Immunity

Article

Formins Regulate the Actin-RelatedProtein 2/3 Complex-Independent Polarizationof the Centrosome to the Immunological SynapseTimothy S. Gomez,1 Karan Kumar,1 Ricardo B. Medeiros,3 Yoji Shimizu,3 Paul J. Leibson,1

and Daniel D. Billadeau1,2,*1 Department of Immunology2 Division of Oncology ResearchMayo Clinic College of Medicine, Rochester, MN 55905, USA3 Department of Laboratory Medicine and Pathology, Center for Immunology, Cancer Center, University of Minnesota Medical

School, Minneapolis, MN 55455, USA

*Correspondence: billadeau.daniel@mayo.eduDOI 10.1016/j.immuni.2007.01.008

SUMMARY

T cell receptor (TCR)-mediated cytoskeletal re-organization is considered to be actin-relatedprotein (Arp) 2/3 complex dependent. We there-fore examined the requirement for Arp2/3- andformin-dependent F-actin nucleation during Tcell activation. We demonstrated that withoutArp2/3-mediated actin nucleation, stimulated Tcells could not form an F-actin-rich lamellipod,but instead produced polarized filopodia-likestructures. Moreover, the microtubule-organiz-ing center (MTOC, or centrosome), which rap-idly reorients to the immunological synapsethrough an unknown mechanism, polarized inthe absence of Arp2/3. Conversely, the actin-nucleating formins, Diaphanous-1 (DIA1) andFormin-like-1 (FMNL1), did not affect TCR-stim-ulated F-actin-rich structures, but insteaddisplayed unique patterns of centrosome coloc-alization and controlled TCR-mediated centro-some polarization. Depletion of FMNL1 or DIA1in cytotoxic lymphocytes abrogated cell-medi-ated killing. Altogether, our results have iden-tifed Arp2/3 complex-independent cytoskeletalreorganization events in T lymphocytes and in-dicate that formins are essential cytoskeletalregulators of centrosome polarity in T cells.

INTRODUCTION

Within minutes of antigen-presenting cell (APC) recogni-

tion, T cells undergo cytoskeletal polarization, involving

formation of an F-actin-rich lamellipodium (Bunnell et al.,

2001) and microtubule-organizing center (MTOC) reorien-

tation to the immune synapse (IS) (Kupfer et al., 1987).

Although the mechanisms controlling polarization of

these cytoskeletal elements remain unclear, this restruc-

turing is essential for T cell function (Vicente-Manzanares

Im

and Sanchez-Madrid, 2004). In addition, the speed with

which this cytoskeletal reorganization occurs makes the

T cell-APC recognition process a unique model for study-

ing the molecular mechanisms controlling cytoskeletal

polarization.

TCR-mediated MTOC (or centrosome) reorientation has

been suggested to be regulated by only a few signaling

molecules (Ardouin et al., 2003; Combs et al., 2006; Kuhne

et al., 2003; Martin-Cofreces et al., 2006; Serrador et al.,

2004; Stowers et al., 1995). Functionally, MTOC polariza-

tion is thought to control the positioning of the secretory

apparatus for directed release of lymphokines in T-helper

cells (Kupfer et al., 1991) or cytotoxins in cytolytic cells

(Kupfer and Dennert, 1984). Also, centrosome-plasma

membrane contact in T cells is thought to be necessary

for directed secretion and to be F-actin dependent

(Stinchcombe et al., 2006). However, the putative actin

regulators controlling this centrosome polarization remain

to be identified.

Several studies making use of pharmacologic agents

have demonstrated that F-actin reorganization is crucial

for T cell activation (Campi et al., 2005; Holsinger et al.,

1998; Valitutti et al., 1995). The actin-related protein

(Arp) 2/3 complex, which is directly stimulated by activa-

tors such as Wiskott-Aldrich Syndrome protein (WASP)

and WASP-family verprolin homologous (WAVE) proteins,

nucleates F-actin into an expanding array of individual fil-

aments branching off one another. This Arp2/3 complex-

generated F-actin meshwork is proposed to produce

sheet-like lamellipodia and spike-like filopodia. Even

though filopodia are predicted to arise from the Arp2/3

complex-dependent dendritic network through selective

bundling of Arp2/3 complex-generated filaments (Biya-

sheva et al., 2004), this idea remains controversial (Faix

and Rottner, 2006). In fact, it is now suggested that the

Arp2/3 complex is dispensable for filopodia formation

(Steffen et al., 2006). Indeed, genetic evidence in yeast,

Drosophila, and C. elegans reveals that the Arp2/3 com-

plex is not the sole nucleator for all F-actin-containing

structures (Evangelista et al., 2002; Hudson and Cooley,

2002; Severson et al., 2002; Tolliday et al., 2002). This sug-

gests that F-actin nucleators are specialized, producing

munity 26, 177–190, February 2007 ª2007 Elsevier Inc. 177

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Formins Regulate T Cell Polarity

specific architectural frameworks that coordinate distinct

cellular functions.

Formin family proteins can also nucleate F-actin, and

unlike the Arp2/3 complex, formins nucleate linear F-actin

filaments and are proposed to generate unbranched struc-

tures, such as actin cables, filopodia, and stress fibers

(Faix and Grosse, 2006). Formins are conserved modular

proteins sharing characteristic formin homology (FH) do-

mains (Higgs, 2005). The FH1 domain interacts with the

G-actin binding protein profilin, providing G-actin for fila-

ment assembly, while the adjacent FH2 domain nucleates

F-actin. In yeast, formins participate in microtubule organi-

zation, spindle positioning, polarized cell growth, and con-

tractile ring formation during cytokinesis (Faix and Grosse,

2006). However, the functions of mammalian formins are

not well understood, with recent evidence suggesting roles

in cell motility, adhesion, and microtubule capture and sta-

bilization (Faix and Grosse, 2006; Kobielak et al., 2004;

Wen et al., 2004). In fact, there appears to be a strong,

yet ill-defined, link emerging between mammalian formins

and the microtubule cytoskeleton.

