The Rockefeller University Press $30.00
J. Cell Biol. Vol. 186 No. 1 99–111
Correspondence to Jonathan A. Cooper: firstname.lastname@example.org
B. Wollscheid’s present address is National Center for Competence in
Research Neuro Center for Proteomics, Institute of Molecular Systems Biology,
Swiss Federal Institute of Technology Zurich and University of Zurich, 8093
C. Henderson’s present address is Vaccine and Gene Therapy Institute, Oregon
Health and Science University, Beaverton, OR 97006.
Abbreviations used in this paper: ARH, autosomal recessive hypercholesterol-
emia; CSC, cell surface capture; HFF, human foreskin fibroblast; LDLR, low den-
sity lipoprotein receptor; shRNA, short hairpin RNA; SILAC, stable isotope
labeling with amino acids in cell culture; Tfn, transferrin; TfnR, Tfn receptor.
Endocytosis and exocytosis move membrane proteins between
the cell surface and intracellular compartments, allowing cells
to adapt to changes in their environment. Endocytosis can be
clathrin dependent or independent (Schmid, 1997). Receptors
that undergo clathrin-dependent endocytosis contain endocyto-
sis signals that bind to adapter proteins, including the well-char-
acterized tetramer AP2 and monomeric phosphotyrosine-binding
domain proteins Dab2, Numb, and autosomal recessive hyper-
cholesterolemia (ARH; Traub, 2003). Multiple low affinity in-
teractions between adapter proteins and clathrin then cooperate
to assemble a clathrin-coated pit that invaginates and pinches
off to form an intracellular vesicle.
Dab2 is an NPXY sequence–specific clathrin adapter that
internalizes the low density lipoprotein receptor (LDLR) and
related receptors (Keyel et al., 2006; Maurer and Cooper, 2006).
It forms dynamic complexes with its cargoes and recruits clath-
rin (Morris and Cooper, 2001; Mishra et al., 2002; Keyel et al.,
2006; Chetrit et al., 2009). Dab2 expression is strongly reduced
in many different carcinomas, particularly ovarian and mam-
mary tumors (Mok et al., 1994; Schwahn and Medina, 1998),
and Dab2 loss allows carcinoma cells to resist anoikis (Sheng
et al., 2000; Wang et al., 2001). Dab2 has also been reported
to regulate the migration of various cell types (Hocevar et al.,
2005; Orlandini et al., 2008). It is unclear whether the roles of
Dab2 in cancer and migration stem from its function as an
endocytic adapter or other mechanisms.
Integrins are cell surface receptors for various ECM
components, with different combinations of integrin and
subunits conferring ECM ligand specificity (Hynes, 1992). In-
tegrins act as bistable switches, toggling between an inactive,
unbound state and an active conformation simultaneously able
to bind the ECM and the cytoskeleton (Carman and Springer,
2003). Binding to the ECM and cytoskeleton induces the clus-
tering of active integrins into structures known as focal com-
plexes or adhesions from which signals are generated to
regulate cellular responses. However, unbound integrins are
teomics approach to identify and quantify glycoprotein
cargoes for an endocytic adapter, Dab2. Surface levels
of integrins 1, 1, 2, and 3 but not 5 or v chains
were specifically increased on Dab2-deficient HeLa cells.
Dab2 colocalizes with integrin 1 in coated pits that are
dispersed over the cell surface, suggesting that it regu-
lates bulk endocytosis of inactive integrins. Depletion of
lathrin-associated endocytic adapters recruit
cargoes to coated pits as a first step in endocyto-
sis. We developed an unbiased quantitative pro-
Dab2 inhibits cell migration and polarized movement of
integrin 1 and vinculin to the leading edge. By manipu-
lating intracellular and surface integrin 1 levels, we
show that migration speed correlates with the intracellu-
lar integrin pool but not the surface level. Together, these
results suggest that Dab2 internalizes integrins freely
diffusing on the cell surface and that Dab2 regulates
migration, perhaps by maintaining an internal pool of
integrins that can be recycled to create new adhesions at
the leading edge.
Quantitative proteomics identifies a Dab2/integrin
module regulating cell migration
Anjali Teckchandani,1 Natalie Toida,1 Jake Goodchild,1 Christine Henderson,2 Julian Watts,2 Bernd Wollscheid,2
and Jonathan A. Cooper1
1Fred Hutchinson Cancer Research Center, Seattle, WA 98109
2Institute for Systems Biology, Seattle, WA 98103
© 2009 Teckchandani et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publica-
tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 186 • NUMBER 1 • 2009 100
for Dab2 may be increased on the surface and decreased on
intracellular membranes of Dab2-deficient cells.
We used SILAC (stable isotope labeling with amino acids
in cell culture) labeling for quantification and identified cell
surface–exposed proteins using the recently developed cell sur-
face capture (CSC) technology (Fig. 1 A; Ong et al., 2002;
Wollscheid et al., 2009). Dab2-deficient HeLa cells were gener-
ated by use of a retroviral vector carrying Dab2-specific short
hairpin RNA (shRNA). A control HeLa cell line was generated
in parallel using control shRNA (Fig. 1 B). Dab2-deficient cells
were grown in SILAC media containing [13C6,15N4]l-arginine
and [13C6]l-lysine, and control cells were grown in normal
media (Ong et al., 2002). After five to six generations, the cells
were harvested and mixed, and their surface glycoproteins were
tagged with biocytin hydrazide (Wollscheid et al., 2009). Sub-
sequently, the cells were lysed, and a microsomal pellet was
prepared. Microsomal proteins were trypsinized, and tagged
glycopeptides were affinity purified with streptavidin beads.
N-glycosylated peptides were specifically eluted by using protein
N-glycosidase F. Eluted N-glycosites (formerly N-glycosylated
peptides) were identified and quantified using mass spectrometry–
based proteomics. Of the 100–150 surface glycoproteins de-
tected in two independent experiments, 57 were identified, and
41 were reliably quantified in both experiments (Tables S1 and S2).
13 glycoproteins were reproducibly increased on the surface
of Dab2-deficient cells (i.e., their light/heavy isotope ratios de-
creased in both experiments). 5 of these 13 glycoproteins were
integrins (Fig. 1 C). Surface levels of integrins 1, 1, 2, and
3 increased the most, and integrin 5 increased slightly. Integ-
rin v was not changed (Table S2). Because an effect of Dab2 on
integrins may partly explain the down-regulation of Dab2 in
cancer, we investigated the effect of Dab2 on integrins in detail.
