Content uploaded by Michael Glogauer
Author content
All content in this area was uploaded by Michael Glogauer on Nov 13, 2017
Content may be subject to copyright.
Cytoskeletal remodeling in leukocyte function
Gabriel Fenteany
a
and Michael Glogauer
b
Purpose of review
This review focuses on recent developments in understanding
the roles and regulation of the cytoskeleton in the function of
leukocytes.
Recent findings
New studies have shed light on the regulation and dynamics of
actin and microtubules in leukocytes relevant both to cell
motility generally and to immune function specifically. The roles
of cytoskeletal dynamics in processes such as cell activation,
cell migration, and phagocytosis are being elucidated.
Dramatic progress has been made recently in understanding
the mechanisms of leukocyte directional sensing, polarization,
and chemotaxis.
Summary
Leukocytes need to be activated, polarize, change shape,
move, or phagocytose in response to their environment.
Leukocytes accomplish these processes by remodeling their
cytoskeleton, the active musculoskeletal system of the cell that
is not just the ultimate effector of motile responses but is also
a dynamic framework for subcellular organization and regional
signaling. Active areas of research include the direct and
indirect reciprocal interactions between the cytoskeleton and
the membrane and among cytoskeletal elements. The
pervasive and multi-layered roles played by small GTPases of
the Rho family and phosphoinositides in leukocyte function are
also becoming clearer.
Keywords
cytoskeleton, Rho-family small GTPases, phosphoinositides,
cell polarization and migration, chemotaxis, phagocytosis
Curr Opin Hematol 11:15–24. © 2003 Lippincott Williams & Wilkins.
Introduction
Leukocytes are highly efficient at migration, which al-
lows them to move rapidly and specifically to sites of
infection, inflammation, or tissue damage. Even apart
from their biologic function as the basis of the immune
system, they are invaluable systems for understanding
the signal transduction pathways and cytoskeletal dy-
namics underlying cell shape change and motility in a
fundamental sense. They are exquisitely responsive to
extracellular signals that modulate their activation, po-
larization, chemoattractant-stimulated directed migra-
tion (chemotaxis), or stimulated random migration (che-
mokinesis). Polymorphonuclear leukocytes (neutrophils)
are fast migrating cells extensively used to study the
signaling pathways and cytoskeletal regulation that me-
diate responses to chemoattractants. The other granulo-
cytes, eosinophils and basophils, are less abundant and
less well studied.
Cytoskeletal rearrangements are also critical for the mi-
gratory and other functions of other types of leukocytes.
Macrophages (differentiated monocytes), like neutro-
phils, specialize in the phagocytosis of invading micro-
organisms, a process driven by cytoskeletal remodeling.
Lymphocytes are a functionally diverse group evolved
for antigen-specific immune functions and recruitment
of other white blood cell types, and activation of T lym-
phocytes by antigen-presenting cells is cytoskeleton-
dependent. Finally, although highly specialized for
blood clotting and anucleate, blood platelets result from
the cytoskeleton-mediated fragmentation of megakaryo-
cytes, myeloid cells that share a common progenitor with
leukocytes. Platelet activation is a simplified model of-
ten used to study actin cytoskeletal dynamics and re-
modeling relevant to cell shape change in general.
Actin dynamics and remodeling in
leukocyte function
Cell shape change and migration in leukocytes and other
adherent crawling cells involve the regulated assembly
and cross-linking of actin filaments that support leading-
edge membrane protrusion, coordinated with cycles of
integrin-mediated cell-substratum attachment and de-
tachment, actin disassembly behind the leading edge,
cell body contraction, and retraction of the cell’s trailing
edge [1–16]. These processes are accomplished by the
activities of a range of actin-binding proteins and up-
stream regulators of actin dynamics.
Filamentous actin (F-actin) has a fast-growing or barbed
end (based on its appearance when decorated with myo-
a
Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois, USA,
and
b
Canadian Institutes of Health Research, Group in Matrix Dynamics, Faculty
of Dentistry, University of Toronto, Toronto, Ontario, Canada
Correspondence to Gabriel Fenteany, PhD, Department of Chemistry, University of
Illinois at Chicago, Chicago, IL 60607-7061, USA
Tel: 312 996 8542; fax: 312 996 0431; e-mail: fenteany@uic.edu
Current Opinion in Hematology 2004, 11:15–24
© 2003Lippincott Williams & Wilkins
1065–6251
15
sin head fragments) and a slow-growing or pointed end.
F-actin assembly involves the polymerization of mono-
meric actin (G-actin). In the cell, actin polymerization
only occurs from the barbed end of a filament. The
means to these ends are as follows: de novo nucleation of
new filaments by actin-nucleating proteins, the dissocia-
tion of capping proteins that normally bind the barbed
ends of filaments in the resting state, or the severing of
existing filaments by severing proteins to generate new
barbed ends. Several routes also exist to F-actin cross-
linking, bundling, and disassembly, mediated by differ-
ent proteins. In addition, microtubules interact with the
actin cytoskeleton in a number of ways, playing still
poorly understood roles in cell polarization, migration,
and other processes [17]. There are clearly multiple
mechanisms to arrive at a given change in actin state in
the cell. These mechanisms are not mutually exclusive,
and cells may pursue different options, depending on the
specific cell and situation. Despite the enormous com-
plexity of the system, basic models of the cytoskeletal
dynamics underlying cell migration have emerged to pro-
vide a source of testable hypotheses [1–17].
The migration of leukocytes, like other crawling cells, is
driven by the dynamics of the actin cytoskeleton [18].
This actin-based motility is initiated by signals from the
environment, orchestrating remarkably complex se-
quences of events. For example, migration of a leukocyte
from the bloodstream to a site of infection, inflammation,
or injury entails multiple bidirectional signaling and ad-
hesive interactions between the leukocyte and vascular
endothelial cells, resulting in attachment of the leuko-
cyte to the endothelium near the affected area, rolling
and arrest of the leukocyte, transient loss of cell-cell ad-
hesion between endothelial cells, and finally transmigra-
tion of the leukocyte across the endothelium [19–21].
During activation of T lymphocytes by antigen-
presenting cells, the cytoskeleton is also involved in for-
mation and stabilization of the immunologic synapse be-
tween the antigen-presenting cell and the lymphocyte; it
is required for sustained interaction and appears to pro-
vide a scaffold for signaling complexes to assemble [22–
27]. Phagocytosis in neutrophils and macrophages,
wherein the cells engulf and internalize microbial patho-
gens, is driven by actin rearrangement [18,28,29], as is
endocytosis in general [30]. Furthermore, actin and mi-
crotubule dynamics are central to cell division in all eu-
karyotic cells [31].
Signaling to the actin cytoskeleton
A basic paradigm of cell biology is the transduction of
extracellular signals across the plasma membrane and
into the cytoplasm to initiate changes in the state of the
cell. Pathways involving inositol phospholipids and small
GTPases are of particular importance. Phosphoinositides
have emerged as key modulators of the activity of actin-
regulatory proteins [32]. Small GTPases of the Rho fam-
ily are prenylated membrane-targeted proteins that func-
tion as critical regulators of actin cytoskeletal remodeling
and other cellular responses to stimuli [33–37]. In the
GTP-bound state, small GTPases are active in signaling
to their effectors; they become inactive once they hydro-
lyze the bound GTP to GDP. Their activity is modu-
lated by at least three classes of proteins: the guanine-
nucleotide exchange factors that accelerate exchange of
GDP for GTP and are therefore activating for signaling;
the GTPase-activating proteins that accelerate the in-
trinsic rate of hydrolysis of GTP to GDP and are inacti-
vating for signaling; and the GDP-dissociation inhibitors
that prevent exchange of GDP for GTP and so are also
inactivating for signaling.
Although each Rho-family member is distinct, there is
considerable cross-signaling between different members
of the Rho family [33–37]. Rho, the prototype member,
actually comprises multiple protein isoforms that are
most associated with the formation of contractile bundles
of F-actin and nonmuscle myosin II (nonmuscle acto-
myosin), such as stress fibers, and focal adhesions. Cdc42
is involved in the formation of filopodia (finger-like
membrane protrusions) and the control of cell polarity.