Here we examined the contributions of the Arp2/3 com-

plex and formins to cytoskeletal regulation during T cell

activation. We found that Arp2/3 complex-depleted cells,

which could not form F-actin-rich lamellipodia, still polar-

ized actin-based filopodia and were even capable of

MTOC polarization. In contrast, the formins, Diapha-

nous-1 (DIA1) and Formin-like-1 (FMNL1), did not regulate

F-actin accumulation at the IS, but instead colocalized

with the centrosome and controlled MTOC polarization

and cell-mediated killing. Thus, we found that the Arp2/3

complex and formins distinctly regulated the T cell cyto-

skeleton, and we identified the formins as essential regu-

lators of centrosome polarity during T cell activation.

RESULTS

TCR Stimulation Leads to Distinct Arp2/3

Complex-Independent F-Actin Structures

In order to examine the role of the Arp2/3 complex in T

cells, we generated short-hairpin RNA (shRNA) vectors

to silence both Arp2 and Arp3. Transfection of these vec-

tors into Jurkat T cells led to a substantial depletion of

Arp2 and Arp3 (Figures 1A and 1B). Consistent with stud-

ies in nonhematopoietic cells (Steffen et al., 2006), sup-

pression of either Arp2 or Arp3 with each individual target-

ing vector led to a decrease in expression of other Arp2/3

complex components (Figures 1A and 1B). Thus, the in-

tegrity of the Arp2/3 complex as a whole is dependent

on the presence of either Arp2 or Arp3 in T cells.

Because the Arp2/3 complex is considered essential for

TCR-mediated F-actin reorganization, we first examined

the morphology of TCR-stimulated Arp2/3 complex-

depleted GFP-actin-expressing Jurkat cells spreading af-

ter TCR ligation (Bunnell et al., 2001). GFP-actin Jurkat

cells were transfected with shRNA vectors containing

a separate mCherry transcriptional cassette (Nolz et al.,

2006). Control mCherry-expressing cells spread onto

anti-TCR-coated coverslips in an ordered fashion, forming

178 Immunity 26, 177–190, February 2007 ª2007 Elsevier Inc.

a round, actin-rich lamellipod (Figures 1C; see Movie S1 in

the Supplemental Data available with this article online).

Spreading was maximal by 5 min after initial coverslip

contact, with the central area corresponding to the cell

body visually devoid of GFP-actin (Figure 1C). This was

followed by a 15–20 min retraction phase, characterized

by disassembly of the lamellipod and the return of GFP-

actin to the cell body (Movie S1). Differential interference

contrast (DIC) images indicated retrograde flow and ruf-

fling of the extended lamellipod throughout the spreading

process (Movie S1).

In contrast to control transfectants, neither shArp2- nor

shArp3-transfected cells formed lamellipodia, but instead

displayed a dramatically different phenotype, extending

long, dynamic actin-rich filopodia in response to TCR

stimulation (Figure 1C; Movies S2 and S3). These struc-

tures were not as long-lived as the lamellipod and were

not uniformly produced, with cells displaying distinct mor-

phologies (Figure 1C). Also, Arp2/3 complex-depleted

cells retained substantial GFP-actin in their cell bodies,

yet were seemingly capable of flattening against the acti-

vating coverslips (Movies S2 and S3). In contrast to Arp2-

suppressed cells, shArp3-transfected cells transiently dis-

played periodic membrane bursts occurring between

adjacent filopodial structures (Movie S3), which were

most likely due to inefficient suppression of Arp3. Phalloi-

din staining of control and Arp2-suppressed T cells indi-

cated that the GFP-actin enrichment seen in Figure 1C

indeed correlated with F-actin polymerization (Figures

1D–1F), suggesting that Arp2/3 complex-independent

actin reorganization was responsible for the microspike

formation. In fact, shArp2-transfected cells no longer pro-

duced filopodia upon cytochalasin treatment (not shown),

but did form them upon colchicine treatment (not shown),

indicating an actin-dependent yet microtubule-indepen-

dent mechanism for formation of these filopodia.

In order to more closely visualize the morphological dif-

ferences between control and Arp2-suppressed cells, we

utilized scanning electron microscopy (SEM). Unstimu-

lated control cells did not undergo morphological alter-

ations in response to the coverslip (Figure 1G), while

anti-TCR-stimulated control cells extended flat sheet-

like structures, which apparently displayed filopodia pro-

truding at the leading edge (Figure 1H, left). After spread-

ing completely, control cells were dramatically flattened

against the activating coverslip, with apparent ruffling at

the periphery (Figure 1H, right). Arp2-suppressed cells

sent out filopodial appendages, which extended only

from the area in contact with the coverslip (Figure 1I,

left). SEM also confirmed that shArp2-transfected cells in-

deed flattened against the coverslip (Figure 1I, right).

These data indicated that TCR-induced F-actin polymeri-

zation arises independently of the Arp2/3 complex, mini-

mally in the form of microspikes.

Arp2/3 Complex-Independent Filopodia Polarize

during APC Recognition

Because of the altered morphology of activated Arp2/3

complex-suppressed cells, we examined whether they

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Formins Regulate T Cell Polarity

Figure 1. TCR Stimulation Leads to Distinct Arp2/3 Complex-Independent F-Actin Structures

(A and B) Jurkat cells were transfected with shRNA vectors against Arp2 and Arp3 and analyzed over time via immunoblot for Arp2 or Arp3 expression.

(C) Jurkat cells expressing GFP-actin (green) were transfected with control, shArp2-mCherry, or shArp3-mCherry vectors (red) and stimulated on anti-

TCR-coated coverslips. Representative confocal images are shown.