We measured surface integrin levels by staining fixed,
nonpermeabilized Dab2-deficient and control cells with anti-
integrin antibodies and fluorescent secondary antibodies, fol-
lowed by FACS. HeLa cells express integrins 11, 31, 21,
51 (in approximate order of decreasing abundance), and v5
(Riikonen et al., 1995). Consistent with the CSC proteomic
results, removal of Dab2 caused 1.7–2.5-fold increases in sur-
face levels of integrins 1 and 1, a small increase in 5, and
no change in v5 (Fig. 2, A and B). Because transferrin (Tfn)
receptor (TfnR) endocytosis does not require Dab2 (Maurer and
Cooper, 2006), we measured TfnR steady-state surface levels as
a control. As expected, TfnR surface levels were unaltered by
Dab2 depletion (Fig. 2 B). The observed effects were specific
because surface integrin 1 was also increased when Dab2 was
transiently depleted with siRNAs targeting different sequences
in Dab2 (Fig. S1 A), and integrin levels were rescued by re-
expressing Dab2 (see Fig. 6 C).
To determine whether the increase in steady-state surface
integrin levels resulted from a change in overall abundance,
cells were permeabilized before staining with integrin 1 anti-
body and analyzed by flow cytometry. Total integrin 1 levels
were only slightly increased by Dab2 removal (Fig. 2 C). More-
over, integrin mRNA levels, measured by RT-PCR, were the
same in control and Dab2-deficient cells (Fig. 2 C). These re-
sults suggest that most of the increase in surface integrin 1 is
inactive and diffuse rapidly in the plane of the membrane
(Duband et al., 1988).
Cell migration requires active focal adhesion disassem-
bly and integrin recycling to allow new contacts to form near
the front of the cell (Webb et al., 2004; Jones et al., 2006).
After focal adhesion disassembly, integrins may diffuse or are
actively recycled, via intracellular compartments, to sites of
new adhesion assembly (Bretscher, 1996; Caswell and Norman,
2006). Intracellular integrin trafficking routes and their regu-
lation are becoming understood (Lawson and Maxfield, 1995;
Pierini et al., 2000; Laukaitis et al., 2001; Rappoport and
Simon, 2003). Specifically, integrin recycling can occur
through “short loop,” returning directly from early endosomes
to the nearby cell surface, or “long loop,” passing via a peri-
nuclear recycling compartment and then returning to the cell
surface at distant sites, including the leading edge. In cancer
and epithelial cells, the long-loop pathway is needed for mi-
gration on collagen-coated surfaces and invasion of the colla-
gen matrix (Powelka et al., 2004; Roberts et al., 2004; Li et al.,
2005; Jones et al., 2006). However, the molecules that inter-
nalize integrins and route them to the appropriate recycling
pathway are less clear. Endocytosis of different integrins may
be clathrin dependent or independent, depending on the cell
type and environment (Altankov and Grinnell, 1993; Memmo
and McKeown-Longo, 1998; Upla et al., 2004; Caswell and
Norman, 2006). Importantly, dynamin-dependent integrin
endocytosis may drive focal adhesion disassembly (Ezratty et al.,
2005). However, cells in suspension also internalize integrins,
suggesting that mechanisms for bulk turnover of inactive inte-
grins exist (Bretscher, 1989).
In an unbiased screen for Dab2-modulated receptors that
may explain the role for Dab2 in cancer and cell migration, we
found that depletion of Dab2 slows the endocytosis of several
but not all integrins by HeLa cells. Measurements of specific
integrins revealed that Dab2 regulates the bulk of constitutive
endocytosis of inactive integrin 1 by HeLa cells and human
foreskin fibroblasts (HFFs). Dab2 and integrin 1 colocalize
in clathrin-coated pits at many sites dispersed over the cell
surface, not specifically at adhesion sites, suggesting that Dab2
may trap freely diffusing integrins in coated pits. Dab2-dependent
endocytosis maintains the intracellular pool of integrin 1.
Dab2 also regulates cell migration depending on its endocytic
function. Our data suggest that Dab2-mediated bulk integrin
endocytosis is important to maintain an intracellular pool of
integrin available for recycling to the front of the cell, thus en-
abling cell migration.
Surface levels of specific integrins increase
when Dab2 is absent
We devised a proteomics approach to identify cargoes whose
endocytosis depends on Dab2. We reasoned that the surface
levels of receptors are determined by the balance between ap-
pearance at the surface, by exocytosis of newly synthesized
and recycling receptors, and loss from the surface by shedding
or endocytosis for recycling or destruction. Therefore, cargoes
101DAB2-MEDIATED INTEGRIN TRAFFIC • Teckchandani et al.
with fluorescent secondary antibody. As predicted by CSC and
FACS, Dab2-deficient HeLa cells bound 70–90% more
P5D2 than control cells (Fig. 4 A, time 0; and Fig. S1 C). Most
integrin 1 was spread over the cell surface as small puncta, as
noted previously (Powelka et al., 2004). Focal adhesions were
not detected, which is consistent with the ability of P5D2 to
bind to integrin 1 and block adhesion (Dittel et al., 1993).
Similar results were obtained with noninhibitory anti–integrin
1 antibody TS2/7 (Fig. S2 A). Parallel coverslips that had
been incubated with P5D2 or integrin 1 antibody were
warmed to 37°C and allowed to recycle antibody for various times.
By 30 min (Fig. 4) or 2 h (Fig. S2 A), the amount of integrin
1 or 1 antibody on the surface of control cells had decreased
by 50–55%, whereas that on the surface of Dab2-deficient
cells had decreased by 15–20%, which is consistent with
either decreased endocytosis or increased exocytosis in the
absence of Dab2.
To examine the intracellular localization of integrin 1,
cells were allowed to internalize P5D2 antibody for various
times, cooled on ice, treated with low pH and high salt to re-
move surface antibody, fixed, permeabilized, and visualized
with fluorescent secondary antibody (Fig. 4 B). In control
HeLa cells, integrin 1 was initially detected in a dispersed
population of tiny vesicles (Fig. 4 B, 15 min). It later moved
into larger, perinuclear vesicles (Fig. 4 B, 30 min). As ex-
pected, only 35% as much P5D2 was detected inside Dab2-
deficient cells than control cells, but both types of vesicles
were detected. In both cell types, the dispersed tiny vesicles
also stained with EEA1 (early endosome antigen 1), a marker
caused by altered traffic between the surface and intracellular
pools. The effect of Dab2 1 traffic was not restricted to HeLa
cells because transient knockdown of Dab2 in HFFs also in-
creased the surface but not total level (Fig. 2 D and Fig. S1 B).
