390 | S. Shu et al. Molecular Biology of the Cell
MBoC | ARTICLE
Actin cross-linking proteins cortexillin I and II are
required for cAMP signaling during Dictyostelium
chemotaxis and development
Shi Shua, Xiong Liua, Paul W. Kriebelb, Mathew P. Danielsc, and Edward D. Korna
aLaboratory of Cell Biology, National Heart, Lung, and Blood Institute, bLaboratory of Cellular and Molecular Biology,
Center for Cancer Research, National Cancer Institute, and cElectron Microscopy Core Facility, National Heart, Lung,
and Blood Institute, National Institutes of Health, Bethesda, MD 20892
ABSTRACT Starvation induces Dictyostelium amoebae to secrete cAMP, toward which other
amoebae stream, forming multicellular mounds that differentiate and develop into fruiting
bodies containing spores. We find that the double deletion of cortexillin (ctx) I and II alters
the actin cytoskeleton and substantially inhibits all molecular responses to extracellular cAMP.
Synthesis of cAMP receptor and adenylyl cyclase A (ACA) is inhibited, and activation of ACA,
RasC, and RasG, phosphorylation of extracellular signal regulated kinase 2, activation of
TORC2, and stimulation of actin polymerization and myosin assembly are greatly reduced. As
a consequence, cell streaming and development are completely blocked. Expression of ACA–
yellow fluorescent protein in the ctxI/ctxII–null cells significantly rescues the wild-type pheno-
type, indicating that the primary chemotaxis and development defect is the inhibition of ACA
synthesis and cAMP production. These results demonstrate the critical importance of a prop-
erly organized actin cytoskeleton for cAMP-signaling pathways, chemotaxis, and develop-
ment in Dictyostelium.
For a number of reasons, including ease of cell culture, genetic ma-
nipulation, and experimental design, the social amoeba Dictyostel-
ium discoideum has long been a model system for investigating the
morphological and molecular events of chemotaxis and develop-
ment. Starvation of Dictyostelium initiates a ∼24-h developmental
process that begins with the pulsed secretion of cAMP by a fraction
of the amoebae, toward which neighboring amoebae chemotax
(Chisholm and Firtel, 2004). Interaction of the secreted cAMP with
the G protein–coupled cAMP receptor 1 (cAR1) on the plasma
membranes of neighboring cells initiates a series of molecular and
morphological events (Swaney et al., 2010), including enhanced ex-
pression of cAR1 and adenylyl cyclase A (ACA; Figure 1, ↑cAR1,
↑ACA), cell elongation and polarization (Johnson et al., 1992; Pitt
et al., 1992; Insall et al., 1994), and chemotaxis. Release of Gβγ from
the heterotrimeric G- protein coupled to cAR1 activates myosin II,
mediated by guanylyl cyclase A (GCA) and cGMP; Bosgraff et al.,
2002; Figure 1). Gβγ also activates two synergistic and partially re-
dundant RasC- and RasG-signaling pathways (Lim et al., 2001; Kae
et al., 2004; Sasaki et al., 2004; Bolourani et al., 2006). One pathway
activates target of rapamycin complex 2 (TORC2) and protein kinase
B (PKB), initiating polymerization of actin at the front of the cell (Cai
et al., 2010; Figure 1), which, together with contraction of actomyo-
sin II at the rear, supports chemotaxis toward the aggregation cen-
ters (Kimmel and Parent, 2003).
A second Ras pathway activates phosphatidylinositol 3-kinase
(PI3K) at the cell’s leading edge, which catalyzes the conversion of
phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol
3,4,5-trisphosphate (PIP3), to which cytoplasmic regulator of adeny-
lyl cyclase (CRAC) binds and activates membrane-associated ACA
(Comer et al., 2005; Figure 1). PIP3 also contributes to the TORC2
pathway, which induces actin polymerization (Tang et al., 2011;
Figure 1). TORC2 contributes to activation of ACA (Lee et al., 2005;
Figure 1), and, independent of Gβγ, binding of cAMP to cAR1 leads
Peter Van Haastert
University of Groningin
Received: Sep 13, 2011
Revised: Nov 4, 2011
Accepted: Nov 16, 2011
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E11-09-0764) on November 23, 2011.
Address correspondence to: Edward D. Korn (email@example.com).
Abbreviations used: ACA, adenylyl cyclase; cAR1, cAMP receptor 1; CRAC, cyto-
plasmic regulator of ACA; ctx, cortexillin.
© 2012 Shu et al. This article is distributed by The American Society for Cell Biol-
ogy under license from the author(s). Two months after publication it is available
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
Volume 23 January 15, 2012 Cortexillins are required for cAMP signaling | 391
protein cortexillin I (Fey and Cox, 1999). The
molecular events underlying this phenotype
and a similar phenotype of Dictyostelium
lacking both α-actinin and filamin (gelation
factor, ABP-120), two other actin cross-link-
ing proteins (Rivero et al., 1996), were not
explored, as we now do for Dictyostelium
cortexillin (ctx)-null cells.
