Golgin-160 and GMAP210 play an important role in U251 cells migration and invasion initiated by GDNF
Golgin-160 and GMAP210 play an important role in U251 cells migration and invasion initiated by GDNF
Chuan-Xi Tang 0 2
Lan Luan 2
Lin Zhang 2
Yue Wang 0 2
Xin-Feng Liu 2
Jie Wang 0 2
Ye Xiong 0 2
Dan Wang 2
Lin-Yan Huang 1 2
Dian-Shuai GaoID 0 2
? These authors contributed equally to this work. 2
0 Department of Neurobiology and Anatomy, Xuzhou Key Laboratory of Neurobiology, Xuzhou Medical University , Xuzhou, Jiangsu , China , 2 School of Nursing, Xuzhou Medical University , Xuzhou, Jiangsu , China , 3 Department of Neurosurgery, The Affiliated Hospital of Xuzhou Medical University , Xuzhou, Jiangsu , China , 4 School of Medicine information, Xuzhou Medical University , Xuzhou, Jiangsu , China
1 School of Medical Technology, Xuzhou Medical University , Xuzhou, Jiangsu , China
2 Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute , UNITED STATES
Gliomas are the most common malignant tumors of the brain and are characteristic of severe migration and invasion. Glial cell line-derived neurotrophic factor (GDNF) promotes glioma development process. However, the regulatory mechanisms of promoting occurrence and development of glioma have not yet been clearly elucidated. In the present study, the mechanism by which GDNF promotes glioma cell migration and invasion through regulating the dispersion and location of the Golgi apparatus (GA) is described. Following GDNF treatment, a change in the volume and position of GA was observed. The stack area of the GA was enlarged and it was more concentrated near the nucleus. Golgin-160 and Golgi microtubule-associated protein 210 (GMAP210) were identified as target molecules regulating GA positioning. In the absence of either golgin-160 or GMAP210 using lentivirus, the migration and invasion of U251 cells were decreased, while it was increased following GDNF. It was also found that the GA was decreased in size and dispersed following golgin160 or GMAP210 knockdown, as determined by GA green fluorescence assay. Once GDNF was added, the above phenomenon would be twisted, and the concentrated location and volume of the GA was restored. In combination, the present data suggested that the regulation of the position and size of the GA by golgin-160 and GMAP210 play an important role in U251 cell migration and invasion.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. This study was
funded by the National Natural Science Research
Foundation of China (grant Nos. 81372698,
81402918) and Jiangsu University Natural Science
Foundation funded project (17KJB310015. Dan
Wang: Funding, Lin-Yan Huang: Funding,
Glioma is a heterogeneous, highly complicated central nervous system (CNS) tumor with an
uncertain mechanism of initiation and progression[
], which results in an unfavorable
outcome. The invasion properties of glioblastoma render a radical surgery necessary and are
responsible for its recurrence[
]. In addition, the migration and invasion of glioma cells
experimental guidance, Dian-Shuai Gao: Funding,
project supervision, study conception and design.
severely disrupt brain function, due to the disruption of normal astrocytes, which are lifted up
from blood vessels by glioma cells[
]. So, it remains a holy grail of the migration of glioma
Cell migration is crucial for remodeling and regulating brain function [
], both during the
early development phase[
] and adulthood. What is then the difference between a normal and
a pathological brain? In normal adult brains, cell migration is limited and appears mainly in
the sub ventricular zone and dentate gyrus areas [
]. Stem cells located in these two areas
continuously produce progenitors that migrate and differentiate. Cell migration is also a feature of
malignant gliomas that use the same tortuous route traveled by stem cells[
]. Many molecules,
including glial cell line-derived neurotrophic factor (GDNF), are involved in cell migration.
GDNF contributes to the maintenance of neuronal migration toward the olfactory bulb [
a previous study, Xiong et al reported that GDNF could activate the proN-cadherin mediated
intracellular signal transduction in glioma cells, which promotes the secretion of matrix
metalloproteinase-9 and degrades extracellular matrix[
]. It therefore appears that GDNF plays a
role in promoting cell migration. Several studies have focused only on the cell migration and
the associated signaling molecules activated by GDNF. Rather, little attention has been paid to
the dynamic changes in the movement of the cells themselves.
