α, slows actin filament barbed end elongation, competes ... · elongation, it allows filaments to...

The mouse formin, FRLα, slows actin filament barbed end elongation, competes with capping

protein, accelerates polymerization from monomers, and severs filaments

Elizabeth S. Harris, Fang Li, Henry N. Higgs*

Department of Biochemistry

Dartmouth Medical School

Hanover, New Hampshire 03755

Running Title: Effects of FRLα on Actin Dynamics

* To whom correspondence should be addressed:

Tel.: 603-650-1420; Fax: 603-650-1128

E-mail: [email protected]

Harris et al on actin dynamicsα Effects of FRL

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JBC Papers in Press. Published on February 29, 2004 as Manuscript M312718200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

Formins are a conserved class of proteins expressed in all eukaryotes, with known roles

in generating cellular actin-based structures. The mammalian formin, FRLα, is enriched in

hematopoietic cells and tissues, but its biochemical properties have not been characterized. We

show that a construct composed of the C-terminal half of FRLα (FRLα-C) is a dimer and has

multiple effects on muscle actin, including: tight binding to actin filament sides; partial inhibition

of barbed end elongation; inhibition of barbed end binding by capping protein; acceleration of

polymerization from monomers; and actin filament severing. These multiple activities can be

explained by a model in which FRLα-C binds filament sides, but prefers the topology of sides at

the barbed end (end-sides) to those within the filament. This preference allows FRLα-C to

nucleate new filaments by side stabilization of dimers; processively advance with the elongating

barbed end; block interaction between C-terminal tentacles of capping protein and filament end-

sides; and sever filaments by preventing subunit re-association as filaments bend. Another

formin, mDia1, does not reduce barbed end elongation rate but does block capping protein,

further supporting an end-side binding model for formins. Profilin partially relieves barbed end

elongation inhibition by FRLα-C. When non-muscle actin is used, FRLα-Cs effects are


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largely similar. FRLα-Cs ability to sever filaments is the first such activity reported for any

formin. Since we find that mDia1-C does not sever efficiently, severing may not be a property

of all formins.

INTRODUCTION

Non-muscle cells contain a variety of actin filament-based structures that perform

diverse roles, including: lamellipodia; ruffles; filopodia; microvilli; and sarcomeric contractile

structures including cytokinetic actin rings, and stress fibers. Assembly mechanisms for these

structures are being vigorously investigated. Spontaneous nucleation of actin monomers occurs

very slowly (1), and specific actin-associated proteins that promote rapid actin assembly are

required for creating each actin-based structure. Arp2/31 complex is a well-characterized

nucleation factor, forming networks of branched actin filaments that are present in lamellipodia

and ruffles (2). In contrast, the proteins controlling assembly of many other actin-based

structures have not been identified.

Formins are a conserved class of actin-associated proteins that have been found in all

eukaryotes examined, and accelerate filament assembly independently of Arp2/3 complex (3).


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Two unifying structural features of formins are the Formin Homolgy 1 and 2 (FH1, FH2)

domains, generally found in the C-terminal half of the protein. The FH1 domain contains

proline-rich sequences capable of binding profilin and SH3 domain containing proteins. The

FH2 domain forms a multimeric structure (4,5).

Budding yeast formins Bni1p and Bnr1p are required for the assembly of actin cables and

cytokinetic actin rings in vivo (6,7). Bni1p has barbed end nucleation activity in vitro, for which

the FH2 domain is sufficient (7,8). Bni1p also slows barbed end elongation, while blocking

complete barbed end elongation inhibition by capping protein (4,5,9). Thus, while Bni1p slows

elongation, it allows filaments to elongate in the presence of capping protein. Since capping

protein usually caps newly assembled filaments within seconds, formins may allow prolonged

filament elongation in cells.

In fission yeast, Cdc12 is required for actin ring assembly in vivo. Cdc12 is a barbed end

nucleator when bound to the actin monomer-binding protein profilin. In the absence of profilin,

Cdc12 tightly caps barbed ends, allowing only pointed end elongation (10).

Mammals contain at least 12 formin isoforms. Few have been studied in detail, with

mDia1 (also called DRF1, Dia1, or p140 Dia) being the most characterized to date. In cells,

mDia1 localizes to cytokinetic actin rings, stress fibers, and lamellipodia (11). Overexpression

of constitutively active mDia1 constructs cause increased stress fiber formation (12,13). In vitro,

a construct of mDia1 containing FH1, FH2, and C-terminal domains is a potent actin filament

nucleator (14), several fold stronger than yeast formins. Similar to Bni1p, mDia1 protects barbed


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ends from capping protein {(5) and this study}.

Here we characterize the biochemical properties of the mammalian formin, FRL

(Formin-Related gene in Leukocytes), first identified from mouse spleen as the 1,094 amino

acid FRL± splice variant (15), although a number of other variants exist in the database. We

restrict our current study to the C-terminal region of FRLα (FRLα-C, amino acids 449-1094),

which contains the complete FH1 and FH2 domains, as well as the full C-terminus. In several

assays, a similar construct of the FRLβ splice variant, differing in its C-terminal 30 amino acids

(15), behaves similarly.

FRLα-C is a dimer and has multiple effects on muscle actin. FRLα-C binds filaments

tightly, with an apparent Kd < 0.1 µM. In addition, FRLα-C slows barbed end elongation with

an IC50 of about 2 nM, demonstrating that it binds preferentially to filament barbed ends. This

inhibition of elongation is only partial, and FRLα-C protects the barbed ends from complete

elongation inhibition by capping protein. In pyrene-actin polymerization assays, FRLα-C

accelerates actin polymerization in a concentration dependent manner, with a persistent lag being

observed even at µM FRLα-C concentrations. The polymerization activity of FRLα-C is much

weaker than that observed for mDia1 (14). FRLα-C also severs actin filaments, creating new

barbed ends capable of elongation. Additional experiments with platelet actin demonstrate that

FRLα-C has similar effects on non-muscle actin. We believe that polymerization acceleration

by FRLα-C is due both to weak nucleation and filament severing. In addition, we postulate that


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FRLα-Cs multiple effects on actin dynamics are due to its ability to interact with filament sides,

with a preference for the side of the barbed end.


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EXPERIMENTAL PROCEDURES

DNA Constructs Our construct of FRLα (accession # 215666) and FRLβ (accession # 006466)

was generated by RT-PCR from 300.19 murine Pre-B lymphoma cell RNA. Total RNA was

isolated from exponentially growing cell cultures with TRIzol reagent (Invitrogen), and cDNA

was produced with oligo dT primer and SuperScript II reverse transcriptase (Invitrogen). The

coding region fragments 449-1094 for FRLα (FRLα-C) and 423-1064 for FRLβ (FRLβ-C)

were amplified with PFU DNA polymerase (Stratagene). The PCR product was cloned into

pGEX-KText vector (a gift from Jack Dixon).

