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A new link between the retrograde actin flow and focal adhesions

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J. Biochem. 2014;156(5):239–248 doi:10.1093/jb/mvu053
JB Review
A new link between the retrograde actin flow and
focal adhesions
Received April 18, 2014; accepted July 7, 2014; published online September 3, 2014
Sawako Yamashiro* and Naoki Watanabe
Department of Pharmacology, Kyoto University Faculty of
Medicine, Kyoto, Japan
*Sawako Yamashiro, Department of Pharmacology, Kyoto
University Faculty of Medicine, Yoshida Konoe-cho, Sakyo-ku,
Kyoto 606-8501, Japan. Tel: +81-75-753-4396,
Fax: +81-75-753-4394,
email: yamashiro.sawako.5c@kyoto-u.ac.jp
The retrograde actin flow, continuous centripetal move-
ment of the cell peripheral actin networks, is widely
observed in adherent cells. The retrograde flow is
believed to facilitate cell migration when linked to
cell adhesion molecules. In this review, we summarize
our current knowledge regarding the functional rela-
tionship between the retrograde actin flow and focal
adhesions (FAs). We also introduce our recent study
in which single-molecule speckle (SiMS) microscopy
dissected the complex interactions between FAs and
the local actin flow. FAs do not simply impede the
actin flow, but actively attract and remodel the local
actin network. Our findings provide a new insight into
the mechanisms for protrusion and traction force gen-
eration at the cell leading edge. Furthermore, we dis-
cuss possible roles of the actin flow-FA interaction
based on the accumulated knowledge and our SiMS
study.
Keywords: actin/cell migration/focal adhesion/retro-
grade flow/single-molecule speckle microscopy.
Abbreviations: ApCAM, Aplysia cell adhesion
molecule; FA, focal adhesions(s); FRAP, fluorescence
recovery after photobleaching; qFSM, quantitative
fluorescent speckle microscopy; SiMS, single-molecule
speckle microscopy.
Cell migration is a dynamic, actin-based cellular pro-
cess that is important for many phenomena in multi-
cellular organisms, including development, wound
healing, immunity and tumour metastasis. The
motive power of cell migration is regulated by the
actin cytoskeleton, which generates intracellular force
by actin polymerization or interaction with myosin
motor proteins. Although how cells use intracellular
force in a spatiotemporally organized manner is a
key question in cell migration research, it is challenging
to monitor intracellular force directly in cells. Instead,
monitoring motion of the objects that receive intracel-
lular force may provide a clue to estimate dynamics of
the force.
ß The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
The retrograde actin flow, continuous centripetal
movement of the cell peripheral actin networks, has
been observed in a wide variety of cultured cells,
such as fibroblasts, epithelial cells, leukocytes,
T lymphocytes and neurons (1—5). The physiological
role of the retrograde flow largely remains unclear, al-
though force driving the flow is considered to facilitate
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cell migration. During cell migration, a cell extends the
lamellipodia, which is the sheet-like structure contain-
ing a highly dense, branched actin meshwork (6). The
branched actin meshwork is generated by the Arp2/3
complex that binds to the side of a pre-existing fila-
ment and nucleates a new actin filament (7). In lamel-
lipodia, actin filaments are mostly oriented with their
barbed ends (plus ends) facing the cell edge (8, 9), and
actin polymerization at the edge of lamellipodia gen-
erates force that pushes the membrane forward and
pushes back the actin array inward to drive the retro-
grade flow (10, 11).
During cell migration, the retrograde flow has been
postulated to promote protrusion via linkage between
the lamellipodial actin network and focal adhesions
(FAs) in the clutch model (10). FAs are integrin-con-
taining, multiprotein structures that form mechanical
links between the extracellular substrate and actin
stress fibres [reviewed in (12)]. FAs can also associate
with lamellipodial actin networks via actin-binding FA
components including vinculin, talin and a-actinin. In
the clutch model, strengthening linkage between cell
adhesion molecules and the lamellipodial actin net-
work reduces the speed of the retrograde flow, whereas
addition of new actin subunits continues at the barbed
ends, leading to cell edge protrusions (10). The model
also predicts that when the actin network moving with
the retrograde flow links to FAs, tension is generated
by flow motor(s) onto the substratum (10). Shootin-1,
L1-CAM and N-cadherin have been proposed to func-
tion as a molecular clutch that link between the actin
network and cell adhesion molecules in axon out-
growth and growth cone migration (13—15).