Fibroblast polarization is one of the most important phenomena in directional cell
]. In cell polarization, the Golgi apparatus (GA) is critically involved in directional cell
migration, since GA acts a pivotal part in supplying the membrane components to the leading
edge for membrane protrusion when the cell is moving[
]. The asymmetric distribution
of protrusional activity is a general characteristic of directional motility, which requires
the integrity of GA and microtubules (MTs). Further, the reorientation of GA has an active
role in directed secretion and cell polarity[
]. The ability of the GA to nucleate MTs has
recently been demonstrated, and the molecular machinery involved in the position of GA has
been partly identified. Studies have confirmed that various treatments that disrupt Golgi
architecture are accompanied by an inhibition of cell migration. For example, deletion of
golgin160 or Golgi microtubule-associated protein 210 (GMAP210) led to fragmentation and
disperse of the GA without disassembling microtubule or actin cytoskeletal systems, and
contributed to the inhibition of directional cell migration [
It has been identified GDNF promotes migration and invasion of glioma cells[
changes in the morphology and position of GA were examined following treatment of GDNF.
For this, we used lentivirus to disrupt the expression of golgin-160 or GMAP210, resulting in
the inhibition of U251 cell migration and invasion with or without GDNF. This suggested that
GA is a prerequisite for directed glioma cell migration with GDNF treatment. The aim of the
present study was designed to figure out the unique biology of GDNF on the migration and
invasion of glioma cells, which provides hitherto unrevealed the mechanism from the
perspective of the GA.
Material and methods
Cell culture and GDNF treatment
Human U251 glioma cells [Obio Technology (Shanghai) Corp., Ltd, Shanghai, China] were
cultured in a 37?C, 5% CO2 humidified incubator using Dulbecco?s modified Eagle?s medium
(DMEM, SH30022.01, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) containing
10% fetal bovine serum (FBS, SV30087.02, GE Healthcare Life Sciences), 100U/ml penicillin
and 100U/ml streptomycin. Exponentially growing cells, derived after two or three passages,
were used for the following experiments: i) U251 cells treated with 50ng/ml GDNF (Merck
KGaA, Darmstadt, Germany) for 0, 0.5, 24 or 48h were used to detect the effects of GDNF on
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the stack area of GA; ii) U251 cells treated with various concentrations of GDNF (0, 25, 50,
75ng/ml) for 48h were used to determine the optimal GDNF concentration.
Golgi apparatus green fluorescence assay
U251 cells treated with GDNF were fixed with 4% formaldehyde for 20min at room
temperature and permeabilized with 0.3% Triton X-100 for 20min. Then GOLGI ID Green assay kit
] (ENZ-51028-K100, Enzo Life Sciences, Inc., Farmingdale, NY, USA) was used for
aldehyde-fixed cells staining, in order to localize and quantify the GA. Golgi ID green allowed the
Golgi apparatus to be identified in confocal images.
Immunofluorescence & confocal microscopy
U251 cells treated with GDNF were fixed with 4% formaldehyde for 20min at room
temperature and permeabilized with 0.3% Triton X-100 for 20min. Next, the cells were blocked with
5% goat serum in PBS for 20min, followed by immunostaining with the primary antibodies
(rabbit anti-Golgin-160, ab96080, Abcam, 1:100; Abcam, Cambridge, UK) overnight at 4?C.
After washing with PBS, the cells were incubated with secondary antibody (DyLight
594-conjugated goat anti-rabbit, E032420, 1:500, EarthOx Life Sciences, Millbrae, CA, USA) for 40min
at 37?C in a dark, moist environment. DAPI was applied to stain the nuclei, and fluorescence
images were captured with fluorescence or confocal laser fluorescence microscopes (FV10i,
Olympus Corporation, Tokyo, Japan).
Total protein in each sample was extracted with NP-40 lysis buffer. The protein concentration
in each sample was detected using the BCA protein assay kit (Hyclone-Pierce, Rockford, IL,
USA). The protein samples were separated on an 8% SDS-PAGE gel and then transferred to
PVDF membranes. After blocking by 5% skimmed milk for 1h at room temperature, blots
were incubated with primary antibodies: rabbit anti-Golgin-160 (ab9608; 1:1000; Abcam),
mouse anti-GMAP-210 (sc-135928; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA),
mouse anti-GAPDH (AG019; 1:10000; Beyotime Institute of Biotechnology, Haimen, China)
and anti-actin (1:10000; Santa Cruz Biotechnology, Inc. sc-8432) antibodies at 4?C overnight.
