TIG3 interaction at the centrosome alters microtubule
distribution and centrosome function
Tiffany M. Scharadin1,*, Haibing Jiang1,*, Stuart Martin2and Richard L. Eckert1,2,3,4,`
1Departments of Biochemistry and Molecular Biology;2Marlene and Stewart Greenebaum National Cancer Institute Cancer Center;3Department of
Reproductive Biology;4Department of Dermatology, University of Maryland School of Medicine, 108 N. Greene Street, Baltimore, Maryland 21201,
*These authors contributed equally to this work
`Author for correspondence (email@example.com)
Accepted 2 February 2012
Journal of Cell Science 125, 2604–2614
? 2012. Published by The Company of Biologists Ltd
TIG3 is an important pro-differentiation regulator that is expressed in the suprabasal epidermis. We have shown that TIG3 activates
selective keratinocyte differentiation-associated processes leading to cornified envelope formation. However, TIG3 also suppresses cell
proliferation by an unknown mechanism. Our present studies suggest that cessation of growth is mediated through the impact of TIG3 on
the centrosome and microtubules. The centrosome regulates microtubule function in interphase cells and microtubule spindle formation
in mitotic cells. We show that TIG3 colocalizes with c-tubulin and pericentrin at the centrosome. Localization of TIG3 at the centrosome
alters microtubule nucleation and reduces anterograde microtubule growth, increases acetylation and detyrosination of a-tubulin,
increases insoluble tubulin and drives the formation of a peripheral microtubule ring adjacent to the plasma membrane. In addition,
TIG3 suppresses centrosome separation, but not duplication, and reduces cell proliferation. We propose that TIG3 regulates the
formation of the peripheral microtubule ring observed in keratinocytes of differentiated epidermis and also has a role in the cessation of
proliferation in these cells.
Key words: b-tubulin, Centrosome, EB1-GFP, Keratinocyte differentiation, Microtubules, Pericentrin, Skin cancer, Squamous cell carcinoma, TIG3
The TIG3 tumor suppressor protein was originally discovered as a
growth suppressor in human keratinocytes (DiSepio et al., 1998).
Also known as RIG1 and H-Rev107-2 (Huang et al., 2000; Jiang
et al., 2005; Ou et al., 2008; Tsai et al., 2006; Tsai et al., 2007),
TIG3 displays homology to the human, mouse and rat forms of H-
rev107, and is a member of the H-rev family of class II tumor
suppressors, and the NlpC and P60 superfamily (Deucher et al.,
2000; DiSepio et al., 1998). These proteins include an N-terminal
hydrophilic domain and a C-terminal membrane-anchoring
domain (Deucher et al., 2000; DiSepio et al., 1998). The N-
terminal domain encodes NCEHFV and LRYG sequence motifs
that are conserved among family members (Anantharaman and
level of TIG3 is reduced in hyperproliferative keratinocytes that
are present in psoriatic lesions and skin tumor cells (Duvic et al.,
1997; Duvic et al., 2000; Duvic et al., 2003). Treating psoriatic
lesions with vitamin-A-related ligands increases the level of TIG3,
which is associated with reduced disease severity (Duvic et al.,
1997; Duvic et al., 2000; Duvic et al., 2003).
Absence of TIG3 expression in monolayer keratinocytes is
thought to be necessary for the maintenance of proliferative
potential (Sturniolo et al., 2003; Sturniolo et al., 2005), and TIG3
expression in monolayer cultures causes cell death (Sturniolo
et al., 2003; Sturniolo et al., 2005). TIG3 is present at high levels
in differentiated human keratinocytes in suprabasal epidermis
and in raft cultures (Jans et al., 2008). TIG3-dependent death is
associated with activation of selected differentiation-associated
processes. For example, TIG3 localizes to the plasma membrane
where it interacts with type I transglutaminase, an interaction that
leads to increased transglutaminase activity and cornified
envelope formation (Sturniolo et al., 2003; Sturniolo et al.,
2005). Mutagenesis studies indicate that TIG3 mutants lacking
the C-terminal membrane-anchoring domain are not active
(Deucher et al., 2000; Sturniolo et al., 2003; Sturniolo et al.,
2005). By contrast, N-terminal truncation converts TIG3 into a
protein that causes keratinocyte apoptosis (Jans et al., 2008).
TIG3 also suppresses the proliferation of keratinocytes (Sturniolo
et al., 2003; Sturniolo et al., 2005), although very little is known
about the mechanism of suppression.
The centrosomeis animportant organellelocated adjacenttothe
nucleus, which serves to nucleate microtubule arrays that organize
cytoplasmic organelles and primary cilia in interphase cells, and
form the mitotic spindles during mitosis (Doxsey et al., 2005). It
includes two perpendicularly-oriented barrel-shaped centrioles
surrounded by pericentriolar material (PCM) (Doxsey et al., 2005;
Kreitzer et al., 1999). Centrosome function is essential for cell
survival and cell division. Our studies suggest that association of
TIG3 with the centrosome alters microtubule distribution,
increases microtubule stability, reduces microtubule anterograde
elongation and suppresses daughter centrosome separation. We
propose that TIG3 interaction at the centrosome is a crucial event
in TIG3-dependent cessation of cell proliferation.