To test whether other endocytic proteins regulate integrin
surface levels, we compared the effects of removing Dab2,
clathrin, AP2, ARH, or Numb. Numb was recently reported to
regulate integrin endocytosis in HeLa cells (Nishimura and
Kaibuchi, 2007). Removal of Dab2, AP2, or clathrin but not
ARH increased the surface levels of integrins 1 and 1 to a
similar extent (Fig. 3 A). Combined removal of Dab2 and AP2
caused little further increase in integrin 1 or 1, suggesting
that Dab2 requires AP2 (and/or vice versa) to regulate integrin
levels. Integrin 5 levels depended more on AP2 or clathrin than
Dab2 (Fig. 3 A). In mirror image to Dab2, Numb removal had a
greater effect on integrin 5 than integrin 1 or 1 (Fig. 3 B).
These results suggest that clathrin and AP2 regulate 1, 1, and
5, but Dab2 and Numb regulate partly overlapping subsets
with Dab2 dominant for 1 and 1 and Numb dominant for 5.
Combined removal of Dab2 and Numb had a less than additive
effect on surface levels of integrin 1 (Fig. 3 C), suggesting
either that they are partially redundant or that maximum integrin
levels have been reached. Neither Dab2 nor Numb affected
Dab2 regulates integrin traffic
Control or Dab2-deficient HeLa cells were grown on collagen-
coated coverslips, incubated with P5D2 anti–integrin 1 anti-
body at 4°C, and washed, and surface integrin 1 was detected
Figure 1. Identification of Dab2 cargoes by CSC pro-
teomics. (A) Outline of rationale for the CSC approach.
Black dots represent biotin tags. (B) Western blot analy-
sis shows that Dab2 levels are greatly decreased in
a Dab2-deficient HeLa cell line. (C) Relative surface
abundance changes of glycoproteins measured by
SILAC-based CSC proteomics. Mean and SD of SILAC
ratios (control/Dab2 deficient) for six integrins (pink
squares) and mean SILAC ratios for 35 other proteins
(blue diamonds) that were quantified in two experi-
ments are shown. Glycoproteins increased in both
experiments are in the lower left quadrant (yellow).
con, control; LC, liquid chromatography; MS, mass
spectrometry; PNGaseF, protein N-glycosidase F.
JCB • VOLUME 186 • NUMBER 1 • 2009 102
To monitor integrin 1 traffic more quantitatively, we
chemically labeled surface proteins with a nonpermeable, revers-
ible biotinylation reagent, sulpho-NHS-SS-biotin. After labeling
and removal of excess reagent, we warmed the cells for various
times before cooling again and stripping off surface biotin with a
nonpermeable reducing agent (MesNa). The cells were then
lysed, integrin 1 was immunoprecipitated, and the biotin label
was detected by Western blotting (Fig. 4 D). Integrin 1 entered
control HeLa cells at 1% min1. This is slower than for serum-
stimulated HeLa cells (Powelka et al., 2004), suggesting that we
are measuring basal endocytosis. Internalization of integrin 1
was strongly inhibited in Dab2-deficient cells (Fig. 4, D and E).
of early endosomes (Fig. 4 C). To test whether the larger peri-
nuclear vesicles may be recycling endosomes, we followed
the traffic of Tfn (Powelka et al., 2004). Integrin 1 followed
the same path as Tfn in both Dab2-deficient and control cells
from dispersed tiny vesicles to larger perinuclear vesicles
(Fig. 4 C). As expected from the lack of an effect of Dab2 on
surface TfnR levels (Fig. 2 B), the quantity of internalized
Tfn did not depend on Dab2 (Fig. 4 C). These results suggest
that both control and Dab2-deficient cells internalize integrin
1 into early endosomes and then pass it into perinuclear
recycling endosomes, but endocytosis is impaired in Dab2-
Figure 2. Steady-state surface levels of integrins 1 and 1 but not v increase in Dab2-deficient cells. (A) Fixed, nonpermeabilized control and Dab2-
deficient HeLa cells were analyzed by FACS. (B) Mean and standard error of fluorescent intensity from four independent experiments are shown. (C) FACS
analysis of fixed, permeabilized HeLa cells shows that total integrin 1 only increases slightly on Dab2 removal. RT-PCR for integrin 1 and actin mRNA
shows equal mRNA levels in control and Dab2-deficient cells. Mean and standard error from five independent experiments are shown. (D) HFFs treated with
control or Dab2 siRNA. Western blot showing decreased Dab2 protein and FACS results showing increased surface integrin 1 (mean and standard error
of two independent experiments). Data for total integrin 1 level in Dab2-deficient HFFs are shown in Fig. S1 B. (B and D) Dashed lines indicate the control
levels. #, P < 0.05; *, P < 0.01; and **, P < 0.001 by t test. 2° Ab, secondary antibody control; con, control; ERK, extracellular signal-regulated kinase.
103DAB2-MEDIATED INTEGRIN TRAFFIC • Teckchandani et al.
cells internalized less collagen and removed less collagen from the
surface than control cells (Fig. S2 B). These results suggest that
Dab2 is needed for endocytosis of integrin–collagen complexes.
Dab2 colocalizes with integrin 1
Dab2 localizes to AP2-containing clathrin-coated pits distrib-
uted over the cell surface (Morris and Cooper, 2001). However,
very little integrin 1 colocalized with Dab2 in these structures
Even when reexport was inhibited using primaquine (Reid and
Watts, 1990), control cells internalized integrin 1 more rapidly
than Dab2-deficient cells (Fig. 4, F and G). These results show
that integrin 1 endocytosis is inhibited when Dab2 is absent.
As an independent test of whether Dab2 regulates integrin
traffic, we followed uptake of an integrin 11 ligand, collagen IV
(Hynes, 1992). Cells were plated on fluorescent collagen IV for 2 h
at 37°C and fixed, and collagen was visualized. Dab2-deficient
Figure 3. Effect of ARH, AP2, clathrin, or Numb depletion on surface integrin levels. (A–C) HeLa cells were treated with various siRNAs and then fixed
and analyzed by FACS with anti-integrin or anti-TfnR antibody. Mean values and standard errors from at least three independent experiments are shown.
