Full Report

1
An Investigation of the Activity of the Zinc Transporter
ZIP10 during the Cell Cycle in Breast Cancer
Frederik Gibson
Biomedical Sciences
Dr. Kathyrn Taylor
Breast Cancer Molecular Pharmacology Group,
Cardiff School of Pharmacy and Pharmaceutical Sciences
Word count: 6000

2
Abstract
For over 50 years zinc has been known to be required for the cell cycle to
progress, however the mechanism of action is unknown. Zinc is transported into the
cell cytoplasm across membranes by the ZIP (Zrt-, Irt-like Protein) transporter
family, but the importance of each individual member and how they are regulated
is not well understood. Prion proteins, which have descended from ZIP transporters,
are known to be N-terminally cleaved for activation. N-terminal cleavage of ZIP10
has been observed and ZIP6, which shares many sequence similarities to ZIP10, is
also N-terminally cleaved before activation. Furthermore, zinc influxes into the cell
during the G1 phase of the cell cycle and ZIP10 has been shown, with data available
from whole genome phosphoscreens, to be phosphorylated during G1 and M phase.
This data together suggests an important role for ZIP10 consistent with zinc influx
during G1 and M which may involve N-terminal cleavage and/or phosphorylation.
The aim of this project is to examine N-terminal cleavage of ZIP10 and investigate
its relation to different cell cycle stages. MCF-7 cells were synchronised by serum
deprivation or nocodazole treatment before growth in normal medium, generating
samples at different stages of the cell cycle. ZIP10 antibodies with epitopes in
different regions of ZIP10 were used in western blot, resulting in the detection of
several different ZIP10 fragments. N-terminal fragments were observed, and were
at their highest levels during mitosis and G1 phase. This result is supported by
previous observations of ZIP6. ZIP10 was also shown to be expressed on the plasma
membrane of mitotic cells by immunofluorescence microscopy. Our results provide
novel evidence of the involvement of ZIP10 in mitosis, and provide new therapeutic
targets for use in breast cancer.

3
Introduction
Breast Cancer and Zinc
Breast cancer is the most common cancer in females, totalling 31.2% of all
cancers in women, and has a mortality rate of 24 per 100,000 in the United Kingdom
(Office for National Statistics 2013). The majority of breast cancers are oestrogen
receptor positive (ER+) and require oestrogen for growth (Berger et al. 2012),
therefore in this study the ER+ MCF-7 cell line was used. It is one of the oldest and
most commonly used breast cancer cell lines, and was grown from invasive breast
ductal carcinoma cells isolated from a woman in 1970 (Levenson and Jordan 1997).
Zinc is involved in a vast variety of processes such as the control of gene
transcription, differentiation and metabolism of proteins, nucleic acids,
carbohydrates and lipids (Vallee and Falchuk 1993), is essential to cell cycle
progression (Fujioka and Lieberman 1964) and is a cofactor for more than 300
enzymes (Vallee and Auld 1990). Importantly zinc has also been shown to be
required for cell division (Wang et al. 2013) and cell motility (Takatani-Nakase
2013), two processes closely related to cancer progression; unsurprisingly zinc
concentrations are known to be high in breast cancer (Rehman and Husnain 2014).
Within the cell zinc is present within the structure of proteins and as a small pool of
labile zinc, however it must be carefully controlled to avoid dysregulation as
deficiency causes, among many other things, cognitive impairment, infertility and
compromised immunity, whilst excess is highly toxic (Jeong and Eide 2013). The
danger zinc can pose to the cell thereby leads to intracellular concentrations of labile
zinc being kept in the nanomolar range. Whilst much research has been dedicated
to zinc pathologies, comparatively little research has been undertaken into the role
of zinc as a second messenger (Yamasaki et al. 2007) despite its involvement in many
regulatory pathways, ranging from embryonic development (Yamashita et al. 2004)
to control of the central nervous system (Sensi et al. 2009), via the inhibition of
protein tyrosine phosphatases (Haase and Maret 2005). Zinc is also known to
directly bind STAT3, a molecule involved in mitosis (Kitabayashi et al. 2010). This
indicates that the role of zinc as a second messenger has not been fully explored, and
may well be as important as the cation second messenger calcium.

4
During the cell cycle labile zinc concentrations are not static, instead there
are peaks in early G1 phase and in late G1/S phase caused by zinc influx and release
from intracellular stores respectively, indicating that zinc plays a key role in cell
cycle progression (Li and Maret 2009). Fluctuations in labile zinc concentrations
within the cell can either occur through the release from proteins containing zinc,
triggered by changes in the intracellular redox potential (Maret 2008), or by flux
through the plasma or intracellular membranes facilitated by zinc transporters and
channels (Colvin et al. 2010). There is much still unknown about zinc transport by
these proteins, and it is the aim of this study to more closely examine the activation
of zinc transporter ZIP10.
Zinc Transport and ZIP10
In mammals labile zinc is heavily regulated by metalloproteins such as
metallothioneins (Vašák and Meloni 2011) and two families of transporters; the ZnT
family (SLC30A), which facilitate the movement of zinc from the cytoplasm into
the extracellular space or intracellular compartments, and the ZIP family (Zrt-, Irt-
like proteins) (SLC39A), which facilitate the movement of zinc into the cytoplasm
from the extracellular space or intracellular compartments (Lichten and Cousins
2009). These families share some structural similarities (figure 1A), with both
containing large intracellular loops. However they differ in the number of
transmembrane domains they have; eight for the ZIP family and six for the ZnT
family, and the orientation of their N- and C-termini, facing into the extracellular
space or intracellular compartments in the ZIP family and into the cytosolic space
in the ZnT family (Fukada and Kambe 2011). Both families can be divided into
subfamilies based on sequence similarities, with the 10 members of the ZnT family
forming 4 sub-families (Huang and Tepaamorndech 2013) and the 14 members of
the ZIP family forming several, such as the LIV-1 subfamily (all members share a
HEXPHEXGD motif) (Taylor et al. 2003), which contains some members involved
in cancer progression; ZIP6 (SLC39A6) is involved in breast cancer metastasis
(Manning et al. 1994) whilst ZIP10 (SLC39A10) (figure 1B) is highly expressed in
and promotes migration of metastatic breast cancer cells (Kagara et al. 2007).
Analysis by the Phosida phosphorylation online database indicates that ZIP10 is

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Figure 1: Schematic diagrams of the Zinc Transporters
(A) Diagram of the predicted generic structures of the ZIP (left) and ZnT (right)
families, adapted from Fukada e Kambe (2011). ZIP transporters move zinc into
the cytoplasm from the extracellular space or intracellular compartments, whereas
ZnT transporters move zinc out of the cytoplasm into the extracellular space or
intracellular compartments, as indicated by arrows. Bold lines indicate histidine-
rich areas within the large intracellular loop. (B) Schematic diagram of the
secondary structure of ZIP10, showing the standard eight transmembrane domains
and large intracellular loop, with a very long N-terminus and short C-terminus, as
well as the site of ectodomain shedding proposed by Ehsani et al. (2012).
A
B
ZIP ZnT

6
phosphorylated at S546 during G1 (Gnad et al. 2007), the same time as the zinc
influx during G1 identified by Li and Maret (2009), and as well as during mitosis.
Along with the knowledge that ZIP10 is required for cell cycle progression, as
disruption leads to cell cycle arrest (Kong et al. 2014), these studies indicate ZIP10
activation may be cell-cycle-dependent. Both ZIP6 and ZIP10 have also been linked
to the development of prion proteins, with all likely sharing a common molecular
ancestor (Ehsani et al. 2011). ZIP10 has been shown to undergo post-translational
ectodomain shedding in mouse N2a cells infected with prion proteins, indicating
further study of ZIP10 may aid the discovery of therapeutics for prion diseases
(Ehsani et al. 2012). ZIP6 and ZIP4 are activated by N-terminal cleavage in healthy
cells (Kambe and Andrews 2009; Hogstrand et al. 2013), therefore this study sought
to determine whether ZIP10 was similarly regulated.
Our hypothesis is that ZIP10 is cleaved similarly to ZIP6 and ZIP4, with
cleavage of the N-terminus during mitosis leading to activation of ZIP10 followed
by zinc influx into the cell. We propose that this zinc influx is essential for cell cycle
progression, and therefore that ZIP10 activation is required for mitosis. Our results
indicated that ZIP10 underwent many cleavages, including N-terminal cleavage,
and we investigated whether this occurred in a cell-cycle-dependent manner. This
work carries significance as a greater understanding of the function and regulation
of ZIP10 may lead to the identification of therapeutic targets for a more specific
approach to breast cancer cells highly expressing ZIP10.

