Localization of mRNA allows cells to spatially regulate
translation and thus create functional subregions with distinct
components. ?-actin mRNA is specifically localized in
fibroblasts (Kislauskis et al., 1993; Lawrence and Singer,
1986). Localization of ?-actin protein to the leading edge
where polymerization takes place is presumably dependant on
this mRNA localization (Shestakova et al., 2001). This
localization occurs in response to intracellular signaling
(Latham, Jr et al., 1994) and is seen in a variety of cell types
(Hill and Gunning, 1993; Hill et al., 1994; Hoock et al., 1991).
Study of this mechanism led to the understanding that all
localized mRNAs contain cis-acting elements, mostly located
in the 3?UTR, that are bound by trans-acting factors to direct
localization. In fibroblasts, short fragments of the ?-actin
3?UTR from several species were demonstrated to be sufficient
for localization when expressed in a heterologous construct
(Kislauskis et al., 1994). One of these fragments, a 54-
nucleotide sequence that forms a stem-loop structure, exhibited
most of the localizing activity and has been termed the ?-actin
mRNA zipcode. Trans-acting localization factors that bound to
the ?-actin zipcode were identified and have been referred to
as zipcode-binding proteins. A primarily cytoplasmic 68 kDa
protein, which bound to the zipcode, was called zipcode-
binding protein 1 (ZBP1) and contains several recognizable
regions, including two RNA-recognition motifs (RRM), four
hnRNP K homology (KH) domains as well as potential nuclear
localization and export signals (Ross et al., 1997).
Recently, ?-actin mRNA localization and ZBP1 have been
implicated in metastasis. First, ?-actin mRNA localization has
been shown to be required for directed cell motility (Farina et
al., 2003; Kislauskis et al., 1997), particularly in non-metastatic
cells (Shestakova et al., 1999). Second, reduction of ?-actin
mRNA localization through treatment with antisense
oligonucleotides targeting the zipcode, which disrupted the
interaction between ZBP1 and ?-actin mRNA, has been shown
to convert the behavior of cells with a polarized movement
phenotype to a ‘random walk’ (Shestakova et al., 2001). Third,
MTLn3 (metastatic) cells do not localize ?-actin mRNA and
contain significantly less ZBP1 than MTC (non-metastatic)
cells derived from the same tumor, which do localize the mRNA
(Wang et al., 2004). Fourth, ZBP1 was found to be highly
expressed in rat mammary tumors as well as in human tumors
(Noubissi et al., 2006; Yantiss et al., 2005) but its expression
was shown to be suppressed specifically in the invasive
subpopulation of tumor cells (Wang et al., 2004). These results
suggested that the ability of tumor cells to exhibit amoeboid
movement and, therefore, heightened chemotaxis ability as is
characteristic of metastatic cells (Condeelis et al., 1992)
depended on a random distribution of ?-actin mRNA, whereas
cells that are able to target ?-actin mRNA retain a stable polarity
that would be less responsive to a chemoattractant.
We hypothesized that ZBP1 protein induces ?-actin mRNA
localization (Oleynikov and Singer, 2003), which in turn
suppresses chemotaxis by establishing a persistent polarity,
leading to reduced responsiveness and ability to orient towards
exogenous chemotactic gradients required for cellular
invasiveness and hence metastatic potential. To test this
The interaction of ? ?-actin mRNA with zipcode-binding
protein 1 (ZBP1) is necessary for its localization to the
lamellipod of fibroblasts and plays a crucial role in cell
polarity and motility. Recently, we have shown that low
ZBP1 levels correlate with tumor-cell invasion and
metastasis. In order to establish a cause and effect
relationship, we expressed ZBP1 in a metastatic rat
mammary adenocarcinoma cell line (MTLn3) that has low
endogenous ZBP1 levels and delocalized ? ?-actin mRNA.
This leads to localization of ? ?-actin mRNA, and eventually
reduces the chemotactic potential of the cells as well as their
ability to move and orient towards vessels in tumors. To
determine how ZBP1 leads to these two apparently
contradictory aspects of cell behavior – increased cell
motility but decreased chemotaxis – we examined cell
motility in detail, both in cell culture and in vivo in tumors.
We found that ZBP1 expression resulted in tumor cells with
a stable polarized phenotype, and reduced their ability to
move in response to a gradient in culture. To connect these
results on cultured cells to the reduced metastatic ability of
these cells, we used multiphoton imaging in vivo to examine
tumor cell behavior in primary tumors. We found that
ZBP1 expression actually reduced tumor cell motility and
chemotaxis, presumably mediating their decreased
metastatic potential by reducing their ability to respond to
signals necessary for invasion.