Rac isoforms are most associated with the formation of
membrane ruffles and lamellipodia (broad, sheet-like
membrane protrusions). The terms lamellipodium and la-
mella are often used synonymously, although in many
cases it is important to make a distinction between the
lamellipodium as the protrusive force-generating lead-
ing-edge tip and the lamella as the rest of the protrusion
back to the cell body [38]. Pseudopod and pseudopodium
are terms also used to describe such protrusive struc-
tures, especially in more amoeboid cells such as leuko-
cytes.
Wiskott-Aldrich syndrome, a disease characterized by
impaired cellular and humoral immunity, results from
mutation in the Wiskott-Aldrich syndrome protein
(WASP). WASP is a member of a protein family that
includes N-WASP and Scar/WAVE proteins, with central
roles in stimulating de novo actin nucleation by the actin-
related protein 2/3 (Arp2/3) complex; different members
of the WASP family are activated by either Cdc42 or Rac
[39–41]. WASP (expressed exclusively in hematopoietic
cells) and N-WASP (neural WASP, which, despite its
name, is ubiquitously expressed) are the best character-
ized mechanistically of the WASP-family proteins and
link Cdc42 activation to Arp2/3 complex-dependent ac-
tin nucleation. WASP is an important regulator of actin
remodeling during T cell activation [42] and other actin-
dependent functions, including chemotaxis and phago-
cytosis, in leukocytes [43]. Its importance in leukocyte
function was again recently highlighted by Jones et al.
[44], who further characterized abnormalities in cell po-
larity, chemotaxis, and podosomes (cell-substratum ad-
hesion structures similar to but distinct from focal com-
16 Myeloid biology
plexes, the short-lived precursors of focal adhesions) in
Wiskott-Aldrich syndrome macrophages and found that
induced expression of wild-type WASP restores normal
morphology and behavior. Furthermore, Launey et al.
[45•] discovered that the differentiation of promyelo-
cytic leukemia HL-60 cells is accompanied by changes in
the expression of WASP and Scar1/WAVE1, with differ-
ent changes observed for differentiation into neutrophil-
like versus monocyte/macrophage-like cells. Modulation
of the expression of different members of the WASP
family may be part of the differentiation program of my-
eloid precursors, and different members may have dis-
tinct roles in leukocyte function.
Actin and phagocytosis
Different pathways to actin remodeling are involved in
different types of phagocytosis. In macrophages, phago-
cytosis through complement receptor 3 (CR3) is medi-
ated by Rho, whereas phagocytosis through Fc␥receptor
(Fc␥R) depends on Cdc42 and Rac [46]. Expanding on
these findings, Olazabal et al. [47•] recently found that
Rho-associated kinase (Rho-kinase) and myosin II are
required for phagocytic cup formation through CR3 but
not Fc␥R, which only requires myosin II for the later
step of internalization of phagocytic vesicles (phago-
somes).
The subsequent fate of internalized phagosomes may
also depend on the actin cytoskeleton. A general mecha-
nism for movement of various types of intracellular
vesicles, such as endosomes, lysosomes, and secretory
vesicles, could be the assembly of an F-actin network
behind the vesicle, which resembles a rocket or comet
tail and propels the vesicle forward [48], similar to the
case of the actin-based motility of certain intracellular
pathogens such as Listeria and Shigella [49–51]. Such a
rocket tail alone does not have a “guidance system” and
is not persistently directional as is the movement of
vesicles along microtubules by motor proteins; rocketing
of vesicles could be tied to other cytoskeleton-
dependent motor systems, or its function could be to
simply augment random movement above the slow dif-
fusion rates for large structures. Formation of short-lived
actin-rich rocket tails and motility of latex bead-
containing phagosomes have been observed in macro-
phages [52], suggesting that phagosome movement
through the cytoplasm may be facilitated by actin-based
propulsion. More recently, Southwick et al. [53•] found
that phagosomes and early endosomes, induced by treat-
ment of macrophages with the secretagogue antagonists
lanthanum and zinc, assemble actin-rich rocket tails with
the apparent involvement of a number of known actin-
binding and actin-regulatory proteins, including the
Arp2/3 complex, N-WASP, profilin, VASP, and zyxin.
However, the question of whether these examples of
actin-based rocketing in perturbed macrophages reflect
normal modes of phagosome movement remains to be
answered.
Leukocyte directional sensing,
polarization, and chemotaxis
Much of our understanding of the signaling events from
chemoattractant receptor occupancy to the actin cyto-
skeleton in leukocytes comes from studies in neutrophils
or related cells such as neutrophil-differentiated HL-60
cells. Leukocytes are recruited to sites of infection, in-
flammation, or injury through the process of chemotaxis,
which involves directed cell migration toward extracel-
lular chemoattractants that include bacterial products
such as N-formylated peptides (e.g., formyl-Met-Leu-
Phe or fMLF, but more commonly referred to as fMLP),
C5a (a product of the complement cascade), products of
phospholipid metabolism such as leukotriene B4, and
chemokines such as interleukin-8. Once a chemoattrac-
tant binds to its cell-surface receptor, a series of
membrane/cytoplasmic “directional sensing” events is
triggered that results in the activation of the cytoskeletal
machinery. Remodeling of the cytoskeleton then brings
about transformation of the cells from a roughly spherical
resting state to a polarized asymmetric shape. This mor-
phologic polarization is characterized by a single protru-
sive actin-rich lamellipodium at the leading edge and a
tail structure or uropod at the trailing edge (Fig. 1). Once
polarized, the cell crawls in the direction of the source of
chemoattractant. If the source of chemoattractant moves,
the cell turns and again migrates up the gradient. Al-
though directional sensing,polarization, and chemotaxis are
terms often used without clear distinction between these
linked processes, a recent review discusses the need to
Figure 1. Filamentous actin (F-actin) cytoskeleton of a
stimulated neutrophil
Neutrophil stimulated with chemoattractant and stained with
rhodamine-phalloidin, a fluorescent probe for F-actin. Note the broad
lamellipodial protrusion (arrowheads) at the leading edge and the retracting tail
or uropod (asterisk) at the trailing edge. Neutrophils and other leukocytes
directionally migrate toward a source of chemoattractant such as bacterially
derived N-formylated peptides by the process of chemotaxis. The dynamics of
the actin cytoskeleton provide the force to drive this movement.
Cytoskeletal remodeling in leukocyte function Fenteany and Glogauer 17
precisely define each and distinguish between them ex-
perimentally to build reasonable models for the overall
sequence [54].
Neutrophils polarize and move directionally toward a
chemoattractant source even in very shallow gradients,
corresponding to as little as a 1% difference in chemo-
attractant concentration across the length of the cell [55].
When a source of chemoattractant forming a gradient is
abruptly moved to the opposite side of the dish, the
neutrophil generally does not simply extend a new mem-
brane protrusion in the new direction and reverse in one
step but rather follows the existing lamellipodium at the
leading edge, which reorients gradually as it continues to
displace, so that the cell appears to make a step-wise
U-turn toward the source [56]. In addition, neutrophils
can polarize and migrate randomly by chemokinesis even
when uniform concentrations of chemoattractant are
added, demonstrating that polarity can arise from self-
organization in the activated neutrophil. These issues
will be revisited after a discussion of recent progress on
signaling to the basic machinery of migration, the actin
cytoskeleton.
Rho-family small GTPases in
leukocyte migration
Some of the first evidence for involvement of GTPases
in the regulation of cellular actin polymerization arose
from research with neutrophils [57–59]. Rac itself was
first identified in a neutrophil-differentiated HL-60
cDNA library [60] and in human platelets [61], whereas
Rho was first discovered in Aplysia [62], and Cdc42 first
in yeast [63–65] and humans [66–68]. Different roles for
the Rho-family GTPases in regulation of the actin cyto-
skeleton were subsequently clearly established [69–79].