However, it has been difficult to validate the original
clutch model. To achieve this, it requires simultaneous
Featured Article
measurements of the retrograde flow speed, the actin
elongation rate at the cell edge and the rate of mem-
brane protrusion with changing the strength of the
linkage between FAs and the actin network. In add-
ition, such biological processes are likely to be modu-
lated by intracellular signaling, which is difficult to
distinguish from the physical processes.
Conversely, it has experimentally been suggested
that when the substratum coated with the extracellular
cell adhesion molecule is artificially contacted with the
dorsal cell surface, linking the extracellular cell adhe-
sion molecule to the cortical actin network increases
239
S. Yamashiro and N. Watanabe
tension between a cell and the substratum as well as
membrane protrusion (16). Aplysia cell adhesion mol-
ecule (ApCAM) is a member of immunoglobulin-like
cell adhesion molecules (Ig-CAMs), and its extracellu-
lar region mediates cell—cell adhesion by homophilic
binding. Spectrin and ankyrin are candidates for actin-
linkage molecules for Ig-CAMs via binding to both
actin filaments and cytoplasmic domain of Ig-CAMs
(17). When a silica bead coated with ApCAM is placed
on the most distal dorsal surface of a growth cone of
an Aplysia bag cell neuron and kept in one place using
a glass needle, bending of the needle towards the cell
centre, a decrease in the retrograde flow rate and the
membrane protrusion anterior to the bead occur sim-
ultaneously (16). Bending of the needle indicates an
increase in the tension on the beads. Thus the force
that drives the retrograde flow is presumably trans-
mitted to the extracellular beads accompanied by
strengthening linkage between the actin network and
the cell surface ApCAM. Tethering the actin network
to the substrate via cell surface receptors may com-
monly generate tension on the substrate.
Interestingly, such linkage between ApCAM beads
and the actin network induces accumulation of actin
and other molecules including Src and cortactin in the
vicinity of the beads (16, 18, 19), suggesting additional
intracellular signaling promotes protrusion.
Nascent adhesions, which are the earliest integrin-
containing structures detectable by light microscopy,
are formed in lamellipodia (20, 21). Nascent adhesions
contain major FA components including integrin, pax-
illin and a-actinin, and are roughly 50.25 mm in diam-
eter (22). Most nascent adhesions disassemble rapidly,
but a part of them grow into FAs by increasing their
size and forming more stable association with the
extracellular matrix (ECM) (23). As postulated in the
clutch model, nascent adhesions and mature FAs are
possible to link to the lamellipodial actin network
moving with the retrograde flow (Fig. 1D). However,
how these adhesions interact with the retrograde flow
has been largely unknown. Morphology and distribu-
tion of mature FAs are diverse depending on the ECM
proteins, the types of cells, stiffness of the substrate
and so forth (Fig. 1). Under certain conditions, FAs
are formed within lamellipodia. For instance, a part of
FAs often exist in lamellipodia when XTC cells are
cultured on a glass surface doubly coated with poly-
L-lysine (PLL) and laminin (Fig. 1B). We have recently
elucidated the relationship between the local retro-
grade flow velocity and FAs by direct observation of
individual actin filaments using an improved fluores-
cence single-molecule speckle (SiMS) microscopy (24).
Our study proposes a new idea that mature FAs attract
the retrograde flow in lamellipodial region between the
cell edge and the frontal edge of the FAs and actively
remodel the local actin network.
In this review, we discuss our current understanding
of the physiological significance of the retrograde flow
in cell migration. In particular, we highlight the rela-
tionship between the retrograde flow and FAs. We
introduce imaging techniques to measure the flow velo-
cities and our recent detailed analysis which revealed
how FAs modify the local retrograde flow. Finally, we
240
propose possible concepts that could explain how the
interaction between the retrograde flow and FAs plays
roles in organization of local cytoskeletal structures.
How Do Cells Drive the Retrograde Flow
in Lamellipodia?
Adherent cells form diverse patterns of cytoskeletal
and FA organization depending on the condition of
culture substrates. Figure 1 shows examples of XTC
cells showing distinct spatial arrangement of actin and
FAs on different substrates. When XTC cells adhere to
glass surfaces coated with 1 mg/ml PLL, they form
well-spread lamellipodia and few FAs (Fig. 1A).
