From the
The fascins are a widely distributed family of proteins that
organize filamentous actin into bundles. We have cloned, sequenced, and
expressed the murine homolog. Fascin is most abundant in brain and is
found in other tissues including uterus and spleen. The deduced open
reading frame encodes a protein of 493 amino acids with a molecular
mass of 54,412 Da. Previous solubility problems with bacterially
expressed fascins were overcome by producing the mouse protein as a
fusion with Escherichia coli thioredoxin. A method for
cleaving the fusion protein and for purifying active recombinant fascin
is described. The N-terminal sequence and molecular mass estimated on
SDS gels indicate that recombinant fascin is full-length.
Two-dimensional gel electrophoresis suggests that recombinant fascin is
post-translationally modified in a manner similar to that observed in
mouse brain. Recombinant fascin and the fusion protein are recognized
by monoclonal anti-fascin antibodies and will bundle rabbit skeletal
muscle F-actin in vitro at a stoichiometry of 4.1:1 actin to
fascin. Electron cryomicroscopy images show that the reconstituted
bundles are highly ordered. However, their fine structure differs from
that of echinoid fascin-actin bundles. This structural difference can
be attributed to fascin.
How cells regulate the superorganization of cytoplasmic actin
filaments is an area of active interest. Several actin-associated
proteins have been isolated from cytoplasmic actin gels formed when low
calcium, ATP-supplemented cell extracts are warmed. This approach was
pioneered in studies on the gelation of sea urchin egg cytoplasm by
Kane (1975, 1976). One component of echinoid actin gels is a 55-kDa
protein termed fascin (Bryan and Kane, 1978). Echinoid fascin organizes
actin filaments into bundles in egg microvilli (Otto et al.,
1980) and into the core bundles of filopodia in coelomocytes (Otto and
Bryan, 1981). Until recently, fascin was thought to be restricted to
invertebrates and was considered to be an invertebrate analog of
fimbrin. Recent cloning of fascin cDNAs from Strongylocentrotus
purpuratus demonstrated that fascins are widely distributed
throughout the animal kingdom (Bryan et al., 1993). Sequence
comparison showed that echinoid fascin is homologous to the product of
the Drosophila singed gene (Paterson and O'Hare, 1991),
which is important in both mechanosensory bristle development and
oogenesis. Overton showed that developing bristles contain a central
core of microtubules surrounded by numerous actin filament bundles. In
developing bristles of singed flies, the number of actin
filament bundles is severely reduced, and those that remain are highly
disorganized (Overton, 1967). In addition, the recombinant singed gene product, Drosophila fascin, can produce actin
bundles with the same 12-nm transverse periodicity (Cant et
al., 1994) as seen in echinoid fascin-actin bundles (Bryan and
Kane, 1978). Antibody evidence shows that Drosophila fascin is
present in normal bristles, but missing in the deformed bristles of
singed flies (Cant et al., 1994). Normal
Drosophila oogenesis involves the transfer of cytoplasm from
nurse cells to the oocyte through joining structures called ring
canals. Flies deficient in fascin lack cytoplasmic actin bundles
responsible for centering the nuclei in the cell; in their absence, the
nuclei plug up the ring canals, blocking cytoplasmic transfer (Cant
et al., 1994).
In addition to Drosophila, fascin
homologs have now been cloned from a Xenopus pituitary library
(Holthuis et al., 1994) and a human teratocarcinoma cDNA
library (Duh et al., 1994). Finally, peptide sequence data
(Bryan et al., 1993) reveal that human fascin is identical to
the 55-kDa bundling protein isolated from HeLa cells
(Yamashiro-Matsumura and Matsumura, 1985). To begin characterizing the
function of fascin in mammals, we have cloned the murine homolog from
fetal mouse brain, described its tissue distribution, and identified
isoforms. We have constructed a new expression vector that can be used
to produce large amounts of soluble active fascin. We show that this
material bundles filaments into highly ordered arrays that will be
useful for high resolution structural work.