Much progress has been made in understanding the sig-
naling pathways involving Rho-family proteins that lead
from chemoattractant receptor occupancy to F-actin as-
sembly in neutrophils [80,81]. At least two main path-
ways to actin polymerization lie downstream from the
fMLP receptor [82]. Both pathways depend on phos-
phoinositides, and both also appear to involve Cdc42 or
shared factors. One pathway leads from Cdc42 to de novo
actin nucleation through and the Arp2/3 complex. The
other pathway leads to Rac-dependent actin polymeriza-
tion even when Arp2/3 complex-dependent nucleation is
maximally inhibited with an Arp2/3 complex-seques-
tering fragment derived from N-WASP. While Rac is
known to initiate Arp2/3 complex-dependent actin
nucleation through Scar/WAVE proteins, this is not the
only pathway from activated Rac to actin polymerization.
In the case of the stimulated neutrophil, the Rac-
dependent mechanism appears to involve a major con-
tribution from elongation from existing filament barbed
ends generated through uncapping or severing, or an
Arp2/3 complex-independent nucleation mechanism
(such as through formins) [83,84].
A tale of two Racs
Rac proteins are key regulators not only of the actin
cytoskeleton but also of the NADPH oxidase system in
neutrophils [85]. Using Rac2-deficient mice and neutro-
phils from a patient with a naturally occurring mutation
in Rac2, it has been demonstrated that the Rac2 isoform
is a key regulator of multiple antimicrobial functions,
including cell polarization and chemotaxis, granule se-
cretion, and generation of reactive oxygen species by the
NADPH oxidase complex [86]. However, the impor-
tance of Rac1 in neutrophil function has remained un-
certain. The high degree of homology in the effector
regions of Rac1 and Rac2 has led to the hypothesis that
these two proteins function interchangeably. Using pu-
rified neutrophil membranes and recombinant Rac1 and
Rac2, Heyworth et al. [87] demonstrated that both iso-
forms have equal activity in the reconstitution of super-
oxide production, although Rac2 was more efficient in
the presence of neutrophil cytosol. In permeabilized
neutrophils, dominant-negative mutants of Rac1 and
Rac2 are equally effective at inhibiting fMLP-induced
actin polymerization [82].
A recent study by Li et al. [88•] using Rac2-deficient
neutrophils suggests that Rac1 and Rac2 have discrete
functions inasmuch as activation and signaling profiles
for each isoform in intact neutrophils are unique. The
authors found that four times more Rac2 is activated
compared with Rac1 in fMLP-activated murine neutro-
phils. In addition, they demonstrated using neutrophils
from Rac2-null and Rac2-heterozygous mice that the
level of activated Rac2 is rate-limiting for chemoattrac-
tant-induced actin polymerization, chemotaxis, and su-
peroxide generation. Another study examined the role of
Rac1 in regulating neutrophil functions using selective
deletion of Rac1 in neutrophils [89•]. In contrast to Rac2,
Rac1 was found to be required only for chemotaxis and
not for NADPH oxidase function. Recent work on the
unique roles of these two small GTPases demonstrates
that these proteins localize to different compartments in
the cell: Rac1 localizes to the actin cytoskeleton, whereas
Rac2 localizes to internal membrane compartments
[90,91].
A tail of Rho and Rac too
Rho-kinase activity is required for migration and tail re-
traction in leukocytes [92–94], for example, through
phosphorylation of myosin light chain kinase [92], which
would promote actomyosin bundle formation and con-
traction. Liu et al. [95] recently provided evidence that
RhoA/Rho-kinase signaling also promotes de-adhesion in
Jurkat T lymphoma cells and neutrophils by inhibiting
actin cytoskeleton-dependent cell spreading. Worthy-
lake and Burridge [96•] found that Rho-kinase activity is
18 Myeloid biology
required to restrict integrin activation and membrane
protrusion to the leading edge in monocytes through a
pathway in part involving actin-depolymerizing fac-
tor/cofilin. RhoA/Rho-kinase signaling outside of the
leading edge appears to promote the development of a
single leading edge by limiting adhesion and protrusion
elsewhere. Yoshinaga-Ohara et al. [97] found that Rho
activity is required for maintenance of polarity as well as
tail retraction in neutrophils and that chemoattractant
stimulation results in Rho-dependent dephosphorylation
of moesin, an F-actin cross-linking protein; furthermore,
inhibition of type 1 and type 2A serine/threonine protein
phosphatases prevents uropod retraction.
Using a fluorescence resonance energy transfer-based
biosensor, Gardiner et al. [98••] found that green fluo-
rescent protein conjugates of both Rac1 and Rac2 are
activated in the leading edge of migrating neutrophils.
Surprisingly, however, they are also activated in the tail.
Furthermore, endogenous Rac2 is recruited to the lamel-
lipodium and to a lesser extent the tail following stimu-
lation. Rac activation in response to stimulation with che-
moattractant is greater in adherent compared with
suspended cells (which also become polarized with
stimulation, although still more rounded than adherent
cells); adhesion is particularly important for Rac activa-
tion in the tail. Using dominant-negative mutants, these
authors also found that Rac activity is functionally re-
quired for uropod retraction in addition to leading-edge
extension and maintenance of polarity.
Positive feedback for polarization:
phosphoinositides and Rho-family
small GTPases
Intact inositol phospholipids in the plasma membrane
play critical roles in the regulation of the actin cytoskel-
eton, and it is well established that phosphatidylinositol
(PI) 4,5-bisphosphate [PI(4,5)P
2
] in particular can bind a
range of different actin-binding proteins to promote F-
actin assembly and establish membrane/cytoskeleton
linkages [32]. (These roles are distinct from those of
PI(4,5)P
2
as a substrate for the well-known hydrolytic
pathway catalyzed by phospholipase C.) In addition, in-
tact PI 3,4,5-trisphosphate [PI(3,4,5)P
3
or PIP
3
], the
product of phosphorylation of PI(4,5)P
2
by PI 3-kinases
(PI3Ks), has been implicated in cytoskeletal regulation
and the control of cell polarity, with roles that appear
separate from those of PI(4,5)P
2
[99]. PI3K activity is
opposed by that of PI 3-phosphatases such as PTEN,
and both play roles in the directional sensing and polar-
ization required for chemotaxis [100]. Other phosphati-
dylinositol kinases and phosphatases are also involved in
controlling PIP
3
levels, such as the PI 5-phosphatase
SHIP, which plays roles in both hematopoiesis and ma-
ture leukocyte functions [101]. However, it is not clear
which phosphatase, or phosphatases, is most responsible
for setting levels and distribution of PIP
3
in neutrophils
[102]. Furthermore, little is known about possible func-
tions of other phosphoinositides, including other PI
3-phosphate lipid products of PI3K, in the regulation of
the cytoskeletal dynamics and cell shape change.
Work in the last few years in neutrophils and other sys-
tems such as Dictyostelium has led to the concept that
short-range positive feedback involving PI3K products,
specifically PIP
3
, and activated Rho-family GTPases
leads to amplification of shallow external chemoattrac-
tant gradients to steeper internal signaling gradients that
establish the “front” of the cell (Fig. 2) [102–104]. Sev-
eral alternative models exist, such as the local excitation-
global inhibition model [54,100], and a mature under-
standing may ultimately incorporate elements of
different models and depend on specific cell type.
Weiner et al. [105••] recently provided evidence for key
aspects of the positive feedback model in neutrophil-
differentiated HL60 cells, showing that an increase in
Figure 2. Signaling cascade involved in neutrophil directional
sensing, polarization, and chemotaxis
Positive signaling dominates at the leading edge (steps indicated with black
arrows). Signal amplification converts a shallow external chemoattractant
gradient into a steep internal signaling gradient during directional sensing and
polarization, allowing chemotaxis to occur (reviewed in [102–104]).