Conversely, elongated FAs and spread lamellipodia
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are developed in XTC cells culture on surfaces
coated with 0.1 mg/ml PLL and 5 mg/ml laminin
(Fig. 1B). In these cells, edges of FAs frequently exist
in lamellipodia (Fig. 1B). When XTC cells are cultured
on 10 mg/ml each of PLL and fibronectin-coated cover-
slips, cells form narrow lamellipodia at the cell periph-
ery, FAs inside of lamellipodia, and well-developed
stress fibres associated with FAs (Fig. 1C). In these
cells with distinct cytoskeletal organization, the retro-
grade actin flow ubiquitously occurs in lamellipodia.
Based on the properties of the lamellipodial actin
network with the free barbed-ends facing the cell
edge and intensive polymerization at barbed ends,
actin polymerization pushing against membrane ten-
sion is thought to partly drive the retrograde flow. In
neuronal growth cones, application of cytochalasin B,
which inhibits actin polymerization at barbed ends,
causes receding of the actin network from the entire
peripheral edge toward the cell body with forming a
cell peripheral zone devoid of actin filaments (25).
Thus, the retrograde flow is still driven without actin
polymerization. A similar phenomenon is observed in
sea urchin coelomocytes treated with cytochalasin D
(26). In neural growth cones, the myosin II ATPase
inhibitor blebbistatin decreases the retrograde flow in
peripheral domain that corresponds to lamellipodia
(27), but has little effect on the flow speeds in newly
formed lamellipodia in T cells (28) and XTC cells on a
PLL-coated surface (24). In neural growth cones, the
actin bundles in the peripheral domain appear to be
connected with the actin bundles containing myosin II
in the central area of the growth cone (27), which could
account for the sensitivity of its leading edge actin
retrograde flow to myosin-II inhibition. Currently,
the true flow motors that directly drive the lamelli-
podial actin flow remain to be identified.
Interestingly, dendritic cells, a type of rapidly
migrating leukocytes, seem to switch distinct mechan-
isms for effective migration in various extracellular en-
vironments. For instance, dendritic cells enables to
migrate on ECM-coated adhesive substrate with form-
ing a strong linkage between the actin network and the
substrate, whereas they continue to migrate on non-
adhesive substrate at the same speed without forming
such linkage but with accelerated retrograde flow (3).
In the latter case, when the cells migrate without ad-
hesion, actin polymerization in the leading edge is
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Fig. 1 XTC cells display distinct actin and FA organization on different substrates. Cells express Lifeact-mCherry to visualize actin and EGFP-
vinculin to mark FAs. Lamellipodia is the cell peripheral region that contains actin densely. (A) The cells form well-spread lamellipodia and few
FAs on glass surfaces coated with 1 mg/ml PLL. (B) The cells form elongated FAs and spread lamellipodia on surfaces coated with 0.1 mg/ml
PLL and 5mg/ml laminin. In these cells, edges of FAs frequently exist in lamellipodia. (C) The cells form narrow lamellipodia, FAs inside of
lamellipodia, and well-developed actin stress fibres associated with FAs on surfaces coated with10 mg/ml each of PLL and fibronectin. Note
that to prepare different substrates, the glass surfaces were incubated with the solution containing each substance at the indicated concentra-
tion, and then the excess amount of them was removed and washed away. (D) A schematic diagram of FAs and actin networks at leading edge
of a cell.

accelerated to compensate, which presumably keeps
membrane protrusion and migration velocity constant
(3). Conversely, when Dictyostelium cells are detached
from the substratum by changing the external solution,
the actin filaments display accelerated retrograde flow,
but the detached cells cannot advance pseudopods
(29). Leukocytes are able to migrate flexibly through
three-dimensional environments without tightly adher-
ing to specific substrates (30). Therefore, such migra-
tion mode without clutch and rapid adaptation to
environmental changes might be unique in leukocytes.
Nevertheless, in other cell types, the retrograde flow
and cell adhesion molecules may interact in a complex
manner during cell migration.

