The N-terminal fragment of gelsolin inhibits the interaction of DNase I with isolated actin, but not with the cofilin-actin complex

REGULAR ARTICLE

The N-terminal fragment of gelsolin inhibits the

interaction of DNase I with isolated actin, but not with

the cofilin-actin complex

Deepak Chhabra, Neil J. Nosworthy, Cristobal G. dos Remedios

Muscle Research Unit, Institute for Biomedical Research, School of Medical Sciences,University of Sydney, Australia

Apoptosis is essential in embryonic development, clonal selection of cells of the immune systemand in the prevention of cancer. Apoptotic cells display characteristic changes in morphology thatprecede the eventual fragmentation of nuclear DNA resulting in cell death. Current evidenceimplicates DNase I as responsible for hydrolysis of DNA during apoptosis. In vivo, it is likely thatcytoplasmic actin binds and inhibits the enzymatic activity and nuclear translocation of DNase Iand that disruption of the actin–DNase I complex results in activation of DNase I. In this reportwe demonstrate that the N-terminal fragment of gelsolin (N-gelsolin) disrupts the actin–DNase Iinteraction. This provides a molecular mechanism for the role of the N-gelsolin in regulatingDNase I activity. We also show that cofilin stabilises the actin–DNase I complex by forming aternary complex that prevents N-gelsolin from releasing DNase I from actin. We suggest thatboth cofilin and gelsolin are essential in modulating the release of DNase I from actin.

Received: September 8, 2004Revised: October 29, 2004

Accepted: November 6, 2004

Keywords:

Actin-binding proteins / Allosteric interactions / Apoptosis / DNase I activation / Nativepolyacrylamide electrophoresis

Proteomics 2005, 2, 3131–3136 3131

1 Introduction

Apoptotic cell death is fundamental to the integrity of bio-logical tissues. Breakdown of the regulation of apoptosisresults in abnormal development and tissue remodelling,and contributes to the transformation of cells and theireventual progression to cancer. Regardless of the initiatingstimulus, cells undergoing apoptosis display characteristicmorphological and biochemical changes including mem-brane blebbing, chromosome condensation and fragmenta-tion of nuclear DNA into approximately 200 bp intervals [1,2]. Cleavage of DNA is primarily mediated by DNase I [3–7],although a number of other enzymes including DNase X [8]and DFF40 [9, 10] may be involved in some tissues. There is a

large body of circumstantial evidence that the structuralprotein, actin, forms a 1:1 complex with DNase I and inhibitsits enzymatic activity and nuclear translocation in vivo [11–13] thereby preventing uncontrolled cell death. Recovery ofDNase I activity is mediated by disruption of the actin–DNase I complex through an as yet uncharacterised process.

Actin is the principal component of the eukaryotic cyto-skeleton. Globular monomers of actin (G-actin) reversiblyassemble to form filaments (F-actin) of indefinite length.Regulation of actin dynamics by actin-binding proteins(ABPs) is essential for control of cell shape, cytokinesis andmotility. Furthermore, ABPs, particularly gelsolin and cofi-lin, are thought to be involved in the mechanism of apopto-sis.

Gelsolin is an actin-severing protein that has beenimplicated in both the morphological and biochemicalchanges in apoptotic cells. The protein consists of homolo-gous N- and C-terminal halves each of three domains (seg-ments 1–3 and 4–6). The C-terminal half of gelsolin impartsCa21 sensitivity to the protein. In the presence of Ca21, there

Correspondence: Professor Cristobal G. dos Remedios, MuscleResearch Unit, Anderson Stuart Building F13, University of Syd-ney, NSW, Australia 2006E-mail:[email protected]:161-2-9351-6546

© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de

DOI 10.1002/pmic.200401127

3132 D. Chhabra et al. Proteomics 2005, 2, 3131–3136

is a significant structural rearrangement in the C-terminaldomain of gelsolin that facilitates severing and capping ofactin [14]. Upon binding, the C-terminal half of gelsolin alsoinduces conformational changes in actin [15].