Protein Preparation and Purification - For FRLα-C and FRLβ-C, Rosetta DE3 E. coli

(Novagen) were transformed with expression construct and grown to OD600 0.6-0.8 in TB (12

g/liter Tryptone, 24 g/liter yeast extract, 4.5 ml/liter glycerol, 14 g/liter dibasic potassium

phosphate, 2.6 g/liter monobasic potassium phosphate) with 100 µg/ml ampicillin and 34 µg/ml

chloramphenicol at 37°C. After reduction to 16°C for 30 min, 0.5 mM IPTG was added, and the

cultures were grown overnight. All subsequent purification steps were performed at 4°C or on

ice. Pelleted bacteria were resuspended in EB (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5 mM

EDTA, 1 mM DTT, and 1 pill/50 ml Complete protease inhibitors [Roche]) and extracted by

sonication. After ultracentrifugation, supernatant was loaded onto glutathione-Sepharose 4B


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(Amersham), and washed with EB (without protease inhibitors but with 0.05% thesit). Thrombin

(Sigma T-4265) was added to a 50% slurry of the beads to 10 U/ml, and the suspension was

mixed for 4 hours. Cleaved protein was washed from the column, and thrombin was inactivated

with 5 mM DFP/1 mM PMSF for 15 min, after which DTT was added to a concentration of 10

mM. FRLα-C was further enriched by SourceS15 chromatography (Amersham). Final protein

pools were concentrated with Centricon P-20 (Amicon) and dialyzed in Na50MEPD (50 mM

NaCl, 0.1 mM MgCl2, 0.1 mM EGTA, 2 mM NaPO4 pH 7.0, 0.5 mM DTT). Aliquots were

stored at 4°C or at -70°C, with no resulting change in protein activity levels from either storage

method. MDia1 748-1255 was expressed as lovingly described previously (14). In contrast to

FRLα-C, mDia1 748-1255 lost 90% of its nucleation activity upon freezing, so was stored at

4°C. For human profilin I, BL21 pLysS DE3 E. coli (Novagen) were transformed with

expression construct (gift from Thomas Pollard) and grown to OD600 0.6-0.8 in LB (10g/liter

Tryptone, 5 g/liter yeast extract, 10 g/liter NaCl), with 100 µg/ml ampicillin and 34 µg/ml

chloramphenicol at 37°C. 1 mM IPTG was added and cultures were incubated an additional 4

hours at 37°C. All subsequent purification steps were performed at 4°C or on ice. Pelleted

bacteria were resuspended in EB and extracted by sonication. After ultracentrifugation

supernatant was filtered through 0.45 µm syringe filter and loaded onto Poly-L-proline (Sigma

P-3886) linked to CNBr-activated sepharose (Amersham). Column was washed with buffer 1

(10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT), buffer 2 (buffer 1 with 3


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M urea), and buffer 3 (buffer 1 with 8 M urea) sequentially. Buffer 3 eluate was dialyzed in DB

(2 mM Tris-HCl pH 8.0, 0.2 mM EGTA, 1 mM DTT, 0.01% NaN3) overnight and then for an

additional 2 hours in fresh DB. Profilin was stored at 4°C. Capping protein was a kind gift from

David Kovar and Thomas Pollard. α-actinin (AT01-A) and full-length muscle myosin

(MY02-A) were purchased from Cytoskeleton. Rabbit skeletal muscle actin was purified from

acetone powder (16) and labeled with pyrenyliodoacetamide (17). Both unlabeled and labeled

actin were gel filtered on S200 (18), which was crucial to obtain reproducible polymerization

kinetics. Platelet actin was purchased from Cytoskeleton (APHL95). Before use, actin was

resuspended with water then centrifuged at 100,000 rpm for 30 min at 4°C in TLA-120 rotor

(Beckman). Supernatant was loaded onto a SourceQ 5/5 column (Amersham) equilibrated with

G0 buffer (2 mM Tris-HCl pH 8.0, 0.5 mM DTT, 0.1 mM CaCl2). Actin was eluted with a

linear gradient from G0 to G300 (G0 + 300 mM NaCl), and eluted as a single peak at 250 mM

NaCl. ATP was added immediately to a final concentration of 0.2 mM. Peak fractions were

pooled and actin polymerized by addition of EGTA, MgCl2, and imidazole pH 7.0 to 1, 1, and

10 mM, respectively and incubation at room temperature for 4 hours, then overnight at 4°C.

Polymerized actin was centrifuged at 100,000 rpm for 30 min at 4°C in TLA-120 rotor. Pellets

were resuspended in G buffer (2 mM Tris-HCl pH 8.0, 0.5 mM DTT, 0.2 mM ATP, 0.1 mM

CaCl2, and 0.01% NaN3), pushed through 30 gauge needle, and dialyzed in G buffer for 48

hours. After dialysis actin was centrifuged at 100,000 rpm for 3 hrs at 4°C in TLA-120 rotor.


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Upper 2/3 of supernatant was removed and used for subsequent assays. An alternative

purification process, in which actin was polymerized, depolymerized, then gel filtered over a

Superdex200 10/30 column (Amersham) in G buffer was also performed. Both purification

procedures produced actin monomers with similar polymerization properties and removed

gelsolin and Arp2/3 complex effectively.

Protein Size Analysis Techniques – Gel filtration chromatography was conducted using a

Superdex200 10/30 column (Amersham) calibrated with both high and low molecular weight

standards (Amersham). Stokes radius was calculated following manufacturer’s instructions.

Analytical ultracentrifugation was conducted using a Beckman Proteomelab XL-A and an AN-

60 rotor. For sedimentation velocity analytical ultracentrifugation, 0.7 µM FRLα-C in 100 mM

NaCl, 1 mM MgCl2, 1 mM EGTA, 10 mM NaPO4 pH 7.0, 0.5 mM DTT was centrifuged at

30,000 rpm and 20ÚC, and 220 nm absorbance monitored every two minutes by continuous scan

at 0.003 cm steps. Protein partial specific volume, buffer density, and buffer viscosity were

determined using Sednterp (program by David Hayes & Tom Laue). Scans 1-200 were

analyzed using Sedfit87 (www.analyticalultracentrifugation.com), resulting in one major species

(>90%) centered at 4.2 S with a frictional ratio of 2.02. For sedimentation equilibrium analytical

ultracentrifugation, various concentrations of FRLα-C (0.25, 0.333, 0.5, 0.667. 0.75, 1, and 1.25

µM) in the same buffer as for velocity centrifugation was centrifuged at 7000, 10,000, and


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14,000 rpm for 15, 10, and 10 hours, respectively, at 20°C. Scans at 220 nm and 0.001 cm steps

were recorded every hour. Winmatch software (program by David Yphantis) was used to

confirm equilibrium, and Winreedit software (Yphantis) was used to trim the data. Winnonln

software (Yphantis) was used to fit data. First, individual concentrations at individual speeds

were analyzed, and minor speed-dependent and concentration-dependent changes in apparent

MW were detected. Next, data at multiple concentrations and speeds were fit to a single species,

resulting in an apparent MW of 110 kDa (rmsd = 0.00481). The systematic residual error pattern

of this fit suggested non-ideality (19). Finally, the same multiple concentrations and speeds were

fit to a monomer-dimer equilibrium, with a calculated monomer MW of 71,335 daltons. The

resulting fit (rmsd = 0.00441) had little systematic error, and suggested an apparent Kd for

dimerization of 0.1 µM.

Actin Filament Binding Assays Actin (10 µM) was polymerized for 2 hours at 23°C in

polymerization buffer (G-Mg buffer [2 mM Tris-HCl pH 8.0, 0.5 mM DTT, 0.2 mM ATP, 0.1

mM MgCl2, and 0.01% NaN3] plus 1xKMEI [10 mM imidazole pH 7.0, 50 mM KCl, 1 mM

MgCl2, 1 mM EGTA], followed by addition of 10 µM phalloidin (Sigma P-2141). This actin

stock was diluted to desired concentration in polymerization buffer in the absence or presence of

putative binding proteins. Mixing was conducted in polycarbonate 7 x 20 mm centrifuge tubes

(Beckman 343775) to a final volume of 200 µl. Filaments were pipetted using cut pipetteman


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tips to minimize shearing. After 5 min at 23°C samples were centrifuged at 80,000 rpm for 20

min at 20°C in TLA-100.1 rotor (Beckman). 150 µl of supernatant was removed, lyophilized,

and resuspended in 15 µl SDS-PAGE sample buffer. After removal of the remaining

supernatant, pellets were washed briefly with 200 µl 1XKMEI, then resuspended in 20 µl SDS-

PAGE sample buffer. Supernatants and pellets were analyzed by Coomassie-stained SDS-

PAGE. Similar assays omitting phalloidin or adding 5 mM NaPO4 pH 7.0 were also conducted.