7
Methods
Cell Preparation
Wild-type MCF-7 cells were cultured in RPMI-1640 with 5% foetal calf
serum (FCS), 10 iu/ml penicillin, 10 μg/ml streptomycin, and 2.5 μg/ml fungizone at
37°C in a humidified 5% CO2 atmosphere. Every 3-4 days the cell medium was
changed.
Treatments
1. Cells were synchronised by withdrawing FCS from medium for 24 hours. Normal
medium was then added back for a variable number of hours before harvesting
cells.
2. Cells were synchronised in mitosis by adding 100ng/ml nocodazole (Sigma-
Aldrich, M1404) to cell medium for 20 hours followed by nocodazole withdrawal
for 0-4 hours before harvesting, producing samples with cells synchronised to or
just after mitosis.
3. Cells were transfected with wild-type ZIP10 DNA by incubating a mixture of
5𝜇l/ml lipofectamine 2000 (Invitrogen) and 2.5𝜇g/ml WT ZIP10 DNA
containing plasmid and normal medium without penicillin, streptomycin and
fungizone at room temperature for 20 minutes, before adding to cells at 70%
confluence. 1.5𝜇l butyrate was then added to cells, which were left for 20 hours
before harvesting. The transfected ZIP10 DNA had a V5-tag using vector
pcDNA3.1/V5-His-TOPO (Invitrogen, K4800-01) at the C-terminus.
Cell Lysis and Western Blot
Medium containing non-adherent cells was centrifuged and the resultant
pellet was washed in ice-cold PBS and centrifuged again, both times at 1000rpm for
5 minutes. 100𝜇l lysis buffer (50mM Tris, 150mM NaCl, 5mM EGTA, 1% Triton X-
100, pH 7.6), 2mM Na3VO4, 25mM NaF and 10% Protease Inhibitor Cocktail
(Sigma-Aldrich) were used to re-suspend resultant pellet, and treat adherent cells
before scraping into an Eppendorf and pooling with collected non-adherent cells.
Cells were left on ice to lyse for 1 hour before being centrifuged at 12,000rpm for 12

8
minutes at 4°C. Protein concentration was determined by mixing a 0.5% sample
protein and water solution with BioRad Protein Assay dye and leaving for 1 hour.
A BioRad Microassay Spectrophotometer measured absorbance and compared to
known standards, allowing samples at 1𝜇g/𝜇l of protein to be generated. Western
blot analysis used 12% SDS-PAGE with 35𝜇g of sample/well with antibodies at
stated concentrations (table 1).
Immunofluorescence Microscopy
Cells grown until 70% confluent on 0.17mm glass coverslips. Cells were fixed
in 3.7% (v/v) formaldehyde for 15 minutes, washed in phosphate buffered saline
(PBS) for 10 minutes and permeabilised in 1% BSA, 0.4% saponin in PBS for 15
minutes. Coverslips were blocked using 10% (v/v) normal goat serum, incubated in
primary antibody (table 1) for 1 hour at room temperature, washed with PBS and
incubated in secondary antibody (table 1) in a dark wet chamber for 30 minutes at
room temperature. Coverslips were treated with Vectashield mounting medium with
4,6-diamino-2-phenylindole (DAPI) and viewed on a Leica RPE automatic
microscope using a x63 oil immersion lens. All images were processed with one level
of deconvolution.
Computer Analysis of the ZIP10 sequence
The program ‘Compute pI/Mw’ predicted protein weights (Artimo et al.
2012), the program ‘epestfind’ located potential pest sites (Artimo et al. 2012) and
potential ZIP10 phosphorylation sites were identified by the ELM (Dinkel et al.
2014), NetPhorest (Horn et al. 2014), PHOSIDA (Gnad et al. 2007) and Phosphonet
(Safaei et al. 2011) online databases. The ELM database was also used to identify
potential cleavage sites.
Statistics
Comparisons between means were analysed using a paired two-tailed t-test
(where n=2). All means are compared to a control sample. Statistical significance
was assumed if p<0.05. Values are presented as means of three repeats ± standard
error of the mean (SEM).

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Table 1: Antibodies used for western blot and immunofluorescence
microscopy
Antibody Species
Dilution
Supplier/ ID
IF WB
ZIP10AL Rabbit 1:100 1:1000 Abcam/ 83947
ZIP10HN Rabbit 1:100 1:1000
Sigma-Aldrich/
HPA036512
ZIP10SL Mouse 1:100 1:1000
Sigma-Aldrich/
SAB1401780
ZIP10BN Rabbit 1:100 1:1000 Biogene/ Custom Made
pS10 Histone H3 Rabbit 1:500 1:1000 Cell Signalling/ 3377S
pS10 Histone H3 Mouse 1:500 1:1000 Cell Signalling/ 97062
Cyclin E Rabbit - 1:1000
Santa Cruz
Biotechnology/ sc481
#E1413
Cyclin A Mouse 1:100 1:1000
Santa Cruz
Biotechnology/
sc271682
Cyclin B1 Mouse 1:100 1:1000
Santa Cruz
Biotechnology/ sc245
#F0313
V5 Mouse 1:100 - Invitrogen/ 46 0705
β-actin HRP-
conjugated
Mouse - 1:50000 Cell Signalling/ 5125
Anti-mouse IgG
HRP-
conjugated
Mouse - 1:10000
GE Healthcare/
NZA931
Anti-rabbit IgG
HRP-
conjugated
Rabbit - 1:10000 Cell Signalling/ 7074
Anti-mouse
conjugated
Alexa Fluor 488
Goat 1:1000 - Invitrogen/ A-11001
Anti-mouse
conjugated
Alexa Fluor 594
Anti-rabbit
conjugated
Alexa Fluor 488
Anti-rabbit
conjugated
Alexa Fluor 594
Goat 1:1000 - Invitrogen/ 11037
IF: immunofluorescence microscopy. WB: western blot.