Supplementary material available online at
Key words: Chemotaxis, ZBP1, Intravital imaging, Invasion, Motility
ZBP1 enhances cell polarity and reduces chemotaxis
Kyle Lapidus, Jeffrey Wyckoff, Ghassan Mouneimne, Mike Lorenz, Lillian Soon, John S. Condeelis* and
Robert H. Singer*
Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461-1975, USA
*Authors for correspondence (e-mails: email@example.com; firstname.lastname@example.org)
Accepted 12 July 2007
Journal of Cell Science 120, 3173-3178 Published by The Company of Biologists 2007
Journal of Cell Science
hypothesis, we compared cell motility and chemotactic
response, together with motility, protrusion and
orientation towards vessels in tumors in cells with
different levels of ZBP1 expression. To determine
whether expression of ZBP1 in ‘random-walking’ cells
was sufficient to change them into ‘linear-walkers’, we
examined motility in a stable cell line expressing
ZBP1 derived from random-walking metastatic cells
(MTLn3). In addition, we examined the movement of
cells with or without ZBP1 protein expression in the
presence of chemotactic factors. Finally, the metastatic
cell line expressing elevated levels of ZBP1 was tested
for its ability to invade within mammary tumors to
determine the role these motility changes play in vivo in
an environment where metastasis occurs. Our results
suggest that ZBP1 expression: (1) alters motility
patterns, converting the movement of tumor cells with a
random walking pattern characteristic of invasive cells
into a highly polarized pattern of movement
characteristic of non-invasive cells; (2) interferes with
the ability of tumor cells to move in the direction of a
chemotactic gradient and; (3) in living animals, ZBP1
expression leads to a similar reduction in motility
towards vessels within tumors.
Results and Discussion
Levels of ZBP1 in cell lines
We hypothesized that the differences in polarity, motility
and metastasis seen between random walking, highly
metastatic mammary-tumor-derived cells (MTLn3) and
linear walking, less invasive mammary-tumor-derived
cells (MTC) stem from differences in ZBP1 levels.
Previous work demonstrated that levels of ZBP1 mRNA
differ between these cell lines, although differences in
protein levels had not been directly observed (Wang et al.,
2004). Therefore, we compared ZBP1 protein levels in
these cell lines as well as the cell line we generated
expressing a ZBP1 fusion protein. ZBP1 levels in the
stable cell line referred to as ZBPA in Fig. 1 and ZBP
throughout the rest of this paper are 3.99±1.96 times that
of the parental MTLn3 cell line, and are closer to that of
a less metastatic cell line derived from the mammary
tumor MTC, which has ZBP1 levels 5.42±2.13 times that
of MTLn3 cells and 1.44±0.43 times that of the ZBP1-
expressing MTLn3 stable cells (supplementary material Fig. S1).
ZBP1 increases the polarity of locomotion in tumor cells
Although the specific role of ZBP1 in ?-actin mRNA
localization and metastasis has been described, its role
associating cell motility with metastasis needs to be established.
To determine the effects of ZBP1 on cell motility directly,
metastatic tumor cells (MTLn3) that do not express ZBP1 were
transfected with a GFP-ZBP1-expressing transgene, and two
stable lines were selected from independent transfections. ZBP1
function is unaffected by this fusion with GFP (Farina et al.,
2003). Both transformants and the control cells, expressing GFP
alone, were subjected to extensive motility analysis. Dynamic
imaging analysis software (DIAS) was used to determine
various parameters of cell movement and provide an accurate
picture of cell position and displacement. This provided a
rigorous assessment of the differences between genetically
identical cell populations differing only in ZBP1 expression
levels. Centroid (Fig. 1A) and perimeter (Fig. 1B) plots
demonstrated that ZBP1-induced conversion of a parental
‘random walk’ into a more linear ‘crawling pattern’. These
experiments demonstrated that ZBP1 expression was sufficient
to induce the motility phenotype consistent with a polarized
cell, leading to significantly increased persistence and
directionality in cell motility (Fig. 1C,D). Cell speed was also
calculated from this analysis and for control cells was 0.76±0.15
?m/minute, for ZBPA was 1.05±0.27 ?m/minute and for ZBPB
was 0.79±0.22 ?m/minute. Because cell speed was not reduced
with ZBP1 expression, changes in speed cannot form the basis
for changes in response to chemoattractant or in metastatic
Orientation towards a chemoattractant is reduced with
We suggest that a tumor cell polarized as the result of ZBP1
Journal of Cell Science 120 (18)
Fig. 1. ZBP1 expression causes phenotypic conversion from random walk
to directed movement, increasing persistence and directionality. Movement
of GFP- and GFP-ZBP1-expressing MTLn3 cells (from two independently
derived stable cell lines, indicated as ZBPA and ZBPB) was examined in
5% serum. The total time interval was 30 minutes, and time between
successive frames was 1 minute. (A,B) Plots of cell (A) centroid and (B)
perimeter for six GFP-expressing control MTLn3 cells (CTRL, A and B
top) and six GFP-ZBP1-expressing MTLn3 cells (ZBP, A and B bottom).