241
S. Yamashiro and N. Watanabe
Direct Observation of the Modification of
the Local Retrograde Flow by FAs
The continuous centripetal flow of cell-associated ma-
terial at the cell periphery is one of the most prominent
phenomena in cultured cells, and has long been recog-
nized with advances in microscopic techniques for live
cell imaging. Abercrombie, who pioneered the study of
cell migration [reviewed in (31)], first described the
centripetal movement in chicken fibroblasts by obser-
ving particle transport on the cell surface using time-
lapse phase-contrast microscopy (32). Wang (5) re-
vealed the retrograde flow of the actin networks in
lamellipodia by fluorescence recovery after photo-
bleaching (FRAP) of fluorescently labeled actin.
Later, fluorescent speckle microscopy (FSM) was de-
veloped with advances in high sensitivity cameras with
cooled charge-coupled device detectors (33). FSM
allows quantitative analysis to reveal the velocities of
the retrograde flow with high resolution (4, 24, 34, 35).
Linking the actin network to FAs in lamellipodia
has been predicted to slow the speed of the retrograde
flow locally. However, it has been difficult to detect the
local modification of the actin flow by FAs especially
when FAs are small. In addition, it is challenging to ac-
curately measure the flow velocity of the actin popula-
tion with short lifetime that moves only a short
distance. Here, we introduce (i) several issues which
one should keep in mind to accurately measure the
actin flow velocity in lamellipodia, (ii) our improved
speckle microscopy that overcomes those issues and
(iii) our recent detailed analysis of the local retrograde
actin flow near and above FAs with nanometre
accuracy.
Technical difficulties in measuring actin flow velocity
Many studies have reported the actin flow velocity in
lamellipodia using several microscopic techniques to
reveal molecular dynamics such as FRAP (5), photo-
activation of fluorescence (PAF; 36, 37), quantitative
polarized light microscopy (Pol-Scope; 38) and FSM
(4). However, it is difficult to accurately measure the
flow velocities with the above techniques for follow-
ing reasons. First, actin filament turnover in lamelli-
podia is rapid as nearly one-third of filaments have
short lifetimes of less than 10 s (39). Such short-lived
filaments move only short distances (5100—500 nm),
thereby it requires a high spatial resolution to meas-
ure the flow velocity of these short-lived filaments.
Second, if movements of actin filaments are hetero-
geneous, individual filaments must be tracked to
define the flow. Approaches that monitor a mass
of actin filaments, such as FRAP, PAF and Pol-
Scope are not suited for measuring heterogeneous
actin movements because of their limited spatial
resolutions.
FSM is an approach to monitor dynamics of cyto-
skeletal polymers in living cells with higher resolution
than the above microscopic techniques. The principle
of FSM is that incorporation of fluorescently labeled
subunits at low concentrations provides fiduciary
marks on polymers. FSM, which was originally de-
veloped by Waterman-Storer et al. (33) as a technique
242
to measure microtubule movements and the actin flow,
has been developed in two directions: one is fluores-
cence SiMS microscopy (24, 39) and the other is quan-
titative fluorescent speckle microscopy (qFSM; 40).
In the original SiMS analysis, individual EGFP-
actin molecules are imaged in cells expressing a very
low level of EGFP-actin under the control of the de-
fective cytomegalovirus (CMV) promotor (delCMV;
24, 39). The SiMS analysis enables to dissect complex
actin dynamics, which is potentially overlooked by
other approaches including FRAP, PAF and qFSM.
However, this approach demands some experience to
find cells containing fluorescent probes at optimal
levels. Even with delCMV, the defective promoter,
only a minor population of cells expresses a sufficiently
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low level of EGFP-actin. This technical difficulty
might have limited usage of the method.
qFSM analyses denser fluorophore labels than
SiMS analysis (40). Resolvable fluorescent speckles
containing multiple-fluorophore probes (2—10 fluoro-
phores) provide quantitative information on appear-
ance, disappearance and trajectories through
automatic computational analysis (40). qFSM is a
potent method with high statistical power because it
extracts information from thousands of actin speckles
within one image, yielding a heat map representation
of degree of actin assembly/disassembly in subcellular
areas. However, it could often be difficult to accur-
ately measure the retrograde flow velocity by qFSM,
because automated object tracking may misidentify
individual speckles when they are densely packed. A
remarkable example is that two studies in which re-
searchers analysed the same FSM image datasets con-
cluded differently (41, 42). One qFSM study proposed
that actin filaments with two distinct retrograde flow
rates coexist in lamellipodia of newt lung epithelial
cells (41). This is referred to as the lamella hypothesis
since the slower flow speed is equivalent to that in the
area called lamella behind the back of lamellipodia.