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Results
Characterisation of Cell Cycle Samples
MCF-7 cell samples synchronised by serum-withdrawal and nocodazole
treatment were probed by cell cycle marker antibodies using western blot to
investigate the cell cycle stage each sample was in.
Characterisation of Serum-deprived MCF-7 cells
Cell cycle samples were probed with cyclin E (S phase cyclin), cyclin A (G2
phase cyclin), cyclin B1 (G2/M phase cyclin) and pS10 Histone H3 (mitosis marker)
antibodies to confirm harvesting time points were accurate (figure 2). This
experiment confirmed that certain time points corresponded to specific stages of the
cell cycle, thus in later experiments a single sample could be used to represent each
stage of the cell cycle.
Characterisation of Nocodazole-treated MCF-7 cells
Nocodazole-treated samples were probed with the pS10 Histone H3 antibody
to determine the levels of mitotic cells (figure 3). This experiment showed increased
levels of mitotic cells, indicating that samples had been synchronised to mitosis and
that samples harvested after nocodazole withdrawal were entering G1 phase.
Characterisation of ZIP10 antibodies by Western Blot
To investigate ZIP10 cleavage thoroughly four antibodies with epitopes in
different regions of the protein were used, ensuring any ZIP10 fragments generated
would be identified. The location of the antibodies can be seen in figure 4A, as well
as the most likely cleavage sites; a pest site (peptide sequences high in proline,
glutamic acid, serine and threonine linked to enhanced protein degradation (Reverte
et al. 2001)) located in the middle of the N-terminus and the site of ectodomain
shedding proposed by Ehsani et al. (2012). The antibodies are named after the
protein they bind to, ZIP10, with the two letters in superscript denoting the
company that made them and whether they bind the N-terminus or large
intracellular loop (figure 4B). The antibody epitopes are usefully located: at the far

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Figure 2: Characterisation of Cell Cycle samples
After 24 hours of serum-starvation serum was added back to cell medium to allow
the now synchronised cells to grow before being harvested at the indicated time
points. (A) Membranes were probed for pS10 Histone H3 and Cyclin E, A and B1
antibodies in samples at different cell cycle stages. Protein loading measured by β-
actin. (B) Densitometry of the protein bands generated by each antibody
normalised to β-actin with the stage of the cell cycle of each sample indicated.
0
0.5
1
1.5
2
2.5
3
3.5
4
6hr 7hr 18hr 19hr 27hr 28hr 29hr 30hr 31hr 32hr
Densitometryunits
Cyclin E - S phase Cyclin A - G2 phase
Cyclin B1 - G2/M phase H3 - M phase
G1
phase
S phase G2 phase M phase
A
B

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Figure 3: Characterisation of Nocodazole-treated MCF-7 cells
MCF-7 cells were nocodazole-treated for 20 hours followed by nocodazole
withdrawal for the time indicated. (A) The level of mitotic cells in each sample was
judged by activation of pS10 Histone H3 and protein loading was assessed by β-
actin. (B) Bands quantified by densitometry normalised to β-actin and shown
normalised to control ± SEM of three repeats. A paired t-test was conducted,
significance indicated by * (p<0.05), ** (p<0.01).
Nocodazole
addition (hrs)
- 20 20 2020 20
Nocodazole
withdrawal (hrs)
- - 1 32 4
𝛽-actin
pS
10
Histone H3
- 45kDa
- 17kDa
0
0.5
1
1.5
2
2.5
Densitometryunits
**
Nocodazole
addition (hrs)
- 20 20 2020 20
Nocodazole
withdrawal (hrs)
- - 1 32 4
**
A
B

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Figure 4: Diagram of the location of the ZIP10 antibody epitopes and
cleavage sites
Antibody Epitope Location Predicted Band Size (kDa)
ZIP10BN L46 – E59 N 25, 47, 94
ZIP10HN E146 – P254 N 47, 94
ZIP10AL C514 – W530 LIL 47, 69, 94
ZIP10SL C514 – L622 LIL 47, 69, 94
Schematic diagram of the secondary structure of ZIP10 showing the site of
ectodomain shedding, the location of a pest site on the N-terminus and the epitopes
of all used ZIP10 antibodies, with a table of all possible fragments and their
predicted molecular weights. (A) Antibody epitopes are indicated by coloured boxes.
(B) Table of antibodies with colours corresponding to the colour used in (A)
accompanied by epitope, location and the bands sizes the antibody was predicted to
detect. N: N-terminus. LIL: Large Intracellular Loop.
A
B

14
end of the N-terminus is ZIP10BN, which should identify N-terminal fragments; in
the middle of the N-terminus located over the pest site is ZIP10HN, which should
also identify N-terminal fragments; at the start of the large intracellular loop is
ZIP10AL which should identify any C-terminal fragments; and along half the large
intracellular loop is ZIP10SL, which should also bind C-terminal fragments. Using
the knowledge of the location of cleavage sites and antibody epitopes, it was possible
to predict the size of the fragments which the antibodies should identify when used
in western blot (figure 4B).
Characterisation of Large Intracellular Loop antibodies
MCF-7 cells synchronised to different stages of the cell cycle were analysed
by western blot, with probing by the large intracellular loop antibodies predicted to
detect 47, 69 and 94kDa bands, with the appearance of the small bands confirming
that ZIP10 is cleaved on the N-terminus.
Probing with the ZIP10AL antibody generated a single protein band of
44kDa (figure 5A), a size consistent with a N-terminus cleaved via ectodomain
shedding. Representative bands (figure 5B) were analysed by densitometry,
revealing that the highest (although not significantly high) levels of the fragment
were found during G1 and M phase (figure 5C), suggesting that ZIP10 is cleaved
during mitosis and G1 phase.
Probing by the ZIP10SL antibody produced a single band of 63kDa (figure
6A), an expected band size consistent with half the N-terminus and the entire C-
terminus. As with the previous 44kDa band, densitometry analysis of representative
bands (figure 6B) revealed that the highest (although again not significantly high)
levels were found in the G1 and M phase samples (figure 6C), suggesting that
cleavage at the pest site within the N-terminus also occurs during mitosis and G1.
Characterisation of N-terminal antibodies
MCF-7 cells synchronised to different stages of the cell cycle and probed by
western blot with the N-terminal antibodies were predicted to identify 25, 47 and
94kDa bands, with the appearance of the small bands also indicating that ZIP10 is
cleaved on the N-terminus.

15
Figure 5: Characterisation of the ZIP10AL
antibody by western blot
After serum-deprivation and re-addition selected cell cycle and 0hr samples were
analysed by western blot and probed for ZIP10 by ZIP10AL. (A) Representative blot
showing that only a ZIP10 band at 44kDa was detected. (B) Blot showing protein
bands selected to represent each stage of the cell cycle, with each band taken from
the same larger membrane shown in (A). (C) Ratio of protein banding to 𝛽-actin
quantified by densitometry and shown normalised to 0hr ± SEM of three repeats. A
paired t-test was conducted, significance was not seen (all results p>0.05).
0
0.5
1
1.5
2
2.5
3
3.5
4
0hr G1 S G2 M
Densitometryunits
Cell Cycle Samples
70kDa -
50kDa -
37kDa -
25kDa -
- 44kDa ZIP10
AL
Addition of
Serum (hrs)
0 6 7 8 17 18 25 26 27 28 29 30
- 45kDa 𝛽-actin
𝛽-actin
ZIP10
AL
0hr G1 S G2 MSample
- 44kDa
- 45kDa
B
C
A

16
Figure 6: Characterisation of the ZIP10SL
analysed by western blot and probed for ZIP10 by ZIP10SL. (A) Blot showing that
only a ZIP10 band at 63kDa was detected. (B) Representative 63kDa bands, each
from the same set of samples cut from a larger membrane, 0hr band from different
membrane. Protein loading assessed by 𝛽-actin. (C) Densitometry of protein
banding normalised to 𝛽-actin and shown normalised to 0hr ± SEM of three repeats.
A paired t-test was conducted, significance was not seen (all results p>0.05).
0
1
2
3
4
5
0hr G1 S G2 M
Densitometryunits
Cell Cycle Samples
100kDa -
70kDa -
37kDa -
50kDa - - 63kDa ZIP10
SL
Addition of
Serum (hrs)
6 7 8 18 19 27 28 29 30 31
- 45kDa 𝛽-actin
25kDa -
𝛽-actin
ZIP10
SL
Sample 0hr G1 S G2 M
- 63kDa
- 45kDa
A
B
C