Chemoattractant first binds to a G protein-coupled receptor, which causes
dissociation of the heterotrimeric G protein G
i
into G␣
i
and G␥ subunits. The
released G␥ subunits then stimulate PI3K. The resulting PIP
3
, acting through
specific GEFs (such as P-Rex1 [108], which is also directly stimulated by the
G␥ subunits), activates Rac, which stimulates PI3K, leading to further
generation of PIP
3
and repetition of the cycle [105••,106••]. Rac is the dominant
Rho-family small GTPase involved in this positive feedback loop [107••], while
Cdc42 helps determine the location and stability of the Rac-dependent leading
edge [107••,115••]. The asymmetric distribution of amplified PIP
3
strongly
depends on actin dynamics [106••,107••,109••]. Abbreviations: CA,
chemoattractant; G␣
i
and G␥, heterotrimeric G protein subunits; PI(4,5)P2,
PI(3,4,5)P
3
(PIP
3
), and PI(3,4)P
2
, phosphatidylinositol (PI) lipids phosphorylated
at the indicated positions of the inositol ring; PI3K, PI 3-kinase; PTEN, a PI
3-phosphatase; SHIP, a PI 5-phosphatase; Rac and Cdc42, small Rho-family
GTPases; GEFs, guanine-nucleotide exchange factors; GAPs,
GTPase-activating proteins. Figure courtesy of O.D. Weiner, with modification;
reproduced with permission [102].
Cytoskeletal remodeling in leukocyte function Fenteany and Glogauer 19
PIP
3
activates a Rho-family small GTPase or GTPases,
which in turn stimulates PI3K and the generation of
more PIP
3
. In an accompanying report, Wang et al.
[106••] further confirmed the importance of PI3K and
PIP
3
at the leading edge for polarization and chemotaxis.
Moreover, the normal asymmetric distribution of ampli-
fied PIP
3
following uniform stimulation with fMLP be-
comes symmetric and transient when actin polymeriza-
tion or depolymerization is inhibited with latrunculin B
or jasplakinolide, respectively. This suggests that recip-
rocal interplay between PIP
3
and actin dynamics at the
front/leading edge helps initiate and stabilize the inter-
nal signaling gradient required for cell polarity. Sriniva-
san et al. [107••] subsequently provided evidence that
Rac is the key Rho-family small GTPase regulator of the
PIP
3
-dependent positive feedback loop. Relevantly,
P-Rex1, a GEF for Rac discovered in neutrophils, is ac-
tivated by PIP
3
and the heterotrimeric G protein sub-
units G␥ [108••]. In contrast to the direct role played
by Rac in the positive feedback loop, Cdc42 is critical to
regulating the location and stability of the Rac-initiated
leading edge [107••]. Furthermore, PIP
3
accumulation
induced by expression of a constitutively active Rac mu-
tant is sharply reduced when actin polymerization is in-
hibited. Combined with negative interactions that pre-
vent a leading edge from developing elsewhere (for
example, PTEN, which reduces PIP
3
levels elsewhere,
or actomyosin bundle formation and contraction, which
antagonizes protrusion), these mechanisms may guide
orientation of the cell’s axis of polarity.
An even more recent study further extends these ideas.
Xu et al. [109••] defined divergent, opposing signals gen-
erated by different G protein-coupled receptor systems
involved in polarization of neutrophil-differentiated
HL60 cells (Fig. 3). “Frontness” requires the activity of
heterotrimeric G
i
proteins, PI3K, Rac, and F-actin as-
sembly. “Backness,” in contrast, depends on other G
proteins, G
12
and G
13
, Rho, Rho-kinase, and myosin II.
The backness pathway also leads to decreased sensitivity
to chemoattractant. These two pathways lead to forma-
tion of different actin-based structures in the front (as-
sembly of protruding lamellipodial actin networks) and
back (assembly of contracting actomyosin arrays), and
each negatively regulates formation of the opposite
structure. These mechanisms for self-organizing polarity
would provide explanations for why the leading edge is
more responsive to chemoattractant stimulation than
other parts of the neutrophil, how polarity is generated
even in uniform concentrations of chemoattractant, and
why the neutrophil turns rather than simply reverses di-
rection when confronted with a change in the location of
a chemoattractant gradient’s source.
There are still many questions remaining about the pre-
cise roles of PIP
3
(and possibly other PI3K products) and
different PI3K isoforms in polarization, chemotaxis, and
other neutrophil functions. Deletion of PI3K␥abates
PIP
3
production following stimulation of neutrophils
with fMLP or other chemoattractants and impairs normal
neutrophil chemotaxis and superoxide production [110–
112], although Rac activation and F-actin assembly after
chemoattractant stimulation appear unaffected in sus-
pended neutrophils [111]. PI3K␥-null neutrophils are
able to migrate randomly, albeit at a slower rate than
wild-type cells; they tend to have many small protrusions
around the cell body and exhibit loss of normal leading-
edge colocalization of F-actin and protein kinase B/Akt
[113]. Recently, Sadhu et al. [114•] developed a new,
selective, small-molecule inhibitor of PI3K␦and found
that this PI3K isoform is also required for neutrophil
polarization and chemotaxis, although again not for ran-
dom migration. The authors speculated that PI3K␦could
be responsible for amplifying PIP
3
levels following an
initial burst catalyzed by PI3K␥. Collectively, these stud-
ies suggest that the primary role of PIP
3
in neutrophil
chemotaxis is to control polarization and directional local-
ization of the lamellipodium rather than movement itself.
Recent progress has been made in further defining the
function of Cdc42 in leukocyte polarization. Li et al.
[115••] described a pathway that is essential for neutro-
phil directional sensing, polarization, and chemotaxis in
response to complement factor C5a. This pathway in-
volves the active G protein subunits G␥, p21-activated
kinase 1 (PAK1), the PAK-associated GEF PIX␣, and
Figure 3. A model for how “frontness” and “backness” arise
during neutrophil polarization
“Front”signals generate the leading edge/lamellipodium, whereas “back”signals
generate the trailing edge/uropod (see text and [109••] for details). The positive
and negative interactions indicated may establish and maintain front and back
structures during polarization. The pathway to frontness involves G
i
, PI3K, Rac,
and F-actin assembly (which drives membrane protrusion at the leading edge),
while the pathway to backness involves G
12/13
, Rho, Rho-kinase, and myosin II
(which forms, with F-actin, an array of actomyosin bundles that contract at the
trailing edge). Frontness and backness antagonize one another. This model
could account for the self-organizing nature of neutrophil polarity, explaining how
asymmetry of the cell is generated even in uniform concentrations of
chemoattractant and why neutrophils make U-turns when chemoattractant
gradients are reversed. CA, chemoattractant; R, G protein-coupled receptor; G
i
and G
12/13
, heterotrimeric G proteins; Rac and Rho, small Rho-family GTPases;
PIP
3
, PI 3,4,5-trisphosphate. Figure courtesy of H.R. Bourne, with modification;
reproduced with permission [109••].
20 Myeloid biology
Cdc42. PAKs bind and are activated by Cdc42 and Rac
[116]. In the model supported by Li et al. [115••], G␥
binds PAK1, activating Cdc42 via PIX␣; Cdc42 then ac-
tivates PAK1. A noteworthy feature of this pathway is
that PAK1 appears to function as a scaffold for activation
of Cdc42 before itself being activated by Cdc42. Inter-
estingly, while chemoattractant-induced activation of
Cdc42 occurs even in PI3K␥-deficient neutrophils,
Cdc42 activation is not localized to the leading edge in
these neutrophils, implying that proper localization of
the G␥-PAK1/PIX␣/Cdc42 signaling complex depends
on PI3K␥-catalyzed PIP
3
production. These authors
found that this pathway to Cdc42 activation is required
for excluding PTEN from and limiting new F-actin as-
sembly to the leading edge, consistent with the role as-
cribed to Cdc42 in the aforementioned report by Sriniva-
san et al. [107••]. Such a mechanism may not be limited
to neutrophils, as Ratner et al. [117] recently demon-
strated a role for Cdc42 in ensuring that only a single
lamellipodium is maintained in T lymphocytes.