(C,D) Differences in (C) persistence and (D) directionality were
statistically significant between the populations (*P<0.005, error bars
indicate ± s.e.m.). Persistence is the speed divided by the change in
direction, directionality is the net path length divided by the total path
Journal of Cell Science
ZBP1 reduces chemotaxis
expression will be less able to exhibit chemotaxis in the
direction of an EGF gradient. Instead, it will continue to move
in its polarized direction regardless of the orientation of the
gradient. This would explain why ZBP1 could act as a
metastasis suppressor (Wang et al., 2004).
To test this hypothesis, we examined the effect of ZBP1 on
cell motility in a gradient. Control or ZBP1-expressing MTLn3
cells were serum-starved and imaged in a chamber that
provided a stable linear gradient of EGF (Soon et al., 2005).
DIAS was then used to analyze cell behavior. In this assay,
ZBP1-expressing MTLn3 cells showed a reduced ability to
orient and move in the direction of increasing EGF, relative to
control MTLn3 cells (expressing only GFP), which efficiently
locomoted towards increased concentrations (Fig. 2A,B). The
ZBP1-expressing cells continued to be polarized and move
irrespective of the orientation of the gradient (Fig. 2C). This
confirmed that cells with an inherent polarity are less
responsive to a chemoattractant (Condeelis et al., 2005). These
data are consistent with a stochastic model for motility and
chemotaxis, suggesting that an increase in signal decay time
that may be caused by ZBP1-expression-mediated polarization,
reduces orientation behavior in a gradient if the response time
remains constant (Tranquillo et al., 1988). This model also
suggests that factors affecting orientation in a gradient play an
important role in persistence of motility in the absence of a
gradient. Our results are also consistent with the observation
that invasive tumor cells with low ZBP1 expression are more
chemotactic and invasive in vivo (Wang et al., 2004). This
suggests a motility-based mechanism by which ZBP1
expression could reduce metastasis.
ZBP1-expressing cells can sense and respond to a
Reduced orientation towards a chemoattractant could be either
owing to the inability to sense the chemotactic factor or the
inability to turn towards the chemoattractant because of the
existence of stable cell polarity (Janetopoulos et al., 2004). In
order to differentiate between these possible explanations for
the inhibitory effects of ZBP1 expression on chemotaxis, we
applied EGF directionally from a pipette and analyzed
protrusion and retraction of the cell. When the pipette was
applied in front of the leading edge of a polarized cell, both
parental and ZBP1-expressing MTLn3 cells protruded towards
the stimulus, and retracted from the opposite side (Fig. 3). This
indicated that expressing ZBP1 did not influence the detection
of the gradient. Hence, ZBP1-expressing cells can both sense
and respond to a chemoattractant as long as it is aligned with
the polarity of the cell. Therefore, the reduced chemotaxis of
ZBP1-expressing cells probably resulted from the stable cell
polarity induced by elevated ZBP1 expression.
Protrusion and locomotion in primary tumors are
reduced with ZBP1 expression
MTLn3 cells expressing either ZBP1-GFP or GFP alone were
orthotopically injected into the mammary fat pads of rats and
after growth, tumors were inspected by two photon microscopy.
