20,21]. This finding
suggests that giantin may have another function in addition to its
role in the cis-golgin tether, wherein giantin resides on COPI
vesicles and is linked via a soluble protein, such as p115, to
GM130 on Golgi cisternae for COPI vesicle tethering .
Nocodazole Treatment of Giantin RNAi Cells Resulted
Smaller Golgi Stacks
Given giantin’s localization to various areas of the Golgi
complex, we examined whether depletion of giantin would
influence the morphology of the Golgi apparatus and the rate of
intracellular membrane protein traffic. In order to do this, we
performed siRNA-mediated giantin RNAi. To assess the efficiency
of giantin RNAi, giantin protein levels in serial dilutions of mock-
and siRNA-treated cells were compared by western blotting. As
shown in Figure 2A, giantin protein levels were reduced by more
than 87% in giantin siRNA-treated cells. Of note, giantin protein
levels were reduced by 85–95% in all of the experiments
performed herein. Although giantin was almost completely
depleted in giantin siRNA-treated cells, we did not observe
obvious morphological changes in the Golgi apparatus by
immunofluorescence (data not shown). However, when these
giantin-depleted cells were treated with nocodazole, a microtu-
bule-depolymerizing agent known to transform Golgi ribbons into
Golgi stacks, Golgi stacks in giantin siRNA-treated cells appeared
more dispersed when compared to cells treated with nocodazole
alone (Figure 2B). As shown in the quantitation in Figure 2C and
2D, after nocodazole treatment, giantin siRNA decreased the
average Golgi stack size and increased the average number of
Golgi, each by approximately 60% compared to mock siRNA
control cells. This alteration in the size and number of Golgi stacks
within each cell suggests that Giantin may play a role in organizing
and/or stabilizing Golgi stacks. This phenotype was observed
when a different giantin siRNA was used and was reversed by an
exogenous expression of rat giantin cDNA (siRNA2, Figure S1B).
Since the depletion of giantin influenced the organization of
Golgi stacks in nocodazole-treated cells, we examined if the
depletion of giantin changes the length of Golgi cisternae. Mock
and giantin siRNA- nocodazole-treated cells shown in Figure 2B
were processed for EM. As shown in Figure 3A and quantitated in
Figure 3B, no significant difference was observed in the
appearance of Golgi cisternae, quantitated by the average cisternal
length per stack. Therefore, both EM (Figure 3) and immunoflu-
orescence (Figure 2) data support the concept that giantin may be
involved in the organization of Golgi stacks.
Exogenous Expression of Giantin in Drosophila S2 Cells
Organized their Dispersed Golgi Stacks
Since nocodazole and giantin siRNA-treated cells exhibited
more dispersed Golgi stacks, we hypothesized that giantin may be
involved in the organization of Golgi stacks. Many mammalian
cells are known to have organized Golgi stacks, called Golgi
ribbons, whereas invertebrate cells, e.g., Drosophila cells, which do
not express an apparent giantin homologue, have dispersed Golgi
stacks instead of Golgi ribbons. Therefore, we used the S2 cell line,
derived from Drosophila, to examine the function of giantin in the
organization of Golgi stacks. As shown in Figure 4A, transient
expression of rat giantin cDNA in S2 cells partially organized
Golgi stacks. To quantitate the organization and dispersion of
Golgi stacks, dispersion analysis was performed using a custom
Image J plug-in. Cells were manually selected for analysis and the
GM130 signal was smoothed with a mean filter (radius 2 pixels).