More on membrane/cytoskeleton
interactions
Other lipid components of the membrane should not be
ignored, nor should consideration of the organization of
the membrane. Pierini et al. [118] showed that depletion
of plasma membrane cholesterol inhibits neutrophil po-
larization and chemotaxis by preventing prolonged (but
not initial) activation of Rac and sustained F-actin as-
sembly. This suggests that membrane lipid organization
and structure may be important for persistent signaling
and the maintenance of cell polarity. Moreover, struc-
tural interactions between the membrane and the cyto-
skeleton may synergize with signaling interactions. In an
attempt to characterize the protein components of the
neutrophil membrane skeleton (the cortical network of
F-actin and associated proteins found beneath the
plasma membrane), Nebl et al. [119•] used mass spec-
trometry to identify proteins from detergent-resistant
membrane fractions. They found proteins already known
to be associated with both membrane skeletons and lipid
rafts (detergent-resistant packed lipid domains in the
plasma membrane) from studies in other systems. In ad-
dition, the actin-binding protein supervillin, a compo-
nent of the membrane skeleton, partially colocalizes with
the G
i
protein subunit G␣
i
in the cells. The authors
suggest that these membrane fractions represent lipid
signaling microdomains associated with the membrane
skeleton. This study raises interesting questions about
the relationship between the cortical cytoskeletal net-
work and lipid rafts. Asymmetric organization of the
plasma membrane into discrete lipid domains that have
both signaling and structural relationships with the cyto-
skeleton and cytoplasm is an attractive hypothesis in try-
ing to understand cell polarization and chemotaxis in
leukocytes and other cell types [120].
What of the other cytoskeletal systems?
Microtubules and intermediate filaments constitute the
two other cytoskeletal polymer systems in eukaryotic
cells. While intermediate filament proteins such as vi-
mentin are expressed in leukocytes, knowledge of their
role in leukocyte function is still embryonic and mostly
descriptive. Mor-Vaknin et al. [121] recently showed that
vimentin is secreted by macrophages in response to pro-
inflammatory signals and that extracellular vimentin may
be involved in the killing of bacteria and generation of
reactive oxygen species. More work has been done on
the role of microtubules in leukocyte function, partly
because of the availability of drugs that stabilize or de-
stabilize microtubules. Yet the most fundamental ques-
tion about the role of microtubules in leukocyte migra-
tion is still largely unanswered. What is their role? Part of
the answer will come from studies of actin and its regu-
lation. While there is no doubt that actin is the cytoskel-
etal element most central to cell migration, it is also clear
that the actin cytoskeleton does not function in isolation
from the other cytoskeletal systems. In fact, there is
growing evidence that actin and microtubules engage in
both indirect regulatory and direct structural interactions
that are important to cell polarization and migration, as
well as other cytoskeleton-dependent processes [17].
Reorientation of the microtubule-organizing center and
the microtubules emanating from it occurs during acti-
vation of T lymphocytes by antigen-presenting cells,
where microtubules may play roles in directed secretion
and possibly also in signal transduction, as has been re-
cently reviewed [23,26]. Microtubules also appear to be
involved in regulating cell-substratum adhesion. Evans et
al. [122••] recently showed that dynamic leading-edge
podosomal adhesions in macrophages form by both de
novo assembly/growth and fragmentation of precursor po-
dosomes; the polarized formation and turnover of podo-
somes depends on microtubules, whereas actin turnover
in the podosomes does not. Therefore, microtubules ap-
pear to play a role in the stabilization of podosomes at the
leading edge of macrophages.
In neutrophils, Eddy et al. [123•] discovered that micro-
tubules reorient toward the uropod during polarization
both when cells are plated on fibronectin and when they
are in suspension, and this occurs even in the presence of
the microtubule-stabilizing drug Taxol, suggesting that
microtubule depolymerization is not required for reori-
entation. Microtubules are normally excluded from the
actin-rich lamellipodium; however, treatment with a
myosin light chain kinase inhibitor or the F-actin-
destabilizing agent cytochalasin D causes expansion of
the microtubule array and penetration of the microtu-
bules into the lamellipodium. Myosin II and the actin
cytoskeleton therefore appear to be involved in the es-
tablishment or maintenance of microtubule asymmetry
in the neutrophil. Moreover, pharmacological depoly-
merization of microtubules with nocodazole prior to
Cytoskeletal remodeling in leukocyte function Fenteany and Glogauer 21
treatment with chemoattractant causes approximately
10% of the cells to lose their polarity and extend multiple
lamellipodia accompanied by altered localization of
lamellipodium- and uropod-specific markers. However,
the authors also point out the conflicting results in the
earlier literature on the effects of microtubule-destabiliz-
ing drugs on cell polarization, chemotaxis, and chemoki-
nesis. A more recent contrasting study was published by
Niggli [124•], who found that disruption of microtubules
with colchicine in fact induces cell polarity and random
migration in neutrophils without affecting chemotaxis.
This effect of colchicine involves Rho-kinase activity
and phosphorylation of myosin light chain kinase but is
independent of G
i
and PI3K, showing that colchicine-
and chemoattractant-induced pathways to polarization
and migration can be separated. The subtle roles played
by microtubules in leukocyte function have yet to be
fully elucidated.
Conclusion
The importance of the cytoskeleton in leukocyte func-
tion is incontrovertible. Recent progress toward under-
standing the roles and regulation of cytoskeletal dynam-
ics have helped to illuminate the mechanisms of cell
activation, polarization, migration, and phagocytosis. An
outline is emerging to describe the way extracellular
stimuli, lipids in the membrane, cytoskeletal regulators,
and the cytoskeleton interact to achieve changes in cell
state and shape. However, many more questions remain,
making this field a fruitful one for research in the years
to come.
Acknowledgments
The authors thank O.D. Weiner and J. Taunton for helpful comments.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
•Of special interest
•• Of outstanding interest
1Lauffenburger DA, Horwitz AF: Cell migration: a physically integrated molecu-
lar process. Cell 1996, 84:359–369.
2Carlier MF, Pantaloni D: Control of actin dynamics in cell motility. J Mol Biol
1997, 269:459–467.
3Welch MD, Mallavarapu A, Rosenblatt J, Mitchison TJ: Actin dynamics in vivo.
Curr Opin Cell Biol 1997, 9:54–61.
4Stossel TP, Hartwig JH, Janmey PA, Kwiatkowski DJ: Cell crawling two de-
cades after Abercrombie. Biochem Soc Symp 1999, 65:267–280.
5Borisy GG, Svitkina TM: Actin machinery: pushing the envelope. Curr Opin
Cell Biol 2000, 12:104–112.
6Chen H, Bernstein BW, Bamburg JR: Regulating actin-filament dynamics in
vivo. Trends Biochem Sci 2000, 25:19–23.
7Pollard TD: Reflections on a quarter century of research on contractile sys-
tems. Trends Biochem Sci 2000, 25:607–611.
8Pollard TD, Blanchoin L, Mullins RD: Molecular mechanisms controlling actin
filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct 2000,
29:545–576.
9Wear MA, Schafer DA, Cooper JA: Actin dynamics: assembly and disassem-
bly of actin networks. Curr Biol 2000, 10:R891–R895.
10 Higgs HN, Pollard TD: Regulation of actin filament network formation through
Arp2/3 complex: activation by a diverse array of proteins. Annu Rev Biochem
2001, 70:649–676.
11 Pantaloni D, Le Clainche C, Carlier MF: Mechanism of actin-based motility.
Science 2001, 292:1502–1506.
12 Small JV, Stradel T, Vignal E, Rottner K: The lamellipodium: where motility
begins. Trends Cell Biol 2002, 12:112–120.
13 Welch MD, Mullins RD: Cellular control of actin nucleation. Annu Rev Cell
Dev Biol 2002, 18:247–288.
14 Pollard TD, Borisy GG: Cellular motility driven by assembly and disassembly
of actin filaments. Cell 2003, 112:453–465.
15 Fenteany G, Zhu S: Small-molecule inhibitors of actin dynamics and cell mo-
tility. Curr Topics Med Chem 2003, 3:593–616.
16 Carlier MF, Clainche CL, Wiesner S, Pantaloni D: Actin-based motility: from
molecules to movement. Bioessays 2003, 25:336–345.