ZBP1-GFP could be seen localized at the cell peripheries and
at cell-cell junctions (Fig. 4A). This localization pattern
issimilar to that previously seen for ZBP1 after
immunohistological staining of sections from MTLn3-
generated tumors (Wang et al., 2002). Orientation of cells
towards vessels, cell protrusion and locomotion in primary
tumors have been shown to be crucial steps in the metastatic
cascade, and elevated levels of these parameters are correlated
with metastasis (Wyckoff et al., 2000). To determine whether
reduced metastasis of the ZBP1-expressing cells correlated with
changes in any of these movement patterns, we performed
intravital imaging to compare the movement of ZBP1
expressing and control cells within tumors. Whereas control
cells oriented towards the nearest vessels (Fig. 4B), ZBP1-
expressing cells did not (Fig. 4C). Furthermore, ZBP1-
expressing cells were less protrusive in tumors than control
MTLn3 cells. Finally motility was reduced in ZBP1-expressing
cells. In tumors generated by control cells, motility was
Fig. 2. ZBP1 expression reduces movement in the
direction of a chemoattractive gradient. (A,B) Perimeter
and centroid plots are from (A) GFP-expressing or (B)
GFP-ZBP1-expressing MTLn3 cells when exposed to a
gradient of EGF after starvation. These results are
representative of cell behaviors over the time frame
analyzed (1 hour at 1-minute intervals; * indicates
needle position, which is the source of EGF). (C)
Quantification of cell movement relative to an EGF
gradient. The angle between cell movement and the
direction of applied chemoattractant indicates how well
a cell orients to the chemoattractant, with a smaller
angle suggesting greater alignment of the directions.
This is reflected in our results, which take the cosine of
the angle, and for which a greater alignment would give
a value closer to 1 and lower degree of alignment gives
a lower value, and 0 would indicate average random
motion. MTLn3 cells are efficiently stimulated to move
along the gradient, whereas ZBP1-expressing cells less
efficiently orient and move in response to such a
gradient (*P=0.035, error bars indicate ± s.e.m.).
Journal of Cell Science
observed in 28% of the fields analyzed by time-lapse
microscopy over the course of 20 minutes. By contrast, motility
was seen in only 2% of tumors generated by ZBP1-expressing
cells over the same time period. Strikingly, the ZBP1-expressing
cells also showed greatly reduced orientation towards vessels
when compared with the control cell line (Fig. 4D).
Here, we show that ZBP1 expression is sufficient to convert an
unpolarized cell into one with polarized morphological and
movement phenotypes. Possible mechanisms by which zipcode
interactions can result in these polarized phenotypes include
localized expression of a variety of motility-related mRNAs
(Mingle et al., 2005) (Wells et al., personal communication).
ZBP1 binds such mRNAs, and presumably exerts its effects on
polarity by regulating protein synthesis spatially and
temporally so that motility proteins are made in the right time
and place (Huttelmaier et al., 2005). In this way, the polarized
phenotype can be maintained.
Previously, we have shown that MTLn3 cells induced to
express ZBP1 have a reduced tendency to move through a filter
in response to EGF (Boyden Chamber) and to invade a
microneedle containing this chemoattractant in vivo (Wang et
al., 2004). Our data suggest that the effects of ZBP1 expression
on metastasis are caused by increases in polarity leading to a
reduction in the ability to orient towards the chemotactic
source. These results are consistent with studies in a wide
variety of species showing that exogenous chemotactic signals
must overcome the intrinsic polarity of the cells in order to
affect motility (Devreotes and Janetopoulos, 2003).
Signaling molecules play an important role in relaying
information about extracellular signals to affect cell direction
(Funamoto et al., 2002; Janetopoulos et al., 2001; Yart et al.,
2001). This study suggests a new role for signaling, in
particular for Src, in regulating metastasis by regulating
polarized protein expression. Src levels and Src kinase activity
are increased in a wide variety of cancers, particularly
mammary carcinomas (Jacobs and Rubsamen, 1983;
Ottenhoff-Kalff et al., 1992). In addition, increases in Src
activity are associated with the progression of cancer, and are
higher in metastatic lesions than in primary tumors (Talamonti
et al., 1993; Termuhlen et al., 1993). Src becomes rapidly
activated upon activation of the EGF receptor (Osherov and
Levitzki, 1994). We have shown in previous work that ZBP1
represses the translation of its bound mRNAs, particularly ?-
actin (Huttelmaier et al., 2005). Src phosphorylation on
tyrosine 396 of ZBP1 leads to a release of bound mRNA and
hence activation of ?-actin translation. Therefore, local Src
Journal of Cell Science 120 (18)
Fig. 3. ZBP1-expressing cells can sense and move towards an
oriented gradient. ZBP1-expressing cells are capable of responding
normally to a gradient of EGF, but only when the gradient is applied
in the direction of their intrinsic polarity. A chemoattractant, EGF,
was released from a needle placed in front of the leading edge of the
cell while protrusion and retraction were measured from both the
front and back of the cell. Standardized membrane protrusion is
plotted versus time after the micropipette stimulation. Responses of
ZBP1-expressing cells are not substantially different from controls
expressing only GFP (15 cells were measured for each group, error
bars indicate ± s.e.m.).