Dashed lines indicate the control levels. (A) ARH has no effect; Dab2 depletion increases surface 1 and 1; AP2 or clathrin depletion significantly in-
creases surface 1, 5, and 1. (B) Numb depletion has a bigger effect on surface 5 than on 1 or 1. In contrast, Dab2 depletion has a bigger effect on
surface 1 and 1 than on 5. The Dab2 data are the same as those shown in Fig. 2 B. (C) Effects of separate or combined removal of Dab2 and Numb.
Combined removal of Dab2 and Numb caused little further increase in integrin 1. (A and C) Western blot analysis of HeLa total lysates demonstrates that
target proteins ARH, Dab2, AP2, clathrin heavy chain (CHC), and Numb were greatly reduced by siRNA transfection. As a control, extracellular signal-
regulated kinase (ERK) levels remained constant. (B and C) #, P < 0.05; *, P < 0.01; and **, P < 0.001 by t test.
JCB • VOLUME 186 • NUMBER 1 • 2009 104
Figure 4. Dab2 regulates integrin 1 endocytosis. (A–C) Control (Con) and Dab2-deficient HeLa cells were incubated with anti–integrin 1 antibody
(P5D2) for 30 min at 4°C, followed by warming at 37°C for 0, 15, or 30 min. Mean values and standard errors (20 cells/treatment) of pixel intensities
were measured. (A) Surface antibody on fixed, nonpermeabilized cells was detected with fluorescent secondary antibody. 6-µm flattened z projections of
the entire cell are shown. (B) Surface antibody was removed by acid stripping, and internalized antibody was detected. 6-µm flattened z projections of the
entire cell are shown. (C) Internalized antibody colocalizes with EEA1 at 15 min and with Tfn at 30 min. Dab2-deficient cells internalize less integrin 1
antibody but the same amount of Tfn compared with control cells. (D–G) Control and Dab2-deficient HeLa cells were surface labeled with sulpho-NHS-SS-
biotin at 4°C for 30 min and then warmed to 37°C in the absence (D and E) or presence (F and G) of 2 mM primaquine for the indicated times. Biotin was
removed from surface receptors with MesNa treatment, and cells were lysed and incubated with an anti–integrin 1 antibody. Immunoprecipitates were
analyzed by SDS-PAGE followed by Western blotting with peroxidase-conjugated streptavidin. (E and G) Quantification of three independent experiments.
For each time point, mean values and standard errors are shown.
105DAB2-MEDIATED INTEGRIN TRAFFIC • Teckchandani et al.
Numb- and Dab2-containing clathrin-coated pits are consis-
tent with their partial redundancy for integrin endocytosis
(Fig. 3, B and C).
Dab2-mediated endocytosis regulates
Because integrin recycling is needed for directed cell move-
ment, we tested whether Dab2 regulates cell migration. Previ-
ous studies have shown that depleting Dab2 slows migration of
human umbilical vein endothelial cells and NIH 3T3 cells, but
the mechanism was not clear (Hocevar et al., 2005; Orlandini
et al., 2008). Removing Dab2 inhibited migration on collagen IV,
a ligand for the Dab2-regulated integrin 11 (Fig. 6 A). How-
ever, Dab2 had no effect on migration on vitronectin (Fig. 6 A),
a ligand for the Dab2-independent integrin v5 (Figs. 1 C and
2 B). This suggests that Dab2 may regulate migration by affect-
ing integrin traffic.
Removal of Numb also inhibited migration, although to a
lesser extent than removing Dab2 (Fig. S4). Removing both
(unpublished data). Because coated pits only have a brief half-
life (1–3 min; Puthenveedu and von Zastrow, 2006; Loerke
et al., 2009) and only 1% of integrin 1 is internalized per
minute, only a tiny fraction of the surface integrin may be in
coated pits at any given time. To trap integrins in coated pits, we
cooled cells to 4°C (Sorkin, 2004). Under these conditions, in-
active integrin 1 labeled with antibody P5D2 extensively colo-
calized with Dab2 and clathrin (Fig. 5, A and B). Surprisingly,
the Dab2-containing structures were not located close to focal
adhesions, as visualized by staining fixed, permeabilized cells
with antibodies to vinculin (Fig. 5 C). Rather, Dab2 colocalized
with inactive integrin 1 at distant sites, predominantly on the
dorsal surface of the cell (Fig. 5 A and Fig. S3 A). Thus, Dab2
is localized appropriately to mediate endocytosis of free but not
In contrast to Dab2, Numb localizes to clathrin-coated
structures that are near the leading edge of migrating cells
(Nishimura and Kaibuchi, 2007) and the periphery of non-
migrating cells (Fig. S3 B). The different distributions of
Figure 5. Localization of integrin 1 and Dab2. (A) Confluent HeLa cells plated on collagen IV–coated coverslips were treated with anti–integrin 1
antibody for 1 h at 4°C and then fixed, permeabilized, and stained with anti-Dab2 or anticlathrin antibody. Single 0.2-µm sections at the ventral or dorsal
surface are shown. (B) The number of Dab2 puncta that colocalize with integrin 1 (arrowheads in A) at the ventral and dorsal surfaces were counted.
Mean values and standard errors (two independent experiments and 12 cells/treatment/experiment) are shown. (C) Confluent HeLa cells were fixed,
permeabilized, and stained with anti-Dab2 antibodies. Antivinculin antibodies were used to visualize focal adhesions (arrowhead). Single 0.2-µm sections
at the ventral surfaces of cells are shown. (A and C) The white boxes indicate the enlarged images shown in the insets.
JCB • VOLUME 186 • NUMBER 1 • 2009 106
of collagen, but this was not observed. Second, we lowered total
levels of integrin 1 in Dab2-deficient cells such that their sur-
face integrin 1 levels were the same as those on control cells
(Fig. 7, B and C). However, Dab2-deficient cells with normalized
surface 1 integrin migrated even slower than Dab2-deficient
cells with high surface 1 integrin (Fig. 7 D). In addition, lower-
ing total integrin levels in control cells reduced their migration.