17 Rodriguez OC, Schaefer AW, Mandato CA, et al.: Conserved microtubule-
actin interactions in cell movement and morphogenesis. Nat Cell Biol 2003,
5:599–609.
18 Vicente-Manzanares M, Sancho D, Yanez-Mo M, Sanchez-Madrid F: The leu-
kocyte cytoskeleton in cell migration and immune interactions. Int Rev Cytol
2002, 216:233–289.
19 Worthylake RA, Burridge K: Leukocyte transendothelial migration: orchestrat-
ing the underlying molecular machinery. Curr Opin Cell Biol 2001, 13:569–
577.
20 Luscinskas FW, Ma S, Nusrat A, et al.: Leukocyte transendothelial migration:
a junctional affair. Semin Immunol 2002, 14:105–113.
21 Hordijk P: Endothelial signaling in leukocyte transmigration. Cell Biochem
Biophys 2003, 38:305–322.
22 Cannon JL, Burkhardt JK: The regulation of actin remodeling during T-cell-
APC conjugate formation. Immunol Rev 2002, 186:90–99.
23 Sancho D, Vicente-Manzanares M, Mittelbrunn M, et al.: Regulation of micro-
tubule-organizing center orientation and actomyosin cytoskeleton rearrange-
ment during immune interactions. Immunol Rev 2002, 189:84–97.
24 Sechi AS, Buer J, Wehland J, Probst-Kepper M: Changes in actin dynamics at
the T-cell/APC interface: implications for T-cell anergy? Immunol Rev 2002,
189:98–110.
25 Vyas YM, Maniar H, Dupont B: Visualization of signaling pathways and corti-
cal cytoskeleton in cytolytic and noncytolytic natural killer cell immune syn-
apses. Immunol Rev 2002, 189:161–178.
26 Miletic AV, Swat M, Fujikawa K, Swat W: Cytoskeletal remodeling in lympho-
cyte activation. Curr Opin Immunol 2003, 15:261–268.
27 Samstag Y, Eibert SM, Klemke M, Wabnitz GH: Actin cytoskeletal dynamics
in T lymphocyte activation and migration. J Leukoc Biol 2003, 73:30–48.
28 Castellano F, Chavrier P, Caron E: Actin dynamics during phagocytosis. Se-
min Immunol 2001, 13:347–355.
29 May RC, Machesky LM: Phagocytosis and the actin cytoskeleton. J Cell Sci
2001, 114:1061–1077.
30 Schafer DA: Coupling actin dynamics and membrane dynamics during endo-
cytosis. Curr Opin Cell Biol 2002, 14:76–81.
31 Scholey JM, Brust-Mascher I, Mogilner A: Cell division. Nature 2003,
422:746–752.
32 Yin HL, Janmey PA: Phosphoinositide regulation of the actin cytoskeleton.
Annu Rev Physiol 2003, 65:761–789.
33 Hall A, Nobes CD: Rho GTPases: molecular switches that control the orga-
nization and dynamics of the actin cytoskeleton. Philos Trans R Soc Lond B
Biol Sci 2000, 355:965–970.
34 Schmitz AA, Govek EE, Bottner B, Van Aelst L: Rho GTPases: signaling,
migration, and invasion. Exp Cell Res 2000, 261:1–12.
35 Ridley AJ: Rho GTPases and cell migration. J Cell Sci 2001, 114:2713–
2722.
36 Ridley AJ: Rho family proteins: coordinating cell responses. Trends Cell Biol
2001, 11:471–477.
37 Etienne-Manneville S, Hall A: Rho GTPases in cell biology. Nature 2002,
420:629–635.
38 Cramer LP, Siebert M, Mitchison TJ: Identification of novel graded polarity
actin filament bundles in locomoting heart fibroblasts: implications for the
generation of motile force. J Cell Biol 1997, 136:1287–1305.
39 Takenawa T, Miki H: WASP and WAVE family proteins: key molecules for
22 Myeloid biology
rapid rearrangement of cortical actin filaments and cell movement. J Cell Sci
2001, 114:1801–1819.
40 Suetsugu S, Miki H, Takenawa T: Spatial and temporal regulation of actin
polymerization for cytoskeleton formation through Arp2/3 complex and
WASP/WAVE proteins. Cell Motil Cytoskeleton 2002, 51:113–122.
41 Caron E: Regulation of Wiskott-Aldrich syndrome protein and related mol-
ecules. Curr Opin Cell Biol 2002, 14:82–87.
42 Badour K, Zhang J, Siminovitch KA: The Wiskott-Aldrich syndrome protein:
forging the link between actin and cell activation. Immunol Rev 2003,
192:98–112.
43 Thrasher AJ: WASp in immune-system organization and function. Nat Rev
Immunol 2002, 2:635–646.
44 Jones GE, Zicha D, Dunn GA, et al.: Restoration of podosomes and chemo-
taxis in Wiskott-Aldrich syndrome macrophages following induced expres-
sion of WASp. Int J Biochem Cell Biol 2002, 34:806–815.
•
45 Launay S, Brown G, Machesky LM: Expression of WASP and Scar1/WAVE1
actin-associated proteins is differentially modulated during differentiation of
HL-60 cells. Cell Motil Cytoskeleton 2003, 54:274–285.
Evidence is provided for the possibility that modulation of the expression of differ-
ent members of the WASP family may be part of the differentiation program
of myeloid precursors, with different roles for WASP and Scar1/WAVE1 in leuko-
cytes.
46 Caron E, Hall A: Identification of two distinct mechanisms of phagocytosis
controlled by different Rho GTPases. Science 1998, 282:1717–1721.
•
47 Olazabal IM, Caron E, May RC, et al.: Rho-kinase and myosin-II control phago-
cytic cup formation during CR, but not Fc␥R, phagocytosis. Curr Biol 2002,
12:1413–1418.
Further delineating differences between phagocytosis through different receptors
in macrophages, this study shows that Rho-kinase and myosin II are required for
phagocytic cup formation through CR3 but not Fc␥R, which only requires myosin
II for the later internalization of phagocytic vesicles.
48 Taunton J: Actin filament nucleation by endosomes, lysosomes and secretory
vesicles. Curr Opin Cell Biol 2001, 13:85–91.
49 Cameron LA, Giardini PA, Soo FS, Theriot JA: Secrets of actin-based motility
revealed by a bacterial pathogen. Nat Rev Mol Cell Biol 2000, 1:110–119.
50 Goldberg MB: Actin-based motility of intracellular microbial pathogens. Mi-
crobiol Mol Biol Rev 2001, 65:595–626.
51 Portnoy DA, Auerbuch V, Glomski IJ: The cell biology of Listeria monocyto-
genes infection: the intersection of bacterial pathogenesis and cell-mediated
immunity. J Cell Biol 2002, 158:409–414.
52 Zhang F, Southwick FS, Purich DL: Actin-based phagosome motility. Cell
Motil Cytoskeleton 2002, 53:81–88.
•
53 Southwick FS, Li W, Zhang F, et al.: Actin-based endosome and phagosome
rocketing in macrophages: activation by the secretagogue antagonists lan-
thanum and zinc. Cell Motil Cytoskeleton 2003, 54:41–55.
Phagosomes and early endosomes induced by treatment of macrophages with
lanthanum and zinc are shown to assemble actin-rich rocket tails with recruitment
of a number of known actin-binding and actin-regulatory proteins.
54 Devreotes P, Janetopoulos C: Eukaryotic chemotaxis: distinctions between
directional sensing and polarization. J Biol Chem 2003, 278:20445–20448.
55 Zigmond SH: Ability of polymorphonuclear leukocytes to orient in gradients of
chemotactic factors. J Cell Biol 1977, 75:606–616.
56 Zigmond SH, Levitsky HI, Kreel BJ: Cell polarity: an examination of its behav-
ioral expression and its consequences for polymorphonuclear leukocyte che-
motaxis. J Cell Biol 1981, 89:585–592.
57 Therrien S, Naccache PH: Guanine nucleotide-induced polymerization of ac-
tin in electropermeabilized human neutrophils. J Cell Biol 1989, 109:1125–
1132.