Fig. 4. ZBP1 localizes to the periphery of cells and reduces
motility and orientation towards vessels in living tumors.
(A) ZBP1-GFP-expressing cells imaged in vivo using
multiphoton microscopy show ZBP1 (green; one cell outlined
with dotted line) localized to the periphery of cells and at cell-
cell junctions (arrow). Collagen is imaged by second harmonic
generated polarized light in the multiphoton microscope
(purple, arrowhead). (B) MTLn3 cells transfected with CFP
show elongated cell morphology (arrow) and orientation
towards vessels (black spaces) in a living tumor. (C) ZBP1-
expressing cells show a rounded morphology (arrowhead)
along vessels. ZBP1-expressing cells do not polarize in the
direction of vessels. Bars, 25 ?m. (D) ZBP1 cells are less
motile than cells in tumors generated by MTLn3 control cells.
ZBP1 cells also show decreased orientation towards vessels
than cells in tumors generated by MTLn3 control cells
(*P=0.0003, error bars indicate ± s.e.m.).
Journal of Cell Science
ZBP1 reduces chemotaxis
activation at the membrane following EGF receptor stimulation
would regulate local translation of bound mRNAs that have
localized near the activated Src. This way, ZBP1 might play a
role in the mechanism by which increased Src activity
promotes metastasis. Abnormally high Src activity may lead to
widespread phosphorylation of ZBP1. This would cause bound
mRNAs to be translated in inappropriate locations, resulting in
the conversion of polarized cells to random walkers and
sensitization to chemoattractant gradients. Reduced amounts of
ZBP1 would have the same effect, to preclude polarized
translation, hence resulting in an invasive phenotype. Src
mislocalization from the periphery has been implicated in
malignancy (Verbeek et al., 1996). Therefore, a combination
of sufficient ZBP1 levels to repress translation and
appropriately localized Src activity to spatially activate this
translation near the leading edge would prevent inappropriate
responses to growth factors leading to invasion and metastasis.
Effects in tumors
ZBP1 expression reduces the response of tumor cells to
external signals both in vivo and in vitro, despite the ability of
ZBP1 to enhance motility in the absence of a gradient
(Shestakova et al., 2001). We propose that this is due to an
inherent polarity induced by ZBP1 expression (Oleynikov and
Singer, 2003). These results are also consistent with the
possibility of additional mechanisms by which ZBP1 might
exert these effects, particularly in tumors. Particularly, the
localization of ZBP1 to cell-cell junctions suggests a role in
adhesion that may further limit cell motility within a tumor and
subsequent metastasis. The zipcode dependence of junctional
localization in myoblasts also supports this hypothesis
(Rodriguez et al., 2006). Metastatic cells in vivo become
oriented and exhibit protrusive activity towards blood vessels
in response to vascular chemoattractants (Wang et al., 2002;
Wyckoff et al., 2000). These results extend these previous
studies and show that, whether in the presence of a gradient of
chemoattractant in culture or in a tumor, cells exhibit less
spontaneous protrusive activity when they express ZBP1.
Some of these results appear to contradict other studies
demonstrating that ZBP1-related mRNA-binding proteins are
highly expressed in a variety of tumors (Hammer et al., 2005;
Ross et al., 2001; Yaniv and Yisraeli, 2002), including human
breast cancer (Doyle et al., 2000). It has been suggested that
higher expression levels of such mRNA-binding proteins play,
in fact, a causative role in tumorigenesis (Tessier et al., 2004).
There are several important distinctions between these studies
(Doyle et al., 2000) and those previously published by us
(Wang et al., 2004). The ZBP1-expressing cells can form
tumors as well as wild-type cells, and this is consistent with
observations by others. However, the data presented here
address the effect of ZBP1 expression specifically on the role
of chemotaxis, which affects metastasis rather than
tumorigenesis. In particular, we have noticed that the decrease
in ZBP1 expression observed in tumors occurs only in the
invasive subpopulation of tumor cells, whereas the expression
of ZBP1 in the non-invasive cells remains relatively elevated
in the same tumor (Wang et al., 2004). Since the invasive tumor
cells are a minor fraction of the tumor mass their contribution
to the ZBP1 expression status of the whole tumor would not
be detected by gross studies of expression levels in tumors.