Actin Polymerization by Fluorescence Spectroscopy - A detailed procedure is described in (20).

Unlabeled and pyrene labeled actin were mixed in G buffer to produce an actin stock of the

desired pyrene-labeled actin percentage (5% unless otherwise stated). This stock was converted

to Mg2+ salt by 2 min incubation at 23°C in 1 mM EGTA/0.1 mM MgCl2 immediately prior to

polymerization. Polymerization was induced by addition of 10xKMEI (500 mM KCl, 10 mM

MgCl2, 10 mM EGTA, and 100 mM imidazole pH 7.0) to a concentration of 1x, with the

remaining volume made up by G-Mg. Added proteins were mixed together for 1 minute prior to

their rapid addition to actin to start the assay. Pyrene fluorescence (excitation 365 nm, emission

407 nm) was monitored in a PC1 spectrofluorimeter (ISS, Champaign, IL). The time between

mixing of final components and start of fluorimeter data collection was measured for each assay

and ranged between 10 and 15 seconds.


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Calculating Filament Concentration - Slopes of pyrene fluorescence from polymerization time

courses were determined at the 50% point of polymerization with Kaleidagraph (Synergy

Software, Reading, PA). Slopes were converted initially to filament concentration under the

assumption of unrestricted ATP-actin monomer addition to barbed ends with a rate constant

(K+) of 11.6 µM-1s-1 (1) according to the following equation: F = S/(M0.5 x K+), where F is

filament concentration in µM, S is slope converted to µM/sec, and M0.5 is µM monomer

concentration at 50% polymerization. S is calculated by the equation S = (S x Mt)/(fmax fmin),

where S is raw slope in a.u./sec (a.u. = arbitrary units), Mt is µM concentration of total

polymerizable monomer in µM, and fmax and fmin are fluorescence of fully polymerized and

unpolymerized actin respectively, in a.u. Since FRLα-C slows elongation rate by 80%, filament

concentrations from assays conducted in the presence of FRLα-C were multiplied by 5.

Barbed End Elongation Assays Unlabeled actin (10 µM) was polymerized for at least 1 hr at

23°C, then diluted to 5 µM in the presence of 10 µM phalloidin and centrifuged at 100,000 rpm

for 20 min in a TLA-120 rotor. The pellet was resuspended in 3x polymerization buffer to 5

µM, then sheared by two passes through a 27 gauge needle. 37.5 µL of this mixture was aliquoted

into eppendorf tubes and allowed to re-anneal overnight at 23°C. Polymerization buffer

containing FRLα-C, capping protein, or mixtures of both (37.5 µL total) were added to

filaments, mixed by gentle flicking, and incubated at 23°C for 3 min. In competition assays


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between FRLα-C and capping protein, these two proteins were pre-mixed, then added

simultaneously to filaments. After 3 min at 23°C, 75 µL of 2 µM monomers (5% pyrene, Mg2+

converted) and 8 µM profilin in G-Mg buffer were added to the filaments with a cut p200 tip,

mixed by pipetting up and down three times, and placed into the fluorimeter cuvette.

Fluorescence (365/407 nm) was recorded for 180 sec. Initial elongation velocity was obtained

by linear fitting the initial 100 sec of elongation. Final concentrations in the assay were 1.25 µM

phalloidin-stabilized polymerized actin (0.1 to 0.15 µM barbed ends) and 1 µM monomer. In

competition experiments between capping protein and formins, apparent Kd of formin binding to

barbed end was determined by fitting elongation data to a competition binding model as

described in (20), assuming a Kd of 0.1 nM for capping protein binding to barbed ends.

Re-annealing Assay Actin (10 µM) was polymerized for 2 hours at 23°C in polymerization

buffer, then diluted to 1 µM in polymerization buffer with 1.5 µM rhodamine-phalloidin (Sigma

P-1951). This sample was pulled/pushed four times through a 1 ml syringe with a 27 gauge

needle attached, then 10 µl was immediately aliquoted into tubes containing 10 µl

polymerization buffer with or without 200 nM FRLα-C or 200 nM capping protein. To stop the

reactions, 1 ml fluorescence buffer (25 mM imidazole pH 7.0, 25 mM KCl, 4 mM MgCl2, 1 mM

EGTA, 100 mM DTT, 0.5% methylcellulose, 3 mg/ml glucose, 18 µg/ml catalase, 100 µg/ml

glucose oxidase) was added at various time points. Samples (2 µl) were adsorbed to 12 mm


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round glass coverslips previously coated with 0.01% poly-L-lysine. Filaments were pipetted

with cut pipetteman tips to reduce shearing. Samples were visualized on a Zeiss Axioplan 2

microscope (Carl Zeiss, Thornwood, NY) using 100X 1.4 NA objective and images were

acquired with a Hamamatsu Orca II cooled CCD camera using Openlab software (Improvisions

Inc, Boston, MA). Filament length was measured using Openlab software. At least 2 images

were analyzed for every condition and time point, and all filaments with both ends discernable

were measured on each image, resulting in 200-1400 individual filaments being measured for

each condition depending on filament length.

Severing Assay Using Fluorescence Microscopy - Actin (4 µM) was polymerized for at least 1

hour at 23°C in polymerization buffer. 10 µl aliquots from this stock were carefully pipetted into

eppendorf tubes and incubated 10 min at 23°C. 10 µl of protein or buffer was added, mixed by

gentle flicking, and incubated for the desired time at 23°C. Polymerization buffer (20 µl)

containing rhodamine-phalloidin (1 µM final) was added and samples were immediately diluted

200-fold with fluorescence buffer. Samples (2 µl) were adsorbed to 12 mm round glass

coverslips previously coated with 0.01% poly-L-lysine. Great care was taken during sample

preparation to minimize manual severing. Filaments were only pipetted twice during the

procedure: once after initial polymerization but before addition of FRLα-C or buffer, and once

to apply to coverslips. Also, we used cut pipetteman tips to reduce shearing. Samples were

visualized and images acquired as described above. Filament length was measured using

Openlab software. At least 5 images were analyzed for every condition and all filaments with


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both ends discernable were measured for each image, resulting in 200-1500 individual filaments

being measured depending on filament length.

Dual Color Filament Assay Using Fluorescence Microscopy – Actin (4 µM) was polymerized for

at least 1 hour at 23°C in polymerization buffer. 10 µl of polymerized actin was incubated with

10 µl 200 nM FRLα-C or buffer for 10 min at 23°C then 20 µl rhodamine-phalloidin was added

to 1 µM. Samples were diluted 10-fold with 0.5 µM actin monomers and 0.5 µM Alexa 488-

phalloidin (Molecular Probes A-12379), incubated for 6 min at 23°C, and diluted with 2 ml

fluorescence buffer. Samples (2 µl) were adsorbed to 12 mm round glass coverslips previously

coated with 0.01% poly-L-lysine. Filaments were handled as described for the severing assay.

