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| Proc Natl Acad Sci U S A. 2001 December 18; 98(26): 15009–15013. Published online 2001 December 11. doi: 10.1073/pnas.211556398. | PMCID: PMC64974 |
Copyright © 2001, The National Academy of Sciences Cell Biology Direct real-time observation of actin filament branching mediated
by Arp2/3 complex using total internal reflection fluorescence microscopy Kurt J. Amann * and Thomas D. Pollard *†‡*The Salk Institute for Biological Studies, Structural Biology
Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037;
and †Department of Molecular, Cellular, and
Developmental Biology, Yale University, New Haven, CT 06520 Accepted October 18, 2001. |
Abstract Existing methods for studying actin filament dynamics have allowed
analysis only of bulk samples or individual filaments after treatment
with the drug phalloidin, which perturbs filament dynamics.
Total internal reflection fluorescence microscopy with
rhodamine-labeled actin allowed us to observe polymerization in real
time, without phalloidin. Direct measurements of filament growth
confirmed the rate constants measured by electron microscopy and
established that rhodamine actin is a kinetically inactive tracer for
imaging. In the presence of activated Arp2/3 complex, growing actin
filaments form branches at random sites along their sides, rather than
preferentially from their barbed ends. |
Actin filament polymerization
produces forces that push forward the leading edge of motile cells ( 1),
as well as some subcellular structures ( 2), organelles ( 3), and
intracellular pathogens ( 4). Additionally, once polymerized, actin
filaments serve as substrates for myosin motors and provide mechanical
structure for eukaryotic cells. Elucidating the mechanisms regulating
actin polymerization is crucial to understanding fundamental issues of
cellular structure and motility. Bulk biochemical methods for studying
actin polymerization have been available for more than a half-century
( 5) and were initially useful for determining the conditions required
for shifting the monomer-polymer equilibrium. Later electron
microscopic studies allowed filament elongation rates to be determined
directly by measuring their lengths after elongating for known periods
( 6– 8). More recent studies using fluorescently labeled phalloidin to
visualize filaments have allowed direct visualization of filament
severing ( 9– 11) and have shown the products of annealing ( 12) and
branch formation mediated by Arp2/3 complex ( 13, 14). However,
because phalloidin stabilizes filaments, it is not well suited for
observing filament dynamics. Although actin can be labeled directly
with fluorophores on a reactive cysteine residue near the carboxyl
terminus, visualization of filament elongation by wide field
fluorescence illumination has been limited by the high background
fluorescence from both out-of-focus filaments and particularly the
labeled actin monomers that are required to drive the reaction. We have overcome these obstacles by using total internal reflection
fluorescence microscopy (TIRFM) to visualize individual filaments of
partially labeled rhodamine actin bound to N-ethyl maleimide
(NEM) myosin on the illuminated surface of flow cells. The background
fluorescence from monomers is minimized, because only a thin section of
sample is illuminated ( 15) by virtue of an evanescent wave created at
the surface of the supporting glass slide. NEM inhibits myosin motor
activity but allows actin filament binding, even in ATP ( 16). We
observed actin filament elongation and branch formation mediated by
Arp2/3 complex in real time. Direct measurements of filament growth
confirmed the rate constants measured by electron microscopy ( 6, 8) and
established that rhodamine actin is a kinetically inactive tracer for
imaging. In the presence of activated Arp2/3 complex, growing actin
filaments formed branches at random sites along their sides, rather
than preferentially from their barbed ends, providing further support
for the side-binding model for dendritic nucleation ( 13, 17, 18). |
Materials and Methods Proteins. Actin was isolated from rabbit skeletal muscle ( 19) and further
purified by gel filtration on Sephacryl S-300. To label actin on
Cys-374 with rhodamine, 60 μM actin was polymerized and DTT was
removed by dialysis for 2 h against 2 mM Tris ![[center dot]](corehtml/pmc/pmcents/middot.gif) HCl (pH 8.0),
50 mM KCl, 1 mM MgCl 2, 200 μM ATP. A 5-fold
molar excess of 5′-rhodamine-maleimide (Molecular Probes) was added and
mixed at 4°C overnight. Labeled actin was centrifuged at 300,000
× g for 30 min. Unpolymerized, labeled actin in the
supernatant was dialyzed into Ca-G buffer (2 mM Tris HCl, pH
8.0/500 μM DTT/200 μM ATP/100 μM
CaCl 2), gel-filtered on Sephacryl S-300, and
after a 2-h incubation in Ca-G buffer containing 50 mM KCl again
gel-filtered with the same buffer. Labeling stoichiometry was
determined by using a molar extinction coefficient for rhodamine of
80,000 at 543 nm, and actin concentrations were determined by
SDS/PAGE–Coomassie blue staining and densitometry and by
Bradford assay, using unlabeled actin as the standard. In all cases,
the final purified actin was >99% labeled. By sedimentation
equilibrium analytical ultracentrifugation, purified rhodamine actin
was monomeric under both polymerizing and nonpolymerizing conditions.