Since we have focused particularly on the stage in which tumor
cells migrate and prepare for intravasation – a prerequisite for
metastasis – we can determine effects of ZBP1 that may be
particularly crucial for outcome prediction and treatment of
cancer patients with regard to metastasis.
Materials and Methods
Cell culture and cell lines
MTLn3 cells (rat mammary adenocarcinoma cell line) stably expressing EGFP-
FLAG-ZBP1 were generated as previously described (Wang et al., 2004). MTLn3
cells stably expressing ECFP were generated as previously described (Sahai et al.,
2005). Cells were grown in ?-modified Eagle’s medium containing 5% fetal bovine
serum, and the antibiotics penicillin and streptomycin as previously described
(Bailly et al., 1998; Segall et al., 1996).
Analysis of expression levels
Cell lysates were diluted with SDS sample buffer, boiled for 5 minutes and separated
on SDS-PAGE. Proteins were transferred to Hybond ECL membranes (Amersham)
by wet blotting. Primary rabbit polyclonal antibody raised against full-length His-
tagged recombinant ZBP1 was used at 1:4500 and primary mouse monoclonal
antibody against ?-tubulin (Rockland) was used at 1:500. Secondary anti-mouse
Alexa Fluor 680 (Invitrogen Molecular Probes) was used at 1:10,000 and anti-rabbit
IRDye 800 (Rockland) was used at 1:5000. Signal was visualized using the Odyssey
Infrared Imaging System (Li-Cor) and analyzed using IPLab software (BD
Motility in serum
To determine motility characteristics, cells were imaged in their growth medium on
35-mm glass-bottomed MatTek dishes prepared as previously described on a
microscope with a computer-controlled CCD camera as described previously
(Lorenz et al., 2004). Multiple fields were collected concurrently using a macro that
cycles stage positions, and images were collected using IPlab software (BD
Biosciences) and analyzed using a combination of NIH image (developed by the
National Institute of Health available at http://rsb.info.nih.gov/nih-image/) to trace
cell perimeters and DIAS software (Solltech) to analyze persistence (speed divided
by the direction change), and directionality (net path length divided by total path
Response to chemoattractant
Cells were starved and EGF (25 nM) was introduced through a micropipette as
previously described (Mouneimne et al., 2004). In the Soon Chamber Assay, cells
were plated on coverslips on glass bottom dishes, and stimulated by a gradient of
EGF produced by release from the micropipette at the side of a dam (Soon et al.,
2005). Cells were imaged on an inverted Olympus IX-70 microscope (Olympus)
every minute for 1 hour for this assay, whereas in the micropipette assay cells
were stimulated and imaged every minute for 10 minutes. Analysis of motility in
the Soon Chamber Assay was also performed using DIAS, by tracing cell
perimeter, calculating centroid position at each time point, and determining the
cosine of the angle between a line connecting the centroid movement between
timepoints and a line connecting the centroid to the tip of the micropipette at the
side of the dam.
After starvation and micropipette stimulation with EGF as described above,
membrane protrusion was monitored by time-lapse microscopy. Quantification
designated the side facing the EGF stimulation as front side and the side facing
away as the back side. Front and back protrusions were measured along a line
passing through the centroid and the tip of the micropipette. Measurements recorded
at 30-second intervals, after introduction of the micropipette, were calculated using
ImageJ (http://rsb.info.nih.gov/ij/), and were all standardized over the values of the
same cell at 0 seconds (immediately before stimulation). Standardized
measurements were averaged and plotted versus the time after stimulation.
MTLn3-ZBP1 or MTLn3-GFP cells were injected into the mammary fat pads of
female Fisher 344 rats or SKID mice to derive tumors. After 3-4 weeks of growth
rats were placed under isoflurane anesthesia and the tumor was exposed using a
simple skin flap surgery. The animal was then placed onto an inverted Olympus IX-
70 multiphoton microscope (Olympus), using a 20? objective and time-lapse
images were acquired. Approximately three fields of each tumor were imaged for
20-30 minutes each. These procedures have been previously described in detail
(Wyckoff et al., 2000).
Time lapse movies from tumors were reconstructed with Image J and directly
evaluated for cell extension, retraction and locomotion as described (Wyckoff et al.,
Journal of Cell Science
3178 Download full-text
This work was supported by NIH grant AR41480 to R.H.S. and
NIH grant CA100324 to J.C. We thank Shailesh Shenoy for his help
with the figures, and Amber Wells and Stefan Huttelmaier for their
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Journal of Cell Science 120 (18)
Journal of Cell Science