Images were taken through both rhodamine and FITC filters. Filament length was measured

using Openlab software. For each individual filament with both ends discernable, rhodamine and

Alexa 488 labeled segments were measured separately. At least 2 images were analyzed for each

condition, totaling approximately 150 filaments.

Kinetic Severing Assay Using Time-lapse Microscopy - Method adapted from (10,21). Actin

(4 µM) was polymerized for 2 hours at 23°C in polymerization buffer, then rhodamine-

phalloidin was added to label 50% of polymerized actin. Filaments were diluted to 0.067 µM in

modified fluorescence buffer (25 mM imidazole pH 7.0, 25 mM KCl, 4 mM MgCl2, 1 mM


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EGTA, 100 mM DTT, 0.5% methylcellulose, 6 mg/ml glucose, 36 µg/ml catalase, 200 µg/ml

glucose oxidase) + 5 mg/ml BSA. Coverslips (30 x 22 mm) were mounted onto glass slides with

two pieces of double stick tape, forming a perfusion chamber. NEM-treated myosin was diluted

to 50 nM in high salt buffer (50 mM Tris pH 7.5, 600 mM NaCl) and 70 µl was perfused into

chambers for 1 min. Chambers were washed once with 70 µl high salt buffer (50 mM Tris-HCl

pH 7.5, 600 mM NaCl, 1% BSA) and once with 70 µl low salt buffer (same except with 150 mM

NaCl). 70 µl labeled actin filaments were perfused into chambers using a cut pipetteman tip and

incubated 5 min. Chambers were washed once with fluorescence buffer + 5 mg/ml BSA to

remove any unbound filaments. Time-lapse recording was initiated and, after the first image

was acquired, 70 µl FRLα-C (diluted to 200 nM in modified fluorescence buffer with 5 mg/mL

BSA) was perfused into chamber. Only filaments clearly breaking in the middle, resulting in two

discernable pieces that could be tracked, were counted as severing events. Thirteen sequences

for both control and FRLα-C were analyzed, and the sequences containing the highest and

lowest number of severing events for each condition were excluded.

Western blotting of actin preparations - Gel-filtered actin from rabbit muscle, or platelet actin

after initial resuspension or additional purification, was Western blotted at 2 µg on PVDF

(Millipore) against antibodies to human gelsolin (monoclonal GS-2C4 from Sigma), the

ARPC1b subunit of Arp2/3 complex, human profilin I, human cofilin (gifts from Thomas

Pollard), human WASp (22)and capping protein α2 subunit (monoclonal antibody 5B12.3 was


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developed by John Cooper and obtained from the Developmental Studies Hybridoma Bank

developed under the auspices of the NICHD and maintained by The University of Iowa,

Department of Biological Sciences, Iowa City, IA 52242). Blots were developed by

chemiluminescence and stained for protein by Amido Black.

RESULTS


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FRLα-C is a dimer - We expressed a C-terminal construct of FRLα (FRLα-C, amino

acids 449-1094) as a glutathione S-transferase (GST) fusion protein in bacteria (Fig. 1A-B).

After cleavage from GST, FRLα-C elutes with an apparent Stokes radius of 71 Å by gel

filtration, consistent with a spherical particle of 530 kDa (Fig. 1C). Sedimentation velocity

analytical ultracentrifugation reveals a single species of 4.2S and a frictional ratio of 2.02 (Fig.

1D). Equilibrium analytical ultracentrifugation of eight FRLα-C concentrations at three speeds

results in curves that best fit a monomer-dimer equilibrium with an apparent Kd of 0.1 µM (Fig.

1E). These data suggest that FRLα-C might dimerizes in a reversible fashion. The high

frictional ratio suggests that this dimer is elongated, not spherical.

FRLα-C binds muscle actin filament sides and barbed ends - We first characterized

FRLα-Cs effects on muscle actin then performed additional targeted experiments on non-muscle

actin. FRLα-C binds tightly to pre-formed actin filaments, since 0.4 µM phalloidin-stabilized

polymerized actin quantitatively pellets 0.2 µM FRLα-C (Fig. 2A). A comparable construct of

mDia1 binds much more weakly, with an apparent Kd of 3 µM {Fig. 2B; (14)}. As with mDia1

(14), filament binding by FRLα-C is unaffected by the presence of phalloidin or of filament-

bound phosphate (not shown). FRLβ-C binds filaments with similar affinity to FRLα-C (Fig.

2B).

We tested FRLα-Cs effect on barbed end elongation from phalloidin-stabilized actin


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filaments. FRLα-C slows elongation five-fold, as indicated by lower slopes of pyrene-actin

fluorescence compared to actin alone (Fig. 3 inset). The half maximal concentration for this

effect is 2 nM (Fig. 3).

We hypothesized that, similar to yeast formins (4,5,10), FRLα-C may partially block

filament barbed ends resulting in the reduced elongation rate. To examine this possibility further

we tested filament annealing in the presence of FRLα-C, since annealing requires free barbed

ends (23). Phalloidin-stabilized filaments were sheared through a 27 gauge needle, then allowed

to re-anneal in the absence or presence of FRLα-C or capping protein and observed by

fluorescence microscopy. FRLα-C slows filament re-annealing (Fig. 4B,E,H) which becomes

evident at early time points (Fig. 4J). This inhibition, however, is not complete. For comparison,

capping protein allows no significant re-annealing over a 24 hour period (Fig. 4C,F,I).

FRLα-C accelerates polymerization from muscle actin monomers - We tested FRLα-Cs

effect on actin polymerization kinetics by pyrene-actin polymerization assays. FRLα-C

accelerates polymerization of 4 µM actin monomers (Fig. 5A), but with a considerable lag before

new filament production even at µM FRLα-C concentration. We calculated filament

concentration using slopes at 50% polymerization and assuming an elongation rate of 3.62

µm-1sec-1, based on rates of 11.16 and 1.3 µm-1sec-1 for barbed and pointed ends,

respectively (1), and from the 80% inhibition of barbed end elongation by FRLα-C. FRLα-C

enables formation of 10 nM filaments maximally, with a ratio of about 1:5 of filaments


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produced:FRLα-C dimer in the linear range (Fig. 5B). FRLα-C decreases the polymerization

lag five-fold, where lag is defined as the time to reach 10% polymerization (Fig. 5C). FRLβ-C

produces a similar effect (data not shown).

FRLα-Cs ability to accelerate polymerization is dependent on the concentration of actin

monomers (Fig. 5D). Decreasing monomer concentration over a range from 4 µM to 1.5 µM

causes an exponential decrease in the concentration of filaments assembled (Fig. 5E), while the

time required to reach 10% polymerization increases linearly (Fig. 5F).

Profilin modulates FRLα-Cs activities on muscle actin - Profilin is a highly abundant

actin monomer binding protein that inhibits actin nucleation and prevents monomer addition to

filament pointed ends (24). Profilin binds poly-proline sequences in the FH1 domain of formins,

and mDia1 containing the FH1 domain can use profilin-bound actin monomers for nucleation

(14). Consequently we tested profilin’s effect on elongation from actin filaments and

polymerization from monomers in the presence of FRLα-C.

A profilin concentration that binds >95% of the monomer reduces FRLα-Cs inhibition of

elongation from five-fold to two-fold (Fig. 6A). A similar effect occurs with FRLβ-C (Fig.

6B). Profilin has no measurable effect on capping protein-induced elongation inhibition under

these conditions (Fig. 6B).

