Download Cell migration: mechanisms of rear detachment and the formation of

Eur. J. Cell Biol. 83 (2004); 717 ± 724
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Cell migration: mechanisms of rear detachment and
the formation of migration tracks
Gregor Kirfel1), Alexander Rigort, Bodo Borm, Volker Herzog
Institute of Cell Biology, University of Bonn, Bonn, Germany
Migration tracks; Keratinocytes; Integrins; Cytoskeleton;
Extracellular matrix
Cell migration is central to many biological and pathological
processes, including embryogenesis, tissue repair and regeneration as well as cancer and the inflammatory response. In
general, cell migration can be usefully conceptualized as a cyclic
process. The initial response of a cell to a migration-promoting
agent is to polarize and extend protrusions in the direction of
migration. These protrusions can be large, broad lamellipodia
or spike-like filopodia, are usually driven by actin polymerization, and are stabilized by adhering to the extracellular matrix
(ECM) via transmembrane receptors of the integrin family
linked to the actin cytoskeleton. These adhesions serve as
traction sites for migration as the cell moves forward over them,
and they must be disassembled at the cell rear, allowing it to
detach. The mechanisms of rear detachment and the regulatory
processes involved are not well understood. The disassembly of
adhesions that is required for detachment depends on a
coordinated interaction of actin and actin-binding proteins,
signaling molecules and effector enzymes including proteases,
kinases and phosphatases. Originally, the biochemically regulated processes leading to rear detachment of migrating cells
were thought not to be necessarily accompanied by any loss of
cell material. However, it has been shown that during rear
detachment long tubular extensions, the retracting fibers, are
formed and that ™membrane ripping∫ occurs at the cell rear. By
this process, a major fraction of integrin-containing cellular
material is left behind forming characteristic migration tracks
that exactly mark the way a cell has taken.
1)
Corresponding author: Dr. Gregor Kirfel, Institute of Cell Biology,
University of Bonn, Ulrich-Haberlandstr. 61a, D-53121 Bonn, Germany, e-mail: [email protected], Fax: ‡ 49 228 735 302.
Integrins and the formation of cellsubstrate adhesions
Cell migration is a complex process that leads to translocation
through the co-ordinated sequence of distinct functions such as
actin assembly-driven lamellipodia protrusion (Fig. 1), formation of cell-substrate adhesion at the tips of lamellipodia,
actomyosin-powered contraction of the cell body, and detachment of the cell rear (Stossel, 1993; Sheetz, 1994; Lauffenburger
and Horwitz, 1996; Mitchison and Cramer, 1996). Cell adhesion
and attachment to extracellular matrix (ECM) is critical for cell
migration and mediated by low-affinity transmembrane glycoprotein adhesion receptors including members of the heterodimeric integrin family (Buck and Horwitz, 1987; Ruoshlahti
and Pierschbacher, 1987; Hynes, 1992; Sonnenberg, 1993). The
integrins are heterodimers of two type I membrane proteins,
the a and b chains, with large ligand-binding extracellular
domains and short cytoplasmic domains. Cell-substrate adhesions are discrete transmembrane regions in which the extracellular domains of integrins are bound to ECM ligands such as
fibronectin and the cytoplasmic domains are linked by bridging
molecules to cytoskeletal components, either microfilaments or
intermediate filaments. Microfilament-associated adhesion
sites, also referred to as focal complexes and focal adhesions
(Kaverina et al., 2002), are highly dynamic structures, whereas
intermediate filament-associated adhesion sites, also referred
to as hemidesmosomes (Yamada et al., 1996), function in the
more stable anchorage of epithelial cells to their substrate
(Jones et al., 1998). During migration, cell-substrate adhesions
become only transiently detectable (Dunlevy and Couchman,
1993; Matsumoto et al., 1994) and they must be continuously
formed at the cell front and disrupted at the cell rear for a cell to
move.
It has been shown that the dynamic behavior of integrins in
cell-substrate adhesion structures at the rear of the cell is
important in governing cell migration and that, at least in some
cell types, the rate of this rear release determines the migration
velocity (Chen, 1981). Thus, the rate of locomotion of
Dictyostelium cells is limited by their ability to detach at the
rear (Jay et al., 1995), and for Chinese hamster ovary (CHO)
0171-9335/04/083/11-12-717 $30.00/0
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718 G. Kirfel et al.