However, the other study failed to detect such slowly
migrating fluorescent actin speckles in lamellipodia
using several speckle tracking approaches (42). The
contentious subject raised over the fidelity of qFSM
has been still an unsettled issue (42, 43).
Tracking the retrograde flow at nanometre scales
by new SiMS microscopy
We recently developed a new, user-friendly method,
which achieves the SiMS imaging with the highest spa-
tiotemporal resolution (24). The outline of the new
method is shown in Fig. 2A. We use an actin probe
labeled with a fluorescent DyLight dye (Thermo Fisher
Scientific Inc.) on lysine side chains. To deliver
DyLight (DL)-actin into cells, we use electroporation,
which enables incorporation of DL-actin into the cyto-
plasm at a low density suitable for SiMS microscopy
(Fig. 2B and C; 24). With our electroporation method,
almost 100% of cells are labeled with DL-actin mol-
ecules at a similar density. In cells, DL-actin efficiently
incorporates into cellular actin networks, and espe-
cially, DL549- and DL550-actin show greatly im-
proved photostability and brightness. We took
advantage of these properties of DL-actin to increase
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Fig. 2 New SiMS microscopy using DyLight-actin (DL-actin) is user-friendly and enables in vivo nanometre-scale displacement analysis.
(A) Outline of the new SiMS method. (B) Images of DL549-actin speckles in lamellipodia of XTC cells. Time-lapse images paneled at 10 s
intervals in the area (square) are shown on the right. Bar, 10 mm. (C) An image of DL549-actin with fast tracking method. The image was
acquired with a 100 ms exposure time and a full 100-W mercury excitation. Bar, 5 mm. (D) Displacement plot of the central position of a short-
lived DL549-actin speckle in lamellipodia in the series of fast tracking images. The velocity was calculated from a linear fit. Modified from
Yamashiro et al. 2014 (24).

the accuracy of displacement measurements of actin
speckles by devising two strategies. First, the unatte-
nuated 100-W mercury-arc lamp is used for illumin-
ation. Mercury-arc has a strong emission peak at
546 nm, which enables monitoring of DL-actin SiMS
with a high signal-to-noise ratio even with short ex-
posure time. Typically, we acquire images at 100 ms
intervals (fast tracking imaging) for the short dur-
ation of 10 s (Fig. 2C). Note that special care must
be taken to minimize the photodamage by restricting
the illuminated area and duration of the fast tracking
imaging in live cells. A decrease in the retrograde flow
shows a visible sign of the photodamage (24). With
our microscope system, we restrict duration for the
fast tracking imaging within 10 s. Second, subpixel
localization of the SiMS centroid is determined
using the two-dimensional Gaussian fit model of
Speckle TrackerJ software (44). Under this condition,
the centroid of DL-actin SiMS can be resolved with a
localization error of 8—8.5 nm (24). Thus in vivo nano-
metre-scale robust velocity measurement of SiMS
whose displacement is only 100—150 nm (Fig. 2D),
i.e. within the diffraction limit of light microscope,
is achieved.
Using the new SiMS method, we, for the first time,
succeeded in the measurement of the flow rates of actin
speckles including short-lived species (lifetime 510 s),
which move only a few hundred nanometre distance
(24). In lamellipodia of XTC cells shortly after spread-
ing on PLL-coated coverslips, all DL-actin SiMS
including short-lived species flowed at uniform
speeds, suggesting that lamellipodial actin filaments
form a tightly connected network regardless of individ-
ual filament lifetimes. Thus, the lamella hypothesis (41)
does not apply in lamellipodia of XTC cells.
New SiMS microscopy dissects local retrograde
flow near FAs
Using the improved SiMS microscopy, we analysed
how FAs influence local retrograde flow in lamellipo-
dia in detail (24). As shown in Fig. 1B, when XTC cells
are spread on a substrate coated with PLL and lam-
inin, nascent adhesions and mature FAs are formed in
flat lamellipodia, and a part of FAs frequently exist in
lamellipodia of the cells. Thus, we used the cells under
the condition to study the relationship between the
retrograde flow in lamellipodia and FAs. Nascent ad-
hesions are the first observable adhesive structures,
which are small and highly dynamic (12). In SiMS
analysis with DL-actin, the speeds of actin speckles
flowing over nascent adhesions are similar to those
of speckles that flowed near the nascent adhesions
(Fig. 3A). In our study, the speed of DL-actin speckles
flowing over nascent adhesions was 97.3 ± 0.06% of
that of speckles flowing near but outside of the adhe-
sions (n = 15 cases of comparison, four cells; 24).