17
Probing by the ZIP10HN antibody produced ZIP10 bands at 26kDa and
54kDa (figure 7A). The epitope of the ZIP10HN antibody straddles a potential pest
site within the N-terminus, thus may bind both or a single half of the N-terminus if
cleavage occurs at the pest site. The 26kDa band corresponds to half of the N-
terminus, likely the N-terminal half as the half proximal to the C-terminal fragment
has a predicted size of 22kDa, supporting the hypothesis that cleavage at the pest
site occurs. However, it could be the proximal half as Ehsani et al. (2012) have
shown that the N-terminus undergoes four glycosylations which the size predictions
do not take into account, causing an underestimation of the fragment size.
Glycosylation is also likely the reason why the other 54kDa fragment is larger than
47kDa, the predicted size of the full N-terminus. Representative bands (figure 7B/C)
were quantified by densitometry with significantly high levels seen in the G1 and M
phase samples (figure 7D).
Probing by the ZIP10BN antibody identified multiple bands over a vast
range of sizes (figure 8A). By probing samples again with the pre-immune serum of
the ZIP10BN antibody, any pre-existing proteins in the sample prior to the
generation of the ZIP10BN antibody bound its corresponding protein, thus revealing
the 37kDa and 45kDa bands as false positive ZIP10 bands (figure 8B). They were
discounted in all further ZIP10BN western blots, allowing designation of the 26kDa
and 68kDa bands as true ZIP10 bands (figure 9A). Representative bands were
quantified by densitometry (figure 9B/C), with both showing high levels in G1
phase, as well as in G2 and M phase for the 26kDa and 68kDa bands respectively
(figure 9D). The appearance of the 26kDa band supports the data generated by the
ZIP10HN antibody, indicating both antibodies may be binding to the N-terminal
half of the N-terminus. The 68kDa band is more perplexing however, as a fragment
of this size was not predicted for the N-terminal antibodies, indicating cleavage of
ZIP10 may occur in the large intracellular loop.
ZIP10 is cleaved into multiple fragments during the cell cycle
These results have identified various bands of different sizes, with some
corresponding to predicted fragments whereas others were unexpected (band sizes
summarised in table 2). The identification of the 44kDa C-terminal and 54kDa

18
Figure 7: Characterisation of the ZIP10HN
analysed by western blot and probed for ZIP10 by ZIP10HN. (A) Full membrane
showing ZIP10 banding at both 26kDa and 54kDa. (B/C) Representative banding
for each protein band. Each presented band from the same set of samples cut from
a larger membrane, 0hr band was from a different membrane. Protein loading
assessed by 𝛽-actin. (D) Ratio of protein banding to 𝛽-actin quantified by
densitometry and shown normalised to 0hr ± SEM of two (for 26kDa bands) or three
(for 54kDa bands) repeats. A paired t-test was conducted, significance indicated by
* (p<0.05).
0
2
4
6
8
0hr G1 S G2 M
Densitometryunits
Cell Cycle Samples
54kDa
70kDa -
50kDa -
37kDa -
25kDa - - 26kDa ZIP10
HN
Addition of
Serum (hrs)
0 6 7 18 19 27 28 29 30 31 32
- 45kDa 𝛽-actin
- 54kDa ZIP10
HN
100kDa -
150kDa -
G1 S G2 M0hr
𝛽-actin
26kDa
Sample
𝛽-actin
54kDa
G1 S G2 M0hrSample
0
20
40
60
80
0hr G1 S G2 M
Densitometryunits
Cell Cycle Samples
26kDa
*
A
B C
D

19
Figure 8: Characterisation of the ZIP10BN
After 24 hours of serum withdrawal, serum was added back for a different number
of hours for each sample, as indicated. These samples were then probed with
ZIP10BN and the pre-immune serum of the ZIP10BN antibody. (A) Full blot showing
all the main bands identified by the ZIP10BN antibody. Protein loading assessed by
𝛽-actin. (B) Membranes were also incubated in the pre-immune serum of the
ZIP10BN antibody to identify any non-ZIP10 bands. Protein loading assessed by 𝛽-
actin.
ZIP10BN antibody
- 45kDa 𝛽-actin
37kDa -
25kDa -
20kDa -
15kDa -
Addition of
Serum (hrs)
6 7 18 19 27 28 29 30 31 32
50kDa -
70kDa -
100kDa -
- 26kDa
- 68kDa
- 45kDa
- 37kDa
ZIP10BN Pre-immune serum
37kDa -
25kDa -
20kDa -
15kDa -
Addition of
Serum (hrs)
6 7 18 19 27 28 29 30 31 32
- 45kDa 𝛽-actin
50kDa -
70kDa -
100kDa -
- 45kDa
- 37kDa
A
B

20
Figure 9: ZIP10 probed by ZIP10BN
through the cell cycle
Selected cell cycle and 0hr samples were analysed by western blot and probed for
ZIP10 by ZIP10BN. (A) Full blot with true ZIP10 bands labelled. (B/C)
Representative banding for each protein band. Each presented band from the same
set of samples cut from a larger membrane, 0hr band from different membrane.
Protein loading assessed by 𝛽-actin. (C) Ratio of protein banding to 𝛽-actin
quantified by densitometry and shown normalised to 0hr ± SEM of three repeats. A
paired t-test was conducted, significance indicated by * (p<0.05).
0
2
4
6
8
10
0hr G1 S G2 M
Densitometryunits
Cell Cycle Samples
26kDa
- 45kDa 𝛽-actin
37kDa -
25kDa -
20kDa -
15kDa -
Addition of
Serum (hrs)
6 7 18 19 27 28 29 30 31 32
50kDa -
70kDa -
100kDa -
- 26kDa ZIP10
BN
- 68kDa ZIP10
BN
0
1
2
3
4
5
0hr G1 S G2 M
Densitometryunits
Cell Cycle Samples
68kDa
*
B C
D
*
A

21
Table 2: List of ZIP10 antibodies with predicted and observed band sizes
Antibody Location Predicted Band Size (kDa) Observed Band Size (kDa)
ZIP10BN N 25, 47, 94 26, 68
ZIP10HN N 47, 94 25, 54
ZIP10AL LIL 47, 69, 94 44
ZIP10SL LIL 47, 69, 94 63
Table of ZIP10 antibodies accompanied by epitope location and the predicted and
observed bands sizes of each antibody. N: N-terminus. LIL: Large Intracellular
Loop.
N-terminal fragments clearly indicate that N-terminal cleavage occurs, and that
these fragments were both at their highest levels during mitosis and G1 phase
indicates that the cleavage is cell-cycle-dependent. The predicted pest site cleavage
is also heavily supported as the 26kDa band detected by the N-terminal antibodies
can only be generated by pest site cleavage. Identification of 26kDa and 63kDa
fragments shows that cleavage at the pest site can occur both before and after full
N-terminal cleavage, so whether this cleavage has an active role is unclear. Possibly
the N-terminus is first cleaved at the pest site and then cleaved again to activate
ZIP10 fully, as the 26kDa fragment detected by ZIP10BN does show increased
amounts (although not statically high levels) of the fragment during G2 phase. The
ZIP10BN antibody also identified a 68kDa fragment, the highest amounts of which
were found during mitosis. However, a fragment of this size indicates that ZIP10 is
also cleaved at the large intracellular loop within the C-terminus. There are
potential pest and cleavage sites within the large intracellular loop (all potential
cleavage sites summarised in table 3), although the ‘epestfind’ program scores the
pest sites as less likely to actually be there compared to the site on the N-terminus.
Furthermore, a cleavage within the large intracellular loop, the active site of the
transporter, is especially unlikely as the cleavage would have to occur before any N-
terminal cleavage and thus before the transporter had been activated. This may
instead be a symptom of a limitation of this antibody in western blot, as due to the
very high number of bands a false positive may have been used despite attempts to
eliminate them.