Profilin inhibits FRLα-Cs acceleration of polymerization from monomers in a

concentration-dependent manner (Fig. 6C-E). As with decreasing monomer concentration,


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increasing profilin reduces FRLα-C-induced filament concentration exponentially (Fig. 6D) and

increases time to 10% polymerization linearly (Fig. 6E).

FRLα-C competes with capping protein for the barbed end of muscle actin filaments -

Capping protein tightly binds filament barbed ends preventing monomer addition, whereas

FRLα-C inhibits elongation 50% in the presence of profilin. We asked whether FRLα-C could protect

barbed ends against capping protein, similar to Bni1 and mDia1 (4,5). When FRLα-C and

capping protein are added simultaneously to phalloidin-stabilized filaments, FRLα-C increases

elongation rate in a concentration-dependent manner and with an apparent Kd for barbed ends of

3.2 nM (Fig. 7A). Thus, FRLα-C and capping protein compete for barbed end binding.

MDia1 does not slow elongation but competes with capping protein – In contrast to other

formins, mDia1 has no effect on barbed end elongation (Fig. 7B). This experiment was

conducted with an mDia1 construct lacking the FH1 domain and in the presence of high profilin

concentration to block mDia1’s potent nucleation activity (14). Surprizingly, mDial blocks

barbed end capping by capping protein, with similar potency to FRLα-C (2.6 nM apparent Kd

for barbed ends, Fig. 7B).

FRLα-C severs muscle actin filaments - Using a fluorescence microscopy assay, we find

that FRLα-C severs actin filaments. When FRLα-C is incubated with actin filaments in


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suspension followed by stabilization with rhodamine-phalloidin, a marked decrease in filament

length is observed (Fig. 8A-B, G). When rhodamine-phalloidin is added to filaments prior to

FRLα-C incubation, severing is inhibited (Fig. 8D-E). We were concerned that this effect was

artifactual, due to FRLα-Cs barbed end binding ability, and since barbed end capping proteins

slow re-annealing of sheared filaments {Fig. 4C,F,I; (22)}. Therefore we took great care during

sample preparation to minimize filament shearing during manipulation (see Methods). As a

confirmation of our methods, filament length was not significantly affected by the presence of

100 nM capping protein (Fig. 8C) or α-actinin (Fig. 8F). Measurements of filament length were

highly reproducible. Since incubation of filaments with FRLα-C occurs in solution, the

possibility that surface-bound filaments might be artifactually destabilized by an effect of

FRLα-C side binding on filament helical pitch is not an issue.

Severing depends on FRLα-C concentration and incubation time (Fig. 8H-I). Optimal

conditions are 200 nM FRLα-C incubated with filaments for 10 min (Fig. 8H), which results in

a median filament length of 2.2 µm, with 46.8% of filaments shorter than 2 µm. Incubation of

buffer with filaments for 10 min results in a median filament length of 6.7 µm, and only 4.61%

of filaments under 2 µm. The largest filaments are severely depleted by FRLα-C, with filaments

> 10 µm going from 48% to 14%.

To monitor barbed end elongation from severed filaments, we performed a dual filament

assay similar to those previously described (25). Polymerized actin was incubated with buffer

(Fig. 9A) or 200 nM FRLα-C (Fig. 9B), then equi-molar rhodamine-phalloidin was added.


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Subsequently, additional monomers and Alexa 488-phalloidin were added and the new

monomers allowed to elongate. Thus, the original filaments were labeled with rhodamine-

phalloidin, while the newly elongated segments of these filaments were labeled with Alexa 488-

phalloidin. A concentration of 0.5 µM actin monomers was added to minimize pointed end

growth. Rhodamine- and Alexa 488-labeled segments of each filament were measured

separately. Filaments incubated with buffer had median lengths of 8.16 µm and 7.24 µm for

rhodamine-labeled and Alexa 488-labeled ends, respectively, while the numbers for filaments

incubated with FRLα-C were 3.87 and 3.92 µm. Thus, FRLα-C severs pre-formed filaments,

then slows barbed end elongation.

To observe severing activity of FRLα-C more directly, we employed time-lapse

fluorescence microscopy. Rhodamine-labeled actin filaments (1:2 molar ratio of

phalloidin:actin) were tethered to coverslips by NEM-treated myosin. After perfusion of

FRLα-C into chambers, severing could be observed as the appearance of gaps or large breaks in the

filaments over several frames (Fig. 10, arrows). Severing was scored only when a clear gap or

break in the middle of a filament occurred during observation, and when both resulting pieces

could be observed for several subsequent frames. While overall shortening of filaments was

observed with FRLα-C addition, these events were not scored since they may have been due to

photobleaching or movement of filaments out of the plane of focus. A total of 50 severing

events were observed with FRLα-C, while 13 events were observed for buffer alone.


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Severing by FRLα-C increases the concentration of muscle actin filament ends If

FRLα-C severs filaments, then the increase in filament concentration should be reflected in an increased

elongation rate upon addition of monomers. When FRLα-C is incubated with pre-formed

filaments, followed by dilution with 0.5 µM pyrene-actin monomers, we observe a slight

decrease in elongation rate compared to filaments alone (Fig. 11). However, this decrease

actually represents a 4-fold increase in filament concentration, since FRLα-C decreases barbed

end elongation five-fold.

FRLα-Cs effects on platelet actin To examine FRLα-Cs effects on platelet actin, we

purchased platelet actin (approximately 85% β-actin and 15% γ-actin non-muscle isoforms)

from Cytoskeleton (Denver, CO), then performed additional purification procedures (see

Methods). This additionally purified platelet actin was used for all subsequent experiments.

FRLα-C slows barbed end elongation from platelet actin filaments two-fold (Fig. 12A, closed

circles) compared to the five-fold reduction we observe with muscle actin (Fig. 3). The apparent

Kd for this effect is similar to the apparent Kd with muscle actin. Profilin does not reduce

FRLα-C inhibition of elongation from platelet actin (data not shown), in contrast to its effect on muscle

actin. FRLα-C relieves elongation inhibition by capping protein on platelet actin (Fig. 12A,

closed circles), with a similar apparent Kd to that observed on muscle actin.

We also tested FRLα-Cs ability to accelerate polymerization from platelet actin


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monomers. While 4 µM platelet actin monomers polymerize more rapidly than 4 µM muscle

actin monomers (compare Fig. 5A to Fig. 12B) the effects of FRLα-C are similar (Fig. 12B) and

the same trends in concentration of filaments produced and reduction in lag time are observed

(data not shown).

Finally, we tested FRLα-Cs ability to sever platelet actin filaments. Incubation of 500

nM FRLα-C with platelet actin filaments results in a reduction of median filament length from

9.77 µm to 2.34 µm (Fig. 12C-D). Thus, FRLα-C produces qualitatively similar results on

muscle and non-muscle actin, with the largest difference being on elongation rate.

DISCUSSION


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In this study, we find FRLα-C to be a dimer that has multiple effects on muscle actin.

FRLα-C binds tightly to actin filament sides. In addition, FRLα-C binds and partially occludes

actin filament barbed ends as evidenced by: its five-fold inhibition of barbed end elongation, its

inhibition of filament re-annealing, and its inhibition of complete barbed end capping by

heterodimeric capping protein. While not slowing barbed end elongation, mDia1 strongly

inhibits capping protein, further evidence of barbed end binding by formins. Profilin partially

relieves FRLα-Cs inhibition of elongation actin filaments. FRLα-C also accelerates

polymerization from actin monomers, an effect that might be partially due to its ability to sever

filaments. In addition, we find FRLα-C has similar effects on non-muscle actin: it inhibits

barbed end elongation two-fold, inhibits complete barbed end capping by capping protein,

accelerates polymerization from monomers, and severs. This study is the first to demonstrate

severing by any formin.