Combining results from several experiments in which total inte-
grin 1 levels were reduced by siRNA in either control or Dab2-
deficient cells, we found no correlation between surface integrin
levels and migration (r = 0.055; Fig. 7 E). Instead, there was
an excellent correlation between migration rate and internal in-
tegrin levels (r = 0.977; Fig. 7 F). These results mean that the
slow migration of Dab2-deficient cells is unlikely to be caused
by excess adhesion and more likely caused by the altered intra-
Dab2 is reported to have signaling functions (Xu et al.,
1998; Wang et al., 2001; Zhou et al., 2003). Dab2 absence
may alter signaling from focal adhesions and thus affect
focal adhesion assembly or disassembly. We measured the ac-
tivities of Src and FAK using phosphoepitope antibodies. If
anything, the activities of Src and FAK were increased in
Dab2-deficient HeLa cells plated for 45 min on collagen IV
(Fig. S5). This suggests that Dab2 is not required for focal
If Dab2-mediated integrin internalization is important
for trafficking integrins to the leading edge, we predicted that
Dab2-depleted migrating cells may fail to polarize their inte-
grins toward the leading edge. We examined control and
Dab2-deficient cells migrating into a scratch wound (Fig. 7,
Dab2 and Numb caused a greater inhibition than removing
either alone. These results showed an inverse correlation between
cell surface integrin 1 level and migration speed (r = 0.988;
Fig. S4), again suggesting that inhibiting integrin endocytosis
If Dab2 stimulates migration by affecting endocytosis, a
form of Dab2 that does not support endocytosis should not
support migration. The p67 splice form of Dab2 lacks two
clathrin-binding sites and one of two AP2-binding sites pres-
ent in the other form, p96, and unlike p96, it does not associate
with coated pits and is defective for LDLR endocytosis (Morris
and Cooper, 2001; Mishra et al., 2002; Maurer and Cooper,
2006). T7 epitope–tagged Dab2 p96 or p67 was transiently
expressed in Dab2-deficient HeLa cells. Reexpression of Dab2
p96 but not p67 rescued normal integrin levels and migration
(Fig. 6, C and D). This suggests that Dab2 regulates migration
via its effect on endocytosis. We reasoned that Dab2 may reg-
ulate migration by changing surface integrin levels, integrin
signaling, or integrin recycling.
Cell-substrate adhesion affects migration in opposing
ways: adhesion must be strong enough to provide traction but not
so strong as to be an anchor (Palecek et al., 1997). We used two
approaches to alter the adhesive forces of Dab2-deficient cells.
First, we varied adhesion by use of different collagen concentra-
tions. Decreasing or increasing collagen concentration decreased
or increased the migration rate of both control and Dab2-
deficient cells, but Dab2-deficient cells always migrated more
slowly than the controls (Fig. 7 A). If lack of Dab2 had slowed
cell migration by causing excess adhesion, we would have ex-
pected that their migration would increase on lower concentrations
Figure 6. Dab2-mediated migration requires the p96-specific exon. (A and D) Migration through filters coated with collagen IV (A and D) or vitronectin
(A) toward 10% FBS was measured using a Boyden chamber. Cells passing through triplicate membranes were counted and averaged. The collagen IV
results in A show mean and standard error of four independent experiments. *, P < 0.01. (B) Drawing of p96 and p67 forms of Dab2 showing known
binding sites. (C and D) Surface integrin 1 levels (C) and migration (D) for Dab2-deficient HeLa cells reexpressing vector alone or T7-tagged mouse p96
or p67. Mean values and standard errors from two independent experiments are shown. Dashed lines indicate the control levels. (E) Western blots show
Dab2 knockdown and reexpression of T7-tagged mouse p96 or p67. Con, control.
DAB2-MEDIATED INTEGRIN TRAFFIC • Teckchandani et al.
Discovery-driven quantitative analysis of cell surface glycopro-
teins and antibody analysis showed that Dab2 regulates the sur-
face levels and endocytosis of specific integrins. In HeLa cells,
Dab2 has the greatest effects on the surface levels of integrins
1, 1, 2, and 3 but not 5 or v. In the case of integrin 1,
the absence of Dab2 increases the surface level and reduces the
G and H). We found that migrating control cells are asym-
metric, with an increased concentration of integrin 1 and the
focal complex protein vinculin along the leading edge. How-
ever, Dab2-deficient cells are isotropic, with points of vincu-
lin and integrin 1 scattered around the entire periphery.
These results are consistent with a failure of polarized rout-
ing of integrin 1 to the leading edge, perhaps caused by re-
Figure 7. Dab2-mediated endocytosis regulates cell migration. (A and D) Migration through filters coated with collagen IV toward 10% FBS was measured
using a Boyden chamber. *, P < 0.01; **, P < 0.001. (A) Quantification of two independent experiments performed in triplicate. For each point, mean
values and range are shown. (B and C) Integrin 1 levels were reduced in Dab2-deficient cells using 1-specific siRNA. The mean fluorescence intensity
of surface integrin 1 was measured by flow cytometry. (B) FACS histogram from a representative experiment. (C) Means and standard errors from four
independent experiments are shown. (D) Migration of control and Dab2-deficient cells with high and normalized 1 surface levels. Mean and standard
error from four independent experiments are shown. (C and D) Dashed lines indicate the control levels. (E and F) Correlation between migration and
surface integrin level (r = 0.055; E) or intracellular integrin pool (r = 0.977; F). Each point shows the mean and SD of triplicate migration measurements
and a single surface integrin measurement on the same cells, normalized to a simultaneous control (control HeLa, no 1 siRNA migration = 1.0). Surface
integrin 1 levels were measured by FACS. Intracellular integrin 1 levels were calculated from the measured surface level and the steady-state ratio of the
intracellular and surface levels (Fig. 2 and Fig. S2 A). Control HeLa cells: surface 1 = 45%, intracellular 1 = 55%, intracellular/surface = 1.22; Dab2
shRNA cells: surface 1 = 83%, intracellular 1 = 17%, intracellular/surface = 0.20. Dashed lines are best fit to the data. (G and H) Scratch wound assay.
Cells were stained with vinculin (G) or integrin 1 (H). Representative single 0.2-µm sections at the ventral surface of leading edge cells from one of two
independent experiments are shown. The arrow indicates the direction of migration, and the white boxes indicate the enlarged images shown in the insets.
Vinculin and integrin 1 (arrowheads) concentrated at the leading edge of control but not Dab2-deficient cells. (I) Model for the role of Dab2 in integrin
endocytosis and cell migration. Dab2 mediates endocytosis of 1 integrins from coated pits scattered randomly over the cell surface and maintains an
internal pool of integrins. This pool is important for efficient migration because it supplies integrins for recycling to the leading edge and thus allows the
polarized formation of new focal complexes and focal adhesions. Con, control.