TIG3 localizes to the centrosome
As shown in Fig. 1A, TIG3 distributes at three main locations in
cells; the plasma membrane, punctate intracellular foci and at a
Journal of Cell Science
perinuclear location. The perinuclear location is identified by
arrows in Fig. 1A. We showed previously that the hydrophobic
C-terminus of TIG3 is required for correct TIG3 subcellular
localization (Deucher et al., 2000). Indeed, Fig. 1A shows that a
mutant lacking the C-terminus, TIG3 (1–134), displays a diffuse
cytoplasmic distribution and does not display perinuclear
accumulation. We have not studied this mutant further in the
present studies, because it is inactive and has no ability to
modulate cell function (Jans et al., 2008; Sturniolo et al., 2003;
Sturniolo etal., 2005). Because
proliferation, we are particularly interested in the intense
staining that is observed adjacent to the nucleus (Fig. 1B,
arrows). This location suggests that TIG3 interacts with the
centrosome. To test this possibility, we stained cells to detect
TIG3, the centriole protein, c-tubulin and a centrosome marker,
pericentrin. Fig. 1B shows that TIG3 staining surrounds c-tubulin
Fig. 1. TIG3 distributes to a pericentrosomal location. (A) Normal human foreskin keratinocytes were infected with 10 MOI of tAd5-TIG3(1–164) or tAd5-
TIG3(1–134) in the presence of 5 MOI Ad5-TA, and after 24 hours the cells were fixed and stained with antibodies against TIG3. The arrows indicate TIG3
perinuclear accumulation. Cell counting reveals TIG3 colocalization with c-tubulin and pericentrin in 9362% of TIG3-positive cells (mean 6 s.d. for 25 cells
counted on each of four separate experiments). TIG3(1–134) does not display a perinuclear distribution. Scale bars: 10 mm. (B) TIG3 colocalizes with c-tubulin
and pericentrin. Cells were infected as above, and at 24 hours post-infection, cells were stained with anti-TIG3 (red) and anti-c-tubulin, anti-pericentrin, anti-
GM130, anti-calnexin, or anti-mannose 6 phosphate receptor (M6PR) (green) antibodies and nuclei were visualized with Hoechst 33258. Arrows indicate TIG3 at
the centrosome (red). Scale bars: 10 mm. (C) TIG3 localizes to the centrosome. Localization of TIG3 to various organelles was calculated using the Manders’
overlap coefficient (0 indicates no colocalization, 1 indicates 100% colocalization) using confocal imaging. There is a strong tendency for TIG3 to localize to the
centrosome, as detected by colocalization with c-tubulin and pericentrin, but TIG3 also localizes, to a lesser extent, to the ER (calnexin) and Golgi complex
(GM130 and M6PR). M6PR overlap is statistically less than centrosome overlap and statistically more than GM130 and calnexin overlap (P,0.007). TIG3 does
not localize to the nucleus.
TIG3 impacts microtubule function2605
Journal of Cell Science
and pericentrin (arrows) (Nigg and Raff, 2009). Quantitative cell
counts revealed centrosomal localization of TIG3 in 9362% of
TIG3-expressing cells. Additionally, cells were stained with the
Golgi markers GM130 and mannose-6-phosphate receptor
(M6PR), and calnexin, an endoplasmic reticulum marker.
GM130, M6PR and calnexin stained structures in the vicinity
of the centrosome, and some of this staining colocalized with
TIG3 staining. The Manders’ overlap coefficient was calculated
to determine the extent of TIG3 localization to these organelle
markers (Fig. 1C). TIG3 displays high localization to the
centrosome markers and low localization to the nucleus. In
addition, some TIG3 localizes to M6PR, GM130 and calnexin.
Thus, TIG3 colocalizes mostly with the centrosome but some
TIG3 also localizes with the Golgi complex and endoplasmic
TIG3 alters microtubule distribution and stability
As part of the search for a centrosome-specific role for TIG3, we
examined the TIG3 impact on microtubules. The centrosome is a
crucial controller of microtubule function during mitosis and in
interphase cells (Doxsey et al., 2005). Therefore, we assessed
whether localization of TIG3 to the centrosome perturbs the
distribution of microtubules. Fig. 2 compares the microtubule
network in cells infected with an empty vector (EV) and cells
infected with a TIG3 vector. Cells infected with an empty vector
display a typical microtubule network, which includes a
perinuclear halo organized at the centrosome that sweeps
around the nucleus and out to the cell periphery (Fig. 2, left).
By contrast, in TIG3-expressing cells, the microtubules distribute
as a broad band at the cell periphery linked to the centrosome by
thin microtubule threads (Fig. 2, right).
These findings indicate that TIG3 influences microtubule
distribution, and suggest that it also influences other microtubule
properties. We used four approaches to assess the impact of TIG3
on microtubule stability. First, we examined the effect of
nocodazole on microtubule integrity. Microtubules in TIG3-
negative (EV) cells are distributed throughout the cell (Fig. 3A).
Nocodazole treatment produces diffuse high-intensity staining,
which is typical of dissociated microtubules, and, as expected,
withdrawal of nocodazole results in the formation of asters (sites
of microtubule reassembly) at the centrosome (arrows) (Fig. 3A).
By contrast, in TIG3-expressing cells, microtubules concentrate
at the centrosome and in a thick band at the cell periphery with
thin microtubule projections linking these locations (Fig. 3B).
These microtubules display a different response to nocodazole.
Nocodazole treatment eliminates the peripheral tubulin band in
most cells, but the centrosome-localized asters (arrows) survive
nocodazole treatment (Fig. 3B, right panel). The merged images
show TIG3 (arrows) accumulation at the centrosome (Fig. 3B,
left panel). To provide quantitative information, we infected cells
with tAd5-EV or tAd5-TIG3 and after 24 hours counted the
number of cells displaying a peripheral tubulin ring, tubulin at the
centrosome or tubulin at both locations. In untreated cells,
microtubules localized to the centrosome are visible in
19.863.1% of cells and no cells have a peripheral microtubule
ring (Fig. 3C). This value is probably an underestimate, as the
asters can be obscured in cells with robust microtubule staining.