58 Sarndahl E, Lindroth M, Bengtsson T, et al.: Association of ligand-receptor
complexes with actin filaments in human neutrophils: a possible regulatory
role for a G-protein. J Cell Biol 1989, 109:2791–2799.
59 Downey GP, Chan CK, Grinstein S: Actin assembly in electropermeabilized
neutrophils: role of G-proteins. Biochem Biophys Res Commun 1989,
164:700–705.
60 Didsbury J, Weber RF, Bokoch GM, et al.: rac, a novel ras-related family of
proteins that are botulinum toxin substrates. J Biol Chem 1989, 264:16378–
16382.
61 Polakis PG, Weber RF, Nevins B, et al.: Identification of the ral and rac1 gene
products, low molecular mass GTP-binding proteins from human platelets.
J Biol Chem 1989, 264:16383–16389.
62 Madaule P, Axel R: A novel ras-related gene family. Cell 1985, 41:31–40.
63 Johnson DI, Jacobs CW, Pringle JR, et al.: Mapping of the Saccharomyces
cerevisiae CDC3, CDC25, and CDC42 genes to chromosome XII by chro-
mosome blotting and tetrad analysis. Yeast 1987, 3:243–253.
64 Bender A, Pringle JR: Multicopy suppression of the cdc24 budding defect in
yeast by CDC42 and three newly identified genes including the ras-related
gene RSR1. Proc Natl Acad SciUSA1989, 85:9976–9980.
65 Johnson DI, Pringle JR: Molecular characterization of CDC42, a Saccharo-
myces cerevisiae gene involved in the development of cell polarity. J Cell Biol
1990, 111:143–152.
66 Polakis PG, Snyderman R, Evans T: Characterization of G25K, a GTP-
binding protein containing a novel putative nucleotide binding domain. Bio-
chem Biophys Res Commun 1989, 160:25–32.
67 Munemitsu S, Innis MA, Clark R, et al.: Molecular cloning and expression of a
G25K cDNA, the human homolog of the yeast cell cycle gene CDC42. Mol
Cell Biol 1990, 10:5977–5982.
68 Shinjo K, Koland JG, Hart MJ, et al.: Molecular cloning of the gene for the
human placental GTP-binding protein Gp (G25K): identification of this GTP-
binding protein as the human homolog of the yeast cell-division-cycle protein
CDC42. Proc Natl Acad SciUSA1990, 87:9853–9857.
69 Chardin P, Boquet P, Madaule P, et al.: The mammalian G protein rhoC is
ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin
microfilaments in Vero cells. EMBO J 1989, 8:1087–1092.
70 Paterson HF, Self AJ, Garrett MD, et al.: Microinjection of recombinant p21
rho
induces rapid changes in cell morphology. J Cell Biol 1990, 111:1001–
1007.
71 Stasia MJ, Jouan A, Bourmeyster N, et al.: ADP-ribosylation of a small size
GTP-binding protein in bovine neutrophils by the C3 exoenzyme of Clos-
tridium botulinum and effect on the cell motility. Biochem Biophys Res Com-
mun 1991, 180:615–622.
72 Morii N, Narumiya S: ras oncogene-related small molecular weight GTP-
binding protein, rho gene product and botulinum C3 ADP-ribosyltransferase.
Nippon Yakurigaku Zasshi 1992, 99:191–203.
73 Morii N, Teru-uchi T, Tominaga T, et al.: A rho gene product in human blood
platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-
ribosyltransferase on platelet aggregation. J Biol Chem 1992, 267:20921–
20926.
74 Ridley AJ, Hall A: Distinct patterns of actin organization regulated by the small
GTP-binding proteins Rac and Rho. Cold Spring Harb Symp Quant Biol
1992, 57:661–671.
75 Ridley AJ, Paterson HF, Johnston CL, et al.: The small GTP-binding protein
rac regulates growth factor-induced membrane ruffling. Cell 1992, 70:401–
410.
76 Ridley AJ, Hall A: The small GTP-binding protein rho regulates the assembly
of focal adhesions and actin stress fibers in response to growth factors. Cell
1992, 70:389–399.
77 Kozma R, Ahmed S, Best A, Lim L: The Ras-related protein Cdc42Hs and
bradykinin promote formation of peripheral actin microspikes and filopodia in
Swiss 3T3 fibroblasts. Mol Cell Biol 1995, 15:1942–1952.
78 Li R, Zheng Y, Drubin DG: Regulation of cortical actin cytoskeleton assembly
during polarized cell growth in budding yeast. J Cell Biol 1995, 128:599–
615.
79 Nobes CD, Hall A: Rho, rac and cdc42 GTPases: regulators of actin struc-
tures, cell adhesion and motility. Biochem Soc Trans 1995, 23:456–459.
80 Cicchetti G, Allen PG, Glogauer M: Chemotactic signaling pathways in neu-
trophils: from receptor to actin assembly. Crit Rev Oral Biol Med 2002,
13:220–228.
81 Zhelev DV, Alteraifi A: Signaling in the motility responses of the human neu-
trophil. Ann Biomed Eng 2002, 30:356–370.
82 Glogauer M, Hartwig J, Stossel T: Two pathways through Cdc42 couple the
N-formyl receptor to actin nucleation in permeabilized human neutrophils.J
Cell Biol 2000, 150:785–796.
83 Evangelista M, Zigmond S, Boone C: Formins: signaling effectors for assem-
bly and polarization of actin filaments. J Cell Sci 2003, 116:2603–2611.
84 Wallar BJ, Alberts AS: The formins: active scaffolds that remodel the cyto-
skeleton. Trends Cell Biol 2003, 13:435–446.
85 Dinauer MC: Regulation of neutrophil function by Rac GTPases. Curr Opin
Hematol 2003, 10:8–15.
86 Ambruso DR, Knall C, Abell AN, et al.: Human neutrophil immunodeficiency
Cytoskeletal remodeling in leukocyte function Fenteany and Glogauer 23
syndrome is associated with an inhibitory Rac2 mutation. Proc Natl Acad Sci
U S A 2000, 97:4654–4659.
87 Heyworth PG, Bohl BP, Bokoch GM, Curnutte JT: Rac translocates indepen-
dently of the neutrophil NADPH oxidase components p47
phox
and p67
phox
.
Evidence for its interaction with flavocytochrome b558. J Biol Chem 1994,
269:30749–30752.
•
88 Li S, Yamauchi A, Marchal CC, et al.: Chemoattractant-stimulated Rac acti-
vation in wild-type and Rac2-deficient murine neutrophils: preferential activa-
tion of Rac2 and Rac2 gene dosage effect on neutrophil functions. J Immunol
2002, 169:5043–5051.
A detailed characterization of the level of activity and relative contribution made by
Rac2 during neutrophil function.
•
89 Glogauer M, Marchal CC, Zhu F, et al.: Rac1 deletion in mouse neutrophils
has selective effects on neutrophil functions. J Immunol 2003, 170:5652–
5657.
The first study to investigate the specific roles played by Rac1 in neutrophil func-
tions using primary neutrophils.
90 Michaelson D, Silletti J, Murphy G, et al.: Differential localization of Rho
GTPases in live cells: regulation by hypervariable regions and RhoGDI bind-
ing. J Cell Biol 2001, 152:111–126.
91 Dib K, Melander F, Axelsson L, et al.: Down-regulation of Rac activity during

2
integrin-mediated adhesion of human neutrophils. J Biol Chem 2003,
278:24181–24188.
92 Niggli V: Rho-kinase in human neutrophils: a role in signalling for myosin light
chain phosphorylation and cell migration. FEBS Lett 1999, 445:69–72.
93 Alblas J, Ulfman L, Hordijk P, Koenderman L: Activation of RhoA and ROCK
are essential for detachment of migrating leukocytes. Mol Biol Cell 2001,
12:2137–2145.
94 Worthylake RA, Lemoine S, Watson JM, Burridge K: RhoA is required for
monocyte tail retraction during transendothelial migration. J Cell Biol 2001,
154:147–160.