ECM ligands (Fig. 2b) and their cytosolic adaptor proteins as
well as proteases cleaving the adhesion complex including the
cytosolic cysteine protease calpain (Fig. 2b), matrix proteases
(Fig. 2c) and sheddases of the ADAM family (Fig. 2d).
Contractility-promoted release
Fig. 1. Scanning electron micrograph of an epidermal keratinocyte
showing the characteristic polarized morphology of cells migrating on
fibronectin-coated surfaces after stimulation with a motogenic mediator such as the epidermal growth factor (EGF). At the cell front
numerous filopodia (white arrows) and lamellipodia (white asterisks)
are formed by membrane protrusion. These membrane protrusions
must adhere to the substrate for allowing transformation of cellular
contraction forces into cell body translocation. At the cell rear, where
the cell must detach during migration, long tubular extensions, the
retracting fibers (white arrowheads), are formed. N ˆ nucleus. Black
arrow ˆ direction of migration. Bar, 20 mm.
cells transfected with aIIb3 integrins the rate of translocation
on fibronectin-coated surfaces is approximately equal to the
rate of cell rear detachment (Palecek et al., 1998).
If the cytoskeletal contractility exceeds the adhesive strength of
the focal contacts, contractile forces might directly promote
detachment from the substrate during migration (Fig. 2a). In
permeabilized cells ATP-induced contractility that usually is
provided by actin-myosin interactions appears to promote the
disassembly of focal adhesions (Crowley and Horwitz, 1995).
Recent evidence suggests that in fibroblast cells a myosin
transport might pull filaments centripetally inward (Cramer
et al., 1997; Galbraith and Sheetz, 1997). In ultra-structural
studies, it was found that there is a graded polarity of actin
filaments, with barbed ends orientated outward at the cell front
and rear, and a mixed polarity in the cell center (Cramer et al.,
1997). Measurements of traction forces have shown that the
direction of the forces exerted against the substrate agree with
the structural information: forces are directed inward at the
front and the rear of the cell and they oscillate towards the cell
center. Together, these studies (Cramer et al., 1997; Galbraith
and Sheetz, 1997) indicate that cytoskeletal contraction orientated centripetally generates forces that contribute to the
detachment of cell adhesion sites at the cell rear.
For migration to occur, cytoskeletal contractility needs to be
regulated between the front and the rear of the cell. Evidence
for a spatially regulated contraction comes from studies that
indicate that myosin II may help the cell rear to detach from the
substrate and to retract (Jay et al., 1995). Furthermore, traction
forces exerted against the substrate are much higher in the rear
of the cell when a thickened tail forms (Galbraith and Sheetz,
1997). This suggests that spatiotemporarily regulated contraction contributes to the process of rear detachment during cell
migration.
Mechanisms of rear detachment
Regulation of rear detachment by
transient changes of Ca2‡ levels
Breakage of cell-substratum attachment needed to allow
locomotion can, in principle, occur either by intracellular
disruption of the cytoskeleton-integrin linkage (Fig. 2b) or by
extracellular release of the integrin-matrix linkage (Fig. 2a, c,
d). The kinetic model for integrin-mediated adhesion release
during migration predicts two distinct detachment phenomena
integrating the biochemical and biophysical interactions between integrins, the cytoskeleton and the ECM that affect rear
retraction and linkage dissociation mechanisms (Palecek et al.,
1998). In the first, detachment is extremely rapid, dominated by
integrin-ECM dissociation and it occurs when high forces are
generated or when adhesiveness is low. In the second, detachment is much slower, dominated by integrin-cytoskeleton
dissociation occurring at low forces and high adhesiveness
(DiMilla et al., 1991; Huttenlocher et al., 1995; Sheetz, 1994).