By contrast, 43.864.7% of TIG3-positive cells have centrosome-
anchored tubulin filaments, and a microtubule peripheral ring is
present in 45.965.5% of TIG3-positive cells. The majority of
these cells (39.464.6%) are microtubule-positive at both
locations. Few microtubule asters and no rings are detected in
nocodazole-challenged EV cells, but 49.869.5% of TIG3 cells
retain a microtubule aster (Fig. 3C). During recovery from
nocodazole, asters are present in 47.063.4% of EV cells and
35.565.4% of TIG3 cells. In addition, the microtubule ring
reforms in 31.262.2% of TIG3 cells. An interesting finding is
that ,50% of all TIG3 cells lose immunologically detectable
microtubules (Fig. 3B,C, Loss). To assess the reason for this loss,
we prepared extracts for immunoblot detection of b-tubulin.
These studies revealed no change in the level of b-tubulin,
indicating that the actual level of b-tubulin per cell is not reduced
(Fig. 3D). These findings suggest that microtubule status is very
different in EV and TIG3 cells.
As a second method to assess microtubule status, we measured
the amount of a-tubulin in the insoluble (pellet) fraction.
Previous studies indicate that polymerized or stabilized a-
tubulin distributes in the 15,000 g pellet fraction (Onishi et al.,
2007). At 24 hours after tAd5-EV or tAd5-TIG3 infection,
keratinocytes were harvested, total extract and pellet fraction
were prepared, and a-tubulin level was monitored in each
fraction. These experiments show a substantial increase in the
level of a-tubulin that is present in the pellet fraction in TIG3-
positive cells (Fig. 4A). As a third method, we measured the
effect of TIG3 on a-tubulin acetylation and detyrosination.
Detyrosination of a-tubulin, to form glu-a-tubulin, is associated
with increased microtubule stability, as is acetylation of a-tubulin
Fig. 2. Impact of TIG3 on microtubule distribution. Keratinocytes were
infected with an empty vector or TIG3-encoding virus, and after 24 hours
fixed and stained with anti-b-tubulin (green) and anti-TIG3 (red) antibodies.
The presence of TIG3 results in accumulation of b-tubulin in a band at the cell
periphery (green). The arrow indicates accumulation of TIG3 at
Journal of Cell Science 125 (11)2606
Journal of Cell Science
(Bulinski and Gundersen, 1991; Kreitzer et al., 1999; Maruta
et al., 1986; Thyberg and Moskalewski, 1999). Fig. 4B shows
that TIG3 expression leads to increased levels of acetylated a-
tubulin and glu-a-tubulin. To determine whether the acetyl-a-
tubulin is localized to a particular region of the microtubule
network, we stained EV and TIG3 cells with antibodies against
distributed throughout the cell in EV cells. In TIG3-positive
cells it is distributed in a ring at the cell periphery and at the
centrosome. Anti-b-tubulin staining is included to confirm
microtubule distribution (Fig. 4C). Monitoring acetyl-a-tubulin
distribution in individual cells (Fig. 4D) reveals that acetyl-a-
tubulin distributes at the cell periphery and centrosome in TIG3-
positive cells. Thus, the level of acetylated-a-tubulin is increased
in TIG3-positive cells and is present in the tubulin network at
both the centrosome and peripheral ring, and the level of glu-a-
tubulin is also increased. These studies suggest that microtubules
are stabilized in cells that express TIG3.
As a fourth approach, we determined whether TIG3 affects
microtubule growth using the microtubule plus end binding
protein EB1–GFP to monitor anterograde microtubule extension
(Dixit et al., 2009; Piehl et al., 2004; Piehl and Cassimeris, 2003).
EB1–GFP binds specifically to the growing plus end of
microtubules and can be used to trace movement of the leading
tip of the microtubule as it grows towards the cell periphery
(Dixit et al., 2009; Piehl et al., 2004). Keratinocytes were
transfected with pEB1-GFP in the presence of pcDNA3 or
pcDNA3-TIG3. At 18 hours post-transfection, the cells were
monitored for EB1–GFP distribution by fluorescence confocal
microscopy. EV cells display robust plus end microtubule growth
(Fig. 5, EV). By contrast, TIG3-expressing cells display
substantial EB1–GFP accumulation in the vicinity of the
centrosome (arrows) with reduced plus-end growth towards the
cell periphery. These results suggest that TIG3 reduces
anterograde microtubule extension and that extension of many
microtubules is halted before extension is complete. In addition,
EB1–GFP appears to label multiple foci in the vicinity of the
centrosome, suggesting that the structure of the centrosome
nucleation site(s) have changed.
Impact of TIG3 on centrosome function
The centriole and centrosome play a crucial role at all stages of
the cell cycle (Lim et al., 2009; Loncarek et al., 2008; Sekine-
Suzuki et al., 2008). Centrosomes replicate simultaneously with
nuclear DNA during S phase (Doxsey et al., 2005) and during
prophase of mitosis, and the daughter centrosomes separate and
move to opposite poles of the mitotic cell (Doxsey et al., 2005;
Lim et al., 2009; Loncarek et al., 2008). Because of the role of
centrosomes and microtubules in this process, an obvious
expectation is that TIG3 might impede these processes. Indeed,
our studies suggest that TIG3 interferes with centrosome
separation. Keratinocytes were infected with TIG3-expressing
virus, and after 24 hours they were stained with anti-TIG3 and
anti-c-tubulin antibodies. Fig. 6A shows that centrosomes
separate (rectangle) in TIG3-negative cells. By contrast,
daughter centrosomes appear to be closely spaced and not
Fig. 3. TIG3 expression alters the sensitivity of microtubules to
nocodazole. Keratinocytes were infected with empty (EV) (A) or
TIG3-encoding adenovirus (B). After 24 hours, dishes of cells were
treated with vehicle (untreated), treated with 10 nM nocodazole for
2 hours (nocodazole), or treated with nocodazole for 2 hours
followed by 1 hour recovery in nocodazole-free medium (recovery).