Local maxima were found within the resulting data and the list of
local maxima was thresholded using Image J’s build-in IsoData
thresholding algorithm. Finally, the average nearest neighbor
distance was calculated for each cell and tabulated as a measure of
dispersion. As shown in Figure 4B, the average nearest neighbor
distance between Golgi stacks is 70% shorter in S2 cells highly
expressing rat Giantin (n=,20, P,0.01). These data strongly
support the concept that giantin is involved in the organization of
Giantin RNAi Affected Anterograde Transport and
Surface Glycosylation Patterns
To determine whether giantin RNAi influences trafficking, we
performed a Vesicular stomatitis virus G protein (VSV-G)
transport assay. VSV-G-tsO45 is a temperature-sensitive mutant
of the G protein from vesicular stomatitis virus. It has been used
widely for transport assays because it accumulates in the ER at a
restrictive temperature of 40uC and moves to plasma membranes
after a shift to the permissive temperature of 32uC. As shown in
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Figure 5A, plasma membrane-associated VSV-G-tsO45 increased
upon treatment with giantin siRNA. The secretion of another
anterograde cargo, secretory alkaline phosphatase (SEAP) was
increased more than twice by treatment with giantin siRNA
(Figure 5B). This phenotype was also observed when other giantin
siRNAs were used and was reversed by exogenous expression of
rat giantin cDNA (siRNA2, Figure S1C).
To gain insights into giantin RNAi-associated alterations in
anterograde transport, we used lectin staining to examine cell
surface glycosylation patterns. Since gylcosylation occurs mainly in
the Golgi apparatus, changes in the cell surface glycosylation
pattern may reflect changes in glycosylation in the Golgi
apparatus. The sugar-binding specificities of lectins used herein
include Con A (Concanavalin A), terminal alpha-linked mannose;
peanut agglutinin (PNA), galactosyl (beta-1,3) N-acetylgalactosa-
mine; wheat germ agglutinin (WGA), N-acetylglucosamine, and
sialic acids on glycoproteins. It is known that succinylated WGA
(SucWGA) does not bind to sialic acid, unlike the native form, but
retains its specificity toward N-acetylglucosamine. Therefore, the
use of native WGA and succinylated form can distinguish between
sialylated glycoconjugates and those containing only N-acetylglu-
cosamine structures. As shown in Figure 5C, giantin siRNA- and
mock-treated cells exhibited similar surface expression patterns of
Con A-, PNA- and SucWGA-reactive glycans. However, the
WGA-reactive glycans were slightly but significantly increased in
giantin siRNA-treated cells, suggesting that giantin affected the
surface expression of sialyl glycans.
Given the cis-golgin tether model, one might predict that
depletion of giantin would lead to the aberrant trafficking of Golgi
proteins into untethered cytoplasmic COPI vesicles. However, this
was not observed in our study. Instead, we found that the
organization of Golgi stacks in nocodazole-treated cells was
disrupted upon depletion of giantin (Figure 2) without changing
the cisternal lengths of the Golgi stacks (Figure 3). In particular,
the nocodazole fragmented Golgi ministacks were further
dispersed by depletion of giantin. Furthermore, exogenous
expression of giantin cDNA in Drosophila cells induced a relative
clustering of their normally dispersed Golgi stacks, supporting the
notion that giantin plays a role in Golgi organization (Figure 4,
summarized in Figure S2).
One possible interpretation of this data is that giantin depletion
disrupted Golgi stack clustering. The fact that the length of
nocodazole treated Golgi ministacks did not change after giantin
depletion suggests that lateral Golgi ministack fragmentation did
not occur. Therefore, the Golgi dispersion induced by giantin
depletion is likely due to decreased stack clustering, leading to
more and smaller Golgi stacks when counted by a global
thresholding method (Figure 2).