Next, blots were incubated with corresponding goat anti-rabbit (1:3000; Santa Cruz
Biotechnology, Inc.) and goat anti-mouse (1:5000; Santa Cruz Biotechnology, Inc. sc-362257,
sc362277) secondary antibodies for 2h at room temperature in the dark. Finally, the membranes
were scanned using Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA) and
quantified with ImageJ 1.48v (National Institutes of Health, Bethesda, MD, USA). GAPDH
was used as the internal reference protein.
Golgin-160 and GMAP-210 knockdown in U251 cells by lentiviral RNA
pLKD-CMV-R&PR-U6-shRNA vectors were generated using the RNAi sequences (human
Golgin-160: 5-GGAGATGAAGACCAAACAT-3, GMAP210:
5-GCAGTTGACACAACTTATA3; control: 5-TTCTCCGAACGTGTCACGT-3) and transfected into DH5? cells. The packaged
lentivirus containing supernatant was collected and then used to infect U251 cells. Through
screening with puromycin for one week, the knockdown of Golgin-160 (KD-Golgin-160) and
GMAP210 (KD-GMAP-210), as well as their negative control cells (control), were obtained
and stably expressed the Golgin-160-, GMAP210- and vector- specific RNAis (Control),
respectively. The blank group was made up of normal U251 cells that did not undergo any
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Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from U251 cells and its concentration and quality were measured
using an ultraviolet spectrophotometer (OD-1000, BioDee Biotechnology Co. Ltd, Bejing,
China). cDNA was synthesized through reverse transcription of RNA. Next, quantitative PCR
was used to determine the mRNA expression of Golgin-160 and GMAP210. PCR conditions
were as follows: i) 95?C for 30s; ii) 40 cycles: 95?C for 5s, 60?C for 34s; iii) 95?C for 1min, 55?C
for 1min; iv) 81 cycles: 55.0?C-95.0?C, with every cycle increasing the setpoint temperature by
0.5?C and resting for 4s. Actin was used as the internal reference gene. The relative mRNA
expression levels of the target genes were calculated using the 2-??Ct method. The primer
sequences were as follows: Actin forward: 5-TTCTACAATGAGCTGCGTG-3 and reverse,
5-CTCAAACATGAT CTGGGTC-3; Glogin-160 forward: 5-GGAAACACACTTGCAGTCGT-3
and reverse, 5-TCTTC TGCTTCTGTTCCGTG-3; GMAP-210 forward: 5-GGAGGAGATGG
AGCAGTTGT-3 and reverse, 5-CCACAGATTGCTGATTTGGTC-3.
Cell proliferation assays
Cell proliferation was detected via the Cell Counting Kit-8 assay (CCK-8; Dojindo
Laboratories, Kumamoto, Japan).The U251 cells were seeded into 96-well plates at a density of 5x103
cells/ well. The first CCK8 assay was performed when cells treated with non-serum were
starved for 12 h. The above-mentioned results were regarded as starting value (0 h).
Meanwhile, different treatment conditions were carried out as follows: serum-free group,
serumfree + DNA inhibitor +GDNF group, serum-free + DNA inhibitor group, serum-free + GDNF
group, DMEM+ serum group. The concentration of GDNF was 50ng/ml. DNA inhibitor is
mitomycin C (No.10107409001, Roche). 10?l CCK8 dye was added to the wells and incubated
for 60min at 37?C. The optical density was assessed at a wavelength of 450 nm with UVmax
kinetic microplate reader (Molecular Devices, Wokingham, UK). Then we checked the CCK-8
results at the indicated time-points (0,12h and 48h) and recorded the 60min OD450 value.
Wound healing assay
Cell migration was evaluated by wound healing assay. U251 cells were seeded in six-well plates
(106/well) and cultured until confluence. Following culture with serum-free DMEM for 12 h
to avoid the effect of cell proliferation, cells were wounded manually using a 20-?l pipette tip.