(D–I) Control vector-transfected Jurkat cells were bound to poly-L-Lysine-coated coverslips (D and G). Control-transfected (E and H) or shArp2-trans-

fected (F and I) Jurkat cells were spread onto poly-L-Lysine/anti-TCR-coated coverslips. The cells from (D)–(F) were then stained with phalloidin. The

cells from (G)–(I) were then analyzed by SEM. In (H) and (I), the left image is predicted to be early in the spreading process, whereas the right image is

late stage. Arrows in (H) identify protruding microspikes in the early-spreading T cell.

could respond to APCs with polarized F-actin. To analyze

this physiological response, control and suppressed T

cells were allowed to form conjugates with superantigen

(SEE)-pulsed NALM6 or Raji B cells. As expected, control

GFP+ cells flattened against the stimulating NALM6 cells,

showing a robust band of polarized F-actin at the IS (Fig-

ure 2A). Interestingly, conjugates were found between

GFP+ Arp2- and Arp3-suppressed cells and SEE-pulsed

NALM6 cells, and upon analysis, these pairs frequently

displayed polarized F-actin. However, similar to the filopo-

dial structures observed in shArp2- and shArp3-sup-

pressed cells that were activated on coverslips, Arp2-

and Arp3-depleted cells produced disorganized F-actin

rich projections over the B cell surface (Figure 2A). To de-

termine whether costimulation altered this response by

Arp2- and Arp3-depleted cells, we also used SEE-pulsed

RAJI cells, which express B7, the costimulatory ligand that

Im

binds CD28 on T cells. We found that although control

GFP+ cells flattened tightly against the RAJI cells, the

Arp2- and Arp3-suppressed T cells responded with similar

polarized F-actin-based protrusions (Figure 2A).

We next formed conjugates between control or shArp2-

transfected T cells and SEE-pulsed Raji cells, and we im-

aged them by SEM (representative conjugates are shown

in Figures 2B and 2C). In many cases, control cells main-

tained strong contacts with APCs, with the lamellipod ex-

tending over the APC surface (Figure 2B). In contrast,

Arp2-depleted T cells did not produce lamellipodia or dis-

play impressive morphological alterations. Actually, these

cells showed less contact with APCs, extending ‘‘finger-

like’’ projections throughout the cell-cell contact zone

(Figure 2C). These structures were also apparent in confo-

cal images with shArp2- and shArp3-suppressed cells

(Figure 2A). These data further support the notion that

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Formins Regulate T Cell Polarity

Figure 2. Arp2/3 Complex-Independent Filopodia Polarize during APC Recognition

(A) Jurkat cells were transfected with control, shArp2-GFP, or shArp3-GFP vectors (green). Each population was conjugated with SEE-pulsed/CMAC-

stained NALM6 or Raji cells (blue) and stained with phalloidin (red).

(B and C) Control-transfected (B) or shArp2-transfected (C) Jurkat cells were conjugated with SEE-pulsed Raji cells and analyzed by SEM.

polarized F-actin reorganization at the IS is not exclusively

regulated by the Arp2/3 complex.

TCR-Mediated MTOC Polarization Is Arp2/3

Complex Independent

Since Arp2/3 complex-depleted T cells retained the ability

to polarize F-actin structures, we next examined MTOC

reorientation toward APCs, a hallmark of T cell polariza-

tion. We found that TCR-mediated MTOC repositioning

is Arp2/3 complex independent, with shArp2-GFP- and

shArp3-GFP-transfected cells efficiently polarizing their

MTOC and microtubule system to face APCs (Figures

3A and 3B). Thus, while depletion of the Arp2/3 complex

regulates activated T cell morphology, it does not control

the formation or polarization of all F-actin-based struc-

tures or MTOC reorientation toward the IS.

Arp2/3 Complex-Dependent F-Actin Regulates

Integrin Activation and TCR Internalization

TCR-mediated b2-integrin activation is considered actin

dependent and is essential for T cell-APC conjugation

(Kinashi, 2005). To examine whether b2-integrin activation

180 Immunity 26, 177–190, February 2007 ª2007 Elsevier Inc.

directly requires Arp2/3 complex-mediated F-actin, we an-

alyzed conjugate formation in cells suppressed for Arp2

and Arp3. We found that shArp2- and shArp3-transfected

cells formed conjugates with SEE-pulsed B cells less fre-

quently than did control transfectants (Figure 3C) and

that single Arp2 or Arp3 suppression yielded similar results

(not shown). Because b2-integrin-mediated conjugation

was Arp2/3 complex dependent, we next investigated

whether TCR-stimulated shArp2- or shArp3-transfected

cells were capable of binding fibronectin, a b1-integrin-

dependent event. As shown in Figure 3D, GFP-expressing

Arp2- and Arp3-suppressed cells also demonstrated di-

minished TCR-mediated fibronectin adhesion.

Additionally, the Arp2/3 complex may participate in re-

ceptor internalization (Engqvist-Goldstein and Drubin,

2003). As shown in Figure 3E, basal TCR surface expres-

sion was unaffected without Arp2 or Arp3. However, Arp2-

and Arp3-depleted cells displayed substantial TCR inter-

nalization defects (Figure 3F), even in spite of the fact

that microtubules, which are suggested to regulate TCR

internalization (Barr et al., 2006), still polarize in the ab-

sence of the Arp2/3 complex. We next analyzed the ability

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Formins Regulate T Cell Polarity

Figure 3. Arp2/3 Complex-Dependent F-Actin Polymerization Does Not Control MTOC Polarity, but Regulates Integrins and TCR

Internalization

(A and B) Jurkat cells were transfected with control, shArp2-GFP, or shArp3-GFP vectors (green) (A). Each population was conjugated with unpulsed

or SEE-pulsed/CMAC-stained Raji cells (blue), labeled with anti-aTubulin (red), and scored for MTOC polarization (B). Arrows indicate MTOC position.

(C) Jurkat cells were transfected with control or with both shArp2-GFP and shArp3-GFP vectors (green), and incubated with unpulsed or SEE-pulsed/

PKH26-stained NALM6 B cells (red). Conjugation efficiency was determined by flow cytometry.

(D) Jurkat cells were transfected as in (A) and analyzed for fibronectin binding. The percent adhesion (GFP-negative, low, and high) in each sample

was determined by flow cytometry.

(E) Jurkat cells were transfected as in (A) and stained on ice with anti-CD3-PE, and cell-surface expression of the TCR was analyzed on GFP+ cells.