Giantin may mediate Golgi stack clustering via lateral
membrane trafficking between Golgi stacks. Previous work has
shown that lateral trafficking is necessary for Golgi stack biogenesis
[22,23]. If true, this may explain an observed increase in
trafficking of anterograde cargo in Giantin depleted cells, since
inhibiting lateral trafficking may increase the flux through forward
trafficking pathways. To address, this possibility, further experi-
ments involving model organisms that contain simple Golgi
architecture, such as T. brucei, may be warranted. Another possible
mechanism for giantin mediated Golgi stack clustering involves
direct molecular interactions from one Golgi stack to another. To
test such a hypothesis further experiments may seek to precisely
measure the minimum inter-Golgi stack distance. If this distance is
highly consistent, it would argue in favor of a fixed-length direct
Finally, our data showed an increased rate of trafficking of
anterograde cargo associated with alterations of cell surface
glycosylation patterns in giantin-depleted cells (Figure 5). One
could imagine that the increase in WGA-reactive glycans in
giantin-depleted cells indicates an increase in trans-Golgi located
terminal sugar modification (sialylation), which is likely to cause
slower protein trafficking through the sialyltransferase-containing
compartment. However, our results did not support this concept.
It would be interesting to analyze the correlation between
glycosylation patterns and trafficking in future. Additionally, it is
unclear whether the absence of giantin in Drosophila confers an
evolutionary advantage via alterations to its glycosylation path-
ways. Therefore, future research will seek to investigate the
mechanism by which changes in trafficking and Golgi organiza-
tion alter the sugar modification of glycoproteins and glycolipids.
Materials and Methods
Antibodies and Lectins
Monoclonal anti-GM130 (BD Biosciences, San Jose, CA),
polyclonal anti-giantin (, and ab24586; Abcam, Cambridge,
MA), monoclonal anti-giantin [(Dr. Adam Linstedt (Carnegie
Mellon), anti-gamma tubulin (GTU-88; Sigma, St. Louis, MO),
anti-dGM130 (ab30637, Abcam), and anti-VSV-G (VG; Dr. Ira
Mellman (Genentech)] were the antibodies used in this study. All
FITC-labeled lectins and HRP-conjugated secondary antibodies
were purchased from Vector Laboratories (Burlingame, CA) and
Pierce (Rockford, IL), respectively. All Alexa-labeled secondary
antibodies and Hoechst were purchased from Invitrogen (Carls-
Plasmids and siRNAs
VSV-G-tsO45-SP-YFP (VSV-G-YFP) and FLAG-tagged rat
giantin were provided by Dr. Derek Toomre (Yale University, CT)
and Dr. Yoshio Misumi (Fukuoka University, Fukuoka, Japan)
[25,26], respectively. Giantin cDNA from rats was subcloned into
the pMT/V5-His A vector (Invitrogen, Carlsbad, CA) for
expression in S2 cells. Giantin siRNAs  and eGFP (mock
RNAi) were purchased from Dharmacon (Lafayette, CO), Santa
Cruz (Santa Cruz, CA), and Qiagen (Valencia, CA). siRNA1:59-
Cell Culture and Transfection
HeLa cells (CCL-2, ATCC, Manassas, VA) were grown in
Dulbecco’s modified Eagle’s medium supplemented with 10%
FBS (Invitrogen, Carlsbad, CA) at 37uC. Transfection of HeLa
cells with plasmids and siRNAs was performed using Lipofecta-
mine LTX (Invitrogen) and RNAiMAX (Invitrogen), respectively,
according to the manufacturer’s instructions. For depolymeriza-
tion of microtubules, cells were treated with nocodazole (0.2 mg/
ml) (Sigma, St Louis, MO) for 30–50 min .
S2 cells (RCB1153, RIKEN BRC through the National Bio-
Resource Project of the MEXT, Tsukuba, Ibaragi, Japan) were
Figure 1. Giantin localized to buds, vesicles, and cisternal rims. (A) HeLa cells were processed for cryo-electron microscopy and
immunolabeled for giantin. Bar, 200 nm. (B) Quantitation shows that giantin was localized primarily to buds, vesicles, and cisternal rims.
Giantin and Golgi Stacks
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grown in S2 cell medium supplemented with 10% FBS
(Invitrogen, Carlsbad, CA) at 28uC. Transfection of S2 cells with
plasmids was performed by the calcium phosphate method, with
subsequent induction of expression by CuSO4, according to the
manufacturer’s instruction (Invitrogen).