Recombinant fascin was incubated with
3.5 or 7 µM rabbit skeletal muscle F-actin at actin/fascin
molar ratios of 40, 20, 12.5, 10, 7.8, 6.5, 5.5, 5, 4, 2.5, 2, and
1.5:1 in 10 mM Na
Fascin has been assumed to be an invertebrate actin
filament-bundling protein. However, the recent cloning of sea urchin
fascin (Bryan et al., 1993) indicates that this family of
proteins is not related to other known actin-binding proteins and is
far more diverse than previously thought. Homologs have been identified
in Drosophila, humans, Xenopus, and now mice. This
organismal diversity and the wide tissue distribution suggest that
fascins may be a major organizing factor for actin filament bundles
including fibroblast stress fibers and microspikes (Yamashiro-Matsumura
and Matsumura, 1986), neuronal growth cone filopodia,
A practical problem in studying
recombinant fascins has been the insoluble nature of the bacterially
expressed protein. This is in contrast to native echinoid fascin, which
is easily soluble under a range of solution conditions (Bryan and Kane,
1978, 1982). We have used a novel thioredoxin expression plasmid
(LaVallee et al., 1993) to produce mouse fascin as a soluble
fusion protein. We have taken advantage of the fact that fascin is
relatively resistant to digestion by
A
comparison of multiply aligned fascin sequences reveals several peptide
motifs that are absolutely conserved. These motifs presumably are
regions essential for fascin function, and we are attempting to map
F-actin-binding sites on fascin. Our preliminary results suggest that
the conserved N-terminal region may be critical for bundling since the
C-terminal 39-kDa proteolytic fragment of fascin generated by
The bundles formed by mouse fascin appear to be morphologically
different from those isolated from echinoid sources (Bryan and Kane,
1982) or reconstituted from Drosophila fascins (Cant et
al., 1994). One major difference is that the transverse bands seen
in bundles formed by vertebrate fascin are not perpendicular to the
bundle axis as the echinoid bands are. The periodicity patterns also
differ. The 33-36-nm periodicity described by Kane (1976) is
visible in all of these bundles. The constant 11-12-nm
periodicity of echinoid and Drosophila bundles has not been
observed in reconstituted mouse fascin bundles. The stoichiometries of
bundles formed by the three fascins are, however, quite similar, with
estimated actin/fascin ratios of 4.6:1 for echinoid bundles, 4.3:1 for
Drosophila bundles, and 4.1:1 for murine bundles. These
numbers are consistent with a model of fascin-actin bundles (DeRosier
et al., 1977; DeRosier and Censullo, 1981) that proposes one
cross-link/4.5 actin subunits if each cross-link is a fascin monomer.
This model is based on negatively stained images and depends critically
on the values of the actin filament helical parameters. The x-ray
structures for G-actin (Kabsch et al., 1990) and F-actin
(Holmes et al., 1990) have refined the measurements of these
parameters. The numbers used for F-actin in the DeRosier model differ
by
Using electron cryomicroscopy,
we are currently refining structural models for both the echinoid
fascin and reconstituted mouse r-fascin bundles. At this time, the
reasons for the structural differences are not clear since the proteins
themselves, the sources of F-actin used for reconstitution, and the
solution conditions used to produce echinoid, Drosophila, and
murine bundles were all different. Since the bundles produced from
murine and echinoid fascins retain their individual morphology
independent of the species of F-actin used for reconstitution, we
conclude that fascin directs which class of bundle structure is formed.
The invertebrate structure with a banding pattern perpendicular to the
filament axis is seen in echinoid and Drosophila bundles. The
vertebrate structure is defined by tilted transverse bands as seen in
reconstituted murine fascin-actin bundles. The invertebrate pattern has
been seen clearly in situ and has been used to localize
fascin. The differentiation of a novel murine bundle pattern should now
provide a similar means to localize fascin to vertebrate fascin-actin
bundles in situ.
We thank Dr. George Mosialos for providing the human
fascin cDNA and peptide sequence and Dr. Fumio Matsumura for the
anti-human fascin monoclonal antibodies. We also thank Dr. Wah Chiu,
Dr. Michael Schmid, Donna Turner, and Joanita Jakana for help and
expertise with transmission electron microscopy and electron
cryomicroscopy.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Materials
Restriction enzymes were from
Pharmacia Biotech Inc., Sequenase was from United States Biochemical
Corp., and common chemicals were from Sigma and Life Technologies, Inc.
Monoclonal antibodies for human fascin were kindly provided by Drs.