The cells were then fixed and stained with anti-b-tubulin (green),
anti-TIG3 (red) antibodies or both, and then stained with Hoechst
33258. The images were acquired with a confocal microscope. Scale
bars: 10 mm. The arrows show the centrosome. (C) The number of
cells displaying a peripheral microtubule ring (Ring), a visible
MTOC (i.e. centrosome), a peripheral ring and a visible MTOC
(Both), and absence of visible microtubules (Loss) were counted. A
total of 25 cells were counted in each of four coverslips, and the
values are given as the mean percentage of the total 6 s.d., n54. ND,
not detected. (D) Keratinocytes were infected with the indicated
virus, and after 24 hours were treated as above with nocodazole.
Extracts were prepared for detection by immunoblot analysis of
b-tubulin and b-actin.
TIG3 impacts microtubule function2607
Journal of Cell Science
separated in TIG3-positive cells (arrows). In fact, centrosome
separation is rarely observed in TIG3-positive cells. Cell
counting of centrosome status in EV and TIG3 cells reveals
centrosome separation in 1662% of TIG3-negative cells, but in
,1% of cells that express TIG3 (Fig. 6A).
TIG3 could either delay or prevent centrosome separation. To
distinguish between these possibilities, we monitored the impact
of TIG3 on centrosome separation as a function of time. In EV-
infected cells, separated daughter centrosomes are observed at
time points where cells are undergoing division, including 16, 24
and 48 hours (Fig. 6B). The number of cells displaying separated
centrosomes ranges from 23.864% to 10.365% in EV-infected
cells. The number is slightly lower in cells observed at 72 hours,
as these cells are nearly confluent. By contrast, the number of
cells with separated centrosomes is markedly reduced in cells that
express TIG3 (Fig. 6C). These findings suggest that TIG3 does
not delay centrosome separation, but prevents it. We also
duplication by counting the number of EV cells and TIG3-
expressing cells that contain duplicated centrosomes. Fig. 7A
shows that centrosome duplication is not significantly altered in
cells that express TIG3. Fig. 7B shows a TIG3-positive cell
displaying centrosome localization of TIG3 (left panel), and an
expanded higher magnification image shows the duplicated
centrosomes (right panel).
Next, we assessed the role of microtubules in regulating
centrosome separation. Our goal was to assess whether TIG3
through an impacton
microtubules. To investigate this, we monitored the impact of
the microtubule disruptor, nocodazole, on centrosome separation
in control (EV) and TIG3-expressing cells. In EV-infected cells,
nocodazole treatment decreased the number of cells that
displayed separated centrosomes from 2067% to 1468%, a
number that returned to 2766% following the removal of
nocodazole (Fig. 8, recovery). These findings indicate that an
intact microtubule network is necessary for efficient centrosome
separation, and that microtubule status influences centrosome
function. By contrast, centrosome separation was observed in
,2% of TIG3-expressing cells and this was not reduced further
by treatment with nocodazole.
Suppressing centrosome separation is expected to impede cell
cycle progression. To assess this, keratinocytes were infected
with tAd5-TIG3 or tAd5-EV, and after 24 hours they were
incubated for 4 hours with bromodeoxyuridine (BrdU). BrdU is
incorporated into DNA specifically during new DNA synthesis in
S phase. Parallel cultures were stained to detect phosphorylated
histone H3, an M phase marker. Increased histone H3
phosphorylation is observed in mitosis (Goto et al., 1999;
Tapia et al., 2006). Fig. 9A shows the reduction in the number of
BrdU-positive nuclei (green) from 1461% in TIG3-negative
cells to 1.160.3% in TIG3-positive cells. Similarly, TIG3-
expressing cells display parallel reduction in the mitosis marker,
phosphorylated histone H3 (Fig. 9A). This suggests that TIG3
reduces both S and M phase cell cycle progression. We
performed immunoblot analysis to determine if these changes
are due to a change in the level of cell cycle control proteins. No
Fig. 4. Increased tubulin modification in TIG3-positive cells.
(A) TIG3 promotes the accumulation of insoluble a-tubulin.
Keratinocytes were infected with EV or TIG3-encoding virus, and at
24 hours post-infection total and pellet fractions were collected and
characterized for a-tubulin content by immunoblot analysis with anti-a-
tubulin antibodies. The distribution of b-actin was assessed as an internal
control. These studies show that TIG3 increases the accumulation of
insoluble a-tubulin. (B) Glu-a-tubulin and acetyl-a-tubulin levels
increase in TIG3-positive cells. Cells were treated with EV or TIG3
virus for 24 hours, and extracts were prepared for detection by
immunoblot analysis of the indicated epitopes. (C) Acetyl-a-tubulin is
present in the peripheral microtubule ring in TIG3-positive cells. Cells
were treated with EV or TIG3 virus for 24 hours, and fixed and stained
to detect acetyl-a-tubulin (red) and b-tubulin (green). Colocalization of
acetyl-a-tubulin and b-tubulin is indicated by orange and yellow. Scale
bars: 10 mm. (D) At 24 hours post-infection with tAd5-EV or tAd5-
TIG3, cells were fixed and acetyl-a-tubulin distribution was assessed by
staining with anti-acetyl-a-tubulin antibodies and counting the number
of cells displaying a peripheral microtubule ring (Ring), a visible MTOC
(i.e. centrosome), a peripheral ring and a visible MTOC (Both), and
absence of visible microtubules (Loss). A total of 25 cells were counted
on each of four coverslips and the values are given as the mean
percentage of the total 6 s.d. ND, not detected.