HeLa cells grown on coverslips were fixed with 3.7% PFA in
PBS for 15 min, and permeabilized with 0.1% Triton X-100 in
PBS for 5 min at room temperature. Next, the cells were blocked
with 4% BSA in PBS for 15 min, and incubated for 15 min with
primary antibodies diluted in 4% BSA in PBS. The cells were
Figure 2. Giantin RNAi dispersed Golgi mini-stacks generated by nocodazole treatment. (A) Equal amounts (lanes 1 and 2) and serial
dilutions (lanes 3–5) of total cell lysates from giantin siRNA- and mock-treated cells were loaded and subjected to immunoblotting to determine the
degree of knockdown; giantin (upper panel), gamma tubulin (lower panel). Giantin protein levels were reduced by 85%–95% in all giantin siRNA-
treated cells in the experiments presented herein. Giantin siRNA- and mock-treated cells were incubated with nocodazole (0.2 mg/ml) for 45 min, and
then subjected to indirect immunofluorescence for gianitn (red), GM130 (green), and nuclei (blue). GM130 staining is shown in (B). Insets in (B) are the
merged images of all three colors. Normal sized cells were selected and quantified; the sizes of GM130-positive dots are shown (C). Bars in (C)
represent SD of the average sizes of GM130-positive dots in a cell (n=,20 cells).
Figure 3. Giantin RNAi did not change the cisternal lengths of Golgi mini-stacks generated by nocodazole treatment. (A) Nocodazole,
giantin siRNA-, and mock-treated cells were processed for electron microscopy. Bar, 333 nm. (B) Average length of Golgi cisternae in a Golgi stack in
approximately 10 different cells. Bars represent SEM (n=,10 cells).
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washed three times with PBS, and incubated for 15 min with
secondary antibodies conjugated to Alexa fluorophores (Invitro-
gen). After washing the cells, coverslips were mounted on
microscope slides and viewed using an FV1000 confocal micro-
scope (Olympus, Tokyo, Japan). For immunofluorescence of S2
cells, cells were stripped from the culture dish by gentle pipetting
and placed on 0.5 mg/ml Con A-treated coverslips . After
incubation for 1 h at 28uC, the coverslips were treated as
described above. Images were quantified using the Image J
VSV-G Transport Assay
The VSV-G transport assay was performed as previously
described [9,30]. In brief, HeLa cells were transfected with siRNA.
After 72 h, cells were transfected again with the VSV-G-YFP
plasmid and incubated at the restricted temperature of 40uC
overnight. After shifting to the permissive temperature of 32uC,
cells were incubated for 90 min and processed for immunofluo-
rescence to label cell surface VSV-G.
SEAP Transport Assay
The SEAP transport assay was performed as previously
described . In brief, HeLa cells stably expressing SEAP were
transfected with siRNAs. After 90 h, the cells were washed and
fresh culture media were added. After 6 and 24 h, respectively,
culture supernatants were collected and processed for SEAP
activity measurement using the Phospha-light system (Applied
Biosystems). Data are presented as a secretion index, which is the
ratio of SEAP activity detected in the culture supernatant at 6 h to
that detected at 24 h.
Immunogold labeling with anti-giantin and electron microscopy
were performed as previously described [9,20] at the Yale
University Cell Biology and Okayama University Medical School
EM core facilities.
FACS analysis was performed as previously described . In
brief, cells were detached from culture dishes by short trypsin-
Figure 4. Exogenous expression of giantin in Drosophila S2 cells organized their dispersed stacks. (A) Rat giantin cDNA was transiently
transfected into S2 cells. The cells were processed for immunofluorescence imaging of dGM130 (Golgi marker, in red), rat giantin (in green), and
nuclei (blue). The asterisk marks the rat giantin-expressing cell. Bar, 10 nm. (B) Dispersion analysis was performed by measuring the average nearest
neighbor distance between segmented GM130-positive Golgi stacks with or without rat Giantin expression.