Gelsolin and DNase I simultaneously bind to actin in anoncompetitive manner to form a gelsolin–actin–DNase Iternary complex [16–18]. Despite the fact that gelsolin doesnot disrupt the actin–DNase I complex, it is a proposed acti-vator of DNase I in vivo. This is further supported by changesin the intracellular distribution [19] and expression [20] ofgelsolin preceding the progression to apoptosis. Gelsolin iscleaved between segments 3 and 4 during apoptosis to gen-erate an N-terminal fragment (N-gelsolin) that functions in-dependently of Ca21 to destroy normal cell architecture [21].

Cofilin is the principal effector of actin dynamics insidecells and its interaction with actin is primarily regulated byphosphorylation [22, 23]. During times of cell stress, cofilinand actin cotranslocate into the nucleus [24]. Like gelsolin,cofilin forms a ternary complex with actin and DNase I, butthis interaction is positively cooperative [25] and is influ-enced by cations and nucleotides [26].

In this report we use native (nondenaturing) PAGE toqualitatively analyse the effect of apoptotic N-gelsolin andcofilin on the interaction between actin and DNase I. Native-PAGE is a powerful tool for analysing molecular interactionsin vitro. This technique has been used extensively to studythe interactions of actin with ABPs including DNase I [25–27], gelsolin [28] and cofilin [25, 26, 29].

2 Materials and methods

2.1 Actin preparation

Actin was prepared from an acetone-dried powder of rabbitskeletal muscle according to the method of Spudich and Watt[30] with slight modifications as described by Barden and dosRemedios [31]. The final concentration of monomeric actinin G-buffer (2 mM Tris pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP)was determined from its OD290, where E0.1% = 0.63/cm [32].

2.2 Expression and purification of DNase I

JM105 Escherichia coli transformants were grown at 377C inLB medium (USB, Cleveland, OH, USA) containing 50 mg/mL ampicillin. Protein expression was induced when thecells were in log phase of growth (OD600 < 0.6/cm) by addi-tion of isopropyl-b-D-thiogalactopyranoside (IPTG) to a finalconcentration of 1 mM. After 3 h growth, the cells were har-vested by centrifugation, resuspended in 100 mL of DNase-lysis buffer (10 mM Tris pH 8.0, 50 mM NaCl, 2 mM CaCl2)and disrupted using a French press. The lysate was clarifiedby centrifugation at 10 000 6 g for 1 h and applied to a Q-Sepharose (Amersham Biosciences, Uppsala, Sweden) col-umn and eluted with a 0.05–0.35 M NaCl gradient in lysis

buffer. Fractions containing DNase I were concentratedusing Macrosep Pall 10 K Omega centrifugal concentrators(Pall, Ann Arbor, MI, USA), followed by passage through aSephacryl S-200 (Amersham Biosciences) column in lysisbuffer. Concentrated DNase I was dialysed for 24 h againstG-buffer. Protein concentration was determined from theOD280, where E0.1% = 1.1/cm [11].

2.3 Fluorescent labelling of DNase I

DNase I was labelled on a cysteine residue by overnightincubation at 47C with a three-fold excess of fluorescein con-jugated to iodoacetamide (IAF). The reaction was stopped byaddition of DTT to a final concentration of 5 mM. Excess labelwas removed by overnight dialysis against 10 mM PIPES pH6.8 followed by passage through a Sephadex G-25 (Amers-ham Biosciences) column.