JCB • VOLUME 186 • NUMBER 1 • 2009 108
L2 and 51 have no effects on their endocytosis (Vignoud
et al., 1994; Fabbri et al., 1999). Importantly, Bretscher (1992)
noted that different integrin 1 dimers had different endocytic rates
depending on their chains. Therefore, it is possible that the integ-
rin chains rather than the chains may be recognized for endo-
cytosis. Indeed, the differential endocytosis of chains by Dab2
and Numb implies that adapters may distinguish different com-
plexes based on cytoplasmic domains. An alternative explana-
tion, that Dab2 selects integrins for endocytosis only when they are
disengaged from the cytoskeleton, is discussed next.
Focal adhesion disassembly and
Our evidence suggests that Dab2 mediates endocytosis of integ-
rins that are not attached to the cytoskeleton. Altered integrin 1
levels and endocytosis were detected with an antibody that binds
the inactive conformation (Dittel et al., 1993) and does not ac-
cess focal adhesions (Fig. 4). Moreover, we detected integrin 1
in Dab2-containing pits even on the top of the cell (Fig. 5 A). In
addition, Dab2 had a greater effect on surface levels of integrin
11 when cells were plated on plastic or fibronectin than colla-
gen, the ligand for 11 (unpublished data). Integrins diffuse
rapidly over the cell surface if they are not attached to the cyto-
skeleton (Duband et al., 1988). Dab2 can mediate uptake of
ECM ligands (Fig. S2 B). Together, these results imply that Dab2
internalizes integrins that are disengaged from the cytoskeleton
whether or not they are bound to ECM proteins.
A role for Dab2 in endocytosis of disengaged integrins is
consistent with the possible binding of Dab2 to the NPXY se-
quences of integrin chains. These sequences are known to be
critical for binding the focal adhesion protein talin and thereby
creating cytoskeletal attachments (Tadokoro et al., 2003). Talin
release would be needed to expose the NPXY before Dab2
could bind. This would explain a selectivity of Dab2 for dis-
In addition to this mechanism, it is clear that integrin endo-
cytosis also occurs at sites of focal adhesion disassembly. In
highly migratory cells, or in cells in which microtubules are dis-
rupted and then allowed to repolymerize in synchrony, focal com-
plexes are targeted by microtubules, which bring in the endocytic
protein dynamin and stimulate focal adhesion disassembly
(Kaverina et al., 1999; Ezratty et al., 2005). Recent evidence sug-
gests that Dab2 may also be involved, targeting focal adhesions that
are disassembling at the leading edge of migrating HT1080 cells
(Chao and Kunz, 2009). However, in HeLa cells, there appears to
be a division of labor: Numb is localized near focal adhesions at
the periphery and leading edge (Nishimura and Kaibuchi, 2007),
and it may be involved in the synchronized endocytosis of inte-
grins from disassembling adhesion sites, whereas Dab2 mediates
bulk endocytosis of disengaged integrins.
Regulation of migration
We and others have found that Dab2 removal inhibits migra-
tion of several types of cells (Hocevar et al., 2005; Orlandini
et al., 2008; Chao and Kunz, 2009). The mechanism may vary
in different cell types. One possible mechanism, based on
overexpression experiments, is that Dab2–integrin complexes
intracellular pool, with no change in total level. The shift is
caused by a decreased rate of endocytosis. Dab2 colocalizes
with inactive integrin 1 at clathrin-coated pits that are evenly
scattered over the cell surface, so Dab2 likely mediates endo-
cytosis of freely diffusing integrin 1 that is not engaged with the
actin cytoskeleton or the ECM. After endocytosis, integrin 1 is
recycled via the perinuclear recycling compartment. In the ab-
sence of Dab2, integrin 1 and vinculin are not polarized to the
leading edge, and cell migration is inhibited. Therefore, we pro-
pose that Dab2 promotes migration by maintaining an internal
pool of integrin 1 that can be recycled to the surface to create
new adhesion sites (Fig. 7 I).
Dab2 as an endocytic adapter for integrins
Integrins 1, 1, and 5 were up-regulated on the surface of cells
lacking clathrin or AP2, but only integrins 1 and 1 were up-
regulated when Dab2 was absent (Figs. 1 and 2 and Fig. S1 A).
This suggests that Dab2 is selective. Because ARH and Numb
are structurally similar to Dab2, we tested whether these endo-
cytic adapters regulate integrin surface abundance. ARH had no
detectable role, but Numb had a major influence on integrin 5
and small effects on integrins 1 and 1 (Fig. 3). This suggests
that Dab2 and Numb may have partially separate but overlapping
roles. Numb was previously shown to mediate endocytosis of a
subpopulation of surface integrins 1 and 3 (Nishimura and
Kaibuchi, 2007). Both Dab2 and Numb regulate cell migration
(Fig. S4; Nishimura and Kaibuchi, 2007). Numb localizes near
the front, ventral surface of migrating cells (Nishimura and
Kaibuchi, 2007) and around the edge of stationary cells (Fig. S3 B),
whereas Dab2 is found in most coated pits scattered over the cell
surface (Fig. 5 A). This suggests that Numb may regulate the up-
take of integrins 1 and 5 near the leading edge and that Dab2
may internalize integrins 1, 1, 2, and 3 elsewhere on the
cell surface. The partial overlap between Dab2 and Numb in
integrin traffic resembles the parallel effects of Dab2 and ARH
in LDLR endocytosis (Keyel et al., 2006; Maurer and Cooper,
2006) and may explain why dab2 and numb mouse mutants do
not show more pleiotropic phenotypes (Zhong et al., 2000; Morris
et al., 2002; Maurer and Cooper, 2005).
How are integrins selected for endocytosis? Integrin 1
becomes trapped in Dab2-containing coated pits at low temper-
ature (Fig. 5 A), suggesting a physical interaction. Integrin chains
contain one or more NPXY sequences related to the sequences
in lipoprotein receptors that bind the Dab2 phosphotyrosine-
binding domain (Morris and Cooper, 2001; Mishra et al., 2002).
Indeed, Dab2 and integrin 1 have been coimmunoprecipitated
from untreated or TGF-–treated murine mammary epithelial
cells (Prunier and Howe, 2005), suggesting direct binding.
However, we and others (Chetrit et al., 2009) have been unable
to coimmunoprecipitate Dab2 with integrin 1 from HeLa cells.