These results suggest that nascent adhesions barely
link to the lamellipodial actin networks moving along
the retrograde flow (24).
In contrast, mature FAs locally modify the retro-
grade flow in several ways. (i) In the centre of FAs,
actin speckles move slower than those flowing in other
parts of lamellipodia (Fig. 3B, the region surrounded
by a white break line). (ii) In the cell peripheral region
between the cell edge and the frontal edge of FAs, the
retrograde flow biases towards FAs (Fig. 3B). (iii)
Also, in the cell peripheral region between the cell
edge and the frontal edge of FAs, the actin speckles
move at faster speeds than those elsewhere in lamelli-
podia (Fig. 3B). Detailed local flow speed analyses
show that the speckles flowing into the FA area

243
S. Yamashiro and N. Watanabe

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Fig. 3 Effects of nascent adhesions and mature FAs on the local retrograde flow at the cell periphery. (A) A speed and trajectory map of the
DL549-actin speckles in lamellipodia containing nascent adhesions. The inset indicates images of EGFP-vinculin before (green) and after (red)
acquisition of the DL549-actin movie. Lines indicating the trajectories and average speeds of DL549-actin speckles observed within a 100-s-time
window are shown in an image of EGFP-vinculin. Nascent adhesions have little effect on local retrograde flow at the cell periphery. (B) An
average speed and trajectory map of DL549-actin speckles in lamellipodia containing a mature FA. A white dotted line outlines the FA. Lines
indicating the trajectories and average speeds of DL549-actin speckles as in A. (C) A local speed and trajectory map of representative DL549-
actin speckles in the boxed region in (B). Circles indicate locations of speckles in each frame. Local actin flow speeds were measured with a 5-s-
time window (five frames), and the circles corresponding to the intermediate time point (the third frame) were coloured according to speed.
Green arrows indicate the direction of the retrograde flow. Asterisks indicate the speckles slowed down at the frontal edge of FA. Arrowheads
indicate the speckles migrating beside FA. An open arrowhead indicates a speckle migrating beside FA and changing its direction. Modified from
Yamashiro et al. 2014 (24).

often gradually slow down as the speckles approach
to FAs (Fig. 3C, the speckle trajectories with aster-
isks). Based on (i)—(iii), mature FAs appear to accel-
erate and gather the retrograde flow in front of FAs
and locally drag the actin flow at the frontal edge of
the FAs. (iv) The speckles migrating beside FAs move
at a constant speed (Fig. 3C, the speckle trajectories
with filled and open arrowheads), and sometimes
change their direction without changing the speed
(Fig. 3C, the speckle trajectory with open arrowhead),
suggesting that they dodge the FAs. Presumably, the
mature FAs physically impede the actin flow. (v) The
speckles migrating on the lateral edge of FAs occa-
sionally move away from the side of FAs with chan-
ging their speeds irregularly (Fig. 3C, the speckle
trajectories coloured with light blue). The actin net-
work in the lateral edge of FAs might be partly
connected to and pulled out by the actin networks
outside of the FAs. We reproducibly observe the
above features of local retrograde flow near mature
FAs in XTC cells (24).
The observed modification of the actin flow velocity
by FAs must involve transformation of the actin net-
work near and within FAs. The fast-moving actin net-
works flowing towards FAs are transformed into slow-
moving networks. This transformation is presumably
caused by engagement between the actin network and
FAs, or local changes in force to drive the actin flow.
Conversely, part of the actin network flowing towards
the side of FAs seems to dodge the FAs and flow at an
unaltered speed. The rapid turnover of lamellipodial
actin networks might facilitate the flexible transform-
ation of the network locally to adapt for obstruction
by FAs.