The remaining cells were rinsed with PBS and cultured with serum-free DMEM. Photographs
were taken at a magnification of x100 at 0, 24 and 48 h to monitor cell migration across the
wound. The decrease in wound area was measured using IPP software.
Live cell imaging assay
Live cell motility imaging was captured using the Olympus IX81 inverted microscope with a
new UIS2 optical system. U251 cells were seeded in six-well plates (106/well) and cultured
until 80% confluence. Following culture with serum-free DMEM for 12 h, cells were wounded
manually using a 20-?l pipette tip. Meanwhile, GDNF was added and the concentration was
50ng/ml. Images were acquired every 20 mins in the phase starting from 6h to 48h.
Transwell matrigel invasion assay
Serum-free DMEM (100?l and 700?l) were respectively put into the top and bottom chamber
of 24-wel plates Transwell chamber for 2h at 37?C, and then removed. After the chamber was
coated with Matrigel for 4h, cells (3?104) cultured with serum-free DMEM for 12h were
seeded into the top chamber and incubated with DMEM containing 10% FBS for 24h at 37?C.
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Fig 1. The stack area of the GA was increased over time fllowing GDNF treatment. (A) U251 cells treated with 50 ng/ml
GDNF for 0.5, 24 and 48 h and the control group were stained and imaged to detect the GA. Golgi-ID green staining
showing the GA (green), nucleus (blue) and merged images (bar = 50 ?m). (B) Quantitative analysis of the area of the GA is
presented as the mean ? standard deviation of three independent experiments. Compared with the control group, the
average area of the GA body was increased by GDNF treatment at 24 and 48 h ( P<0.05). GA, Golgi apparatus; GDNF, glial
cell line-derived neurotrophic factor.
Cells were fixed with 4% formaldehyde and stained with crystal violet staining solution. The
invading cells were photographed (magnification, ?200) and counted.
After confocal images were acquired, the number of fluorescent objects per cell was
determined using a fixed threshold and the ?Analyze Particle? plugin in Image Pro Plus (IPP)
software. The stack area and fluorescence intensity of Golgi objects were determined for each cell
using IPP. The integrated optical density (IOD) was calculated by dividing the GA
fluorescence intensity by the stack area. We defined the distance from the GA margin to the center of
the nucleus by selecting the best fitting circle around the nucleus. Then the radius of the circle
was measured at the same standard scale.
All analysis was performed with the SPSS 19.0 (SPSS, Inc., Chicago, IL, USA) and expressed as
the mean ? standard deviation (SD). An independent sample t-test was used to determine
significant differences in the mean values between two groups. Multiple comparisons between
groups were performed using one-way analysis of variance followed by the pairwise
comparison (LSD) test for statistical analysis. P<0.05 was considered to indicate a statistically
1. GDNF increased the stack area of the GA in U251 cells
To explore the effects of GDNF on Golgi apparatus, Golgi-ID green staining was performed to
determine the changes of the GA in U251 cells treated with 50ng/ml GDNF for 0, 0.5h, 24h or
48h. The results showed that fragmented GA was reduced, and the GA concentrated and
gathered near the nucleus in U251 cells following GDNF treatment for different time points (Fig
1A). Compared to non-GDNF-treated U251 cells (0h), the average stack area of the GA
increased significantly and was more concentrated around the nucleus in U251 cells treated by
GDNF for 24 and 48h (Fig 1B, P<0.05).
2. GDNF increased the expression of golgin-160 and GMAP-210 in U251 cells
To confirm the results above and further determine the best concentration of GDNF, western
blot was used to detect the expression of golgin-160 and GMAP-210 in U251 cells treated by
different concentration of GDNF (0, 25, 50, 75ng/ml) for 48h. The results showed that protein
expression of golgin-160 and GMAP-210 were elevated in the GNDF treatment groups (Fig
2A), which was consistent with the increase in the stack area of the GA by Golgi-ID green
staining (Fig 2C). The expression of golgin-160 and GMAP-210 were the highest in U251 cells
treated with 50ng/ml GDNF (Fig 2B, P<0.001), and the stack area of the GA achieved the
highest level when cells were treated with 50 ng/ml GDNF (Fig 2D, P<0.05). No statistical
difference was observed among the 25, 50 and 75ng/ml treatment groups.