To ensure that rhodamine actin was competent for copolymerization
with unlabeled actin, before use rhodamine actin was polymerized with a
1:1 ratio of unlabeled actin for 3 h at 4°C, and centrifuged at
400,000 × g for 30 min. Pellets, containing ~40%
rhodamine actin, were suspended in Ca-G buffer and dialyzed against the
same buffer for 2 days. Actin was again centrifuged in Ca-G buffer
before use. Rabbit skeletal muscle myosin was inactivated by incubation
with 1 mM NEM for 40 min at 22°C, and the reaction was quenched with
25 mM DTT. NEM myosin was stored in 50% glycerol and diluted to 100
μg/ml in Mg-G buffer containing 500 mM KCl before use. Arp2/3
complex from bovine thymus ( 20), Acanthamoeba profilin-I
( 21), and recombinant human Scar-I WA ( 18, 22) were prepared by D.
Kaiser, The Salk Institute. Microscopy. An Olympus IX-70 inverted microscope ( 18) was modified for TIRF
illumination. A 100-mW Kr/Ar laser (Melles Griot, Carlsbad, CA)
emitting 40 mW in each of 488- and 568-nm emission lines through a 3×
beam expander (Melles Griot no. 09 LBZ 001) was used as the light
source. D488/10X and D568/10X bandpass filters (Chroma Technology,
Brattleboro, VT) were used as excitation filters. A 100-mM focal length
plano-convex lens focused the beam through a 40 × 23.5 mm
Pellin-Broca prism (CVI Laser, Livermore, CA) on a layer of
glycerol on flow cells above a 1.35 numerical aperture, ×100 objective
lens. Flow cells were constructed by mounting a 24 × 60-mm no. 0
coverslip perpendicularly across a standard 25 × 75 × 1-mm
microscope slide, separated by two 5-mm-wide strips of Parafilm,
stretched tightly and placed 5 mm apart. The resulting chambers
measured ~30 μm in height and held a volume of 5 μl. Filament
polymerization was observed by adding partially labeled monomeric actin
to polymerization buffer and immediately flowing the solution into the
cell. The microscope could be focused and images were captured
typically within 30 s after inducing polymerization. Image and Data Analysis. Exposures of rhodamine-actin samples (100–300 ms) were
collected by using a Hamamatsu C4742-95 cooled charge-coupled device
camera. Images were processed and movies were produced by using
METAMORPH 4.5 (Universal Imaging, Media, PA).
Filament lengths could be accurately measured to ~250 nm ( 13).
Elongation rates were determined by measuring filament lengths from at
least six frames separated by at least 15 s each. Linear fits were
made to the plots of length versus time, with the slope representing
the elongation rate. Elongation rates were converted from
μm s −1 to s −1 by
dividing by 370 actin monomers per micrometer. Correlation coefficients
for single filament elongation plots exceeded 0.95, confirming that
elongation rates were constant. Because calculation of elongation rates
depended on accurate measurement of length changes, rather than
absolute lengths, the fluorescence halo effect did not affect the
results. As a result, length changes of as little as 100 nm could be
measured. Movies were prepared in metamorph, using Cinepak
Codec compression, and are shown at ×30 time compression. |
Results and Discussion TIRFM revealed individual filaments in flow cells containing 1
μM actin filaments labeled with equimolar rhodamine-phalloidin,
whereas no filaments were discernable by epifluorescence because of the
high background fluorescence. Similar results were found when filaments
of 40% rhodamine actin stabilized with unlabeled phalloidin were
examined by epifluorescence and TIRFM. When 1 μM polymerized 40%
rhodamine actin was examined by TIRFM without phalloidin, individual,
surface-bound filaments were observed. Although some background
fluorescence was present with TIRFM, it was significantly less than the
epifluorescence background (data not shown). TIRFM allowed direct, real-time imaging of elongating actin filaments
(Fig. 1). The robustness of the assay is
best appreciated by viewing movies of filament elongation (Movie
1, which is published as supporting information on the PNAS web
site, www.pnas.org). The elongation rate depended on both the
concentration of actin and the fraction of rhodamine-labeled actin. At
actin concentrations lower than 0.3 μM, elongation was too slow to
measure accurately, whereas at concentrations greater than 2 μM, the
high spontaneous nucleation rate produced filaments too numerous to
resolve. Between 0.3 and 2 μM, however, the number of filaments per
field varied roughly in proportion to the concentration. Significant
growth was observed only at only one end, the barbed end (Fig. 1).