(F) Jurkat cells were transfected as in (A), stained on ice with anti-CD3-PE, crosslinked for the indicated times at 37�C, incubated in cold stripping

buffer (removing surface antibodies), and analyzed by flow cytometry for TCR internalization in GFP+ cells.

Bars in (B)–(D) and (F) represent mean ± SD from three independent experiments.

Immunity 26, 177–190, February 2007 ª2007 Elsevier Inc. 181

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Formins Regulate T Cell Polarity

Figure 4. Formins Are Expressed in T Cells

RT-PCR for mRNA expression of FMNL1-3 and DIA1-3 (A). Formins were immunoprecipitated (B and D) or immunoblotted in whole-cell lysates (C and

E) to analyze expression.

of shArp2- and shArp3-transfected cells to signal effi-

ciently after TCR ligation. So far, the role of the actin cyto-

skeleton in regulating calcium-related signaling events

and mitogen-activated protein kinases (MAPKs) in lym-

phocytes has been controversial (Hao and August, 2005;

Holsinger et al., 1998; Rivas et al., 2004; Valitutti et al.,

1995). Interestingly, our studies indicate that Arp2- and

Arp3-suppressed Jurkat cells display normal TCR-medi-

ated PLC-g1 phosphorylation and Ca2+ mobilization (Fig-

ures S1A and S1B). Also, we found prolonged ERK phos-

phorylation in the absence of Arp2 or Arp3 (Figure S1A),

which may be a direct result of diminished TCR internaliza-

tion. Thus, although the Arp2/3 complex was not required

for MTOC and F-actin polarization or certain TCR-medi-

ated signaling events, integrin activation and TCR internal-

ization were diminished. This indicates that F-actin gener-

ated independently of the Arp2/3 complex may have

distinct roles from Arp2/3 complex-nucleated F-actin

during T cell activation.

Formins Display Distinct Cytoskeletal Colocalization

in T Cells

To identify potential Arp2/3 complex-independent mecha-

nisms for F-actin polarization in T cells, we examined

182 Immunity 26, 177–190, February 2007 ª2007 Elsevier Inc.

formins because of their suggested role in filopodia forma-

tion. Formin expression in T cells has not been analyzed,

although lymphocytes have been demonstrated to ex-

press FMNL1 (Favaro et al., 2006). Indeed, we found

FMNL1 mRNA and protein to be expressed in both Jurkat

and peripheral blood human CD4+ (hCD4+) T cells (Figures

4A and 4B), with primary T cells expressing more protein

than Jurkat cells (Figure 4C). In contrast, while mRNA for

the other formin-like family members (FMNL2 and

FMNL3) could be detected, very little protein was immu-

noprecipitated from Jurkat or hCD4+ T cell lysates (Figures

4B and 4D). However, we detected FMNL2 and FMNL3

protein in nonimmune cell types (Figures 4B and 4D). Ad-

ditionally, of the Diaphanous family members (DIA1, DIA2,

and DIA3), DIA1 appeared to be the predominant isoform

expressed in T cells (Figures 4A and 4E).

We next analyzed the localization of these formins by

immunofluorescence. We found fringe-like localization of

FMNL1 at the leading edge of the lamellipod in spreading

T cells (Figure S2B). This FMNL1 accumulation was also

apparent at the edge of F-actin structures, which formed

during APC recognition (Figure 5A), along with a distinct,

‘‘ring-like’’ localization of FMNL1 within the T cell body

(Figure 5B). Upon further analysis, we found that this

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Formins Regulate T Cell Polarity

Figure 5. FMNL1 and DIA1 Display Distinct Cytoskeletal Colocalization in T Cells

Jukat cells were conjugated to SEE-pulsed and CMAC-stained Raji cells (blue) and costained with phalloidin and either anti-FMNL1 (A and B) or anti-

DIA1 (G and H). Jurkat-Raji conjugates were costained with anti-aTubulin and either anti-FMNL1 (C) or anti-DIA1 (I). hCD4+ T cells were costained

with anti-aTubulin and either anti-FMNL1 (D) or anti-DIA1 (J). hCD4+ T cells were conjugated to superantigen cocktail-pulsed Raji cells and stained

with anti-aTubulin and either anti-FMNL1 (E) or anti-DIA1 (K). Jurkat-Raji conjugates were costained with anti-gTubulin to label the centrosome

(arrows) and either anti-FMNL1 (F) or anti-DIA1 (L). Arrows in (A)–(E) and (G)–(K) indicate points of distinct formin localization.

FMNL1 ring reoriented along with the MTOC to face stim-

ulating APCs at later time points (Figure 5C). Moreover,

a similar FMNL1 ring polarized with the MTOC in hCD4+

T cells (Figures 5D and 5E). Interestingly, we identified

that the centrosome was positioned precisely within the

center of this FMNL1 ring structure (Figure 5F).

In contrast to FMNL1, DIA1 was enriched in a fine line

surrounding the lamellipod of spreading T cells (Fig-

ure S2A). In conjugates, DIA1 did not display remarkable

accumulation with F-actin, although it was within

I

F-actin-rich areas (Figure 5G). However, like FMNL1,

DIA1 displayed a distinct point of localization within the

T cell body (Figures 5G and 5H), and upon closer analysis

appeared to be in a ‘‘starburst’’ pattern overlaying the

MTOC and microtubules (Figure 5I). Additionally, DIA1 co-

localized with the MTOC in primary hCD4+ T cells (Figures

5J and 5K) and was also found surrounding the centro-

some (Figure 5L). Moreover, DIA1 localized throughout

the Arp2/3 complex-independent filopodia, whereas

FMNL1 was frequently enriched at the tips (Figure S2). In

mmunity 26, 177–190, February 2007 ª2007 Elsevier Inc. 183

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Formins Regulate T Cell Polarity

184 Immunity 26, 177–190, February 2007 ª2007 Elsevier Inc.

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Formins Regulate T Cell Polarity

contrast to DIA1, DIA2 was not found to localize with

F-actin-rich T cell lamellipodia or filopodia (not shown),

but did similarly surround the centrosome (Figure S3).