Dissociation of adhesion sites depends on highly regulated
events that are mediated by mechanical contraction forces
(Fig. 2a) but also by a variety of tyrosine kinases and
phosphatases that modulate the affinity of integrins for their
Intracellular Ca2‡ regulates many of the molecular processes
that are essential for cell movement including the production of
actomyosin-based contractile forces, the regulation of the
structure and the dynamics of the actin cytoskeleton, and the
formation and disassembly of cell-substratum adhesions (Sjaastad and Nelson, 1997). Waves of increased intracellular free
Ca2‡ levels have been implicated in the release of migrating
neutrophils where the Ca2‡/calmodulin-dependent phosphatase calcineurin has been shown to be responsible for the
detachment of integrin-mediated cell adhesion and the recycling of integrins to the cell front (Maxfield, 1993; Hendey et al.,
1992). Besides calcineurin, the calpain proteases (see below)
might also represent a direct link between the mechanism of
rear detachment and cellular Ca2‡ levels. However, in most
cases, it is still unknown how the transient and highly localized
changes of Ca2‡ levels (Ca2‡ transients) are generated. In fish
keratocytes Ca2‡ transients have been reported to arise from
the activation of stretch-activated Ca2‡ channels, which triggers
an influx of extracellular Ca2‡ (Lee et al., 1999).
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Fig. 2. Schematic illustration summarizing the different strategies of
rear detachment. Mechanical forces generated by actomyosin-driven
contraction are thought to contribute to the dissociation of substrate
adhesions at both, cytosolic and extracellular sites (a). The cytosolic
dissociation of cell-substrate adhesions can be performed by the calpain
cysteine proteases, by phosphorylation/dephosphorylation of cytosolic
Rear detachment by modulation of
integrin affinity for their ECM ligands
The binding of ECM ligands such as fibronectin to the
extracellular portion of integrins leads to conformational
changes in the receptors and to integrin clustering which is a
prerequisite for integrin activation (Miyamoto et al., 1995).
Although integrins themselves do not have any catalytic
activity, signals are transmitted outside-in by activated integrins
through direct and indirect interactions with many partners of
integrins including direct and indirect integrin-actin crosslinkers such as talin and vinculin, tyrosine kinases such as FAK
and Src family members, phosphatases and modulators of small
GTPases (Schwartz and Ginsberg, 2002; Giancotti, 2000). Thus,
integrin clustering can initiate intracellular signals such as
tyrosine phosphorylation and the activation of small GTPases
that regulate the formation and strengthening of adhesion sites
Mechanisms of rear detachment during cell migration 719
adapter proteins and by posttranslational modification of integrins or
adapter proteins (b). Extracellular dissociation of cell-substrate adhesions can be achieved by proteolytic cleavage of matrix constituents
mediated by matrix proteases (c) or by shedding of matrix receptors
such as integrins by specific sheddases leaving parts of the receptors on
the substrate (d).
and the organization and the dynamics of the cytoskeleton
(DeMali et al., 2003; Sieg et al., 2000). Activated integrins
preferentially localize to the leading edge, where new adhesions
form (Kiosses et al., 2001). Integrin affinity is modulated in
large part by interactions at the integrin cytoplasmic tail that
trigger alterations in the conformation of the extracellular
domains (Fig. 2b). The cytoskeletal linker protein talin promotes integrin activation by binding to a subset of integrin bsubunit tails and disrupting a-b-subunit tail interactions (Cram
and Schwarzbauer, 2004). The binding capacity of integrins can
also be modified by posttranslational modifications of the
cytoplasmic domains (Fig. 2b). For example, integrin a4
phosphorylation on serine blocks the binding of paxillin, a
adaptor protein involved in signaling (Liu et al., 2002). The
phosphorylation of a4 integrin at the leading edge and the
consequent release of bound paxillin are required to maintain
stable lamellipodia of T cells migrating on ligands for integrin
720 G. Kirfel et al.
a4b1 (Han et al., 2003). For neutrophils migrating on vitronectin the release of avb3 integrins and their recycling to the
leading edge has been reported to depend on the activity of
calcineurin, a calcium-calmodulin activated protein phosphatase type 2B (Lawson and Maxfield, 1995). Blocking of
calcineurin has been shown to result in reduced neutrophil
migration on vitronectin by reducing the efficiency of detachment (Hendey et al., 1992).
Rear detachment by modulation of
affinity between integrins and the
cytosolic adaptors
Besides the proteolytic cleavage of cytosolic adhesion components, their phosphorylation/dephosphorylation by kinases and
phosphatases has been implicated in the mechanisms involved
in rear release (Fig. 2b) (Larsen et al., 2003). In neutrophils the
plasma membrane-actin cross-linker moesin has been shown to
be modulated by phosphorylation/dephosphorylation in a
spatiotemporal fashion (Yoshinaga-Ohara et al., 2002). Video
microscopy showed that dephosphorylation of moesin preceded rear release. Calyculin A, an inhibitor of type 1 and type 2A
serine/threonine phosphatases suppressed moesin dephosphorylation and impaired rear release in a dose-dependent manner
(Yoshinaga-Ohara et al., 2002). Phosphorylation of moesin is
thought to be regulated downstream of the small GTPase Rho
since its inhibition by C3 exoenzyme impairs phosphorylation
and inhibits rear release. It has also been shown that gradual
loss of tyrosine phosphorylation of FAK coincided with
disruption of focal adhesions and conversion to a motile
phenotype (Matsumoto et al., 1994).