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Fig. 4 Possible roles of gathering and collecting action of mature FAs on the retrograde flow in front of FAs. In the Model-I, the retrograde flow
recruits FA components which bind directly or indirectly to the lamellipodial actin network to FAs. In the Model-II, FAs increases the traction
force and extend the region at the frontal edge of FAs by modification of the retrograde flow. In the Model-III, the retrograde flow remodels the
lamellipodial actin network with the barbed-ends facing the cell edge to anti-parallel actin bundle in stress fibres. When part of an actin filament
near the barbed end links to a FA, the pointed end part of the filament flows faster than the other part of the filament, thereby turns and the
filament align along the orientation of the actin flow (left). Conversely, when part of an actin filament near the pointed end links to a FA, the
barbed end part of the filament rotates so that the pointed end of the filament is directed toward the cellular edge (right). In this way
lamellipodial actin filaments might be reorganized to anti-parallel bundles. In the Model-IV, mechanical stress caused by collision of the actin
network accumulates in the forepart of FAs. In this model, such mechanical stress promotes disassembly of actin filaments locally, whereas part
of actin filaments oriented along the flow direction are stabilized by tension in FAs.

Possible Roles of Gathering and Collecting
Action of FAs on the Retrograde Flow
Based on the above observations, we conclude that
mature FAs attract the flow in front and actively re-
model the local actin network. Although the mechan-
ism for modification of the retrograde flow by FAs is
still unclear, we propose three possible roles of the
actin flow-FA interaction based on our SiMS study
(24) and the knowledge from previous studies.
The retrograde flow may recruit components of FAs
The most possible role of the retrograde flow is to re-
cruit FA components to FAs for maintenance (Fig. 4,
Model-I). In mature FAs, a constant turnover of FA
components, such as vinculin and paxillin, on time-
scales of seconds has been demonstrated using FRAP
(45). The retrograde flow streaming into FAs could
effectively supply FA components including actin-
binding proteins, which bind directly or indirectly to
the lamellipodial actin network. In addition, the retro-
grade flow may remodel ECM via binding to integrins
in front of FAs. The ECM is a dynamic structure
undergoing remodeling processes, which are involved
in morphogenesis and diseases including tissue fibrosis
and cancer invasion [reviewed in (46) and (47)].
Indeed, live imaging with fluorescent fibronectin
during cell migration in Xenopus embryonic tissue ex-
plants shows that rearrangement of fibronectin fibrils
occurs as the cells migrate (48). These possibilities
should be addressed by observing behaviours of
the representative FA components and ECM in front
of FAs. The SiMS microscopy would be a suitable
approach to address such possibilities.
The retrograde flow modified by FAs may increase the
traction force at the frontal edge of FAs
Deceleration of the retrograde flow in the region where
the flow runs into FAs may result from the linkage
between the actin network and mature FAs. In this
case, traction force on the substratum can be exerted
by the retrograde flow-FA interaction. Dynamic trac-
tion force within individual mature FAs on the sub-
stratum has been analysed using high-resolution

245
S. Yamashiro and N. Watanabe
traction force microscopy (49, 50). An inverse relation-
ship between the retrograde flow speed and traction
force on the substratum in lamellipodia and lamella-
containing FAs have been demonstrated using combin-
ation of FSM and traction force microscopy (49).
Furthermore, the traction force decreases but remains
in the blebbistatin-treated cells in which the retrograde
flow remains in lamellipodia, whereas the actomyosin
contractility in stress fibres is inhibited (49). These ob-
servations suggest that the traction force is generated
when the actin network moving with the retrograde
flow links to FAs. Several modeling studies presumed
a ‘stick-slip’ motion in which the actin network-FA
interaction occurs intermittently (51, 52). It is also pre-
dicted that rubbing nascent adhesions by the actin net-
work moving with the retrograde flow reinforces the
link between integrins and actin filaments through
modifying tension-sensitive molecules include talin,
p130Cas, fibronectin and integrin (12, 53), thereby pro-
motes growth of nascent adhesions into FAs. These
models assume that FAs simply produce friction to
the retrograde actin flow.
Conversely, it has not yet been demonstrated rigor-
ously whether the retrograde flow promotes growth of
nascent adhesions in cells. Inhibition of actin polymer-
ization by cytochalasin D diminishes both the retro-
grade flow and nascent adhesions (53). However, since
cytochalasin D diminish the structure of the actin net-
work at the lamellipodium tip (25, 26), the effect on
nascent adhesions might be caused by the collapse of
the surrounding actin structure.