FMNL1 and DIA1 Do Not Control TCR-Induced

F-Actin Accumulation at the IS

Because FMNL1 and DIA1 displayed unique cytoskeletal

localization patterns and potentially regulate Arp2/3 com-

plex-independent F-actin polymerization, we generated

shRNA-targeting vectors against FMNL1 and DIA1 (Fig-

ures 6A and 6B). Use of these suppression vectors

abrogated centrosomal staining by both anti-FMNL1

and anti-DIA1, demonstrating the specificity of the anti-

body staining (Figure S4). We next examined whether

suppression of these formins affected Arp2/3 complex-

independent filopodia formation. Like control, shFMNL1a-

mCherry-, shFMNL1b-mCherry-, and shDIA1-mCherry-

transfected cells spread normally (Figures S5A–S5D).

Likewise, GFP-actin cells cotransfected with shArp2-

mCherry and shFMNL1 or shDIA1 vectors (individually or

combined) produced microspikes upon activation (Fig-

ures S5E–S5I). Also, shFMNL1- and shDIA1-transfected

cells efficiently formed conjugates and displayed normal

F-actin accumulation at the IS (Figures 6C–6E). Moreover,

FMNL1- and DIA1-depleted T cells showed normal PLC-

g1, ERK, and calcium responses after TCR stimulation

(Figures S1C and S1D). Thus, although these formins lo-

calized in the lamellipod of spreading cells and to the filo-

podia of Arp2- or Arp3-suppressed cells, their depletion

did not affect the formation of these F-actin structures or

signaling. This suggested that FMNL1 and DIA1 function

distinctly, because they do not impact Arp2/3 complex-

regulated cellular processes, such as lamellipod formation

and integrin activation.

As a means to further analyze whether formins produce

Arp2/3 complex-independent filopodia, we studied the

role of lymphocyte-expressed ENA and VASP proteins

(EVL; ENA/VASP-like and VASP; vasodilator-stimulated

phosphoprotein). This family of proteins is proposed to

be necessary for formin-dependent filopodia formation

through their bundling of formin-mediated F-actin (Schir-

enbeck et al., 2006). In spreading cells, EVL was accumu-

lated at the very leading edge and colocalized with F-actin

within the lamellipod, whereas VASP primarily localized

only to the edge of the lamellae (Figure S6). In addition,

EVL was within Arp2/3 complex-independent filopodia,

while VASP localized to the tips (Figure S6). Interestingly,

shEVL-mCherry-, shVASP-mCherry-, as well as doubly

shEVL-mCherry- and shVASP-mCherry-transfected cells

spread normally (Figures S7A–S7D). Likewise, GFP-actin

cells cotransfected with shArp2-mCherry and shEVL or

shVASP vectors (individually or both) efficiently formed fi-

lopodia (Figures S7E–S7I). Thus, Arp2/3 complex-inde-

pendent filopodia formation in T cells is also EVL and

VASP independent.

FMNL1 and DIA1 Regulate MTOC Polarization

Recent evidence implicates F-actin reorganization in es-

tablishing T cell centrosomal polarity (Stinchcombe

et al., 2006). Given that formins colocalize with the T cell

centrosome, we examined their role in TCR-mediated

MTOC polarization. In contrast to Arp2/3 complex deple-

tion, which did not affect MTOC reorientation (Figures 3A

and 3B), loss of FMNL1 or DIA1 did reduce MTOC polari-

zation (Figures 7A and 7B). Thus, although FMNL1 and

DIA1 did not affect the formation of F-actin-based struc-

tures at the IS, they do regulate centrosome polarization

in T cells.

Centrosome polarization is essential for the directed re-

lease of granules during cytotoxic T cell (CTL)-mediated

killing, so we were next interested in the ability of

siFMNL1- and siDIA1-transfected primary hCD8+ T cell

clones to kill target cells. Like hCD4+ T cells, hCD8+ T cells

expressed FMNL1 and DIA1, which colocalized with the

MTOC (Figure S8). However, we found that primary hu-

man T cells (CD4+ and CD8+) also highly expressed a

smaller 80 kDa variant of DIA1 (Figure S8C), which along

with the larger variant was specifically depleted with

siDIA1 (Figure 7C). Interestingly, CTL clones did not ex-

press DIA2 (Figure S8C). In a redirected cytotoxicity as-

say, we found decreased target cell lysis by FMNL1-

and DIA1-depleted CTLs, while combined suppression

of these formins substantially reduced CTL-mediated kill-

ing (Figure 7C). This suggested that the diminished centro-

somal polarization in the absence of FMNL1 and DIA1

negatively affects CTL function.

So far, regulation of TCR-mediated MTOC polarization

has been shown to involve FYN, ZAP70, LAT, SLP76,

and VAV1 (Ardouin et al., 2003; Kuhne et al., 2003;

Martin-Cofreces et al., 2006). These components are

known to be required for numerous signaling pathways

downstream of the TCR including the activation of rho

family GTPases. Importantly, regulation of the formin pro-

teins is primarily thought to occur through interactions

with these small GTPases, with DIA1 being regulated by

RHOA-C (Faix and Grosse, 2006) and FMNL1 predicted

to be RAC1 regulated (Yayoshi-Yamamoto et al., 2000).

However, we found that in addition to binding GTP-bound

RAC1 (Figure 7D), FMNL1 also directly and specifically in-

teracted with GTP-RHOA (Figure 7E), but not GTP-CDC42

(Figure 7F). It is surprising that DIA1 and FMNL1 are poten-

tially regulated through interactions with RHO and RAC,

Figure 6. FMNL1 and DIA1 Do Not Regulate Integrin Activation or the Accumulation of F-Actin at the IS

(A and B) Jurkat cells were transfected with shDIA1 (A) or shFMNL1 (B) vectors, and lysates were immunoblotted as indicated.

(C) Jurkat cells were transfected with control, shDIA1-GFP, or shFMNL1-GFP vectors (green) and incubated with unpulsed or SEE-pulsed/PKH26-

stained NALM6 B cells (red), and the percent of GFP+ T cells in conjugates was determined by flow cytometry.