Journal of Cell Science 125 (11)2608
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change in the level of cell cycle regulatory proteins was observed
in TIG3-expressing cells (Fig. 9B), suggesting that the reduced
cell proliferation is not due to changes in the expression of cell
We previously reported that TIG3 reduces cell survival (Jans
et al., 2008; Scharadin et al., 2011; Sturniolo et al., 2003);
however, little is known about the mechanism of growth
suppression. In the present study, we demonstrate that TIG3
localizes to the centrosome, as shown by colocalization with the
centriole and centrosome markers, c-tubulin and pericentrin. The
centrosome is a 1–2 mm diameter organelle located adjacent to
the nucleus – it includes two perpendicularly oriented barrel-
shaped centrioles surrounded by the PCM. The staining pattern of
TIG3 suggests that it interacts with the PCM; moreover, the
interaction is relatively specific. Some TIG3 localizes to the
Golgi complex (GM130, M6PR) and endoplasmic reticulum
(calnexin), but the main site of TIG3 interaction is at the
centrosome. The centrosome is required for cell division and cell
survival, as it serves to nucleate polarized microtubule arrays,
which organize cytoplasmic organelles and primary cilia in
interphase cells, and it also forms the mitotic spindles during
mitosis (Doxsey et al., 2005). The PCM includes hundreds of
proteins, including many large scaffold proteins that function as
regulatory-protein docking sites (Doxsey et al., 2005), and the c-
tubulin ring complexes that are responsible for microtubule
nucleation (Doxsey et al., 2005; Kreitzer et al., 1999). The
centrosome and the microtubule system are intimately connected,
and deficiencies in either microtubule or centrosome function can
reduce cell survival (Mazzorana et al., 2011; Rusan and Rogers,
2009). Moreover, because the centrosome and microtubules
reciprocally influence the function of each other, we studied the
impact of TIG3 on both the centrosome and the microtubules.
TIG3 alters microtubule distribution, covalent modification
One feature we observe is a striking impact of TIG3 on
microtubule nucleation. EB1–GFP is a probe that labels the
growing ends of microtubules and is useful for studying
nucleation (Piehl et al., 2004; Piehl and Cassimeris, 2003). In
TIG3-positive cells, EB1–GFP intensely labels the microtubule-
organizing center (MTOC), which suggests that TIG3 does not
observed. First, there appear to be multiple sites of nucleation,
suggesting that the nucleation sites have been rearranged, and,
second, the nucleated microtubules do not appear to elongate
much beyond the region surrounding the centrosome. This
suggests that although nucleation is ongoing, the fate of the
nucleated microtubules is different from that of microtubules in
A second feature is that the microtubules show an unusual
microtubules extend around the nucleus and project in a
continuous network to the cell periphery. In TIG3-expressing
cells, microtubules accumulate as a ring at the cell periphery,
leaving thin microtubule threads to connect this structure to the
centrosome. It has been shown that the microtubule network is
reoriented in suprabasal keratinocytes such that it distributes to
the cell periphery (Lechler and Fuchs, 2007) and it is at least
possible that TIG3 drives this rearrangement, because it is
expressed in suprabasal epidermis (Jans et al., 2008; Sturniolo
et al., 2003; Sturniolo et al., 2005).
Microtubule accumulation at the cell periphery could also be
due to microtubule nucleation at alternate sites (e.g. at the cell
periphery). However, our experiments using EB1–GFP suggest
that the centrosome is maintained as the primary nucleation site
in TIG3-positive cells. Alternatively, this change in microtubule
distribution could be the result of differences in microtubule
anchoring. A specific set of proteins is involved in anchoring the
negative end of microtubules to the centrosome. Ninein is a
centrosome protein involved in this process (Dammermann and
Merdes, 2002; Delgehyr et al., 2005; Mogensen et al., 2000), and
is shown to redistribute to the plasma membrane in differentiated
keratinocytes (Lechler and Fuchs, 2007). This redistribution
results in the formation of a cortical microtubule ring in these
cells, which is reminiscent of the peripheral ring that we observe.
It is also possible that TIG3 catalyzes release of the microtubule
minus endfrom thecentrosome,
accumulation. Although defining a precise mechanism will
require further investigation, the present studies show that
TIG3 alters the distribution of microtubules.
A third feature is the impact of TIG3 on microtubule response
to nocodazole. In control cells, microtubule asters completely
dissociate following treatment with nocodazole. By contrast,
centrosome-associated asters persist in TIG3-expressing cells
treated with nocodazole. The possibility that this is due to a high
rate of nucleation is supported by the EB1–GFP labeling studies,
which show a high rate of nucleation, and by studies showing
However, differences are
Fig. 5. TIG3 reduces anterograde microtubule growth. Normal
keratinocytes growing in glass-bottom dishes were transfected with 1 mg of
EB1–GFP encoding plasmid in the presence of 2 mg of pcDNA3 (empty
vector, EV) or pcDNA3-TIG3. After 18 hours, EB1–GFP fluorescence was
detected using an Olympus FluoView FV1000 laser confocal microscope. The
arrows indicate pericentrosomal distribution of EB1–GFP-labeled
microtubules in two representative TIG3-positive cells. Scale bars: 10 mm.
TIG3 impacts microtubule function2609
Journal of Cell Science
rapid expansion of asters upon removal of nocodazole. However,
increased maintenance of asters could also be the result of
enhanced microtubule stability.