2.4 Expression and purification of cofilin

Recombinant avian embryonic skeletal muscle cofilin wasexpressed as a GST-fusion protein. BL21(DE3) E. coli trans-formants were cultured, induced to express protein and har-vested as described in Section 2.2. The cell pellet was resus-pended in 100 mL of PBS (0.35 M NaCl, 2 mM KCl, 10 mM

Na2HPO4, 2 mM KH2PO4, pH 7.2) and disrupted using aFrench press. The lysate was clarified by centrifugation andthe supernatant was applied to a glutathione Sepharose 4Bcolumn (Amersham Biosciences) equilibrated with PBS. Thecolumn was washed with 2 vol of thrombin buffer (50 mM

Tris pH 8.0, 2.5 mM CaCl2 and 50 mM NaCl). The matrix wassuspended in 100 U of thrombin/L of LB culture mediumand incubated overnight at 47C. Cofilin was eluted with 2 volof thrombin buffer and dialysed overnight against 10 mM

PIPES pH 6.8. Cofilin was then further purified on a Mono-S(Amersham Biosciences) cation exchange column and elutedwith a 0–0.5 M gradient of NaCl. Purified cofilin was dialysedfor 24 h against G-buffer. Protein concentration was deter-mined from the OD280, where E0.1% = 0.93/cm [33].

2.5 Expression and purification of N-gelsolin

BL21(DE3) E. coli transformants were cultured, induced toexpress protein and harvested as described in Section 2.2,with the exception that everything was done at room tem-perature (<237C). The cell pellet was resuspended in 100 mLof gelsolin-lysis buffer (10 mM PIPES pH 6.8, 0.1 M EDTA,0.1 M EGTA) and disrupted using a French press. The lysatewas clarified by centrifugation at 13 000 6 g for 1 h, appliedto an S-Sepharose (Amersham Biosciences) column andeluted with a 0–1 M NaCl gradient in gelsolin-lysis buffer.Fractions containing N-gelsolin were concentrated using10 K concentrators, followed by passage through a SephacrylS-200 column in gelsolin-lysis buffer containing 50 mM


Proteomics 2005, 2, 3131–3136 3133

NaCl. N-gelsolin was further purified by application to aMono-S cation exchange column and eluted with a 0–1 Mgradient of NaCl. Purified N-gelsolin was dialysed for 24 hagainst G-buffer. Protein concentration was determinedfrom the OD280, where E0.1% = 1.09/cm.

2.6 Effect of N-gelsolin on the actin–DNase I complex

An actin–DNase I complex was formed by incubating DNaseI (60 pmol) with excess actin (150 pmol). Increasing amountsof N-gelsolin (16, 32, 80, 160 and 240 pmol) were then titrat-ed into a mixture of actin–DNase I and actin. Aliquots weretaken and analysed using native (nondenaturing) PAGE.

Native PAGE was performed using a Bio-Rad Mini-PRO-TEAN® II system (Richmond, CA, USA) and a disconti-nuous Tris-glycine buffer system [34] with omission of SDSfrom all solutions. The stacking gel comprised 4% acryl-amide in 80 mM Tris-glycine pH 6.8 and the separating gelcomprised 10% acrylamide in the same buffer adjusted topH 8.8. The gels were bathed in running buffer (25 mM TrispH 8.3, 192 mM glycine, 0.2 mM ATP) and samples were runat 120 V for 90 min at room temperature.

As these gels were performed under nondenaturingconditions, noncovalent interactions between proteins werenot disrupted. Consequently, migration of isolated proteinsand protein complexes was determined by their charge–density ratio and conformation, rather than their apparentmolecular weights as they are in SDS-PAGE.

Gels were stained with 1% w/v CBB R-250 in 40% v/vethanol, 7% v/v acetic acid and destained in 40% v/v ethanoland 7% v/v acetic acid. Due to the poor staining of DNase Iby CBB in these gels, changes in the migration of IAF–DNase I were used to analyse actin–DNase I interactions inthe presence and absence of N-gelsolin. Native-PAGE ofIAF–DNase I (100 pmol) was performed in the presence andabsence of actin (160 pmol) and in the presence of both actinand N-gelsolin (160 and 100 pmol, respectively). To thismixture, N-gelsolin (100 pmol) was added. IAF–DNase wasvisualised using a UV-302 nm transilluminator (UVItec,Cambridge, England).