(D and E) Jurkat cells were transfected as in (C), conjugated with unpulsed or SEE-pulsed Raji cells (blue), labeled with phalloidin, and scored for

F-actin at the IS (D).

Bars in (C) and (D) represent mean ± SD from three independent experiments.

Immunity 26, 177–190, February 2007 ª2007 Elsevier Inc. 185

Immunity

Formins Regulate T Cell Polarity

186 Immunity 26, 177–190, February 2007 ª2007 Elsevier Inc.

Immunity

Formins Regulate T Cell Polarity

yet MTOC polarization in T cells has been suggested to be

CDC42 dependent (Stowers et al., 1995). However, this

CDC42 dependency for MTOC polarity was shown utiliz-

ing overexpression of dominant-negative CDC42, which

may have pleiotropic affects on signaling. Thus, we specif-

ically depleted RAC1 and CDC42 with shRNA and ana-

lyzed TCR-mediated MTOC polarization. Interestingly,

RAC1 suppression, but not CDC42 suppression, resulted

in diminished MTOC polarization in Jurkat cells (Fig-

ure 7G). This suggested that RAC1, possibly through its

effects on FMNL1, is directing TCR-mediated movement

of the MTOC to the IS.

DISCUSSION

In this study, we found that TCR-stimulated Arp2/3 com-

plex-depleted cells still polarized F-actin in the form of fi-

lopodial protrusions and were capable of MTOC polariza-

tion. In contrast, DIA1 and FMNL1 did not regulate F-actin

polarization but instead controlled MTOC polarity. Thus,

the Arp2/3 complex does not exclusively govern TCR-

mediated cytoskeletal reorganization.

Upon stimulation, Arp2- and Arp3-suppressed T cells

form atypical filopodia. However, these structures proba-

bly normally arise during activation but are obscured by

global actin reorganization events within the Arp2/3 com-

plex-driven meshwork. Indeed, Arp3-suppressed T cells

displayed remnants of lamellipod formation, which tran-

siently originated and spread between Arp2/3 complex-

independent spikes. Also, SEM images revealed apparent

filopodial tips protruding from the lamellipod edge in

spreading cells. In fact, microspikes have been described

buried within the lamelipodial meshwork in fibroblasts

(Small, 1981). Thus, Arp2/3 complex-independent filopo-

dia formation might concomitantly direct Arp2/3 com-

plex-mediated lamellipod formation and is likely to be

mechanistically important in cooperating with Arp2 and

Arp3 for lamellipodial production.

Interestingly, it was recently reported that depletion of

hematopoietic protein-1 (HEM-1), a member of the

WAVE2 complex, induced similar microspikes at the lead-

ing edge of stimulated HL-60 cells (Weiner et al., 2006).

Our data predict that this is due to diminished WAVE2-

dependent Arp2/3 complex nucleation activity and proba-

bly does not result from an independent HEM-1-regulated

event. Interestingly, WAVE2 depletion abrogates T cell

spreading and does not lead to the generation of micro-

spikes (Nolz et al., 2006). This may result from the inability

of WAVE2-suppressed T cells to efficiently mobilize Ca2+

I

through calcium-regulated activated calcium (CRAC)

channels (Nolz et al., 2006), because Arp2/3 complex-

independent filopodia formation is calcium dependent

(data not shown). Alternatively, the WAVE2 complex may

be necessary for other actin nucleators to function or lo-

calize in T cells. Indeed, HEM-1 was found to associate

with formin family members (Weiner et al., 2006). More-

over, we find that WAVE2 localized to the edges of micro-

spikes produced in Arp2/3 complex-depleted T cells, as it

does at the edge of the T cell lamellipod (Figure S9; Nolz

et al., 2006; Zipfel et al., 2006). Interestingly, we do not

see WASP or WIP localizing to these same filopodia (not

shown). Thus, it is possible that the WAVE2 complex local-

izes to the edges of filopodia and may even participate in

their formation through an association with formins or

other actin-nucleating complexes. Subsequently, WAVE2

could promote the Arp2/3 complex-dependent F-actin

polymerization required for filling between microspikes,

resulting in the formation of lamellipodia.

FMNL1 and DIA1 localize within F-actin-rich structures,

suggesting their involvement in F-actin polarization. Nev-

ertheless, these formins did not appear to participate in

actin dynamics (lamellipodia or filopodia) that lead to F-

actin polarization at the IS. It remains possible that Arp2/

3 complex-independent filopodial formation results from

another T cell-expressed formin or that there is functional

redundancy between different formin family members. It is

also interesting that depletion of EVL and VASP, which are

proposed to be required for the generation of formin-

mediated filopodia (Schirenbeck et al., 2006), did not af-

fect the formation of filopodia in the absence of Arp2 or

Arp3. Thus, either formins do not require an association

with these actin-bundling proteins to promote the forma-

tion of filopodia in T cells, or formins are not the major reg-

ulator of microspike formation in Arp2/3 complex-sup-

pressed cells. It is possible that other actin nucleators,

such as SPIR proteins, could regulate these spikes.

Interestingly, although there may be functional compen-

sation between formins in the generation of filopodia in the

absence of Arp2 or Arp3, loss of FMNL1 or DIA1 individu-

ally affects MTOC reorientation, suggesting that, at least in

this regard, they are not functionally redundant. In fact, it is

clear that these formins display strikingly different patterns

of centrosome colocalization, which may provide the ba-

sis for why they each independently regulate MTOC polar-

ization. It is already thought that formins are essential for

actin-dependent processes leading to polarity of the yeast

MTOC (Faix and Grosse, 2006). Thus, microtubule regula-

tion may be a general property of this protein family in

Figure 7. FMNL1 and DIA1 Control TCR-Mediated MTOC Polarization

(A and B) Jurkat cells (A) were transfected with shRNA-GFP vectors as indicated and conjugated with unpulsed or SEE-pulsed/CMAC-stained Raji B

cells (blue). The conjugates were stained with anti-aTubulin (red) and scored for MTOC polarization (B). Arrows indicate MTOC position.