An additional interesting response to nocodazole treatment is
the preferential loss of the peripheral tubulin ring, a structure
specifically present in TIG3 positive cells. Treatment with
nocodazole results in a complete loss of this structure. It is not
clear why this ring structure forms in TIG3-positive cells because
microtubule elongation appears to cease close to the centrosome.
However, these rings reappear during recovery from nocodazole
treatment, suggesting that ring formation is not a one-time
response to TIG3. An additional feature is that microtubules are
not visible by immunostaining in ,50% of TIG3-positive cells.
This is despite the fact that the level of tubulin has not decreased,
as measured by immunoblot analysis. The absence of signal is not
likely to be due to epitope masking, as the reduction is observed
with each of three antibodies that detect different tubulin epitopes
(rabbit anti-b-tubulin, mouse anti-b-tubulin and mouse anti-
We also investigated whether TIG3 expression covalently
modifies microtubules to alter stability. Microtubule stability is
regulated by the post-translational modification of a-tubulin. One
mechanism is the removal of the a-tubulin C-terminal tyrosine
(Kreitzer et al., 1999). This process is controlled by an
unidentified tubulin carboxypeptidase, which removes tyrosine
to expose glutamic acid; the modified product is called glu-a-
tubulin (Kreitzer et al., 1999). Tubulin tyrosine ligase is an
enzyme that catalyzes tyrosine replacement to regenerate
tyrosinated a-tubulin. Tyrosinated a-tubulin subunits turn over
in 3 to 5 minutes; detyrosinated tubulin (glu-a-tubulin) turns over
in 3 to 5 hours (Kreitzer et al., 1999). Thus, microtubules that are
enriched with glu-a-tubulin are highly stable. Acetylation of a-
tubulin is also associated with enhanced stability (Hammond
et al., 2008), as is the accumulation of microtubules in the
Fig. 6. Impact of TIG3 on centrosome
separation. (A) TIG3 inhibits centrosome
separation. Normal human keratinocytes were
infected with tAd5-TIG3 at a level that infects a
subset of cells. At 24 hours post infection, cells
were stained with anti-TIG3 (red) and anti-c-
tubulin (green) antibodies, and nuclei were
visualized with Hoechst 33258. Arrows show the
c-tubulin at the centrosome (green). TIG3 is the
red fluorescence. The rectangle indicates
separated daughter centrosomes in a TIG3-
negative cell. The plot indicates the percentage of
control and TIG3-positive cells with separated
centrosomes (6 s.e.m., 50 cells were counted in
each of the four experiments). Differences are
significant as determined by Student’s t-test,
P,0.005. Scale bars: 10 mm. (B,C) Normal
human keratinocytes were infected with tAd5-EV
or tAd5-TIG3 at 0 hours and cultures were
harvested at 16 hours, 24 hours, 48 hours and
72 hours post-infection, and were fixed and
stained as in Fig. 6A. The arrows indicate
centrosomes. The numbers indicate the percentage
of cells displaying separated daughter
centrosomes. The values are the mean 6 s.d.,
n550 cells counted. Similar results were observed
in three separate experiments. Scale bars: 10 mm.
Journal of Cell Science 125 (11) 2610
Journal of Cell Science
insoluble fraction in lysates. One possibility is that microtubule
accumulation at the cell periphery in TIG3-positive cells is due to
increased acetylation of a-tubulin and increased formation of
perhaps associated with
glu-a-tubulin. We detected glu-a-tubulin and acetyl-a-tubulin in
control cells and increased levels in TIG3-positive cells. Acetyl-
a-tubulin was detected in TIG3-positive cells at the centrosome
and in the peripheral tubulin ring. Moreover, we observed
increased levels of insoluble tubulin in TIG3-positive cells,
which is also an indicator of increased microtubule stability; thus,
TIG3 appears to increase microtubule stability. An important
question is how the impact of TIG3 on microtubules might
influence cell survival and centrosome function. In this respect,
centrosome and microtubule functions are linked together. It is
known that the centrosome influences microtubule function, and
it is also known that agents that influence microtubule turnover or
stability, such as paclitaxel, influence centrosome function (Lanzi
et al., 2001; Mazzorana et al., 2011). Thus, we propose that the
one way that TIG3 reduces cell survival and halts proliferation is
through its impact on microtubule distribution and stability, and
that this also impacts the centrosome.
TIG3 halts daughter centrosome separation
The localization of TIG3 to the centrosome could be expected to
impact on centrosome separation. Indeed, centrosome separation
is impeded in TIG3-positive cells, and this is associated with
reduced cell proliferation as measured by reduced incorporation
of BrdU into DNA (an S-phase event), and reduced levels of
phosphorylated histone H3 (an M-phase marker). In control
cultures, 15% of cells are BrdU positive and 10% display high-
level phosphorylated histone H3 staining. By contrast, in TIG3-
expressing cultures, these levels are suppressed to ,1%,
suggesting an impact of TIG3 on cell cycle progression. This is
not associated with changes in expression of cell cycle control
proteins associated with G1, S or G2/M, suggesting that it
uniformly impacts all phases of the cell cycle.