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EDTA treatment, and then blocked with PBS containing 0.2%
FBS for 30 min. After blocking, cells were incubated with
fluorescein-labeled lectins (Vector Laboratories) for 30 min on
ice. After washing, cells were analyzed by FACS Calibur (BD
To quantitate the organization and dispersion of Golgi stacks,
dispersion analysis was performed using a custom ImageJ plug-in.
Cells were manually selected for analysis and the GM130 signal
was smoothed with a mean filter (radius=2). Local maxima were
found within the resulting data and thresholded using ImageJ’s
build-in IsoData thresholding algorithm  to remove back-
ground local maxima. Finally, the average nearest neighbor
distance  was calculated for each cell and tabulated as a
measure of dispersion.
caused the giantin RNAi phenotype that was reversed by
an exogenous expression of rat giantin. (A) Equal amounts
of total cell lysate from siRNA2- and mock-treated cells were
loaded, and then subjected to immunoblotting to determine the
degree of giantin knockdown; giantin (upper panel), gamma
tubulin (lower panel). Giantin siRNA2 reduced giantin protein
levels by approximately 80%. (B) HeLa cells were transfected with
or without giantin siRNA2. After 72 h, a rat giantin expression
plasmid was transfected into one batch of siRNA2-transfected
cells. After a further 24 h of incubation, the cells were incubated
with nocodazole (0.2 mg/ml) for 45 min, and then subjected to
indirect immunofluorescence as described in Figure 2. Cells with
normal sizes were selected and quantified; the average sizes of
GM130-positive dots are shown. Bars represent SD (n=,20
cells). (C) SEAP was increased by siRNA2. HeLa cells stably
expressing SEAP cDNA were transfected with or without giantin
siRNA2. After 72 h, rat giantin cDNA was transfected to one
batch of siRNA2-transfected cells. After a further 24 h, cells were
Other human giantin siRNA (siRNA2) also
washed, aliquots of culture supernatants were collected after
another 6 and 24 h, and then phosphatase activities were
measured as described in Figure 5. The ratio of the activities 6
and 24 h after washing are shown in the graph. Bars represent SD
(n=3). Inset in (C) shows giantin protein levels in the samples
obtained by western blotting. Giantin protein levels in the samples
were normalized using gamma tubulin levels and their ratios are
(upper diagram) is known to be fragmented by Nocodazole, a
microtubule-disrupting agent, and transformed into separated
ministacks. We show that Nocodazole fragmented Golgi minis-
tacks are further dispersed by the depletion of Giantin. Giantin’s
contribution to Golgi ministack aggregation may be through direct
inter-stack bridging or vesicular trafficking.
Working model. The mammalian cell Golgi ribbon
We thank Drs. Graham Warren, Ira Mellman, Susan Ferro-Novick, Fred
Gorelick and the Warren/Mellman/Ferro-Novick group, and members of
the Research Core for Interdisciplinary Sciences in Okayama University
for discussion and support. We also thank Drs. Adam Linstedt, Derek
Toomre, Catherine Rabouille, Yoshio Misumi for generously providing
reagents, and Ms. Morven Graham, Ms. Masumi Furutani, Dr. Joerg
Malsam, Dr. Mitsuko Hayashi-Nishino and the late Marc Pypaert for
electron microscopy, and Ms. Risa Matsumoto and Dr. Tomoka Kawase
for technical assistance provided. This work was performed under the
Cooperative Research Program of ‘‘Network Joint Research Center for
Materials and Devices’’.
Conceived and designed the experiments: SY YH AS. Performed the
experiments: MK JM AS. Analyzed the data: MK TG SY AS. Contributed
reagents/materials/analysis tools: TG SY AS. Wrote the paper: TG SY
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