22
Taken together these results provide strong evidence that ZIP10 is cleaved
in a cell-cycle-dependent manner during mitosis and G1 phase. This indicates that
ZIP10 may have some role in mitosis, so to explore this further MCF-7 cells were
treated with nocodazole, an agent which inhibits microtubule polymerisation (Head
et al. 1985), synchronising cells in mitosis and allowing a more detailed assessment
of the levels of ZIP10 in and directly after mitosis.
Table 3: List of all Potential Cleavage Sites
Position Domain Potential Cleavage Protein
25-26 N Signal Peptidase Complex
41-45 N SKI1
84-88 N SKI1
93-95 N N-Arginine Dibasic Convertase
148-150 N NEC1/NEC2
206-219 N Pest Site
232-234 N NEC1/NEC2
306-312 N Proprotein convertase 7
310-312 N NEC1/NEC2
~405 N Ectodomain Shedding Site
516-520 LIL SKI1
542-544 LIL N-Arginine Dibasic Convertase
576-594 LIL Pest Site
562-576 LIL Pest Site
608-612 LIL Caspase-3
608-612 LIL Caspase-7
633-637 LIL SKI1
636-638 LIL N-Arginine Dibasic Convertase
824-828 C SKI1
A list of all cleavage sites identified by computer analysis of the ZIP10 sequence,
listed in order of position accompanied by the domain of ZIP10 in which they occur
and the potential cleavage protein if known. Pest sites in bold. N: N-terminus. LIL:
large intracellular loop. C: C-terminus.

23
The Characterisation of ZIP10 in Nocodazole-treated MCF-7 cells
Having established that ZIP10 is cleaved during mitosis and G1 phase, MCF-
7 cells were treated with nocodazole for 20 hours followed by growth in normal
medium for 0-4 hours before harvesting, followed by western blot analysis using the
ZIP10BN, ZIP10HN, ZIP10AL and ZIP10SL antibodies. Nocodazole inhibits
microtubule polymerisation, thus affected cells cannot form mitotic spindles
(Akhshi et al. 2014) causing arrest in pro-metaphase. Since G1 phase begins after
mitosis, cells synchronised in this way allow investigation of ZIP10 activity during
and directly after mitosis leading into G1 phase.
ZIP10 levels decrease after mitosis in Nocodazole-treated cells
Probing with the N-terminal antibodies ZIP10BN and ZIP10HN revealed that
the levels of N-terminal ZIP10 fragments decreased. The 26kDa band of ZIP10BN
(figure 10A) and 54kDa band of ZIP10HN (figure 10B) were quantified by
densitometry, with both bands displaying downward trends in the ZIP10 levels in
the hours after mitosis, as well as indicating the level of ZIP10 in the control samples
was equal to or greater than the amount in mitotic samples (figure 10C),
contradicting the previous western blot results.
Probing with the intracellular loop antibodies ZIP10AL and ZIP10SL showed
that the levels of C-terminal ZIP10 fragments stayed relatively static, with no
significant difference in ZIP10 between any of the samples. The 44kDa band of
ZIP10AL (figure 11A) and 63kDa band of ZIP10SL (figure 11B) were quantified by
densitometry, with both showing slight upward trends in ZIP10 levels and control
samples also having similar or only slightly smaller levels of ZIP10 than in mitotic
samples (figure 11C), also contradicting the previous results.
Despite differing from the previous western blot results which indicated
ZIP10 levels were high in G1 phase, these results do still support the hypothesis that
ZIP10 is N-terminally cleaved. Furthermore, the downward trend in the N-terminal
fragment levels after mitosis suggests that after ZIP10 activation the cleaved N-
terminus is quickly degraded. The location of a pest site within the N-terminus
supports this, as proteins containing pest sites usually undergo rapid degradation
(Reverte et al. 2001). That the levels of the 44kDa fragment do not significantly

24
Figure 10: Investigation of N-terminal cleavage in Nocodazole-treated
MCF-7 cells
MCF-7 cells were treated with nocodazole for 20 hours before being harvested after
the indicated number of hours. Non-nocodazole-treated cells were used as control.
Samples were then analysed by western blot and probed for ZIP10 by ZIP10BN and
ZIP10HN. (A/B) Representative membranes of ZIP10 probed by ZIP10BN and
ZIP10HN showing 26kDa and 54kDa bands respectively. Protein loading assessed
by 𝛽-actin. (C) Ratio of protein banding to 𝛽-actin quantified by densitometry and
shown normalised to control ± SEM of three repeats. A paired t-test was conducted,
significance indicated by * (p<0.05), ** (p<0.01).
𝛽-actin
26kDa
Nocodazole
addition (hrs)
Nocodazole
withdrawal (hrs)
54kDa
𝛽-actin
20 20 20 20 20-
- 1 2 3 4-
ZIP10
BN
ZIP10
HN
20 20 20 20 20-
- 1 2 3 4-
0
0.2
0.4
0.6
0.8
1
1.2
Densitometryunits
ZIP10HN – 54kDa band
0
0.2
0.4
0.6
0.8
1
1.2
Densitometryunits
ZIP10BN – 26kDa band
*
** **
*
Nocodazole
addition (hrs)
Nocodazole
withdrawal (hrs)
20 20 20 20 20-
- 1 2 3 4-
*
*
*
20 20 20 20 20-
- 1 2 3 4-
A B
C

25
Figure 11: Investigation of ZIP10 cleavage by intracellular loop
antibodies in Nocodazole-treated MCF-7 cells
MCF-7 cells were treated with nocodazole for 20 hours before being harvested after
the indicated number of hours. Non-nocodazole-treated cells were used as control.
Samples were then analysed by western blot and probed for ZIP10 by ZIP10AL and
ZIP10SL. (A/B) Representative membranes of ZIP10 probed by ZIP10AL and
ZIP10SL showing 44kDa and 63kDa bands respectively. Protein loading assessed by
𝛽-actin. (C) Ratio of protein banding to 𝛽-actin quantified by densitometry and
shown normalised to control ± SEM of three repeats. A paired t-test was conducted,
significance was not seen (all results p>0.05).
𝛽-actin
63kDa
ZIP10
AL
ZIP10
SL
𝛽-actin
44kDa
Nocodazole
addition (hrs)
Nocodazole
withdrawal (hrs)
20 20 20 20 20-
- 1 2 3 4-
20 20 20 20 20-
- 1 2 3 4-
0
0.5
1
1.5
2
2.5
Densitometryunits
ZIP10AL - 44kDa band
0
0.5
1
1.5
2
2.5
3
Densitometryunits
ZIP10SL - 63kDa band
Nocodazole
addition (hrs)
Nocodazole
withdrawal (hrs)
20 20 20 20 20-
- 1 2 3 4-
20 20 20 20 20-
- 1 2 3 4-
A B
C

26
change indicates that the activated C-terminal fragment of ZIP10 remains in the
plasma membrane, mediating zinc influx during mitosis and G1 phase. Taken
together this suggests that ZIP10 activated during mitosis is either degraded, if a
N-terminal fragment, or remains in the plasma membrane, if a C-terminal fragment.
ZIP10 expression may then increase again during G1 phase to mediate the zinc
influx identified by Li and Maret (2009), explaining the high levels of ZIP10 seen in
G1 phase for the cell cycle samples.