FRLα-C is an elongated dimer, in agreement with biochemical data for a Bni1p FH2

construct (amino acids 1348-1750) (5). A slightly longer Bni1p FH2 construct appears

tetrameric (4). Possibly, the core FH2 region forms a dimer, while more C-terminal sequences

induce higher order oligomerization for some formins. Since our construct contains the entire C-

terminus, this region of FRLα does not appear to affect higher order oligomerization with high

affinity. Our equilibrium analytical ultracentrifugation results suggest that FRLα-C

dimerization may be reversible. Since reversible dissociation of a dimer is difficult to detect

definitively, the details of dimerization equilibrium await further experimentation. If FRLα-C is


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in monomer-dimer equilibrium, binding to the filament side or barbed end may stabilize the

dimeric state.

The ability of formins to inhibit barbed end dynamics has previously been demonstrated,

and a gradient of inhibition has emerged. Cdc12 completely inhibits barbed end elongation (10),

FRLα-C inhibits elongation from muscle actin filaments 80% (this study), Bni1p inhibits 25%

to 50% (4), and mDia1 causes no inhibition (this study). To allow elongation at a reduced rate,

formins may either change the twist of the filament helix by side binding, or bind near the barbed

end in a manner that inhibits access to monomers. We prefer the latter explanation, since low

concentrations of formin are sufficient for elongation inhibition. In addition, three formins,

mDia1 (14), Bni1 (4), and FRLα (data not shown) inhibit barbed end depolymerization

To slow barbed end elongation, formins must move with the advancing barbed end.

Models for processive capping by Bni1p have been proposed by Zigmond et al (4) and Mosely et

al (5), which predict that Bni1p moves along with the barbed end as it elongates. This effect is

predicted to be dependent on multimerization since at least one formin subunit must remain

bound while the other moves to a new barbed end actin subunit. Processive capping seems even

more likely for FRLα-C, since it binds so tightly to actin filament sides. Our observation that

low FRLα-C concentrations inhibit elongation (IC50 of 2 nM) suggests that FRLα-C must bind

preferentially to the barbed end. If FRLα-C bound with equal affinity to the side and barbed

end, then higher concentrations then those observed would be required to inhibit elongation.


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We propose a side ratchet model for FRLα-C binding to elongating barbed ends (Fig.

13). FRLα-C binds to both actin filament sides and barbed ends, but barbed end binding is

preferred. FRLα-C does not sit on the end, but binds the side of the two barbed end subunits

(end-side binding). End-side binding partially occludes access by monomers, slowing

elongation (Step 1a). Different formins occlude the barbed end to different degrees, resulting in

the gradient of elongation inhibition observed (Cdc12>FRLα>Bni1>mDia1). When a monomer

does add, the interaction between one FRLα-C subunit and the actin subunit (now the

penultimate subunit) weakens, allowing the FRLα-C subunit to release and re-bind the new

barbed end subunit. The other FRLα-C subunit remains bound to its actin subunit, thus

maintaining association with the actin filament (Step 2a). The other formin subunit then

advances analogously (Step 3). Our model extends those of Mosley et al (5) and Zigmond et al

(4) in that we propose formin binding to the sides of barbed end subunits, instead of directly on

the barbed end itself.

The reason for formin binding preference at the end-side over the sides is unclear. We

agree with Zigmond et al (4) that binding is probably not affected by actin subunit nucleotide

state, since both FRLα-C (data not shown) and mDia1 (14) slow barbed end depolymerization

from ADP-actin filaments. We postulate that the end-side preference arises from topology

differences between end-sides and sides.

Profilin bound monomers alter FRLα-Cs effect on elongation from muscle actin

filaments, reducing elongation inhibition by > 50%. This effect suggests that profilin binds to


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FRLα-C since addition of profilin-actin to barbed ends would be more inhibited by FRLα-C

then would free actin if profilin did not associate. FRL has been shown to associate with profilin

in vitro and in vivo, and this interaction occurs specifically through it’s FH1 domain (15).

Profilin probably increases elongation rate by providing a closely apposed monomer that better

overcomes partial barbed end occlusion by FRLα-C.

FRLα-C protects filament barbed ends from complete elongation inhibition by capping

protein. This inhibition of capping protein has a similar concentration dependence as elongation

inhibition, supporting our model that FRLα-C remains associated at the barbed end as the

filament is elongating, and is not binding and releasing with each new monomer addition. One

would expect FRLα-C to be less effective at blocking capping protein than at inhibiting

elongation if FRLα-C were continuously dissociating and re-associating. The X-ray crystal

structure of capping protein (26) has lead to development of a model in which two C-terminal

“tentacles” each contact two or three subunits at the barbed end in an “end-side” manner (27).

This information supports our side ratchet model, since end-side binding by formin would

inhibit end-side tentacle binding. The fact that mDia1 does not inhibit elongation but inhibits

capping protein at concentrations similar to those of FRLα-C (<5 nM) is remarkable, and

supports the end-side binding model.

As proposed by Zigmond et al (4) and by Mosley et al (5), the ability to protect against

capping protein provides a mechanism for prolonging the elongation phase of filaments. While

in vitro FRLα-C slows down elongation, in vivo filaments protected against capping protein


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would elongate efficiently, while unprotected filaments would get capped immediately (within 1

second) by capping protein. This possibly provides a mechanism for making longer filaments

found in filopodia and microvilli as opposed to the short branched filaments in

lamellipodia/ruffles (28).

FRLα-C severs filaments, a property not previously demonstrated for formins. This property

may be unique to FRLα-C, since preliminary data suggest mDia1 does not sever. Our

hypothesis is that the severing mechanism is related to the side binding and barbed end binding

properties. In the absence of free barbed ends, FRLα-C binds to the filament side (Step 1b).

The filament then flexes due to its normal thermal motion, creating space for FRLα-C to bind as

it does at the filament barbed end (Step 2b). When the filament flexes back FRLα-C occludes

part of the barbed end, preventing re-association and causing severing. After FRLα-C severs, it

remains associated with the new barbed end as it elongates (Step 2a, bottom). In contrast to

FRLα-C, mDia1-C does not sever efficiently, which may be due to two properties that are dissimilar

to FRLα-C: 1) weak side binding, and 2) lack of barbed end occlusion.

Severing is probably a major contributor to FRLα-Cs acceleration of polymerization

from monomers we observe in vitro for the following reasons. The lag before polymerization,

even with high concentrations of FRLα-C, suggests that FRLα-C must wait for filaments to

nucleate spontaneously before amplifying filament production. FRLα-Cs effect on

polymerization is highly dependent on free actin concentration. Filaments produced by FRLα-C

decrease exponentially with either decreasing monomer concentration, or increasing


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concentration of profilin. Severing may not be the only contributor to polymerization

acceleration in vitro, since FRLα-C might also enhance nucleation similar to other formins.

FRLα-Cs partial inhibition of re-annealing may also accelerate polymerization in vitro by

keeping filament number high.

We postulate that nucleation is also due to end-side binding, which causes stable

dimerization (Fig. 13). Other nucleators are thought to operate by stabilizing actin dimers, but

do so by binding the dimer end and not the side. Arp2/3 complex may form a dimer mimic,

allowing elongation toward the barbed end (2). Capping protein binds dimers in a barbed end

manner, allowing elongation toward the pointed end (29). Formins might be a third variety of

nucleators, stabilizing dimers from the side and potentially allowing elongation in both

directions.