2.7 Effect of N-gelsolin on the cofilin–actin–DNase I

ternary complex

Cofilin (110 pmol), DNase I (60 pmol) and actin (150 pmol)were combined to yield mixtures of cofilin–actin complex,actin–DNase I complex, cofilin–actin–DNase I ternary com-plex and free actin. To this mixture, increasing amounts ofN-gelsolin (16, 32, 50, 80 and 180 pmol) were titrated.Samples were analysed using native-PAGE as described inSection 2.6.

3 Results

3.1 Effect of N-gelsolin on the actin–DNase I complex

Figure 1 shows a 10% polyacrylamide gel run under non-denaturing conditions demonstrating the effect of N-gelsolinon the actin–DNase I complex. Lane 1 contains 150 pmolactin. The addition of ATP to the running buffer preservedactin in the monomeric state and promoted its migration as adistinct band [25, 26]. In the absence of ATP, assembly ofactin results in a distinct laddering pattern due to a hetero-genous mixture of actin oligomers. Lane 2 contains 50 pmolN-gelsolin. No protein band is seen here, possibly becauseN-gelsolin has a high pI (<7.3), and so will have little or nonet charge under the running buffer conditions (pH 8.3).Consequently, N-gelsolin does not migrate as a distinct band.Lane 3 contains 60 pmol DNase I. Recombinant DNase Imigrates as a single band that is poorly stained with CBB.Lane 4 contains 150 pmol actin mixed with 50 pmol N-gel-solin. This results in the formation of a new, intensely-stained band, the N-gelsolin–actin complex. As expected, thiscomplex predictably has a lower charge/density ratio andthus migrates slower than actin alone. Lane 5 contains150 pmol actin mixed with 60 pmol DNase I. This results inthe formation of a new, intensely-stained band, the actin–DNase I complex that migrates between actin alone andDNase I. Lanes 6–10 contain constant amounts of actin andDNase I as in lane 5, in the presence of increasing amounts(16, 32, 80, 160 and 240 pmol) of N-gelsolin. The progressive

Figure 1. 10% native-PAGE geldemonstrating the effect of increas-ing concentrations of N-gelsolin onthe interaction between actin andDNase I. The contents of each laneare described in the text. The effectof increasing N-gelsolin is to dis-sociate the actin-DNase I complex(lanes 6–10).



addition of N-gelsolin to the actin–DNase I complex con-taining free actin produces a band of increasing intensitycorresponding to an N-gelsolin–actin complex. This is con-sistent with a decrease in intensity of the band correspond-ing to the actin–DNase I complex. Interestingly, the intensityof the band corresponding to uncomplexed actin was notsignificantly diminished until the actin–DNase I complexwas consumed by N-gelsolin. Thus, our results demonstratethat N-gelsolin binds to actin–DNase I in preference touncomplexed actin, suggesting that the affinity of N-gelsolinfor the actin–DNase I complex is higher than for actin alone.

Figure 2 demonstrates changes in the migration ofDNase I (100 pmol) in the presence of either isolated actin(160 pmol) or with both actin and N-gelsolin (160 and100 pmol, respectively). Lane 1 demonstrates the migrationof IAF–DNase I as a double band near the bottom of the gel.Lane 2 demonstrates the change in migration of IAF–DNaseI upon addition of actin to form an actin–IAF–DNase I com-plex. Addition of N-gelsolin to the actin–DNase I complex(lane 3) results in migration of IAF–DNase I at a rate corre-sponding to isolated IAF–DNase I (lane 1). These data con-firm that N-gelsolin disrupts the actin–DNase I interaction.

Figure 2. UV transillumination of a native-PAGE gel demonstrat-ing the migration of fluorescent IAF-DNase I. Lanes 1, 2 and 3respectively contain IAF-DNase I alone, IAF-DNase I-actin com-plex, and IAF-DNase I-actin-N-gelsolin. Addition of N-gelsolin toan actin-DNase I complex results in release of IAF-DNase I (lane 3).