(C) hCD8+ T cells were transfected with siRNA against FMNL1 and/or DIA1 and then used in a redirected cytotoxicity assay. Data were analyzed by the

Student’s t test, *p < 0.05 and **p < 0.0002.

(D–F) Purified FLAG-tagged RAC1 (D), CDC42 (E), or RHOA (F) was loaded with GDP or GTPgS and then analyzed for binding to GST control or

GST-FMNL1 GBD (GTPase binding domain). GST-PAK-GBD was used as a positive control for binding to GTP-loaded RAC1 and CDC42, whereas

RHOTEKIN-GBD was used for RHOA.

(G) Jurkat cells were transfected with the indicated shRNA-GFP vectors and then analyzed for MTOC polarization as in (A) and (B).

Bars in (B), (C), and (G) represent mean ± SD from three independent experiments.

mmunity 26, 177–190, February 2007 ª2007 Elsevier Inc. 187

Immunity

Formins Regulate T Cell Polarity

eukaryotic cells, with a formin network controlling the ac-

tin-dependent processes that allow microtubule position-

ing. In fact, recent studies established the importance of

formins in microtubule stabilization and suggest their

role in migration through possible effects on microtubule

polarization (Eng et al., 2006; Yamana et al., 2006). There-

fore, even though the polarization of recognizable actin

structures is unaffected by loss of FMNL1 and DIA1, we

propose that these proteins each control distinct actin

processes that are not as readily observed but are neces-

sary for centrosome positioning toward the IS.

Recently, it was suggested that the centrosome must

contact the plasma membrane at the IS to allow T cell se-

cretion (Stinchcombe et al., 2006). In this study, they pro-

posed that the CDC42-binding actin-regulatory protein

IQGAP1, which interacts with microtubule plus-end

complexes, may provide the force for positioning the

centrosome by linking actin-dependent processes to mi-

crotubule ends. In support of this, we found that shRNA-

depletion of IQGAP1 affects MTOC polarization (T.S.G.

and D.D.B., unpublished observation), suggesting that

these plus-end complexes are, in fact, essential for centro-

some polarity in T cells. It is of interest that formin family

proteins may also bind microtubule plus-end complexes

(Faix and Grosse, 2006), and therefore might be required

for their movement. Thus, it is likely that formin-dependent

and formin-independent processes acting along microtu-

bules and directly at the MTOC will participate in TCR-me-

diated centrosome polarity and cell-mediated cytotoxicity.

Surprisingly, although dominant-negative expression

studies have indicated the importance of CDC42 in MTOC

polarization during T cell activation (Stowers et al., 1995),

we find that depletion of RAC1, but not CDC42, abrogates

this process in T cells. While it is possible that the residual

expression of CDC42 after suppression is sufficient to

drive MTOC reorientation, this finding is interesting be-

cause DIA1 is RHO regulated, FMNL1 interacts with

RHOA and RAC1, and IQGAP1 binds RAC1 (in addition

to CDC42). However, we must note that in one report,

FMNL1 was suggested to be CDC42 regulated (Seth

et al., 2006). In this study they could not detect GTP-

RAC1 or GTP-CDC42 binding to FMNL1, so they instead

analyzed relief of FMNL1 autoinhibition by using a large

molar excess of CDC42 and truncated FMNL1 domains.

Thus, this may not reflect a physiological mechanism of

FMNL1 regulation. Further studies are needed to define

the signaling pathways linking formin-mediated actin reg-

ulation to microtubule dynamics in T cells. In particular, the

generation of reagents to specifically target and detect

different members of the very homologous rho family

(RHOA-C) GTP-binding proteins will help in dissecting

the pathways linking formins to the TCR.

In contrast to MTOC polarization, b2 integrin activation

appears to be specifically Arp2/3 complex dependent

and does not rely on FMNL1 or DIA1. This is interesting be-

cause WAVE2 controls integrin activation in T cells (Nolz

et al., 2006), and we observe a similar b2 integrin defect

between Arp2/3 complex- and WAVE2-suppressed cells,

suggesting that WAVE2 regulation of b2 integrins is Arp2/3

188 Immunity 26, 177–190, February 2007 ª2007 Elsevier Inc.

complex dependent. However, the b1 integrin defect of

WAVE2-depleted cells seems more severe than that

seen with loss of either Arp2 or Arp3 (not shown) (Nolz

et al., 2006). Interactions between the WAVE2 complex

and formins may be important in this regard. In fact, it

was recently demonstrated that formins might predomi-

nantly regulate b3 integrins (Butler et al., 2006), suggesting

that integrin subsets might also be separately regulated

through distinctly formed actin frameworks. Further dis-

section of the mechanisms by which distinct pools of F-

actin are regulated to control both polarity and integrin

function is necessary to fully understand these subtle

complexities.

Altogether, we demonstrate that the Arp2/3 complex is

not the sole regulator of TCR-mediated cytoskeletal dy-

namics, and we find that formins control the dynamic cen-

trosomal polarization necessary for T cell function and cel-

lular cytotoxicity. Through the continued characterization

of the distinct regulatory mechanisms that collaboratively

control cytoskeletal restructuring, we can begin to func-

tionally dissect the specific contributions of the diverse

F-actin architectural frameworks and their roles in estab-

lishing cell polarity.

EXPERIMENTAL PROCEDURES

Reagents and Plasmids

Reagents were from Sigma unless otherwise specified. Anti-CD3

(OKT3) was from the Mayo Pharmacy and anti-ZAP70, anti-PLCg1,

and anti-WAVE2 have been previously described (Gomez et al.,

2005; Nolz et al., 2006). Anti-Arp3 and anti-CDC42 (BD Transduction

Laboratories), anti-Arp2 (clone H-84; Santa Cruz), and anti-ARPC2

and anti-RAC1 (clone 23A8; Upstate Biotechnology) were used in

this study. In addition, polyclonal antibodies against DIA1 and DIA2

(Bethyl Laboratories) were used. We obtained monoclonal anti-

FLAG, anti-aTubulin, and anti-gTubulin from Sigma. We also used an-

tibodies against ERK1/2, pERK1/2 (T202/Y204), and pPLCg1 (Y783)

from Cell Signaling Technology. Antisera against FMNL1, FMNL2,

and FMNL3 were generated by immunizing rabbits with KLH-conju-

gated synthetic peptides (Cocalico Biologicals; Table S1). Antibodies

against EVL(AA261-340) and VASP(AA231-325) were generated simi-

larly, but with GST-fusion proteins. Anti-CD33PE was from BD Immu-

nocytometry Systems. The pFRT-H1p, pCMS3.eGFP.H1p, and

pCMS3.mCherry.H1p shRNA vectors have been described (Gomez

et al., 2005; Nolz et al., 2006). RT-PCR was performed as described

(Nolz et al., 2006). See Table S1 for RT-PCR primers and shRNA target-

ing sequences.