In our SiMS study, mature FAs appear to actively
remodel the local actin network to accelerate and
gather the retrograde flow in front of FAs (24). This
act of the FAs would lead to increase in traction force
and extend the region in where traction force is exerted
at the frontal edge of FAs (Fig. 4, Model-II). If a new
technique to measure traction force at growing adhe-
sion structures with sufficient resolution is developed,
we might be able to evaluate the correlation between
change in the flow speeds, traction force and accumu-
lation of FA components during assembly of FAs.
Modification of the retrograde flow by FAs may
remodel lamellipodial actin filaments to stress fibres
As described in Introduction, lamellipodia contain a
dense, branched meshwork of actin filaments with
their barbed ends directed towards the leading edge
of the cell (6). Part of lamellipodial actin filaments
might be reorganized into FA-associated actin stress
fibres, which are composed of bundled actin filaments
associated with myosin II and actin cross-linking pro-
teins. Actin stress fibres contain anti-parallel filaments
that contribute to myosin II-dependent contractility.
We propose a model in which the FA-modulated
local retrograde flow promotes the redirection of la-
mellipodial filaments into anti-parallel filament bun-
dles. In our SiMS study, actin filaments above
mature FA moved at slow speeds probably due to
the friction between actin filaments and FAs, whereas
actin filaments at the side of FAs flows at fast speeds
(Fig. 3B; 24). As shown in Fig. 4 (Model-III), when
part of an actin filament near the barbed end links to a
246
FA, the pointed end part of the filament turns and the
filament align along the orientation of the retrograde
flow (Fig. 4, Model-III, left). Conversely, when part of
an actin filament near the pointed end links to a FA,
the barbed end part of the filament might rotate so that
the pointed end of the filament is directed towards the
cell edge (Fig. 4, Model-III, right). These actin fila-
ments oriented in an anti-parallel manner may be
cross-linked to form actin bundles in stress fibres.
Mechanical stress caused by collision of the actin
network moving at a fast flow speed in front of FAs
and that moving at a slow speed over FAs could accu-
mulate at the frontal edge of FAs (Fig. 4, Model-IV).
Such mechanical stress may promote disassembly of
actin filaments locally. Currently, there is neither bio-
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chemical nor cell biological evidence whether mechan-
ical stress promotes disassembly in the Arp2/3
complex-mediated actin meshwork. Nevertheless, we
recently show that mechanical stress applied by micro-
needle manipulation causes a rapid actin monomer in-
crease in XTC cells (54). Mechanical stress also
increases the level of actin-associated AIP1, which
enhance actin-depolymerizing factor (ADF)/cofilin-
mediated actin disassembly (54). In addition, a previ-
ous qFSM study reported that actin—myosin II
contraction enhances actin disassembly in keratocytes
(55). Conversely, part of actin filaments, especially
these oriented along the retrograde flow direction in
the forepart of FAs, can be stabilized by tension. A
biochemical study suggests that tension along an actin
filament reduces the binding of ADF/cofilin, thereby
preventing the filament from being severed by those
proteins (56). In this way, actin filaments flowing
into FAs might survive and form actin bundles.
It is intriguing to see how actin structures are re-
modeled accompanied by the modulation of the local
retrograde flow. The combination of the SiMS micros-
copy and the EM tomography would serve to address
the model (Fig. 4, Model-III). The recent electron tom-
ography is capable of distinguishing polarity of actin
filaments in cells (8), which should help examine the
orientation of actin filaments near/in FAs.
Conclusion
We have summarized current knowledge regarding the
relationship between the retrograde actin flow and
FAs. FAs do not simply impede the local retrograde
flow, but actively attract and remodel the local actin
network. Our findings provide a new insight into the
mechanisms for protrusion and traction force gener-
ation at the cell leading edge. Further studies using
our new SiMS microscopy and combination with
other microscopic techniques will elucidate how cells
generate intracellular force and transmit the force to
the extracellular matrix during migrating.
Acknowledgements
This work was supported by NEXT program grant LS013 from the
Cabinet Office, Government of Japan (N.W.), a grant from Takeda
Science Foundation (N.W.) and a Grant-in-Aid for Scientific
Research on Innovative Areas Grant Number 00624347 from the
Ministry of Education, Science, Sports and Culture of Japan (S.Y.).

Conflict of Interest
None declared.
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