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Fig 2. Increased expression of golgin-160 and GMAP210 and increased stack area of the GA were both induced by GDNF in a dose- dependence manner.
(A and B) Western blotting detected the expression of golgin-160 and GMAP-210 in U251 cells treated by 0, 25, 50 and 75 ng/ml GDNF for 48 h. The expression
of golgin-160 and GMAP-210 was increased most significantly following treatment 50 ng/ml GDNF ( P<0.001). (C and D) Immunofluorescence analyzed the
stack area of the GA in U251 cells treated with 0, 25, 50 and 75ng/ml GDNF for 48 h. The GA area in U251 cells was increased following GDNF treatment, and it
was significant following treatment with 50 ng/ml ( P<0.05; bar = 50 ?m). GMAP-210, Golgi microtubule-associated protein 210; GA, Golgi apparatus; GDNF,
glial cell line-derived neurotrophic factor.
3. GDNF promoted U251 cell migration and invasion influenced by
depletion of golgin-160 or GMAP210
Based on our previous study [
] and the determination of the optimal treatment time and
dosage of GDNF, we speculated that GDNF-facilitated U251 cell migration would be regulated by
golgin-160 and GMAP-210. To test this speculation, wound healing and transwell invasion
assay were conducted. The results showed that the gap-filling rate in the GDNF treatment
group was higher than that in the control group, following scraping for 24h (Fig 3A, P<0.05).
Furthermore, the difference in the percentage of gap-filled was more significant at 48h (Fig
P<0.01). The Transwell invasion assay results showed that the number of penetrated
cells after treatment with 50ng/ml GDNF for 24h increased, as compared with control group
(Fig 3C), which was consistent with the results of our previous study [
]. To improve the
Fig 3. U251 cell migration was promoted by GDNF. (A) The effect of GDNF on U251 cell migration was assessed by wound healing assay ( P<0.05, P<0.01,
bar = 100?m). (B) The effect of GDNF on U251 cell invasion was assessed by Transwell invasion assay ( P<0.001; bar = 100 ?m). GDNF, glial cell line-derived
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Fig 4. Effects of golgin-160 knockdown on U251 cell migration and invasion. (A) Golgin-160 was knocked down by lentivirus, and its expression was assessed
by western blotting and RT-qPCR ( P<0.001). The effect of golgin-160 knockdown on U251 cell (B and C) migration and (D and E) invasion was assessed by
wounding healing and cell invasion assays. Cell migration and invasion were inhibited by the depletion of golgin-160, and GDNF could improve it (##P<0.05 and
P<0.01; bar = 100 ?m). RT-qPCR, reverse transcription quantitative polymerase chain reaction; GDNF, glial cell line-derived neurotrophic factor.
persuasion of results, whether cell proliferation interferes with migration was explored
additionally. As shown in S1 Fig, when cells were treated with non-serum, the proliferation was
decreased significantly compared with serum group. Indeed, cell proliferation was improved
only when GDNF was added ( P<0.05). Rather, once DNA inhibitor was used, even adding
GDNF could not increase proliferation. As a whole, the effect of GDNF on cell proliferation
was almost negligible through numerical analysis. Meanwhile, DNA inhibitor inhibited
proliferation ultimately, which was little difference in proliferation from serum-free group. All the
above detailed data were shown in the S1 Table. More plausibly, short-time time-lapse live
imaging of cell motility was performed (S2 Fig), which was used to identify that GDNF could
enhance cell motility in a spatially controlled manner absolutely. The results suggested that
GDNF promoted cell motility obviously, which was consistent with S1 Fig ( P<0.05). Video
data was separately uploaded as supplementary result (S1 Video). From the video, cell motility
was significantly increased by GDNF; the wound healing was quicker than control group.
Based on these, we made a brief summary: The ability of GDNF promoting cell migration
could not be masked by cell proliferation.