27
Characterisation of ZIP10 expression using Fluorescence Microscopy
An investigation by fluorescence microscopy of ZIP10 expression through
the cell cycle in MCF-7 cells transfected with wild-type ZIP10 containing a V5-tag
on the C-terminus and MCF-7 cells treated with nocodazole for 20 hours was
undertaken. This allowed analysis of both when during the cell cycle as well as where
in the cell ZIP10 was expressed. Staining with a V5 antibody detected any full
length or C-terminal ZIP10 fragments, allowing identification of the location of
active ZIP10. The same four ZIP10 antibodies used for western blot; ZIP10BN,
ZIP10HN, ZIP10AL and ZIP10SL, were used again to reveal any disparity in cell cycle
expression or location of specific ZIP10 fragments.
Characterisation of ZIP10 antibodies using Fluorescence Microscopy
Staining with the V5 antibody shows ZIP10 is only expressed on the plasma
membrane (figure 12). To ensure the ZIP10 antibodies were detecting ZIP10 they
were co-stained with the V5 antibody in transfected cells, with only those that co-
localised identifying ZIP10. Only the ZIP10BN antibody co-localised with the V5
antibody on the plasma membrane (figure 12A), with the ZIP10AL and ZIP10HN
antibodies both failing to co-localise (figure 12B/C), suggesting the ZIP10BN
antibody may be the only functional ZIP10 antibody for use in fluorescence
microscopy. However, all identified transfected cells were in interphase, so to
investigate ZIP10 expression in mitosis nocodazole-treated cells were stained with
cell cycle marker and ZIP10 antibodies.
Through co-staining the ZIP10AL (figure 13A) and ZIP10BN (figure 13B/C)
antibodies with cyclin B1 (activation required for G2/M transition (Maller et al.
1989)) and pS10 Histone H3 (activation essential for mitosis (Crosio et al. 2002)) it
was found that both bound ZIP10 on the plasma membrane of mitotic cells.
Staining in non-mitotic cells by ZIP10BN (figure 13C) indicates ZIP10 may also have
a role during interphase, possibly G1 or G2 phase as indicated by the western blot
results.

28
Figure 12: ZIP10 is expressed on the plasma membrane
Fluorescence microscopy images showing MCF-7 cells transfected with wild-type
ZIP10 before staining with a ZIP10 antibody conjugated to Alexa Fluor 594 (red)
and a V5 antibody conjugated to Alexa Fluor 488 (green), with nuclei
counterstained with DAPI (blue). (A) The ZIP10BN antibody co-localised with the
V5 antibody on the plasma membrane, showing that the ZIP10BN antibody works.
(B/C) The ZIP10AL and ZIP10HN antibodies both did not co-localise with the V5
antibody, indicating they may not be useful for fluorescence microscopy. (A/B/C)
All show V5 only binds the plasma membrane of cells.
DAPI ZIP10 Antibody V5 Antibody Merge
A
B
C
ZIP10BN
ZIP10AL
ZIP10HN

29
Figure 13: ZIP10 is expressed strongly in Mitotic cells
Fluorescence microscopy images showing MCF-7 cells treated with nocodazole for
20 hours before co-staining with a cell cycle marker antibody conjugated to Alexa
Fluor 594 (red) and a ZIP10 antibody conjugated to Alexa Fluor 488 (green), with
nuclei counterstained with DAPI (blue). White arrows indicate mitotic cells, orange
arrows indicate cells in G2 phase. (A) The ZIP10AL antibody only co-localised with
the cyclin B1 (G2/M phase cyclin) antibody in mitotic cells, with the ZIP10AL
antibody exhibiting perinuclear staining in some non-mitotic cells. (B) The ZIP10BN
antibody co-localised with the cyclin B1 antibody, staining all three cells, with the
bottom two undergoing mitosis and the third just entering prophase. (C) Clearly
displays the co-staining of the mitotic cells by the pS10 Histone H3 and ZIP10BN
antibodies. The ZIP10BN antibody also seems to stain some other non-mitotic cells,
DAPI Cell Cycle
Marker
ZIP10 Antibody Merge
B
C
Cyclin B1
pS10 Histone H3
ZIP10BN
ZIP10BN
A Cyclin B1 ZIP10AL
Cell Cycle Marker

30
ZIP10HN staining shows that it cannot bind ZIP10 on the plasma membrane,
however it does bind to the nuclear membrane of all interphase cells (figure 14).
Binding of the nuclear membrane is a phenomenon common to all four ZIP10
antibodies, and since ZIP10 is never expressed on the nuclear membrane this
binding is extremely unlikely to be a true positive, indicating instead that the
antibodies are binding other proteins localised to the nuclear membrane.
Finally, staining with the ZIP10SL antibody clearly shows that in no cells,
mitotic or otherwise, does ZIP10SL bind the plasma membrane, or indeed anything
strongly (figure 15). This indicates that for use in fluorescence microscopy the
ZIP10SL antibody is not functional.
ZIP10 is cleaved on the Plasma Membrane
As well as proving that ZIP10 is expressed on the plasma membrane, the co-
staining of the V5 and ZIP10BN antibodies also supports the hypothesis that N-
terminal cleavage of ZIP10 occurs on the plasma membrane as opposed to the
endoplasmic reticulum. Due to the location of the ZIP10BN epitope at the far end of
the N-terminus if any cleavage occurred the fragment excised would contain the
epitope of the ZIP10BN antibody. Staining with the ZIP10BN antibody only
identifies ZIP10 on the plasma membrane, therefore full length ZIP10 is expressed
on the plasma membrane.
ZIP10 is present on the Plasma Membrane of non-mitotic cells transfected with
wild-type ZIP10
That only the N-terminal antibody ZIP10BN bound the transfected ZIP10
whilst the other N-terminal antibody ZIP10HN and the intracellular loop antibody
ZIP10AL did not (figure 12), despite ZIP10AL binding endogenous ZIP10 in mitotic
cells (figure 13A), indicates that there is either a disparity between the transfected
and endogenous ZIP10 or between ZIP10 in mitotic and non-mitotic cells. The
likelihood of a difference between transfected and endogenous ZIP10 is very low,
however there is a possible explanation for why ZIP10AL would bind ZIP10 in
mitotic but not non-mitotic cells. If ZIP10 is activated during mitosis a change in

31
Figure 14: the N-terminal antibody ZIP10HN
does not bind ZIP10
20 hours before staining with a cell cycle marker antibody conjugated to Alexa Fluor
594 (red) and the ZIP10HN antibody conjugated to Alexa Fluor 488 (green), with
nuclei counterstained with DAPI (blue). White arrows indicate mitotic cells. (A)
The cyclin A (G2 phase cyclin) antibody does not significantly co-stain with the
ZIP10HN antibody, as the antibody bound the nuclear membrane of most interphase
cells. (B) Shows that ZIP10HN does not stain mitotic cells, with no co-localisation
with the pS10 Histone H3 antibody at all.
DAPI Cell Cycle
Marker
ZIP10 Antibody Merge
A Cyclin A ZIP10HN
B pS10 Histone H3 ZIP10HN
Cell Cycle Marker

32
Figure 15: the Large Intracellular Loop antibody ZIP10SL
does not bind
ZIP10
20 hours before staining for pS10 Histone H3 conjugated to Alexa Fluor 488 (green)
and ZIP10SL conjugated to Alexa Fluor 594 (red), with nuclei counterstained with
DAPI (blue). The merged image shows only weak and negligible staining of the
nuclear membrane for ZIP10SL, with no staining of mitotic cells.
DAPI pS10 Histone H3
ZIP10SL Merge