Moreover, although Dab2 binds well to integrins 3 and 5
in vitro, it binds poorly to 1 (Calderwood et al., 2003). The
importance of the NPXY sequence in integrin chains for endo-
cytosis is controversial. A conservative NPXF mutant integ-
rin 1 appears to function and traffic normally in keratinocytes
but not fibroblasts (Czuchra et al., 2006; Pellinen et al., 2008),
whereas nonconservative mutations of the NPXY signals in
109DAB2-MEDIATED INTEGRIN TRAFFIC • Teckchandani et al.
Cruz Biotechnology, Inc.), rabbit anti-Dab2 (Santa Cruz Biotechnology, Inc.),
mouse anti-Dab2 (BD), mouse anticlathrin (X22; Abcam), mouse antiadaptin
(clone AP.6; EMD), mouse anti-T7 (EMD), mouse anti-TfnR (Ab-1; Abcam),
rabbit anti–phospho FAK (pY576; Invitrogen), rabbit anti-FAK (Santa Cruz
Biotechnology, Inc.), rabbit anti–phospho Src (pY416; Cell Signaling Technol-
ogy), rabbit anti-Numb (C29G11; Cell Signaling Technology), rabbit anti-
ARH (gift from L. Traub, University of Pittsburgh, Pittsburgh, PA), rabbit
anti-Fyn (FYN3; Santa Cruz Biotechnology, Inc.), mouse antivinculin (hVIN-1;
Sigma-Aldrich), mouse anti-EEA1 (BD), rabbit anti-EEA1 (Thermo Fisher Sci-
entific), and mouse anti–extracellular signal-regulated kinase (BD). The mouse
hybridoma cell line (LP-016) expressing monoclonal Src antibody (LA074)
was a gift from J. Meisenhelder (Salk Institute for Biological Studies, La Jolla,
CA), and the 327 anti-Src mouse monoclonal antibody was provided by
J. Brugge (Harvard Medical School, Boston, MA). Alexa Fluor–tagged sec-
ondary antibodies were purchased from Invitrogen.
The CSC method for suspension cells was previously described (Wollscheid
et al., 2009). The following modifications were made for adherent cells
and quantitative analysis (Ong et al., 2002). Control and Dab2-deficient HeLa
cell lines were grown in normal and heavy lysine/arginine-supplemented
media (Invitrogen), respectively, for five to six population doublings. When
just confluent, cells were removed from 10 15-cm plates using 2 mM EDTA
in PBS for 10 min at 37°C, harvested by centrifugation, and allowed to re-
cover in suspension in the respective media at 37°C for 30 min. Equal cell
numbers were then combined and washed once with cold PBS and once
with cold labeling buffer (PBS containing 0.1% BSA and 20 mM Pipes, pH
6.7). All subsequent steps were performed at 0–4°C. Cells were oxidized
with 1.2 mM NaIO4 in 40 ml of labeling buffer for 30 min, washed in cold
PBS, and incubated in 25 mM biocytin hydrazide (Biotium, Inc.) in 10 ml
of labeling buffer for 2 h. Cells were washed twice, resuspended in 20 ml
of hypotonic buffer (10 mM Tris HCl, pH 7.5, and 0.5 mM MgCl2) for 10 min,
and broken with 20 strokes of a tight Dounce homogenizer. A postnuclear
supernatant was centrifuged in the SW41 rotor (Beckman Coulter) at
35,000 rpm for 1 h. Membrane pellets were resuspended and recentri-
fuged to remove cytoplasmic contamination. Subsequent steps of dissolving
in Rapigest, digesting with Endo Lys-C and trypsin, binding to streptavidin
beads, washing at high pH, and eluting bound N-glycosylated peptides
with protein N-glycosidase F have been described previously (Wollscheid
et al., 2009). Samples were analyzed using mass spectrometers (QTOF
[Agilent Technologies]; Finnigan LTQ-FT [Thermo Fisher Scientific]). Pep-
tides were identified by using the SEQUEST algorithm (Eng et al., 1994) in
combination with Peptide Prophet (Nesvizhskii et al., 2003), searching for
peptides containing aspartic acid instead of asparagine in NXS/T glyco-
sylation signals. ASAP (automated statistical analysis of protein abun-
dance) ratios were determined (Li et al., 2003) and curated manually.
Steady-state surface and total integrin levels were measured by flow cytom-
etry. Cells were detached using 10 mM EDTA-PBS for 10 min at 37°C,
washed with PBS, pelleted by centrifugation, and fixed in cold 4% para-
formaldehyde-PBS for 20 min. Cells were incubated with anti-integrin or
TfnR antibodies for 1 h at room temperature. To measure total integrin, cells
were permeabilized with 0.1% Triton X-100 in PBS for 5 min at 25°C be-
fore incubating with anti-integrin antibodies. Surface or total antibody was
quantified by FACS analysis after staining with an Alexa Fluor 488 goat
anti–mouse secondary antibody. Profiles were gated on intact cells, based
on morphology, and mean fluorescent intensity was obtained. For rescue
experiments, profiles were gated on GFP-positive cells, and surface anti-
body was quantified after staining with an Alexa Fluor 647 goat anti–
mouse secondary antibody.
Knockdown experiments in HeLa cells and primary HFFs were performed
as described previously (Maurer and Cooper, 2006). Cells were trans-
fected with 50 pmol of a pool of four siRNA oligonucleotides specific for
Dab2, clathrin, AP2 µ2, or ARH (Thermo Fisher Scientific) using Oligo-
fectamine (Invitrogen) on days 1 and 3 and analyzed on day 5. Numb
siRNA (Thermo Fisher Scientific) was given on days 1 and 3, and cells were
analyzed on day 6. Total cell lysates were analyzed by immunoblotting to
show that target protein levels were significantly reduced compared with a
protein loading control (Fig. 3).