To determine whether the golgin-160 or GMAP210 contributes to the effect of GDNF on
migration and invasion of U251 cells, we constructed lentivirus and stable cell line was
screened by the puromycin. The western blot and RT-PCR experiments showed that
knockdown of golgin-160 (Fig 4A) or GMAP210 (Fig 5A) had been successful. Simultaneously, cell
migration was evaluated by scratch wound assay. The results demonstrated that
down-regulation of golgin-160 or GMAP210 could decrease cell migration (Figs 4B, 4C, 5B and 5C
P<0.01,). When stable line cells resumed treating with GDNF, their migration ability was
improved, as compared with that of cells in the knockdown golgin-160 (or GMAP210) group.
However, their ability was still worse than that of cells in the control group at 48 h (#P<0.05),
while there was no difference between these two groups at 24h (Figs 4C and 5C). Subsequently,
cell invasion was explored in vitro. Similarly, regardless of the protein that was knocked down,
cell invasion was impaired (Figs 4D and 5D; P<0.01,). In addition, the number of penetrated
cells was increased with the action of GDNF (Figs 4E and 5E; #P<0.05).
4. Depletion of GMAP210 yields more fragments in the GA and drives it
away from the nucleus
Based on the confirmation that cell migration and invasion were inhibited by the depletion of
golgin-160 or GMAP210, changes in the GA, which is one of the most important organelles in
regulating cell migration and invasion, were observed. Immunofluorescence was performed to
localize golgin-160 protein, a member of the Golgi family localized to the GA. The results
showed that the fluorescence intensity of golgin-160 was clearly decreased (Fig 6A and 6B),
and the difference was statistically significant ( P<0.01). In addition, as compared with
control group, the distinct fluorescent Golgi mini-objects were increased by the depletion of
GMAP210. Fig 6C shows the dispersed Golgi membranes and fragments. Next, the distance
from GA to the nucleus was measured. It was found that golgin-160 or GMAP210 knockdown
blocked the peri-centrosomal positioning of the GA[
]. The results showed that the distance
from the distal edge of GA to the nuclear center was increased, due to the decreased expression
of GMAP210 (Fig 6D and 6E, P<0.05). Knockdown cells depleted by either golgin-160 or
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Fig 5. The depletion of GMAP-210 inhibited U251 cell migration and invasion, while GDNF improve it. (A) GMAP-210 was knocked down by lentivirus and
its expression was assessed by western blotting and RT-qPCR ( P<0.001). The effect of GMAP-210 knockdown on U251 cell (B and C) migration and (D and
E) invasion was assessed by wounding healing and cell invasion assays. Cell migration and invasion were inhibited by the depletion of GMAP-210, and GDNF
could improve it (#P<0.05 and P<0.01; bar = 100 ?m). GMAP-210, Golgi microtubule-associated protein 210; GDNF, glial cell line-derived neurotrophic
factor; RT-qPCR, reverse transcription quantitative polymerase chain reaction.
GMAP210 behaved in a similar manner, which was verified by the results presented in the
5. GA Agglutination level was affected by golgin-160 and the agglutination
level was improved in U251 cells following GDNF treatment
To confirm the complete morphology of Golgi, the effect of exogenous GDNF on the GA
when golgin-160 was knocked down in U251 cells was also explored through Golgi-ID green
staining. The results proved consistent with those of glogin-160 staining above. It was verified
that GDNF could promote U251 cell migration and invasion, which required the mediation of
golgin-160 and GMAP210. Since the depletion of golgin-160 generated GA fragmentation and
inhibited migration and invasion, the change in the GA in cells treated with 50ng/ml GDNF
for 48h was observed. Consistent with our speculation, the agglutination level of the GA was
increased by GDNF stimulation (Fig 7A and 7B), which was reflected by the analysis of
integrated optical density (IOD) of the GA ( P<0.001). Consistent with the previous results (Fig
6), the depletion of golgin-160 actuated the GA dispersion but rather may abate agglutination
level of the GA, as compared with the control group (Fig 7A?7C). When GDNF was
replenished based on the knockdown of golgin-160, the agglutination level of the GA was improved
significantly (Fig 7C and 7D), which was reflected by the findings of the GA IOD analysis
( P<0.01). Altogether, the results of the present study demonstrated that GDNF promotes
U251 cell migration and invasion through the mediation of the GA orientation and
agglutination by golgin-160 and GMAP-210.