While the overall effects of FRLα-C on non-muscle actin are similar to those for muscle

actin, there are some important differences. FRLα-C inhibits elongation from platelet actin

filaments less than from muscle actin filaments. We observe a two-fold inhibition, compared to

the five-fold inhibition seen on muscle filaments. Also in contrast to muscle filaments, profilin

does not cause any relief to this inhibition. One possible reason for these differences could be

due to slight differences in the structure of the barbed ends of non-muscle filaments, which may

decrease occlusion of the barbed end by FRLα-C. A second possible reason for these observed

differences could be the presence of contaminating proteins in one of the actin preparations. As

reported by the manufacturer, we detected gelsolin in the platelet actin after preparation


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according to their instructions. In addition, some Arp2/3 complex appeared present in this

preparation. In contrast we did not detect profilin, capping protein, WASp or cofilin. Additional

purification steps appeared to remove both gelsolin and Arp2/3 complex from the platelet actin

preparation (data not shown). However, additional contaminants may persist and affect the

actions of FRLα-C.

It is unclear which biochemical properties of FRLα-C are relevant in cells. The severing

and polymerization acceleration activities are weak, raising doubts that they alone could generate

filaments rapidly enough to support formation of large actin-based cellular structures. However,

other binding proteins may enhance severing and/or nucleation by FRLα-C. Capping protein

inhibition is a potent effect, and certainly is a possibility for creating long filaments in cells.

Acknowledgments We thank Duane Compton for use of his microscope and Larry Myers

and Charlie Barlowe for use of their protein chromatography equipment. We also thank Thomas

Laue (University of New Hampshire) and John Champagne (Wyatt Technology Corp.) for help

with interpretation of analytical ultracentrifugation data, Bruce Goode (Brandeis) and Sally

Zigmond (University of Pennsylvania) for sharing results before publication, and David Kovar

and Thomas Pollard (Yale) for profilin expression vector, capping protein, and several


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antibodies.

This work was supported in part by the Norris Cotton Cancer Center and American

Cancer Society Institute grant IRG-82-003-18. Henry Higgs is also supported by a Pew

Biomedical Scholars award and by NIH grant P20RR16437 from the COBRE Program of the

National Center for Research Resources.


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FOOTNOTES

1The abbreviations used are: Arp2/3, actin-related protein 2/3 complex; BSA, bovine serum

albumin; DTT, dithiothreitol; FH1 and FH2, Formin homology domains 1 and 2; FITC,

fluorescein isothiocyanate; FRL, Formin-related gene in Leukocytes; GST, glutathione S-

transferase; mDia1, mammalian Diaphanous formin; NEM, N-ethylmaleimide; SH3, Src

homolgy 3.


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FIGURE LEGENDS

FIG. 1. FRLα-C is a dimer. A, Bar diagram of FRLα, showing FH1 domain (vertical bars,

amino acids 537-611), FH2 domain (diagonal bars, 627-1006), and alternately spliced C-terminus

(boxes, 1062-1094). FRLα-C construct spans 449-1094. B, Coomassie-stained 15% SDS-

PAGE of proteins used in this study. 1 µg of the following proteins: 1) FRLα-C, 2) muscle

actin, 3) capping protein, 4) profilin, 5) FRLβ-C. C, Superdex200 gel filtration chromatography

of FRLα-C in gel filtration buffer (10 mM NaPO4 pH 7.0, 100 mM NaCl, 1 mM MgCl2, 1 mM

EGTA, 0.5 mM DTT). Peak elution volumes of the following markers are shown along the top:

V = blue dextran 2000 (void); 85 Å = thyroglobulin; 61 Å = ferritin; 52 Å = catalase; 48 Å =

aldolase; 35.5 Å = BSA; 30.5 Å = ovalbumin; 21 Å = chymotrypsinogen; and 16.4 Å = RNaseA.

D, Sedimentation velocity analytical ultracentrifugation of 0.7 µM FRLα-C (peak fraction from

Superdex200 chromatography) in gel filtration buffer. The major species (>90%) sediments at

4.2 S, while a minor species (7%) sediments at 1.8 S. The calculated frictional ratio is 2.02. E,

Sedimentation equilibrium analytical ultracentrifugation of 0.75 µM FRLα-C at 14,000 rpm.

The fit line (dark line) represents fitting to a monomer-dimer equilibrium of the 71,335 dalton

FRLα-C monomer, including data at two other concentrations and speeds (nine data sets total),

and is comparable to fitting of five other concentrations at these speeds. The apparent Kd for

dimerization is 0.1 µM.


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FIG. 2. FRLα and FRLβ bind muscle actin filaments tightly. A, Coomassie-stained SDS-

PAGE of pelleting assays using 0.2 µM FRLα-C and varying concentrations of phalloidin-

stabilized actin filaments. Binding and pelleting were conducted in polymerization buffer at

23ÚC. B, Graph of % FRLα-C (closed circles), FRLβ-C (open circles) or mDia1-C (crosses)

in pellet with varying concentrations of actin filaments. FRLα and FRLβ bind with apparent

Kds of < 0.2 µM, and mDia1 binds with a Kd of 3 µM.

FIG. 3. FRLα slows elongation from muscle actin filament barbed ends. Phalloidin-stabilized

filaments (1.5 µM polymerized actin, about 0.1 nM barbed ends), were mixed with varying

concentrations of FRLα-C for 1 min, followed by addition of 1 µM pyrene-actin monomers

(5% pyrene). Elongation was measured for 180 sec by increase of pyrene fluorescence, and the

elongation rate measured as the slope of this increase. Inset: examples of the raw elongation data

without FRLα-C (closed circles) and with 25 nM FRLα-C (open circles).

FIG. 4. FRLα inhibits filament re-annealing of muscle actin filaments. A-I, 0.5 µM

polymerized actin was stabilized with 0.75 µM rhodamine-phalloidin, sheared through a 27

gauge needle, then allowed to re-anneal in the presence of buffer, 100 nM FRLα-C or 100 nM

capping protein. Samples were diluted at indicated time points, adsorbed to glass coverslips

coated with poly-L-lysine, and viewed by fluorescence microscopy. Scale bar 10 µm. J, Time


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course of re-annealing as judged by increase in median filament length, for filaments alone

(closed circles), with 100 nM FRLα (open circles), or with 100 nM capping protein (closed

squares).

FIG. 5. FRLα accelerates polymerization from muscle actin monomers. A, Pyrene-actin

polymerization assays containing 4 µM monomeric actin (5% pyrene) and the indicated nM

concentrations of FRLα-C in polymerization buffer. B, Plot of filaments produced at 50%

polymerization as a function of FRLα-C. C, Plot of time required to reach 10% polymerization

as a function of FRLα-C. D, Pyrene-actin polymerization assays containing indicated µM

concentrations of monomeric actin (5% pyrene) and 200 nM FRLα-C. % polymerization

represents fluorescence normalized to the scale of 4 µM monomer. E, Plot of filaments

generated at 50% polymerization in the presence of 200 nM FRLα-C (closed circles) or absence

of FRLα-C (closed squares) as a function of monomer concentration. F, Plot of time required to

reach 10% polymerization in the presence of 200 nM FRLα-C as a function of monomer

concentration.