Src and FAK tyrosine kinases are also thought to be involved
in focal adhesion turnover associated with cell migration
(Carragher and Frame, 2004). The selective inhibition of Src
kinase has been shown to decrease the rate constants for focal
adhesion disassembly (Webb et al., 2004). The role of Src in
focal adhesion turnover is also shown in studies on the
activation of oncogenic v-Src. Many components of focal
adhesions, including b1 integrin, tensin, paxillin and FAK
become highly activated upon activation of v-Src resulting in
focal adhesion disassembly and cell-substrate detachment
(Frame et al., 2002). In permeabilized fibroblasts, however, a
rapid breakdown of focal adhesions has been observed upon
the addition of ATP leading to tyrosine phosphorylation of
cytoskeletal components (Crowley and Horwitz, 1995).
Rear detachment by calpains ± cytosolic cysteine proteases
The calpains represent a well conserved family of intracellular
calcium-dependent cysteine proteases (Beckerle et al., 1987).
The two ubiquitous calpains, m-calpain and M-calpain, named
according to their relative requirement for Ca2‡ in vitro, with mcalpain requiring micromolar and M-calpain requiring millimolar levels, have been implicated in adhesion formation and
rear detachment (Fig. 2b) (Bhatt et al., 2002; Huttenlocher
et al., 1997). Due to this dual function calpain must be regulated
differentially in a front versus rear fashion. Calpain binding to
phospholipids has been shown to target calpain to the
membrane and decreases the Ca2‡ requirement thus facilitating
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its activation. Adhesion-relevant targets of calpain are the
adhesion complex components talin, paxillin, a-actinin, ezrin,
and FAK as well as the cytoplasmic domains of b1 and b3
integrin (Du et al., 1995; Cooray et al., 1996; Perrin and
Huttenlocher, 2002). Inhibition of calpain has been shown to
lead to a stabilization of peripheral FA, increased adhesiveness,
decreased detachment and inhibited b1 and b3 integrindependent migration. Reduced expression of calpain has
been reported to be correlated with reduced migration of
CHO cells. Treatment of migrating cells with pharmacological
inhibitors of calpain activity and knock-out studies with calpain
/ fibroblasts result in impaired retraction of the cell rear,
increased tail length and suppression of cell migration (Dourdin et al., 2001; Palecek et al., 1998). Calpain-mediated proteolysis has therefore been proposed as a mechanism for
promoting disassembly of focal adhesion structures, leading to
the turnover of integrin-dependent cell-matrix adhesion that is
needed for cell locomotion (Glading et al., 2002).
Rear detachment by pericellular proteolysis
Recruitment of cell surface proteases such as matrix-metalloproteinases (MMP) and cathepsins, to ECM contacts sites and
localized proteolysis is a key event during invasive migration of
tumor cells. This pericellular proteolysis is also a crucial process
during epidermal wound healing, when keratinocytes from the
wound margin must move along dermal ECM at the interface
between the fibrin clot and the dermal matrix, i.e., the leading
keratinocytes have to proteolytically dissolve and to remodel
the fibrin barrier ahead of them to create a path (Murphy and
Gavrilovic, 1999). The main enzyme for fibrinolysis is plasmin
derived from plasminogen within the clot. Plasmin can be
activated either by tissue-type specific plasminogen activator
(tPA) or urokinase-type plasminogen activator (uPA) (Ossowski and Aguirre-Ghiso, 2000). In migrating keratinocytes
both activators and the receptor for uPA are up-regulated. In
addition, various members of the MMP family (McCawley and
Matrisian, 2001; Seiki, 2002), each of which cleaves a specific
subset of matrix proteins, are also up-regulated by wound-edge
keratinocytes (Grondahl-Hansen et al., 1988, Romer et al.,
1994). MMP-9 can cleave collagens type IV and VII in the
basement membrane and is thought to be responsible for
detaching keratinocytes from their substrate (Salo et al., 1994).