In the present study, it was found that the incubation of cells with GDNF leads to an increased
cell migration and invasion capability. As a result, the GA counts much in regulating
directional cell migration mediated by golgin-160 and GMAP210, which organize Golgi
ministacks. Cell migration is a dynamic and highly complicated cellular process. Deregulation of
migration is the main characteristic of cancer, and glioma is no exception.
Studies have shown that Golgi orientation is important for cell polarization and migration,
as a polarized Golgi supplies membrane components for leading edge protrusion or material
for apical secretion[
]. Based on this study and our previous study [
], we speculated that
GDNF could raise the agglutination level of GA and lead it towards the nucleus. This
hypothesis was confirmed by the present results.
In mammalian cells, the GA is polarized in both structure and function and it is localized to
juxta nuclear zone[
]. The Golgi itself participates in a variety of protein modification
processes as well as the supply of components to the cell membrane. During migration, the
changes of cell membrane polarization accompanied with the continuous and dynamic
changes of the component in the front membrane. Therefore, it is worth determining the GA
position and the direction of cells movement during cell migration. When GDNF was added
to the U251 cells, they secreted more MMP9 [
], which degrades ECM and basement
membrane components and promotes their invasion and metastasis [
]. Both the results of a
previous and this study showed that cell migration was likely influenced by the polarity of the
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Fig 6. Morphological observation of the GA and distance detection from cell nucleus through Immunofluorescence. (A and B)
Immunofluorescent images and analysis of golgin-160, which was considered a Golgi marker protein. Red (golgin-160), blue (nuclear) and merged
images (bar = 50 ?m). Following golgin-160 knockdown, the integrated optical density of golgin-160 was decreased, while the GA profile was still
visible ( P<0.01). (C) Zoom in to the specific cells from A (arrow pointing); it was shown that the increased dispersion level of the GA was due to
the depletion of golgin-160. (D and E) Zoom in to the specific cells from A (rectangle); the distance from the remote margin of the GA to the cell
nucleus was detected using Image Pro Plus. Each dot represents the distance from the GA to the nucleus of different cells. The different values were
plotted (?m); the KD-Golgi-160 group was father from the nucleus ( P<0.05). GA, Golgi apparatus.
position and function of the GA following GDNF treatment, which led to GA reorientation
toward the migration leading edge and secretory trafficking [
]. In addition, GDNF promotes
MMP-9 secretion in U251 cells [
]. We speculated that the secretion of MMP9 was also in the
direction of leading edge, which facilitates cell infiltration. However, this mechanism requires
In the present study, the entire structure of the GA was observed; it appeared to be in a
diffuse state and to have undergone significant changes following the knockdown of golgin-160.
The GA was also found further away from the nucleus. These observations were consistent
with those of the previous study[
]. Numerous studies have confirmed that GA is one of the
most important organelles involved in directional cell migration[
]. It is also associated with
asymmetric cytoskeletal arrangement[
], intracellular organelle localization, membrane
] and polarized cell morphology[
]. What could regulate the Golgi
reorientation? A matter of sparking and intense research was executed. Primarily,
disassembling the GA leads to its failure to orient towards the leading edge, which further damages the
centrosome reorientation . Hurtado et.al reported that both the centrosome and the GA
failed to reorient towards the leading edge, and directional migration was strongly inhibited
by knocking down AKAP450[
]. That is the cause of the destruction of the polarity of the GA
and the centrosome. The centrosome is also known as an microtubule-organizing center
], which determines the positioning of GA [
]through MTs such as tubulin[
A different study also identified that the AKAP450, which is involved in the targeted
positioning, is critical for cellular functioning, particularly in MT nucleation and cell migration[
Astrocytes change from sparsely branched cells to polarized and highly ramified cells [
during development, which requires the polarity of the centrosome and GA. In addition
GDNF plays an important role in astrocyte development. In our study, it was observed that
GDNF could promote glioma cell migration and increase the expression of GA-related
proteins. Following the knockdown of golgin-160 or GMAP210, the polarity orientation of GA
was destroyed and cell migration was inhibited. Even so, what do the golgin-160 and
GMAP210 work on earth in cell migration and whether it has a relationship with cell polarity
of GA or not? Tubulin endows the dynamics and polarity of Golgi apparatus and cell itself,
which makes it produce migration power. Studies have shown that once the microtubules
depolymerize, the Golgi apparatus is not a ribbon vesicle, but is broken up into small heaps
Golgin-160 and GMAP210 are proteins with key roles in GA positioning and function[
The GA has an assembly center of ?/?-tubulin acting as MTOC[
]. Of note, the depletion
of GMAP210 or golgin-160 leads to the failure to recruit ?-tubulin, which causes further
destruction around the centrosome, GA fragmentation and inhibition of cell migration .