For rescue experiments, siRNA specific for human Dab2 (Thermo
Fisher Scientific) was used to deplete cells of Dab2. Cells were trans-
fected with T7-tagged mouse Dab2 on day 4 and analyzed on day 5. For
promote initial contacts between the cell and ECM (Chetrit et al.,
2009). Alternatively, in HT1080 cells, Dab2 may stimulate focal
adhesion disassembly (Chao and Kunz, 2009). However, in HeLa
cells, Dab2 is not located near adhesion sites. Another possibility,
which we favor, is that Dab2 regulates the dynamics of integrin–
ECM contacts as an indirect effect of its role in bulk integrin
endocytosis. Our finding that Dab2 regulates HeLa cell migration
on collagen but not vitronectin is consistent with the specificity
of Dab2 for endocytosis of collagen-binding but not vitronectin-
binding integrins. Moreover, migration on collagen requires the
endocytic activity of Dab2 (Fig. 6). A possible effect of Dab2 on
adhesion is unlikely to be involved because Dab2 promoted mi-
gration regardless of collagen concentration (Fig. 7 A). The ab-
sence of Dab2 could also slow migration if the increased
disengaged integrin on the surface slowed focal adhesion dis-
assembly by a mass action effect. However, decreasing the total
level of integrin 1 did not rescue the migration of Dab2-
deficient cells (Fig. 7 D). Instead, these cells migrated even slower.
These integrin-depleted cells have less internal 1 integrin
available for exocytosis. Two lines of evidence suggest a model,
illustrated in Fig. 7 I, in which Dab2 promotes migration by
maintaining the intracellular integrin pool. First, there is a strong
correlation between migration rate and the level of intracellular
integrin (Fig. 7 F). Second, Dab2-deficient cells fail to polarize
integrins and focal complex components in the direction of mi-
gration (Fig. 7, G and H). Therefore, we propose that Dab2 may
stimulate migration by maintaining an internal store of integrin
that can be recycled to the front of the cell and stabilize the lead-
ing edge (Lawson and Maxfield, 1995; Bretscher, 1996; Jones
et al., 2006). Collectively, with the spatial separation between
focal adhesion disassembly and integrin endocytosis, this means
that endocytosis of disengaged integrins may play an indirect role
in forming new adhesion contacts at the front of the cell. The
model provides a rationale for how constitutive turnover of inac-
tive cell surface integrin could impact the rapid dynamics of focal
adhesion assembly and disassembly at the front of the cell.
Materials and methods
Cells and DNA constructs
HeLa cells and primary HFFs (gift from J. Roberts, Fred Hutchinson Cancer
Research Center, Seattle, WA) were cultured in DME supplemented with
10% FBS and 1% penicillin-streptomycin. HeLa cells were transduced to
stably express control or Dab2 shRNA. A pBabe puro vector with the his-
tone H1 promoter cloned into the second long terminal repeat (Welcker
et al., 2003) was modified by swapping in the hygromycin selection gene
(gift from M. Maurer, Fred Hutchinson Cancer Research Center). Hairpins
to target Dab2 or a control sequence were inserted downstream of the H1
promoter 5-CAAAGGATGTGGGTCAACATT-3. The control sequence tar-
geted was 5-TATGTCAAGTTGTATAGTTA-3.
Retroviral particles were generated by cotransfection of HEK293T
cells with constructs and packaging vector. HeLa cells were infected with
retrovirus and, 48 h later, selected with 250 µg/ml hygromycin. Protein
expression was detected by immunoblotting. For rescue experiments, vec-
tor alone (pCGT) or vector encoding T7-tagged mouse Dab2 (p96 or p67)
was cotransfected with pGL1SuperC GFP expression vector into HeLa cells
using Lipofectamine 2000 (Invitrogen).
The following integrin antibodies were provided by E. Wayner (Fred
Hutchinson Cancer Research Center): inhibitory mouse anti–integrin 1
(P5D2-1), mouse anti–integrin 5 (P1D6-H9), and mouse anti–integrin v5
(P1F6). Other antibodies included mouse anti–integrin 1 (TS2/7; Santa
JCB • VOLUME 186 • NUMBER 1 • 2009 110
resuspended in DME at 250,000 cells/ml. The lower wells of the chamber
were loaded with 10% FBS in DME. An 8-µm pore diameter membrane
(Neuro Probe, Inc.) coated with 4 µg/ml collagen IV or 2 µg/ml vitronectin
separated the bottom and top chambers. Cells were added to the top
wells. The chamber was incubated in a humidified atmosphere of 5% CO2
at 37°C for 12 h. The cells on the top side of the membrane were removed,
and the migrated cells on the bottom side were stained with 0.1% crystal
violet in 20% ethanol and counted by using a light microscope at a magni-
fication of 40.
Scratch wound assay. Dab2 shRNA and control cells were grown at
equivalent density for 24 h on coverslips coated with 4 µg/ml collagen IV.
A single scratch was made with a pipette tip, and cells were allowed to
migrate into the wound for 10 h. Cells were stained with antibodies to
integrin 1 and vinculin and visualized using a 40× oil objective on
a microscope (DeltaVision; Carl Zeiss, Inc.).
Online supplemental material
Fig. S1 shows that steady-state surface levels of integrin 1 increase upon
depletion of Dab2. Fig. S2 shows that Dab2 regulates integrin 11
endocytosis and collagen uptake. Fig. S3 shows that Dab2 and integ-
rin colocalize over the entire dorsal surface, whereas Numb is enriched
at the periphery of the cell on the ventral side. Fig. S4 shows that the
combined removal of Dab2 and Numb caused a greater inhibition of
migration than removing either alone. Fig. S5 shows that FAK and Src
activation increases on Dab2 depletion. Table S1 shows isotope labeling
ratios (ASAP; light/heavy) of 35 cell surface–labeled proteins, excluding
integrins, that were quantified by SILAC and CSC. Table S2 shows surface
integrin quantification by SILAC labeling and CSC of proteins from con-
trol cells labeled with light isotopes and Dab2-deficient cells labeled with
heavy isotopes. Online supplemental material is available at http://www
We gratefully acknowledge reagents, expert advice, and assistance from
M. Maurer, J. Roberts, E. Wayner, H. Qian, C.J. McGlade, J. Ranish, D. Bausch-
Fluck, and the Fred Hutchinson Cancer Research Center Flow Cytometry and
Scientific Imaging resources. We are especially grateful to S. Parkhurst,
E. Mulkearns, T. Parsons, D. Schlaepfer, L. Traub, V. Vasioukhin, and R. Walter
for comments on an early draft of the manuscript.
This research was supported by National Institutes of Health grants R01-
GM66257 (to J.A. Cooper), RO1-AI51344-01 (to J. Watts), and N01-HV-
28179-22 (to B. Wollscheid) and the National Institutes of Health National
Research Service Award GM078776 (to A. Teckchandani).
Submitted: 30 December 2008
Accepted: 12 June 2009
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