33
the conformation of the large intracellular loop, the site of zinc transport, would be
expected, thus the epitope of the ZIP10AL antibody may only be available in the
activated form of ZIP10. Since transfection dramatically increases the basal
expression of the target protein, ZIP10 would have likely been expressed on the
plasma membrane in its non-active form, so the epitope for ZIP10AL may have been
unavailable. That the ZIP10BN antibody was able to bind regardless may be due to
its small epitope and location at the far end of the N-terminus, making it less likely
to become unavailable. A similar explanation can be used to explain why the
ZIP10SL antibody was unable to identify ZIP10 but did identify a ZIP10 fragment
during western blot. Western blot samples are protein solutions and therefore whilst
the epitope of ZIP10SL may have been unavailable in a cell due to the conformation
of ZIP10 or interference by the plasma membrane or other proteins, in solution all
such barriers have been removed and the ZIP10SL antibody would be able to bind
its epitope. Overall this data provides evidence that ZIP10 expression increases and
ZIP10 undergoes conformational change when the cell enters mitosis, supporting
the hypothesis that ZIP10 is involved in mitosis.
Endogenous ZIP10 is present on the Plasma Membrane of Mitotic cells
MCF-7 cells were treated with nocodazole for 20 hours, causing
approximately 20% of all cells to arrest in mitosis, aiding in the visualisation of
ZIP10 in mitotic cells. As can be seen in figure 13 both ZIP10AL and ZIP10BN bind
the plasma membrane of mitotic cells, supporting previous western blot results
showing increased expression ZIP10 during mitosis. Figure 13C also shows that
ZIP10BN bound the plasma membrane of interphase cells, while ZIP10AL only bound
the nuclear membrane (figure 13A). It is interesting to note that ZIP10BN binds the
plasma membrane of G2 cells whereas ZIP10HN does not, and that through western
blot ZIP10BN identified a 26kDa ZIP10 fragment with increased levels during G2
phase whereas ZIP10HN identified a 26kDa fragment with increased levels in mitosis
but not in G2 phase. This indicates that either the antibodies are binding different
halves of the N-terminal fragment in western blot, or that in cells the conformation
of the N-terminus inhibits the binding of the ZIP10HN antibody.

34
Through observation by fluorescent microscopy this study has clearly shown
that ZIP10 is present on the plasma membrane of G2 phase and mitotic MCF-7 cells.
Coupled with the finding through western blot that various ZIP10 fragments have
increased expression during mitosis, this data provides strong evidence that ZIP10
expression increases during mitosis.

35
Discussion
A new role for ZIP10 during Mitosis
This study provides novel evidence for the cell-cycle-dependent activation of
ZIP10 by N-terminal cleavage during mitosis. We propose a dual pathway (figure
16), with full length ZIP10 expressed on the plasma membrane before the N-
terminus of some is cleaved at the pest site during G2 phase, producing a 26kDa
fragment and reducing the remaining ZIP10 protein to 63kDa, followed by
ectodomain shedding of the remaining N-terminus during mitosis, leading to
activation. This is concurrent with some ZIP10 progressing to mitosis without being
cleaved at the N-terminal pest site during G2, and instead undergoing cleavage at
both the pest site and site of ectodomain shedding during mitosis, generating the far
N-terminal 26kDa fragment, the proximal N-terminal (26kDa if also identified by
the ZIP10HN antibody, otherwise predicted to be 22kDa) fragment and the full N-
terminal 54kDa fragment, reducing the remaining ZIP10 protein to 44kDa and
leading to activation.
ZIP10 in Cancer
ZIP10 is known to be involved in a number of cancers, from promoting
migration in breast cancer (Takatani-Nakase et al. 2014) to increased expression in
aggressive renal cell carcinoma (Pal et al. 2014). Similarly it has been shown that
disruption of the function of ZIP10 inhibits cell cycle progression (Kong et al. 2014),
however how the function and regulation of ZIP10 relates to mitosis has not been
investigated in any detail. The findings of this study show that in MCF-7 breast
cancer cells multiple cleavages of ZIP10 occur, that these cleavages are cell cycle-
dependent, that ZIP10 expression is increased during mitosis and G1 phase and that
ZIP10 is expressed on the plasma membrane, supporting the hypothesis that
activation of ZIP10 in the plasma membrane of mitotic cells by N-terminal
cleavage, which facilitates the influx of zinc into the cell and increases survival and
proliferation of the MCF-7 breast cancer cells (Hershfinkel et al. 2007).

36
Figure 16: ZIP10 through the cell cycle
Flow diagram of ZIP10 cleavage and activation through the cell cycle. Scissors
indicate cleavage occurs at this site. Arrows indicate the passage of time through
the cell cycle. The exact size of the proximal N-terminal fragment is unclear, as it is
unknown whether the ZIP10HN antibody identified it as 26kDa or whether ZIP10HN
was only binding the far N-terminal fragment.
Full length
ZIP10
26kDa
Fragment
26kDa
Fragment
54kDa
Fragment
63kDa
Fragment
???kDa
Fragment
Active
44kDa
Fragment
Full length
ZIP10 is
expressed on
the Plasma
Membrane
G2 Phase
-
ZIP10 is
cleaved at the
Pest Site
Mitosis and
G1 phase
-
Activation of
ZIP10

37
Similarities between ZIP10 and other ZIP transporters in the Liv-1 sub-family
Kambe and Andrews (2009) have show that ZIP4 is localised to the plasma
membrane before activation by proteolytic cleavage of the entire N-terminus,
similarly to the N-terminal cleavage of ZIP10 proposed by figure 16.
Research has shown that ZIP6 is also involved in breast cancer progression.
In MCF-7 cells expression of ZIP6 was increased by STAT3, a molecule linked to
metastasis (Bowman et al. 2000), and activated by proteolytic N-terminal cleavage
in the endoplasmic reticulum before trafficking to the plasma membrane where it
mediates zinc influx, activating AKT and inhibiting GSK-3𝛽, leading to the nuclear
localisation of Snail, the repression of E-cadherin expression and promotion of cell
migration (Hogstrand et al. 2013). This process occurs naturally in zebrafish
gastrula organiser cells during epithelial-mesenchymal transition; a critical stage of
embryonic development (Yamashita et al. 2004), and cancerous cells can reactivate
this pathway to metastasize and invade tissues in humans (Taylor et al. 2004). Other
associations between ZIP10 and ZIP6 have been documented; STAT3 co-localises
with ZIP10 in human follicular lymphoma cells, promoting antiapoptotic signalling
(Miyai et al. 2014); ZIP10 is activated similarly to ZIP6, with both undergoing
proteolytic N-terminal cleavage (although our data indicates ZIP10 is activated at
the plasma membrane whereas ZIP6 is activated in the endoplasmic reticulum); and
ZIP10 has been shown to form a heteromer with ZIP6 when both are activated
(Taylor et al. Unpublished). Taken together this evidence suggests that ZIP10 and
ZIP6 activation is strongly linked, indicating that ZIP10 has an as yet undiscovered
role in epithelial-mesenchymal transition or a similar pathway involving STAT3
and ZIP6.
The binding of the nuclear membrane by ZIP10 antibodies
Through use of fluorescence microscopy in MCF-7 cells transfected with wild-
type ZIP10 containing a V5 tag and co-staining slides with V5 and ZIP10
antibodies, it was found that only the ZIP10BN antibody co-localised with the V5
antibody by binding ZIP10 on the plasma membrane. However, the ZIP10AL
antibody also bound ZIP10 on the plasma membrane of mitotic nocodazole-treated
MCF-7 cells, as did the ZIP10BN antibody, indicating that ZIP10AL may only be