Centrosomes are duplicated during the S-phase of the cell
cycle in synchrony with nuclear DNA replication (Doxsey et al.,
2005; Hinchcliffe and Sluder, 2001). During early prophase, the
centrosomes separate and begin the process of migration to
opposite poles of the dividing cell (Doxsey et al., 2005;
Hinchcliffe and Sluder, 2001). Our studies suggest that TIG3
does not inhibit centrosome duplication, but rather inhibits
Fig. 7. TIG3 does not inhibit centrosome duplication. (A) Centrosomes
were counted in 30 EV or TIG3-expressing cells. A slight decrease in cells
displaying duplicated centrosomes is observed in TIG3-expressing cells, but
the reduction was not statistically significant (P,0.41). (B) Images showing
centrosome duplication in a TIG3-expressing cell. Keratinocytes were
infected with TIG3-expressing virus, and after 48 hours the cell was fixed and
stained to detect TIG3 (red), c-tubulin (green) and DNA (blue). The yellow
staining in the left panel (arrow) indicates centrosome-localized TIG3. The
arrows in the right panel show the closely spaced duplicated centrosomes in
the same cell (only c-tubulin staining is shown for clarity). Scale bars: 10 mm.
Fig. 8. Keratinocytes were infected with tAd5-EV or tAd5-TIG3.
After 24 hours, dishes of cells were treated with vehicle (untreated),
10 nM nocodazole for 2 hours (nocodazole), or nocodazole for
2 hours followed by 1 hour recovery in nocodazole-free medium
(recovery). The cells were then fixed and stained with anti-c-tubulin
(green) and anti-TIG3 (red) antibodies, and stained with Hoechst
33258. The images were captured using a confocal microscope.
Scale bars, 10 mm. The arrows indicate the centrosome. The counts
are the number of cells that display a separated centrosome, and are
presented as the mean 6 s.d. derived from the count of ten
TIG3 impacts microtubule function 2611
Journal of Cell Science
centrosome separation and thereby influences cell division.
Indeed, our findings show that the daughter centrosomes are
separated by more than 1.5 mm in 15.9% of control cells, a
number that is consistent with the number of cells in mitosis
(10.2%). By contrast, centrosome separation is observed in ,1%
of TIG3-positive cells, suggesting that TIG3 inhibits centrosome
separation and/or centrosome migration. Time course studies
reveal that TIG3 does not delay but actually halts separation.
Proteins that link the mother and daughter centrosomes during
replication have been identified – these proteins are cleaved to
permit centrosome separation at the appropriate stage of the cell
cycle (Bahe et al., 2005). It is possible that TIG3 impedes the
cleavage of these proteins and, thereby, inhibits centrosome
separation. TIG3 might also inhibit centrosome replication.
However, the number of cells that contained duplicated
centrosomes was not significantly reduced (P,0.41) in cells
expressing TIG3. Thus, TIG3 does not inhibit centrosome
In summary, our studies show that the centrosome is a major
site of TIG3 localization in normal human keratinocytes, and that
this is associated with changes in microtubule distribution,
elongation and covalent modification, and that TIG3 association
at the centrosome suppresses centrosome separation (Fig. 10). In
addition, we suggest that the altered microtubule environment
can feedback to further alter centrosome function. Ultimately, we
argue that these events lead to cessation of cell proliferation and
reduce cell survival.
Materials and Methods
Cell culture and reagents
Primary cultures of human foreskin keratinocytes were cultured in 0.09 mM Ca2+-
containing keratinocyte serum-free medium (KSFM) (Sturniolo et al., 2005). The
rabbit polyclonal antibody against TIG3 has been described (Deucher et al., 2000).
Mouse monoclonal anti-c-tubulin (catalog number sc-17788), rabbit polyclonal
anti-b-tubulin (catalog number sc-9104), rabbit polyclonal anti-cyclin E (catalog
number sc-481), rabbit polyclonal anti-CDK2 (catalog number sc-163), mouse
monoclonal anti-cyclin A (catalog number sc-239), mouse monoclonal anti-cyclin
B1 (catalog number sc245), mouse monoclonal anti-CDK1 (catalog number sc-
54), and rabbit polyclonal anti-CDK4 (catalog number sc-601) antibodies were
Fig. 9. TIG3 suppresses cell division. (A) TIG3 reduces the incorporation of BrdU. Normal human foreskin keratinocytes were infected with empty or TIG3
encoding adenovirus, and after 24 hours they were incubated for 4 hours with BrdU. Cells were then fixed and stained with anti-TIG3 (red), anti-BrdU (green)
antibodies and Hoechst 33258. The plots indicate the percentage of BrdU-positive cells (6 s.e.m., n5 four experiments with 50 cells counted per experiment) and
the percentage of cells that stain strongly for phosphorylated histone H3 (6 s.e.m. with 100 cells counted per experiment). Differences are significant as
determined by Student’s t-test, P,0.01. Scale bars: 10 mm. (B) Cell lysate was collected from keratinocytes at 24 hours after infection with tAd5-EV or tAd5-
TIG3. The cell cycle regulatory-protein level was determined by immunoblot analysis. The numbers indicate the molecular weight (kDa). The phases of the cell
cycle are indicated (G1, S, G2/M).
Fig. 10. Schematic of TIG3 action. TIG3 interacts with the centrosome and
halts centrosome separation during cell division. It also alters microtubule
subcellular distribution, nucleation and stability. These changes result in the
cessation of cell division. Centrosomes (yellow), nuclei (blue), TIG3
localization (orange) and microtubules (green) are shown. The blue outline
represents the plasma membrane. The dashed arrows indicate that the
centrosome and microtubules reciprocally influence the function of the other.