3.2 Effect of N-gelsolin on the cofilin–actin–DNase I

ternary complex

Figure 3 shows a 10% polyacrylamide native gel demon-strating the effects of adding N-gelsolin to the cofilin–actin–DNase I ternary complex. Lane 1 contains 150 pmol actinmixed with 110 pmol cofilin. Excess actin monomer (about40 pmol) migrates as a distinct band near the bottom of thegel. There are two other distinct but lighter bands present.Previous reports [25, 26] have shown these are probablydimers and trimers of actin. Cofilin has a high pI (<8.0) andthus a very low net charge under the running conditions.Consequently, cofilin did not enter the polyacrylamide run-ning gel. The cofilin–actin complex is the dark-staining bandthat migrates essentially as reported previously [25, 26]. Lane2 contains 150 pmol actin mixed with 60 pmol DNase I. As inFig. 1 (lane 5), the actin–DNase I complex produces anintense band that migrates at a slower rate than monomericactin. Lane 3 contains 150 pmol actin mixed with 50 pmol N-gelsolin. As with Fig. 1 (lane 4), the N-gelsolin–actin complexmigrates relatively slowly, and forms a distinct band. Lane 4contains a mixture of 150 pmol actin, 60 pmol DNase I and50 pmol N-gelsolin. This results in the formation of an actin–DNase I band and an N-gelsolin–actin band. Since no otherband is visible, we conclude that DNase I and N-gelsolin donot bind to each other and neither do they form a ternarycomplex. Lane 5 contains a mixture of 150 pmol actin,60 pmol DNase I and 110 pmol cofilin. Three distinct bandsare now clearly seen. The middle and lower bands corre-spond to the cofilin–actin and actin–DNase I complexes,respectively. The upper band is a new cofilin–actin–DNase Iternary complex. Lanes 6–10 contain constant amounts ofactin, DNase I and cofilin as in lane 5, but with increasingamounts (16, 32, 50, 80 and 180 pmol) of N-gelsolin. Thisprogressive addition produces a band of increasing intensitycorresponding to an N-gelsolin–actin complex with a con-comitant decrease in the density of the actin–DNase I com-plex that disappears completely in the presence of excess N-gelsolin. Interestingly, the cofilin–actin binary complex andcofilin–actin–DNase I ternary complex are not markedlyaffected by N-gelsolin until a large excess is added. Note that

Figure 3. 10% native-PAGE geldemonstrating the effect of increas-ing concentrations of N-gelsolin onthe interactions between cofilin,actin and DNase I. The contents ofeach lane are described in the text.The formation of a cofilin-actin-DNase I ternary complex appears toprevent the disruption of the actin-DNase I interaction as seen in Fig. 1.


Proteomics 2005, 2, 3131–3136 3135

Figure 4. Diagram illustrating the effect of cofilin on the N-gel-solin-induced disruption of the actin-DNase I complex: (a) full-length gelsolin and DNase I bind actin in a non-competitivemanner; (b) the binding of N-gelsolin to actin disrupts the actin-DNase I complex; and (c) formation of a cofilin-actin-DNase Iternary complex maintains the interaction between actin andDNase I despite the presence of N-gelsolin. Key to figures: A,actin; D, DNase I; N-gel-N-terminal fragment of gelsolin; C-gel, C-terminal fragment of gelsolin; C, cofilin.

as the gelsolin content increases, the gelsolin–actin complexsplits into two bands, one corresponding to the N-gelsolin–actin complex, and a progressively dense band reflecting for-mation of higher order complexes between N-gelsolin andactin [35]. In short, the presence of N-gelsolin selectivelydissociates the actin–DNase I complex, whereas the cofilin–actin–DNase I ternary complex remains stable in the pres-ence of N-gelsolin.

4 Discussion

The actin cytoskeleton plays a key role in the regulation ofapoptosis. The enzymatic activity and nuclear translocationof DNase I in vivo are thought to be inhibited by its associa-tion with actin [12] and this complex is further stabilised bythe cooperative binding of cofilin [25].