Cell Culture, Transfection, Immunoprecipitation,

GST Pull-Down, and Immunoblot Analysis

Jurkat-E6, hCD4+, RAJI, NALM6, P815, and GFP-actin-Jurkat-E6 cells

were passaged as previously described (Gomez et al., 2006). Human

CD8+ T cells were cloned and cultured as described (Billadeau et al.,

2000). Jurkat cells were transfected with shRNA suppression vectors

as described (Gomez et al., 2006). CD8+ T cells were transfected by

electroporation at 295V with 3.5 3 106 cells and 600 pmol of siRNA

per transfection. For single suppression, 300 pmol control siRNA

and 300 pmol specific siRNA were used, and for double suppression,

300 pmol of each specific siRNA was used together. For stimulation of

Jurkat T cells, OKT3 mAb was used at 5 mg/ml. Immunoprecipitations

(from 750 mg of protein) and lysates (75–100 mg of protein) were pre-

pared and analyzed by immunoblot (Gomez et al., 2005). GST pull-

downs were done with GTPase Binding Buffer (25 mM Tris [pH 7.5],

40 mM NaCl, 1 mM DTT, 30 mM MgCl2, and 0.5% NP-40). GST-fusion

Immunity

Formins Regulate T Cell Polarity

protein (25 mg) was bound to GSH-agarose (30 ml) followed by one

wash. FLAG/His-tagged GTPases were purifed from insect cells by

the FastBac system and Probond resin (Invitrogen) and loaded with

GDP or GTPgS. For loading, 25 nM of purified GTPase was incubated

with 50 mM GDP or GTPgS in GTPase Loading Buffer (20 mM Tris [pH

7.5], 50 mM NaCl, 1 mM EDTA, and 0.1% Triton) for 10 min at 37�C,

and then MgCl2 was added to a final concentration of 2.5 mM. This

GTPase-containing solution was then added to the GST-fusion protein

bound agarose, rotated for 25 min at 4�C, and then washed two times.

Cytotoxicity Assays

The redirected 51Cr-release assays were performed as previously de-

scribed (Billadeau et al., 2000). CD8+ T cells were used 48 hr after

transfection with siRNA and were able to kill P815 cells only in the pres-

ence of OKT3 (1 ng/well). In all cases, spontaneous release did not ex-

ceed 10% of maximum lysis. Lytic units were calculated based on

20% cytotoxicity (Billadeau et al., 2000).

Immunofluorescence and Live Cell Imaging

Immunofluorescence of fixed Jurkat- or hCD4+-containing conjugates

was performed as described (Gomez et al., 2005). For quantification,

50–100 conjugates were chosen randomly, and an individual blinded

to the experiment scored conjugates consisting of one GFP+ T cell

and one blue CMAC-stained B cell. Conjugates showing distinct label-

ing at the cell-cell contact site were scored positive. MTOC polariza-

tion studies were performed similarly, except conjugates were allowed

to form for 30 min and scored positive if the MTOC (based on aTubulin

staining) was against the cell-cell interface. For immunofluorescence

of spreading cells, Jurkat cells were settled for 5 min onto poly-L-

lysine-coated coverslips (Gomez et al., 2006), which were preincu-

bated with or without anti-CD3 (20 mg/ml in PBS) overnight at 4�C.

For live cell imaging, Jurkat cells were imaged spreading onto anti-

TCR-coated coverslips as described (Nolz et al., 2006).

Scanning Electron Microscopy

For scanning electron microscopy (SEM), cells were prepared on glass

coverslips as above (Gomez et al., 2005) but were placed in fixative

(4% formaldehyde and 1% glutaraldehyde in sodium phosphate buffer

[pH 7.3]) at 4�C for up to 36 hr. The coverslips were treated with a series

of solution exchanges, including two phosphate buffer washes fol-

lowed by sequential 60% ethanol, 70% ethanol, 80% ethanol, 95%

ethanol, and 100% ethanol washes. Coverslips were placed into a crit-

ical point dryer while in 100% ethanol for drying, sputter-coated with

gold-palladium, and imaged on a Hitachi H-4700 SEM.

TCR Expression and Internalization

Jurkat cells were transfected with the indicated GFP-shRNA vectors,

and basal TCR expression and TCR internalization were analyzed by

flow cytometry gating on GFP+ cells as described (Gomez et al., 2005).

Adhesion Assays, Conjugate Analysis, and Calcium

Mobilization

Adhesion assays were performed as described (Nolz et al., 2006). Con-

jugate assays were performed as described (Gomez et al., 2005; Nolz

et al., 2006). Calcium mobilization studies were done as previously

described (Gomez et al., 2005).

Supplemental Data

Supplemental Data include nine figures, one table, and three movies

and can be found with this article online at http://www.immunity.

com/cgi/content/full/26/2/177/DC1/.

ACKNOWLEDGMENTS

We would like to thank R.A. Schoon and C.J. Dick for their help with the

cytotoxicity assays. This work was supported by the Mayo Founda-

tion, NIH grant R01-AI065474 to D.D.B., NIH grant F31-AI068624 to

T.S.G., NIH grant R01-CA47752 to P.J.L., and NIH grants R01-

AI038474 and R01-AI031126 to Y.S. The authors declare that they

have no competing financial interests.

Received: October 6, 2006

Revised: December 20, 2006

Accepted: January 8, 2007

Published online: February 15, 2007

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