Filament barbed ends grew steadily at constant rates for at least 5
min, indicating that the monomer pool was not appreciably depleted by
polymerization. The barbed-end elongation rate was directly
proportional to the actin concentration over the range tested, yielding
an elongation rate constant of 5.5
μM −1s −1 for 40%
labeled actin (Fig. 2A).
Extrapolation of the elongation rate to zero actin concentration
yielded a k− of 1.2
s −1, in close agreement with electron
microscopic studies ( 8). Occasionally during the course of observation,
fully formed filaments appeared spontaneously, having elongated in
solution and diffused into the plane of excitation. Because the
filaments appeared so suddenly, such events were easily distinguished
from filament elongation.
 | Figure 1Elongation of actin filaments visualized by TIRFM. Actin monomers (1
μM, 40% rhodamine-labeled) were polymerized in fluorescence buffer
(50 mM KCl/1 mM MgCl2/10 mM imidazole, pH 7.0/1 mM
EGTA/100 μM (more ...) |
 | Figure 2Dependence of barbed-end elongation rates on actin monomer
concentration. Actin filament elongation was observed as described in
Fig. 1. (A) Dependence on total actin concentration with
constant 40% rhodamine-actin. (B) Dependence on
fraction (more ...) |
At a total actin concentration of 1 μM, the elongation rate was
inversely proportional to the fraction of labeled actin (Fig.
2B). The barbed end elongation rate constant,
extrapolated to 100% unlabeled actin, was 8
μM −1s −1, similar
to values determined by electron microscopy ( 6, 8). The elongation rate
constant extrapolated to zero at 0.22 μM total actin, which
corresponded to 0.13 μM unlabeled actin (Fig. 2A).
Similarly, the dependence of the elongation rate constant on the
fraction of rhodamine actin extrapolated to zero growth at 85%
rhodamine actin, which corresponded to 0.15 μM unlabeled actin. These
values agree closely with the established value of 0.1 μM for the
critical concentration at the barbed end. Thus, the elongation rate was
determined solely by the concentration of unlabeled actin. Rhodamine
actin behaved as a kinetically inactive tracer for imaging, likely
because of inefficient incorporation into filaments under
nonequilibrium conditions. Based on these observations we chose 20%
labeled actin for subsequent experiments. TIRFM allowed us to visualize actin filament branch formation
mediated by activated Arp2/3 complex in real time (Fig.
3; Movie 2, which is published as
supporting information on the PNAS web site). Filaments bound to
NEM-myosin on the surface of the TIR flow cell were elongated for 3
min, before adding Arp2/3 complex, the activator Scar-WA, and
additional actin monomers. These mother filaments continued to elongate
at the rates determined by the monomer concentration. The earliest
indications of branch formation were bright spots appearing along the
lengths of mother filaments (Fig. 3 A–C). The bright
spots grew to become bumps protruding from the sides of filaments.
After typically less than 1 min, they became recognizable as branches
emanating from the sides of mother filaments (Fig. 3 A and
B). The branches exhibited the 70° angle characteristic of
Arp2/3 complex-mediated branches, and elongated at the same rate as
mother filament barbed ends. This is further confirmation that the
pointed end of the daughter filament is located at the base of the
branch.
 | Figure 3Actin filament branch formation. Filament elongation of 20% rhodamine
actin was visualized by TIRFM as described in Fig. 1. (A
and B) Actin monomers (1 μM) were visualized
polymerizing for 2 min before addition of 100 nM Arp2/3 (more ...) |
Branches arose both from the sides of filaments that had grown before
addition of Arp2/3 complex and from portions of filaments that had
grown in the presence of Arp2/3 complex. Although the latter
observation could in principle be explained by Arp2/3 complex
incorporating into the growing mother filament, the former is only
reasonably explained by Arp2/3 complex binding to the side of the
mother filaments. Branch density (number of branches per μm mother
filament) varied with the Arp2/3 concentration, but only a relatively
narrow range of concentrations (25–100 nM) produced images suitable
for quantitative analysis. Using 100 nM Arp2/3 complex, branches ( n = 146) arose
from pre-existing mother filaments (161 mother filaments, 3.56 ±
1.71 SD μm in length) at a density of one branch per 4.4 μm. The
sites of branching from mother filaments were essentially random, as
they occurred 2.34 ± 1.5 SD μm from the position of the barbed
end at the time of Arp2/3 complex addition (Fig.