95 Liu L, Schwartz BR, Lin N, et al.: Requirement for RhoA kinase activation in
leukocyte de-adhesion. J Immunol 2002, 169:2330–2336.
•
96 Worthylake RA, Burridge K: RhoA and ROCK promote migration by limiting
membrane protrusions. J Biol Chem 2003, 278:13578–13584.
This study demonstrates that Rho-kinase activity is required to restrict integrin ac-
tivation and membrane protrusion to the leading edge in neutrophils through a
pathway involving cofilin.
97 Yoshinaga-Ohara N, Takahashi A, Uchiyama T, Sasada M: Spatiotemporal
regulation of moesin phosphorylation and rear release by Rho and
serine/threonine phosphatase during neutrophil migration. Exp Cell Res
2002, 278:112–122.
••
98 Gardiner EM, Pestonjamasp KN, Bohl BP, et al.: Spatial and temporal analysis
of Rac activation during live neutrophil chemotaxis. Curr Biol 2002, 12:2029–
2034.
Rac is shown to be activated not only at the leading edge but also in the retracting
tail of migrating neutrophils.
99 Insall RH, Weiner OD: PIP3, PIP2, and cell movement: similar messages,
different meanings? Dev Cell 2001, 1:743–747.
100 Iijima M, Huang YE, Devreotes P: Temporal and spatial regulation of chemo-
taxis. Dev Cell 2002, 3:469–478.
101 March ME, Ravichandran K: Regulation of the immune response by SHIP.
Semin Immunol 2002, 14:37–47.
102 Weiner OD: Regulation of cell polarity during eukaryotic chemotaxis: the che-
motactic compass. Curr Opin Cell Biol 2002, 14:196–202.
103 Rickert P, Weiner OD, Wang F, et al.: Leukocytes navigate by compass: roles
of PI3K␥and its lipid products. Trends Cell Biol 2000, 10:466–473.
104 Bourne HR, Weiner O: A chemical compass. Nature 2002, 419:21.
••
105 Weiner OD, Neilsen PO, Prestwich GD, et al.: A PtdInsP
3
- and Rho GTPase-
mediated positive feedback loop regulates neutrophil polarity. Nat Cell Biol
2002, 4:509–513.
This study provides evidence for a positive feedback loop in amplificationofPIP
3
for establishing neutrophil polarity by showing that an increase in PIP
3
activates
Rho-family small GTPases, which in turn stimulates PI3K and the generationof
more PIP
3
.
••
106 Wang F, Herzmark P, Weiner OD, et al.: Lipid products of PI(3)Ks maintain
persistent cell polarity and directed motility in neutrophils. Nat Cell Biol 2002,
4:513–518.
This is a companion study to the preceding one, providing additional evidence for
the importance of PIP
3
at the leading edge and demonstrating interplay between
PIP
3
and actin dynamics in the establishment and maintenance of cell polarity.
••
107 Srinivasan S, Wang F, Glavas S, et al.: Rac and Cdc42 play distinct roles in
regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis. J Cell Biol
2003, 160:375–385.
This work identifies Rac as the key Rho-family small GTPase involved in the PIP
3
-
dependent positive feedback loop, while Cdc42 is critical to regulating the location
and stability of the Rac-initiated leading edge.
108 Welch HC, Coadwell WJ, Ellson CD, et al.: P-Rex1, a PtdIns(3,4,5)P
3
- and
G␥-regulated guanine-nucleotide exchange factor for Rac. Cell 2002,
108:809–821.
••
109 Xu J, Wang F, Van Keymeulen A, et al.: Divergent signals and cytoskeletal
assemblies regulate self-organizing polarity in neutrophils. Cell 2003,
114:201–241.
This study defines two divergent, opposing pathways that determine “frontness”
(G
i
, PI3K, Rac, and F-actin assembly) and ⬘backness⬘(G
12/13
, Rho, Rho-kinase,
and myosin II) during polarization and may explain neutrophil responses to che-
moattractants.
110 Sasaki T, Irie-Sasaki J, Jones RG, et al.: Function of PI3K␥in thymocyte de-
velopment, T cell activation, and neutrophil migration. Science 2000,
287:1040–1046.
111 Li Z, Jiang H, Xie W, et al.: Roles of PLC-2 and -3 and PI3K␥in chemoat-
tractant-mediated signal transduction. Science 2000, 287:1046–1049.
112 Hirsch E, Katanaev VL, Garlanda C, et al.: Central role for G protein-coupled
phosphoinositide 3-kinase ␥in inflammation. Science 2000, 287:1049–
1053.
113 Hannigan M, Zhan L, Li Z, et al.: Neutrophils lacking phosphoinositide 3-ki-
nase ␥show loss of directionality during N-formyl-Met-Leu-Phe-induced che-
motaxis. Proc Natl Acad SciUSA2002, 99:3603–3608.
•
114 Sadhu C, Masinovsky B, Dick K, et al.: Essential role of phosphoinositide
3-kinase ␦in neutrophil directional movement. J Immunol 2003, 170:2647–
2654.
A selective inhibitor of PI3K␦was developed and used to show that this PI3K
isoform is required for neutrophil polarization and chemotaxis, but not for random
cell migration.
••
115 Li Z, Hannigan M, Mo Z, et al.: Directional sensing requires G␥-mediated
PAK1 and PIX␣-dependent activation of Cdc42. Cell 2003, 114:215–227.
A pathway is described that is essential for directional sensing, polarization, and
chemotaxis involving G␥, PAK1, PIX␣, and Cdc42, with a novel function for PAK1
as a Cdc42 scaffold protein in addition to an effector; in addition, PIP
3
appears to
be involved in the localization of Cdc42 activity.
116 Bokoch GM: Biology of the p21-activated kinases. Annu Rev Biochem 2003,
72:743–781.
117 Ratner S, Piechocki MP, Galy A: Role of Rho-family GTPase Cdc42 in polar-
ized expression of lymphocyte appendages. J Leukoc Biol 2003, 73:830–
840.
118 Pierini LM, Eddy RJ, Fuortes M, et al.: Membrane lipid organization is critical
for human neutrophil polarization. J Biol Chem 2003, 278:10831–10841.
•
119 Nebl T, Pestonjamasp KN, Leszyk JD, et al.: Proteomic analysis of a deter-
gent-resistant membrane skeleton from neutrophil plasma membranes. J Biol
Chem 2002, 277:43399–43409.
A characterization of protein components of the neutrophil membrane skeleton and
provides insight into the relationship between the membrane skeleton and lipid
rafts.
120 Manes S, Ana Lacalle R, Gomez-Mouton C, Martinez AC: From rafts to crafts:
membrane asymmetry in moving cells. Trends Immunol 2003, 24:320–326.
121 Mor-Vaknin N, Punturieri A, Sitwala K, Markovitz DM: Vimentin is secreted by
activated macrophages. Nat Cell Biol 2003, 5:59–63.
••
122 Evans JG, Correia I, Krasavina O, et al.: Macrophage podosomes assemble at
the leading lamella by growth and fragmentation. J Cell Biol 2003, 161:697–
705.
This work demonstrates that dynamic leading-edge podosomal adhesions in mac-
rophages form by both de novo assembly/growth and fragmentation of precursor
podosomes and that polarized formation and turnover of podosomes depends on
microtubules.
•
123 Eddy RJ, Pierini LM, Maxfield FR: Microtubule asymmetry during neutrophil
polarization and migration. Mol Biol Cell 2002, 13:4470–4483.
This study shows that microtubules reorient toward the uropod during neutrophil
polarization and that myosin II and an intact actin cytoskeleton are required for
microtubule asymmetry.
•
124 Niggli V: Microtubule-disruption-induced and chemotactic-peptide-induced
migration of human neutrophils: implications for differential sets of signalling
pathways. J Cell Sci 2003, 116:813–822.
Disruption of microtubules was found to induce cell polarity and random migration
in neutrophils in a manner that depends on Rho-kinase but not G
i
and PI3K.
24 Myeloid biology