Journal of Cell Science 125 (11)2612
Journal of Cell Science
from Santa Cruz (Santa Cruz, CA). Mouse monoclonal anti-b-actin (catalog
number A5441), mouse monoclonal anti-BrdU (catalog number B8424) and mouse
monoclonal anti-acetyl-a-tubulin (catalog number T7451) antibodies were
obtained from Sigma (St. Louis, MO). Rabbit polyclonal anti-phosphorylated
histone H3 (catalog number 06–570) and rabbit polyclonal anti-detyrosinated a-
tubulin (Glu-tubulin) (catalog number AB3201) antibodies were obtained from
Millipore (Billerica, MA). Mouse monoclonal anti-b-tubulin (catalog number
ab11311), mouse monoclonal anti-M6PR (catalog number ab2733), and mouse
monoclonal anti-pericentrin (catalog number ab28144) antibodies were from
Abcam (Cambridge, MA). Rabbit polyclonal anti-a-tubulin (catalog number 2144)
and mouse monoclonal anti-p21 (catalog number 2947S) antibodies were from
Cell Signaling Technology (Danvers, MA). Alexa 488-conjugated goat anti-rabbit
IgG (catalog number A11008), goat anti-mouse IgG (catalog number A11029),
Alexa 555-conjugated goat anti-rabbit IgG (catalog number A21429), goat anti-
mouse IgG (catalog number A21424) antibodies and Hoechst 33258 were from
Invitrogen (Carlsbad, CA). Peroxidase-conjugated donkey anti-rabbit IgG (catalog
number NA934) and peroxidase-conjugated sheep anti-mouse IgG (catalog
number NA931) antibodies were obtained from GE Healthcare (Piscataway, NJ).
Nocodazole was purchased from Calbiochem (Gibbstown, NJ). Mouse monoclonal
anti-GM130 (catalog number 610822), mouse monoclonal anti-calnexin (catalog
number 610524) and mouse monoclonal anti-cyclin D1 (catalog number 554180)
antibodies, and BrdU were purchased from BD Biosciences (Rockville, MD).
Plasmid pEB1-GFP was purchased from Addgene (Cambridge, MA).
tAd5-EV, tAd5-TIG3(1–164) and tAd5-TIG3(1–134) adenoviruses have been
described (Jans et al., 2008; Sturniolo et al., 2003; Sturniolo et al., 2005). The tAd5
cytomegalovirus promoter. This promoter is active in the presence of a
transactivator (TA) protein, which is provided by co-infection with an Ad5-TA
adenovirus (Jans et al., 2008; Sturniolo et al., 2005). tAd5-EV is an empty
adenovirus and tAd5-TIG3 encodes the full-length 164 amino acid TIG3 protein
(Jans et al., 2008; Sturniolo et al., 2005). TIG3(1–134) encodes an inactive mutant
that lacks the C-terminal membrane-anchoring domain (Jans et al., 2008; Sturniolo
et al., 2005). Keratinocyte cultures were incubated with a multiplicity of infection
(MOI) of 10 of tAd5-EV, tAd5-TIG3 or tAd5-TIG3(1–134) in the presence of 5
MOI of Ad5-TA in KSFM containing 6 mg/ml polybrene (catalog number H9268;
Sigma). Cells were fixed and stained for immunofluorescence or extracts were
prepared for immunoblot analysis at 24 or 48 hours post-infection. The Manders’
overlap coefficient was used to measure overlap of TIG3 staining with other
organelle marker-proteins using the JACoP plugin for ImageJ (Bolte and
Cordelie `res, 2006).
operator elementlinked tothe
Keratinocytes growing on coverslips were infected with adenovirus, and after 24 or
48 hours they were washed, fixed with 4% paraformaldehyde in phosphate-
buffered saline (PBS) for 30 minutes, and permeabilized with methanol for
10 minutes at 220˚C. The coverslips were then incubated for 1 hour with
appropriate primary and secondary antibodies. After washing, the cells were fixed
to slides by using Mowiol 4–88 (Calbiochem, Gibbstown, NJ), and fluorescence
was visualized using an Olympus OX81 spinning-disc confocal microscope.
Keratinocytes were infected with 10 MOI of tAd5-EV or tAd5-TIG3(1–164). After
24 or 48 hours the cells were collected in PBS and centrifuged at 500 g for
5 minutes. The cell pellet was dissolved in lysis buffer (Cell Signaling
Technology) supplemented with protease inhibitor cocktail (Calbiochem) and
centrifuged at 20,000 g for 20 minutes. The pellet was washed twice with PBS, and
then re-suspended and boiled in 4% SDS. Electrophoresis was performed on an
equal number of cell equivalents of supernatant (soluble) and pellet (insoluble)
fractions on denaturing and reducing 4–15% polyacrylamide gels for immunoblot
Keratinocytes on coverslips were infected with 10 MOI of tAd5-EV or tAd5-TIG3.
At 24 hours the cells were incubated with 10 mM of BrdU for 4 hours. The cells
were fixed with 4% paraformaldehyde for 30 minutes at 4˚C, washed in PBS
containing 1% Triton X-100, incubated in 1 M HCl for 10 minutes on ice, in 2 M
HCl for 10 minutes at room temperature and 20 minutes at 37˚C. Coverslips were
incubated in 0.1 M borate buffer for 12 minutes at 25˚C, washed with PBS and
stained with antibodies against BrdU or TIG3.
Microtubule nucleation assay
EB1 is a microtubule plus-end binding protein that exchanges continuously and
labels the plus end of microtubule (Piehl et al., 2004; Piehl and Cassimeris, 2003).
Normal keratinocytes, growing in glass bottom dishes, were transfected with 1 mg
of plasmid encoding EB1-GFP fusion protein (EB1–GFP) (Piehl et al., 2004; Piehl
and Cassimeris, 2003) in the presence of 2 mg of pcDNA3 or pcDNA3-TIG3(1–
164) (Jans et al., 2008; Sturniolo et al., 2003; Sturniolo et al., 2005). After
18 hours, EB1–GFP was detected using an Olympus FluoView FV1000 laser
confocal microscope and a 606objective with 3-second intervals between image
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