Dictyostelium ctxI and ctxII—444 and
441 amino acids, respectively—are parallel
dimers with a coiled-coil domain and two
globular heads that contain actin-binding
sites (Faix et al., 1996). Cortexillin I also has
a putative PIP2-binding site at its C-termi-
nus (Faix et al., 1996) and a second, and
stronger, actin-bundling domain in the C-
terminal region that is inhibited by PIP2
(Stock et al., 1999). Of importance, ctxI and
ctxII occur in quaternary complexes with
Rac1 and either one of the Dictyostelium
IQGAP proteins DGAP1 and GAPA (Faix
et al., 2001; Lee et al., 2010; Mondal et al.,
2010). Both cortexillins accumulate in the
cortex of vegetative cells and the cortical
region of spreading cells (Faix et al., 1996),
where, together with myosin II, they bundle
and cross-link actin filaments in an antipar-
allel manner (Schroth-Diez et al., 2009). In
motile cells, both cortexillins are enriched at the leading edge and,
to a lesser extent, at the rear (Faix et al., 1996). Cortexillins also
localize to the cleavage furrow of dividing cells (Faix et al., 1996),
independent of myosin II (Weber et al., 1999), where, together with
myosin II, they increase cleavage furrow stiffness (Girard et al.,
2004; Reichl et al., 2008).
Here we report that both head-to-tail cell streaming of Dictyos-
telium amoebae into multicellular mounds and development of the
mounds to mature fruiting bodies are partially inhibited in ctxA− and
ctxB− cells (ctxA and ctxB are the genes coding for proteins ctxI and
ctxII, respectively) and completely inhibited in ctxA−/B− cells, as they
are in cells expressing Y53A-actin. We found that intracellular and
extracellular cAMP signaling is also impaired in cortexillin-null cells
but in a different way than in Y53A-actin cells. In particular, expres-
sion of both cAR1 and ACA are severely diminished in ctxA−/B− cells
but not in Y53A cells, and translocation of ACA-containing vesicles
to the rear of chemotaxing cells is not impaired in ctxA−/B− cells but
is in Y53A cells. Expression of ACA-yellow fluorescent protein (YFP),
but not expression of cAR1-YFP, in ctxA−/B− cells significantly res-
cues the phenotype of WT cells. Thus, whereas impairment of cell
streaming and development of Y53A-actin cells may be caused pri-
marily by inhibition of ACA vesicle translocation to, and secretion of
cAMP at, the rear of the cell (Shu et al., 2010), inhibition of cell
streaming and development of ctxA−/B− cells probably result princi-
pally from decreased secretion of cAMP due to inhibition of ACA
synthesis. The phenotypes of Y53A cells and ctxA−/B− cells demon-
strate the critical importance of a properly organized actin cytoskel-
eton for cAMP-induced signaling pathways.
First, we confirmed by Western blots that ctxA− cells expressed ctxII
and not ctxI, that ctxB− cells expressed ctxI and not ctxII, and that
ctxA−/B− cells expressed neither ctxI nor ctxII (Supplemental Figure
S1A). Furthermore, we observed that ctxI and ctxII were enriched in
to phosphorylation and activation of extracellular signal regulated
kinase 2 (ERK2), which increases cAMP concentration (Segall et al.,
1995) by inhibiting its hydrolysis by a phosphodiesterase (Maeda
et al., 2004). ACA-containing vesicles translocate to the rear of
chemotaxing cells (Kriebel et al., 2008), where secretion of cAMP
creates a cell-to-cell cAMP signal relay (Kimmel and Parent, 2003;
Figure 1), resulting in head-to-tail streams of cells that aggregate
into tight mounds of 100,000 or more cells in ∼12 h. Over the next
12 h, the multicellular mounds differentiate through several mor-
phological stages, developing into mature fruiting bodies compris-
ing a spore head supported by a stalk. In an appropriate nutritional
environment, spores germinate into amoebae, and the life cycle
Recently we made the serendipitous observation that ectopic
expression of Y53A-actin inhibits cell steaming during cAMP-in-
duced aggregation (although individual cells chemotax normally)
and blocks development beyond the mound stage (Liu et al., 2010;
Shu et al., 2010). The developmental phenotype of Y53A-actin cells
correlates with an inhibition of intracellular and intercellular cAMP-
signaling pathways (Shu et al., 2010), including the trafficking of
ACA vesicles to, and secretion of cAMP at, the rear of chemotaxing
cells. It is highly likely that the underlying cause of these phenomena
is the disorganized actin cytoskeleton of amoebae expressing Y53A-
actin. Whereas wild-type-cell cytoskeletons comprise a mostly ho-
mogeneous array of filaments, cytoskeletons of Y53A-actin cells
contain many shorter filaments and numerous bundles and aggre-
gates of short and long filaments (Shu et al., 2010), similar to the
structures formed by copolymerization of Y53A-actin and WT actin
in vitro (Liu et al., 2010).