Disinhibition of the enzymatic activity of DNase I duringapoptosis may be due to disruption of its interaction withactin, although the mechanism by which this occurs is notyet well understood. Cleavage [36, 37] and hyperpolymerisa-tion [38] of actin have been proposed to activate DNase I, al-

though this has been questioned [39, 40]. Furthermore, gel-solin is reported to increase the activity of DNase I in vitro[41] and has paradoxically been shown to be both an inhibitor[24, 42, 43] and effector [15, 19, 44–46] of apoptosis.

Both actin and gelsolin are known substrates for apoptoticenzymes. Gelsolin is specifically cleaved between domains 3and 4 by caspase-3 to generate an N-terminal fragment (resi-dues 1–352) [44, 47] that, unlike full-length gelsolin, functionsindependently of free Ca21 [44, 46]. Actin is cleaved by theinterleukin-1b-converting enzyme (ICE) family of proteasesto generate a species of actin with reduced polymerisation[48]. Although cleavage of either actin or gelsolin alone canproduce the morphological changes associated with apopto-sis, it is not sufficient to cause DNase I disinhibition and thusfacilitate cell death [19, 36, 37, 39].

Our results demonstrate that the apoptotic N-terminalfragment of gelsolin competes with DNase I for binding toactin. This contrasts with full-length gelsolin that non-competitively binds actin and DNase I to form a gelsolin–actin–DNase I ternary complex [16–18]. We also demonstratethat the disruption of the actin–DNase I interaction byN-gelsolin is partially prevented by the binding of cofilin (seeFig. 4), which has been shown elsewhere to be more stablethan the binary complex [25, 26]. A corollary of this is thatincreased expression of cofilin within the cell may inhibitDNase I activation in vivo and delay apoptotic cell death. Thisis consistent with reports demonstrating upregulation ofcofilin expression in some types of cancer [49].

Actin and DNase I bind with very high affinity (KD <0.05 nM) [50], much higher than the affinity of actin for cofi-lin (KD < 0.5–5 mM) [51, 52]. Despite this difference, ourresults demonstrate that N-gelsolin interacts with the actin–DNase I complex in preference to the cofilin–actin complex.These changes in affinity may be attributed to the absence ofconformational changes within actin, particularly around theDNase I binding locus (subdomain 2), that are normallyinduced by full-length gelsolin [53].

Our proposal of a role for cofilin as a modulator of DNaseI activation accords with a report demonstrating colocalisa-tion of cofilin and DNase I in vivo [54] and the suggestion thatthe cofilin–actin–DNase I complex exists within the nucleus[55]. Our data suggest that for N-gelsolin to disinhibit DNaseI activity, cofilin must first be dissociated from the tight cofi-lin–actin–DNase I ternary complex. This may be achieved bycleavage of the cofilin-binding locus on actin. This sugges-tion is supported by reports demonstrating the cleavage ofthe N-terminal region of actin by apoptotic enzymes in vitro[36, 37, 48].

We propose a model of DNase I activation that requirescleavage of both actin and gelsolin. First, gelsolin is cleavedto yield an unregulated F-actin severing protein. Next, thecofilin-binding domain of actin is cleaved to destabilise theactin–DNase I interaction by preventing the binding of cofi-lin. The net effect of these reactions is an increased actin–DNase I complex that is susceptible to disruption by theN-gelsolin during apoptosis. Alternatively, cleavage of actin or



gelsolin alone would only affect cytoskeletal dynamics with-out activating DNase I. This provides a molecular mechan-ism for the role of N-gelsolin in regulating DNase I activity.

The authors would like to thank Dr. B. A. Connolly (Uni-versity of Newcastle, UK) for his gift of plasmid encoding DNaseI, Dr. Takashi Obinata (Chiba University, Japan) for his gift ofplasmid encoding cofilin and Professor Helen Yin (University ofTexas, USA) for her kind gift of plasmid encoding the N-terminalhalf of gelsolin. This research was supported by a grant from theAustralian Research Council.

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