3E). Because short branches within 0.5 μm of mother
filament barbed ends were most affected by small errors in length
measurement, these branches may be slightly underrepresented in the
data. Because ATP hydrolysis in actin filaments occurs at a rate of 0.3
s −1 ( 23) and P i is
released at a rate of 0.002 s −1 ( 24), the oldest
portions of average mother filaments contained ~85%
ADP-P i-actin and 15% ADP actin. Because of the
~30-s delay between washing out actin monomers and addition of the
branching solution, there was essentially no ATP-actin cap on the
barbed ends at the time Arp2/3 complex was added. Thus, although we
observed no preference for branch formation near barbed ends, the
shallow gradient of ADP-P i to ADP along the
length of mother filaments would not necessarily reveal a subtle
preference for branches forming on the sides of ATP- or
ADP-P i actin subunits relative to ADP subunits. Branches also grew from portions of filaments that grew after the
addition of Arp2/3 complex. From frames captured shortly after
branches became visible, we measured the distance from the branch point
to the barbed end of the daughter filament and both ends of the mother
filament ( n = 154). Because all barbed ends elongated
at the same rate, it was possible by extrapolation back in time to
determine the position of each branch on the mother filament relative
to the barbed and pointed ends of the mother filament at the time that
the branch started to grow. Branches arose on average 1.56 ± 1.42
SD μm from the barbed ends of mother filaments that averaged 4.0
± 2.1 SD μm in length (Fig. 3F). On average,
branches arose on mother filaments 40 ± 19% of the length from
the barbed end of the mother filament. Of 152 branches, 116 arose from
the barbed end half of the mother filament. Thirty six others arose
from the pointed end half of the mother filament, some as much as 10
μm from the barbed end. Two remaining daughter filaments measured
longer than their mothers. Because only ~100 nm (37 subunits) of the
newest growth were composed of primarily ATP actin, with the proportion
declining exponentially toward the pointed end, a preference for branch
formation from ATP filaments relative to ADP-P i
or ADP filaments could underlie the modest preference for branching
near the barbed ends of elongating filaments. Because branches arose, on average, 1.56 μm from the mother filament
barbed end, this means that, on average, the barbed end of the mother
filament elongated past the branch point for 4 min before the branch
started to grow. If Arp2/3 complex incorporated into the mother
filament during its elongation as postulated ( 25), an average 4-min
delay before branch formation would be required to explain this
observation. Experiments monitoring actin polymerization by pyrene
fluorescence ( 18, 22, 26) demonstrated unambiguously that nucleation by
Arp2/3 complex proceeds within seconds in the presence of stimulatory
actin filaments rather than after a substantial lag. Furthermore,
epifluorescence images of filaments grown for less than 4 min in the
presence of activated Arp2/3 complex show well-established branches
( 14, 18). We previously demonstrated that profilin can favor Arp2/3
complex branching by reducing the spontaneous nucleation rate and
nucleation from capping protein ( 13). Using TIRFM, we observed that
profilin greatly increases the density of Arp2/3 complex branching
from mother filaments (Fig. 3 C and D).
When 60-nM activated Arp2/3 complex was added to mother filaments
along with actin monomers and an excess of profilin, numerous branches
appeared as bumps on filament sides (Fig. 3C; Movie 3, which
is published as supporting information on the PNAS web site). The high
efficiency of nucleation rapidly depleted the monomer pool, resulting
in an early arrest of branch elongation. After washing out unbound
components and adding additional actin monomers, the branches resumed
rapid elongation (Movie 4, which is published as supporting information
on the PNAS web site). The resultant branched structures (Fig.
3D) were composed of filaments too numerous to count
accurately, resembling the “dandelion” structures observed by
wide-field fluorescence microscopy at high concentrations of Arp2/3
complex ( 13). TIRFM is a robust tool for analysis of actin dynamics and the proteins
that control them. The ability to observe the assembly and disassembly
of individual filaments in real time will serve as a valuable
complement to existing analytical methodologies. In the present case,
we addressed the mechanism of actin filament branching by activated
Arp2/3 complex. Biochemical, microscopic, and structural analysis
( 13, 17, 18, 27, 28) had indicated that branch formation occurred on
the sides of filaments. Nonetheless, without direct observation of the
events, alternative hypotheses ( 14, 25) were still formally possible.
Our observations show that branches form on the sides of filaments.
TIRFM will similarly allow further insight into and clarification of
the mechanisms of action of a wide variety of actin-associated
proteins. |
|
Acknowledgments We thank Enrique De La Cruz or his critical evaluation of
this manuscript and helpful suggestions. This work was supported by
National Institutes of Health Research Grant GM26338 (to T.D.P.) and
Postdoctoral Fellowship GM20638 (to K.J.A.). |
Abbreviations | NEM | N-ethyl maleimide | | TIRFM | total internal fluorescence microscopy |
|
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