In addition, certain MMPs can bind to integrins, thereby
providing a mechanism for localized matrix degradation
(Chapman et al., 1999). For example the collagen-cleaving
MMP-1 was shown to bind to a2 integrins and to be co-localized
with the collagen-binding a2b1 integrin at the leading edge of
migrating keratinocytes. Since MMP-1 cleavage of collagen
results in the exposure of integrin-binding sites, there might be a
co-operation between matrix receptors and proteinases to
perform an efficient migration on collagens. More recently, the
uPA receptor (Blasi, 1999, Mondino et al., 1999), a glycosyl
phosphatidylinositol-anchored membrane protein (Chapman
et al., 1999), was recognized as a multifunctional protein that
interacts with integrins and, thereby, regulates integrin function
and initiates signaling events that alter cell adhesion, migration
and proliferation (Wei et al., 1996; Ossowski and AguirreGhiso, 2000; Preissner et al., 2000; Simon et al., 2000). Comparable processes of highly localized pericellular proteolysis might
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also contribute to the continuous detachment of the cell rear
during cell migration by weakening the interaction between
integrins and the ECM (Fig. 2c).
Protein shedding and rear detachment
Ectodomain shedding, the proteolytic release of membraneanchored cell-surface proteins, is essential for physiological and
developmental events (Peschon et al., 1998; Yamamoto et al.,
1999). Shedding is mediated by membrane-resident sheddases
of the ADAM (a disintegrin and metalloprotease) protein
family (Werb, 1998; Wolfsberg et al., 1995) and has been
described for a variety of growth factors including TNF-a,
TGF-a and the Alzheimer amyloid precursor protein (APP)
(Black et al., 1997; Blobel, 1997; Buxbaum et al., 1998; Arribas
et al., 1996). The recent finding that collagen XVII/BP 180, an
epithelial adhesion molecule belonging to the group of
collageneous transmembrane proteins (Banyard et al., 2003),
is shed by a member of the ADAM family (Franzke et al., 2002,
2004) points to the involvement of sheddases in the process of
rear detachment (Fig. 2d). Further evidence for sheddasemediated detachment strategies comes from observations
showing that CD44 (Lesley et al., 1993; Lesley and Hyman,
1998), a widely expressed integral membrane glycoprotein that
serves as specific adhesion receptor for the ECM glycosaminoglycan hyaluronan (Aruffo et al., 1990) is shed from the
surface of fibroblasts and monocytes by members of the
ADAM family (Shi et al., 2001). Shedding of CD44 has also
been reported to be critical for the induction of tumor cell
migration (Okamoto et al., 1999). Recently, the shedding of a
55-kDa fragment of b1 integrin has been shown for cardiac
fibroblasts and myoblasts both in vitro and in vivo (Goldsmith
et al., 2003). Although integrins have been shown to associate
with specific members of the ADAM protein family through
the disintegrin domain (Chen et al., 1999), no evidence for
cleavage of an integrin by ADAM proteins has yet been
reported. More recently four ADAMs have been shown to
cleave the ECM proteins fibronectin and collagen IV in vitro
(Alfandari et al., 2001). ADAM-mediated cleavage of ECM
proteins could foster rear detachment during cell migration but
it might also release growth factors, previously bound to ECM,
for downstream signaling.
Integrin loss and migration tracks
Originally, these biochemically regulated processes were
thought to facilitate rear detachment of migrating cells by a
process which does not necessarily induce any loss of cell
material during migration. However, for fibroblasts migrating
in vitro, a large fraction of integrins has been found to be
released from the cell and left on the substratum. In addition,
the formation of migration tracks resulting from the release of
cellular material onto glass surfaces and artificial matrices has
been described for a number of cell types including fibroblasts
of different organisms (Fuhr et al., 1998; Richter et al., 2000),
mammalian thymoma and sarcoma cells, cytotoxic T-lymphocytes and primary chondrocytes (Zimmermann et al., 2001).