FIG. 6. Effects of profilin on FRLα modulation of muscle actin dynamics. A, Profilin decreases

FRLα-C inhibition of barbed end elongation from actin filaments. Elongation assays were

conducted as in Figure 3 in the absence or presence of 4 µM profilin mixed with pyrene-actin

monomer prior to addition to filaments. B, Bar graphs comparing elongation inhibition by 25


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nM FRLα-C (gray), FRLβ-C (diagonal lines), or capping protein (cross hatched bars) under

conditions described in Figure 3 in the absence or presence of 4 µM profilin. C-E, Effect of

profilin on polymerization from actin monomers. C, Pyrene-actin polymerization assays

containing 4 µM monomers (5% pyrene). Monomers were incubated with profilin for 1 minute

before addition of FRLα-C. Actin alone (circles), actin plus 200 nM FRLα-C (squares), actin

plus 4 µM profilin (crosses), actin plus FRLα-C and profilin (triangles). D, Plot of filaments

generated from 4 µM monomers at 50% polymerization in the presence (open circles) or absence

(actin alone, closed circles) of 200 nM FRLα-C as a function of profilin concentration, with the

scales normalized to illustrate the similarity of profilins effect on actin alone and on actin with

FRLα-C. Inset the same data without normalization to show the difference in magnitude with

and without FRLα-C in the presence of profilin. E, Plot of time required to reach 10%

polymerization in the presence (open circles) or absence (closed circles) of 200 nM FRLα-C as

a function of profilin concentration. Data not shown for actin alone plus 4 µM and 8 µM

profilin.

FIG. 7. Competition between formins and capping protein for muscle actin filament barbed

ends. A, Elongation rates of 1 µM pyrene-actin monomers (5% pyrene) from phalloidin-

stabilized actin filaments (1.5 µM, about 0.13 nM barbed ends) in the presence of the indicated

nM concentrations of FRLα-C in the presence of 1 nM capping protein. B, similar experiment


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as in A except with varying mDia1 748-1255 in the absence (closed circles) or presence (open

circles) of 0.6 nM capping protein.

FIG. 8. FRLα severs muscle actin filaments in a time and concentration dependent manner. A-

C, F, 2 µM polymerized actin was incubated with buffer (A), 200 nM FRLα-C (B), 100 nM

capping protein (C), or 100 nM α-actinin (F), for 10 min, then stabilized with 2 µM rhodamine-

phalloidin and immediately diluted 200-fold with fluorescence buffer. D-E, 2 µM polymerized

actin was stabilized with 2 µM rhodamine-phalloidin, then incubated with buffer (D) or 200 nM

FRLα-C (E) for 10 min and diluted 200 fold with fluorescence buffer. After dilution with

fluorescence buffer, samples (A-F) were adsorbed to poly-L-lysine coated glass coverslips and

viewed by fluorescence microscopy. G, Filament length distribution for filaments incubated

with buffer (solid bars) or 200 nM FRLα-C (slashed bars) for 10 min as described for (A-B).

Approximately 8% of filaments incubated with buffer were > 26 µm, while 0.5% of filaments

with FRLα-C were > 26 µm (data not shown). H, 2 µM polymerized actin was incubated with

200 nM FRLα-C for indicated time points, then processed as described above. Percent

filaments < 2 µm (open circles) and median length (closed circles) were plotted vs. time. I, 2 µM

polymerized actin was incubated with varying concentrations of FRLα-C for 10 min, then

processed as described above (closed circles). Also 2 µM polymerized actin was pre-stabilized

with 2 µM rhodamine-phalloidin, then incubated with 200 nM FRLα-C for 10 min, diluted with

fluorescence buffer and viewed as described above (open circles).


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FIG. 9. Muscle actin filaments severed by FRLα elongate slowly. A-B, 2 µM polymerized

actin was incubated with buffer (A) or 200 nM FRLα-C (B) for 10 min, then stabilized with

rhodamine-phalloidin. This mixture was diluted with 0.5 µM actin monomers and Alexa 488-

phalloidin, incubated for 6 min, and further diluted with fluorescence buffer. Samples were

adsorbed to poly-L-lysine coated glass coverslips and viewed by fluorescence microscopy. A,

Median length of rhodamine-phalloidin labeled filaments (red) 8.16 µm, median length of Alexa

488-phalloidin labeled filaments (green) 7.24 µm. B, Median length of rhodamine-phalloidin

filaments (red) 3.87 µm, median length of Alexa 488-phalloidin labeled filaments (green) 3.92

µm. Scale bar 10 µm.

FIG. 10. Time-lapse observation of severing of muscle actin filaments. 0.067 µM filaments

labeled with rhodamine-phalloidin (50% labeling) were perfused into chambers coated with

NEM-treated myosin, incubated for 5 min, then washed with high and low salt buffers and

modified fluorescence buffer + 5 mg/ml BSA. The first image was acquired (0 min), then 200

nM FRLα-C was perfused into chamber and images were acquired every minute for 10 min.

Arrows indicate severing events.

FIG. 11. Severing by FRLα increases the concentration of muscle actin filament ends. Pre-

polymerized actin filaments (2 µM) were mixed with buffer alone or 200 nM FRLα-C for 3 min,

followed by dilution with 1 volume of 1 µM actin monomers (20% pyrene-labeled), and


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fluorescence measured for 3 min. Calculated filament concentrations for filaments alone (11.6

µM-1sec-1 elongation rate constant) and filaments with FRLα-C (2.3 µM-1sec-1) were 0.268 and

1.07 nM, respectively. Data are representative of two experiments conducted on two different

occasions (four experiments).

FIG. 12. Effects of FRLα on platelet actin. A, Elongation rates of 0.5 µM pyrene-actin

monomers (5% pyrene) from phalloidin-stabilized platelet actin filaments (0.125 µM) in the

presence of the indicated nM concentrations of FRLα-C (closed circles) or in the presence of

indicated nM concentrations of FRLα-C and 0.5 nM capping protein (open circles). Results are

presented as elongation rates compared to filaments alone. B, Pyrene-actin polymerization

assays containing 4 µM monomeric platelet actin (5% pyrene) and the indicated nM

concentrations of FRLα-C in polymerization buffer. C-D, Severing assay; 2 µM polymerized

platelet actin was incubated with buffer (C) or 500 nM FRLα-C (D) for 10 minutes, then

stabilized with 2 µM rhodamine-phalloidin and immediately diluted 200-fold with fluorescence

buffer. After dilution samples were adsorbed to poly-L-lysine coated glass coverslips and

viewed by fluorescence microscopy.

FIG. 13. Model for the creation of actin filaments by FRLα. A dimer of FRLα-C can nucleate


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actin filaments by side nucleation. FRLα-C remains bound to the side at the barbed end (the

end-side) as the filament elongates (step 1a). FRLα-C protects the barbed end against capping

protein (step 1a), while still allowing the addition of actin monomers (step 2a). FRLα-Cs

preference for end-sides over sides allows it to walk with the barbed end as new monomers add

by releasing from the penultimate actin subunit and binding the side of the newly added barbed

end subunit (step 2a). FRLα-Cs dimeric state allows one FRLα-C subunit to remain bound

while the other moves to the new barbed end. FRLα-C bound along the length of the filament

severs by preventing re-association of subunits as filaments flex by thermal motion (step 2b),

creating a new barbed end capable of elongation (step 2a bottom).


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Elizabeth S. Harris, Fang Li and Henry N. Higgscapping protein, accelerates polymerization from monomers, and severs filaments

, slows actin filament barbed end elongation, competes withαThe mouse formin, FRL

published online February 29, 2004J. Biol. Chem.

10.1074/jbc.M312718200Access the most updated version of this article at doi:

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