Another study has also indicated that golgin-160 ensures efficient delivery of
protein-processing to the surface from the trans-Golgi network [
]. GMAP210 acts at the endoplasmic
reticulum (ER)-to-Golgi during anterograde trafficking, and is also required for retrograde
trafficking to the ER[
]. That demonstrates the importance of golgin-160 and GMAP210 in
the GA peri-centrosomal positioning and cell migration. GDNF increased the expression of
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Fig 7. Golgi-160 was essential for agglutination level of the GA and GDNF improved it. (A and B) Immunofluorescence and GA agglutination level analysis in the
control group, with or without 50 ng/ml GDNF treatment. Following GDNF intervention, the agglutination level of the GA was more prominent ( P<0.001;
bar = 100 ?m). (C and D) Immunofluorescent images and GA agglutination level analysis in the KD-golgin160 group, with or without 50 ng/ml GDNF treatment. The
integrated optical density of the GA was decreased and its morphology was diffused due to the depletion of golgin-160. GDNF improved the agglutination level
( P<0.01). For better observation and analysis, we zoomed in to the images of selected target cells (red rectangle). Bar = 50?m. GA, Golgi apparatus; GDNF, glial cell
line-derived neurotrophic factor.
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golgin-160 and GMAP210 in U251 cells. However, the specific mechanism of GDNF on the
GA is still unknown. In summary, establishing conditions to test the specific role of GA
positioning on directed secretion and glioma cell polarity following GDNF treatment should be
further investigated. It should be further explored that GDNF regulates GA reorientation and
how it affects cell polarity.
The regulation of the positioning and size of the GA by golgin-160 and GMAP210 plays an
important role in GDNF promoting U251 cell migration and invasion.
S1 Fig. Relative proliferation and migration of U251 cells treated with GDNF, DNA
inhibitor (mitomycin C). (A) Cell proliferation was assessed by CCK8. In serum-free medium,
GDNF increased the proliferation moderately ( P<0.05) compared with other Intervention
conditions. There was no difference in cell proliferation between DNA inhibitor group and
serum-free + DNA inhibitor group.(B and C) Wound healing assay was used for comparison
of cell migration. Both serum-free +GDNF and Serum-free +DNA inhibitor +GDNF groups
performed greater mobility in cell migration than other two groups ( P<0.001). There was
no difference between serum-free +GDNF and Serum-free +DNA inhibitor +GDNF groups.
S2 Fig. Live cell imaging of cell motility. At 6th h after scratching, start recording through
Olympus IX81 inverted microscope with a new UIS2 optical system. The duration of recording
was from 6th to 48th h. 0s represents the starting point of recording (The actual time is 6th h
after the scratching); 12s represents the end point of recording (The actual time is 48th h after
S1 Video. Video data of cell motility in control and GDNF groups.
S1 Table. The OD450 data comparison among different groups (mean?SD).
We would like to express our deep gratitude to the center of neurobiology of Xuzhou Medical
University. In addition, Chuanxi Tang especially wishes to thank his fiance?e- Chunyan Mu
who gave him powerful support over the past year.
Conceptualization: Chuan-Xi Tang, Dian-Shuai Gao.
Data curation: Lan Luan, Lin Zhang.
Formal analysis: Lin Zhang.
Funding acquisition: Dan Wang, Lin-Yan Huang, Dian-Shuai Gao.
Investigation: Lan Luan, Yue Wang.
Methodology: Lan Luan, Yue Wang.
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Project administration: Chuan-Xi Tang, Jie Wang.
Software: Xin-Feng Liu.
Supervision: Dian-Shuai Gao.
Validation: Ye Xiong.
Visualization: Xin-Feng Liu.
Writing ? original draft: Chuan-Xi Tang.
Writing ? review & editing: Lin-Yan Huang.
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