Migration tracks are hardly visible by conventional light
microscopy and have originally been considered matrix fibrils
Mechanisms of rear detachment during cell migration 721
(Halfter et al., 1990). Later, Palecek et al. (1996) showed that
fibroblasts leave behind migration tracks which contain integrin macroaggragates, i.e., integrin-containing membranous
patches (Regen and Horwitz, 1992). These structures apparently result from a process which has also been described as
™membrane ripping∫ during the migration of fibroblasts (Bard
and Hay, 1975; Chen, 1981; Regen and Horwitz, 1992) (for
review see (Lauffenburger and Horwitz, 1996)). The formation
of integrin-macroaggregates by membrane ripping is thought to
depend on the gradual disruption of the actin cytoskeleton
thereby inducing the fragmentation of cylindrical cell extensions into periodic chains of pearls (Bar-Ziv et al., 1999). In
contrast to fibroblasts, cells migrating faster and more persistent such as keratinocytes and neutrophils were believed not to
form migration tracks (Palecek et al., 1996).
Recently, however, it was demonstrated that migrating
keratinocytes leave behind migration tracks (Fig. 3a) containing large numbers of macroaggregates (Fig. 3c) that could be
classified due to their size, distribution within the migration
tracks and their origin (Kirfel et al., 2003). Type I macroaggregates are spherical and tubular structures with a diameter of
about 100 nm which both are arranged like ™pearls on a string∫
and apparently derive from ripping of retracting fibers, i.e. the
tubular membrane extensions at the rear end of keratinocytes
(Fig. 3c). Comparable but in detail different structures had also
been described for fibroblasts, lymphocytes and chondrocytes
(Fuhr et al., 1998; Zimmermann et al. 2001). Dendritically
organized tubular structures as described by Zimmermann
et al. (2001) for these cell types were absent from keratinocyte
migration tracks. The absence of these dendritic structures from
keratinocyte migration tracks was assumed to be due to a
migration velocity-dependent increase in the forces between
cells and substrate. Accordingly, low velocity produces low
forces which might favor the formation of branched structures,
whereas high velocity/high forces abolish branching. Type II
macroaggregates are spherical structures of about 50 nm which
are left behind in form of clusters in the gaps between the
retracting fibers (Fig. 3c) and were assumed to derive from the
fragmentation of former hemidesmosomes. So far, there have
been no reports on comparable structures for other cell types
(for review see (Zimmermann et al., 2001)). By immunolabeling it was shown that type I macroaggregates contain high
amounts of b1 integrin (Fig. 3b) that is a regular component of
focal adhesions and part of the fibronectin and laminin
receptors expressed on the surface of migrating keratinocytes.
Both types of macroaggregates were shown to be linked to the
substratum by regularly arranged fibrous material consisting of
ECM proteins such as laminin and fibronectin (Kirfel et al.,
2003). It can be concluded that the anchorage of the macroaggregate-resident integrins to ECM proteins is maintained
during the rear release process thereby contributing to the
formation of migration tracks.
Keratinocyte macroaggregates lack, however, any cytosolic
proteins including actin and the adhesion complex constituents
talin and vinculin (Kirfel et al., 2003; Rigort et al., this issue).
This points to a cytosolic cleavage mechanism between
integrins and their adaptor proteins that might be mediated
by calpain proteases or by modulators of integrin-adaptor
affinity such as calcineurin.
There is accumulating evidence from current investigations
on keratinocytes that macroaggregates are completely membrane-covered structures that contain besides integrins a
variety of other membrane proteins but also the complete set
722 G. Kirfel et al.
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Fig. 3. Characteristic migration tracks are formed during rear detachment of epidermal keratinocytes pointing away from the cell rear with
its numerous retracting fibers (a). These tracks consist of macroaggregates containing high amounts of cellular b1 integrin as visualized by
immunofluorescence (b). Transmission electron microscopy revealed
that two types of macroaggregates are formed. Type I macroaggregates
are tubular and spherical structures with a diameter of about 100 nm
that are arranged like pearls on a string and seem to derive from
membrane ripping at the tips of retracting fibers (c, arrows). Type II
macroaggregates are spherical structures with a diameter of about
50 nm that are found as clusters between the type I macroaggregates (c,
ovals). Bars: (a, b) 20 mm; (c) 2 mm.
of membrane lipids such as cholesterol and ceramides. The
release of macroaggregates might represent an additional
strategy of rear detachment allowing efficient cell migration.
However, it cannot be excluded that macroaggregates fulfill
additional physiological tasks and might act as guiding structures for trailing cells or represent a special type of paracrine
signaling machinery involved in chemotactic steering of
neighboring cells.
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