Of interest, a developmental phenotype similar to that of
Dictyostelium amoebae expressing Y53A-actin, that is, inhibition of
both aggregation streams and development of mounds to mature
fruiting bodies, had been described for Polysphondylium (a close
relative of Dictyostelium) upon deletion of the actin cross-linking
FIGURE 1: Schematic depiction of cAMP signaling pathways in D. discoideum. cAMP binding to
G protein–coupled cAR1 increases the expression of cAR1 and ACA and the release of Gβγ,
which activate RasC and RasG pathways. Activation of PI3K leads to phosphorylation of PIP2,
and PIP3 brings CRAC to the plasma membrane, activating ACA, which converts ATP to cAMP,
which is secreted at the rear and binds to cAR1 of neighboring cells. Activation of TORC2
activates PKB, which initiates actin polymerization at the front of the cell. TORC2 also
contributes to the activation of ACA, and PIP3 contributes to PKB activation. Gβγ also activates
GCA, which synthesizes cGMP, which, acting through GbpC, contributes to myosin activation
and actomyosin contraction at the rear of the cell. cAMP-bound cAR1 also phosphorylates and
activates ERK2, which contributes to ACA activation. Not all of the intermediates in these
pathways are shown.
400 | S. Shu et al. Molecular Biology of the Cell
between myosin II and the actin crosslinker cortexillin I. Curr Biol 19,
Rivero F, Koppel B, Peracino B, Bozzaro S, Seigert F, Weijer CJ, Schleicher
M, Albrecht, Noegel AA (1996). The role of the cortical cytoskeleton:
F-actin crosslinking proteins protect against osmotic stress, ensure cell
size, cell shape and motility, and contribute to phagocytosis and devel-
opment. J Cell Sci 109, 2679–2691.
Sasaki AT, Chun C, Takeda K, Firtel RA (2004). Localized Ras signaling at the
leading edge regulates PI3K, cell polarity, and directional movement. J
Cell Biol 167, 505–518.
Sasaki AT, Firtel RA (2009). Spatiotemporal regulation of Ras-GTPase during
chemotaxis. Meth Mol Biol 571, 333–348.
Schroth-Diez B, Gerwig S, Ecke M, Hegerl R, Diez S, Gerisch G (2009).
Propagating waves separate two states of actin organization in living
cells. HSFP J 3, 412–427.
Segall J, Kuspa A, Shaulsky G, Ecke M, Maeda M, Gaskins C, Firtel RA,
Loomis WF (1995). A MAP kinase necessary for receptor mediated acti-
vation of adenylyl cyclase in Dictyostelium. J Cell Biol 128, 405–413.
Shu S, Liu X, Korn ED (2003). Blebbistatin and blebbistatin-inactivated
myosin II inhibit myosin II-independent processes. Proc Natl Acad Sci
USA 100, 6499–6504.
Shu S, Liu X, Kriebel PW, Hong MS, Daniels MP, Parent CA, Korn ED (2010).
Expression of Y53A-actin in Dictyostelium disrupts the cytoskeleton and
inhibits intracellular and extracellular chemotactic signaling. J Biol Chem
Stock A, Steinmetz MO, Janmey PA, Aebi U, Gerisch G, Kammerer RA,
Weber I, Faix J (1999). Domain analysis of cortexillin I: actin-bundling,
PIP2-binding and the rescue of cytokinesis. EMBO J 18, 5274–5284.
Svitkina TM, Bulanova EA, Chaga OY, Vignjevic DM, Kojima S, Vasiliev JM,
Borisy GG (2003). Mechanism of filopodia initiation by reorganization of
a dendritic network. J Cell Biol 160, 409–421.
Swaney KF, Huang C-H, Devreotes PN (2010). Eukaryotic chemotaxis: a
network of signaling pathways controls motility, directional sending, and
polarity. Annu Rev Biophys 39, 265–289.
Tang M, Iijima M, Kamimua Y, Chen L, Long Y, Devreotes PN (2011). Disrup-
tion of PKB signaling restores polarity to cells lacking tumor suppressor
PTEN. Mol Biol Cell 22, 437–447.
Tseng Y, Kole TP, Lee JS, Fedorov E, Almo SC, Schafer BW, Wirtz D (2005).
How actin crosslinking and bundling proteins cooperate to generate
an enhanced cell mechanical response. Biochem Biophys Res Commun
Van Haastert PJ, Kien E (1983). Binding of cAMP derivatives to Dictyoste-
lium discoideum cellsActivation mechanism of the cell surface cAMP
receptor. J Biol Chem 258, 9636–9642.
Weber I, Gerisch G, Heizer C, Murphy J, Badelt K, Stock A, Schwartz JM,
Faix J (1999). Cytokinesis mediated through the recruitment of cortexil-
lins into the cleavage furrow. EMBO J 18, 586–594.
Wessels D, Lusche DF, Kuhl S, Heid P, Soll DR (2007). PTEN plays a role in
the suppression of lateral pseudopod formation during Dictyostelium
motility and chemotaxis. J Cell Sci 120, 2517–2531.