38
able to bind ZIP10 in mitotic cells where ZIP10 is activated by cleavage and
possibly undergoes conformation change, thus exposing the otherwise unavailable
antibody epitope. However, in both transfected and nocodazole-treated cells both
the ZIP10BN and ZIP10AL antibodies, as well as the ZIP10HN antibody and the
ZIP10SL antibody (although faintly) bound the nuclear membrane of interphase
cells. Use of the V5 antibody in transfected cells, which can only bind transfected
ZIP10, confirmed that ZIP10 is only expressed on the plasma membrane with
nuclear membranes remaining dark, indicating that the ZIP10 antibodies are all
binding other proteins localised to the nuclear membrane in non-mitotic cells. Using
a Basic Local Alignment Search Tool (BLAST) (Magrane and Consortium 2011) to
compare the epitopes of each ZIP10 antibody to the entire human proteome, it was
found that the antibodies do share some sequence similarities with other proteins
(table 4). The ZIP10HN antibody in particular had a large number of low e-value,
and therefore more likely to be significant, proteins which are expressed in the
nucleus. Which of these many matches, if any, the ZIP10 antibodies were binding
during fluorescence microscopy is unknown, but when comparing the molecular
weights of these matched proteins to the band sizes detected during western blot
there were no matches, thus it can be assumed that those bands did correspond to
actual ZIP10 fragments. Furthermore, the protein bound on the plasma membranes
of mitotic cells is likely to be ZIP10 (rather than the same interfering proteins
released into the cytoplasm due to the breakdown of the nuclear membrane during
mitosis) as the ZIP10BN antibody, which definitely does bind ZIP10 as it co-localised
with the V5 antibody in transfected cells, shows the same binding as the other
antibodies in mitotic cells.
Limitations of the Western Blot Samples
The samples for western blot were synchronised by either 24hour serum-
withdrawal from cell medium or 20hour nocodazole treatment, however these
methods are not directly comparable. Serum-withdrawal causes cells to synchronise
due to zinc deprivation, although in which stage of the cell cycle they arrest is
unknown as previous studies have found that zinc is required for both G1/S and
G2/M progression (Chesters and Petrie 1999). The efficacy of synchronising cells by

39
Table 4: ZIP10 antibody blast search results
Tested
Antibody
Protein
E-
threshold
Subcellular
Location
Weight
(kDa)
ZIP10AL
Serine/threonine-
protein kinase NEK7
5.6
Nucleus,
Cytoplasm
34.6
ZIP10AL
Tyrosine-protein
kinase Lyn
45
Plasma membrane,
Nucleus,
Cytoplasm, Golgi
58.6
ZIP10AL
Putative zinc finger
protein 355P
64 Nucleus 49.7
ZIP10HN
TSC22 domain family
protein 1
0.00011
Nucleus,
Cytoplasm
110
ZIP10HN Protein FAM76B 0.00024 Nucleus 38.7
ZIP10HN
Forkhead box protein
B2
0.0027 Nucleus 45.6
ZIP10SL Protein FAM160B1 1.2 Unknown 86.6
ZIP10SL
Transcriptional
regulator ATRX
4.4 Nucleus 283
ZIP10SL
Transcription
initiation factor IIA
subunit 1
5.2 Nucleus 41.5
ZIP10BN
Nuclear pore
membrane
glycoprotein 210
29 Nuclear membrane 205
ZIP10BN PPP1R9A protein 40 Unknown 122
ZIP10BN Acyl-protein
thioesterase 1
56 Cytoplasm 24.8
Compared the epitopes of each ZIP10 antibody to the entire human proteome. The
expectation value (E) threshold is calculated by comparing the number of matches
in a database to the statically expected number of matches. Proteins with lower E
values are more likely to bind the tested antibody. Size recorded to 3 significant
figures.

40
this method is also uncertain as not all cells are synchronised, with almost every cell
cycle marker having a presence in samples at every time-point (figure 2B).
Nocodazole is more specific because as a microtubule polymerisation inhibitor it
causes cell cycle arrest in mitosis, however as with cells treated with serum
deprivation not all cells are synchronised (Zieve et al. 1980). This is likely the reason
why the repeats for some samples varied significantly, as the stage of the cell cycle
the majority of the unsynchronised cells were in could have affected the level of
ZIP10 observed. A better method may have instead been to use a thymidine block
repeatedly, a process by which the thymidine usually available in cell medium is
replaced by thymidine glycol, an oxidised form of thymidine which inhibits DNA
replication, causing cells to arrest in S phase (Dolinnaya et al. 2013). This would
likely have been more effective at synchronising cell samples and decreased the
variation between cell cycle samples of different sets of repeats, and should be used
in future experiments.
Conclusions
This report contains novel evidence that ZIP10 is involved in the cell cycle
progression of breast cancer cells, and includes a pathway that may provide targets
for therapeutic intervention. Zinc itself cannot be targeted as it is essential for many
processes other than just cell division, but ZIP10 certainly can be. If the activation
of ZIP10 can be inhibiting by blocking the cleavage of the N-terminus, this could
potentially block proliferation in oestrogen-receptor positive breast cancer cells. One
possible method by which this could be achieved would be to treat MCF-7 cells
directly with ZIP10 antibodies, as they may block the cleavage proteins if their
epitopes were directly over or proximal to the cleavage site. This method has already
been tested, with treatment by ZIP10 antibodies preventing progression of MCF-7
cells through mitosis (Nimmanon et al. Unpublished). Repeat analysis in other
systems may confirm these results, so further testing in other cancer cell lines and
in animal models is warranted. Studies of ZIP7 have shown that through
phosphorylation-dependent activation by CK2 ZIP7-mediated zinc influx leads to
the activation of tyrosine kinases, promoting cell division and inhibiting apoptosis
(Taylor et al. 2012). If ZIP10 cleavage and activation are also phosphorylation-

41
dependent an analysis of ZIP10 phosphorylation may uncover a more precise
mechanism by which ZIP10 is regulated throughout the cell cycle. A search of
various phosphorylation databases can quickly show a vast number of potential
phosphorylation sites for ZIP10, especially on the large intracellular loop. The
Phosida database in particular identifies the serine at position 546 as a likely target
for phosphorylation in both G1 and mitosis (table 5). Analysis of the kinases that
may phosphorylate this site and the various other proteins in the pathway may
identify yet to be discovered kinases responsible for ZIP10 cleavage and activation,
presenting other therapeutic targets for breast cancer.
Table 5: Phosida phosphorylation score for S546 through the cell cycle
G1 G1/S Early S Late S G2 M
3.26 0.21 -0.91 -1.17 -1.3 1.2
Table shows highest scores phosphorylation score in G1 and mitosis. Score
calculated by Phosida based on likelihood of phosphorylation.
Acknowledgements
Thank you to Dr Kathyrn Taylor for the opportunity to undertake this
project, for her excellent advice and support and permission to report unpublished
results. Also thank you to Thirayost (Tony) Nimmanon for his guidance and to
everyone at the Breast Cancer Molecular Pharmacology Group for their
encouragement and assistance.

42
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Supporting Information
Detailed Western Blot method
12% SDS-PAGE was run with 35𝜇g of sample/well and transferred to
0.45𝜇m nitrocellulose membrane (GE Healthcare, Amersham protran). Membranes
were stained with Ponceau S to check protein transfer, washed with 1x Tris-buffered
saline containing 0.05% Tween-20 (TBST) and blocked with 5% (w/v) marvel in 1x
TBST for 30 minutes at room temperature. Membranes were washed in 1x TBST
before incubation overnight at 4°C in primary antibody (table 1) in 1x TBST, 1%
NaN3 and 5% Western Blocking Reagent (Roche). Membranes were then washed
thoroughly in 1x TBST and incubated in secondary antibody (table 1) in 1% (w/v)
marvel in 1x TBST for 1 hour at room temperature. Membranes were washed in 1x
TBST before chemiluminescent detection using X-ray film and chemiluminescent
reagents Clarity ECL (Biorad), Pierce ECL (Thermo Scientific), Supersignal West
Femto Maximum Sensitivity Substrate (Thermo Scientific) or Supersignal West
Extended Duration Substrate (Thermo Scientific). Film was processed by a Konica
Minolta SRX-101A developer, photographed, and using AlphaDigiDoc
densitometry software protein banding was quantified and normalised to β-actin.

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