Fumio and Shigeko Matsumura.
Gel Electrophoresis and Western Blotting
Samples
of mouse tissues were homogenized in 50 mM Tris, pH 8.0, 2%
SDS and boiled for 5 min. 20 µg of protein from each sample was
separated by SDS-polyacrylamide gel electrophoresis, blotted onto
nitrocellulose, and incubated with primary anti-fascin antibodies at a
dilution of 1:2000. Blots were developed with
5-bromo-4-chloro-3-indolyl phosphate ( p-toluidine salt)/nitro
blue tetrazolium after incubation with alkaline phosphatase-conjugated
goat anti-mouse antibodies.
Library Screening and Cloning
5 10
phage from a Uni-Zap XR fetal mouse brain cDNA library
(Stratagene, La Jolla, CA) were screened with a 2.7-kilobase piece of
human fascin cDNA kindly provided by Dr. George Mosialos (Mosialos
et al., 1994). Three primary positive clones were obtained;
two gave secondary positive plaques. In vivo excision was
carried out following the manufacturer's instructions. The
resulting 2.7-kilobase cDNAs were sequenced in M13 using the dideoxy
procedure (Sanger et al., 1977). cDNA fragments were subcloned
into M13mp18 or M13mp19 as described (Sambrook et al., 1989).
Sequencing was performed using Sequenase according to the
manufacturer's instructions.
Fascin Expression Plasmid Construction
All
subcloning and expression procedures were performed in Escherichia
coli strain BL21. pTRXFUS (LaVallee et al., 1993), a
thioredoxin-containing plasmid, was obtained from the Genetics
Institute (Cambridge, MA). pAED, carrying a T7 promoter,
was kindly provided by Dr. Don Doering (Whitehead Institute, Cambridge,
MA). The thioredoxin-fascin fusion protein expression vector was
constructed as follows. A 539-base pair N-terminal fragment of murine
fascin cDNA was generated by polymerase chain reaction using the
oligonucleotides CGAAGGTACCGatgACCGCCAACGGCAC and GCCAAGGTGATGAGCGA
(start codon is underlined); digested with KpnI and
SalI; and subcloned in frame into pTRXFUS, creating pTRXFAS-N.
This plasmid was cut with BglII and NcoI, and a
2003-base pair BglII- NcoI fragment containing the
remainder of the fascin open reading frame was inserted into pTRXFAS-N,
creating the plasmid pTRXMFAS. The complete thioredoxin-fascin open
reading frame was then subcloned into pAED
3` of the T7
promoter to produce pMFAST7.
Recombinant Fascin Expression and
Purification
pMFAST7 was transformed into E. coli BL21
cells. These were grown at 37 °C to an A of
0.6, induced with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside, and grown at 30
°C for 6 h. Cells were pelleted; resuspended in 0.4 volume of 20%
(w/v) sucrose in 20 mM Tris-Cl, pH 8.0, 2.5 mM EDTA;
set on ice for 10 min; and then pelleted for 10 min at 15,000
g. This pellet was resuspended in an equal volume of 20
mM Tris-Cl, pH 8.0, 2.5 mM EDTA; set on ice for 10
min; and pelleted again. The supernatant will be referred to as the
``shockate.'' The shockate containing the thioredoxin-fascin
fusion protein was incubated with
-chymotrypsin (1:30 (w/w)
enzyme/substrate) for 3 h at 30 °C. The reaction was stopped by the
addition of diisopropyl fluorophosphate to a final concentration of 0.5
mM. All chromatographic steps were carried out at 4 °C.
The mixture was run over a DEAE-Sephacryl column equilibrated in 10
mM NaPO
, pH 6.8, and eluted with a linear gradient
of 10-500 mM NaCl. Recombinant fascin-containing
fractions were then applied to a hydroxylapatite column equilibrated in
10 mM KHPO
, pH 6.8, and eluted with a linear
gradient of 10-500 mM KHPO
, pH 6.8. The
pooled fractions were dialyzed against 10 mM KHPO
,
pH 6.5, and loaded onto a sulfopropyl-Sephadex column equilibrated in
the same buffer. The run-through fraction containing
r-fascin
(
)
was concentrated over Centriprep 30
microconcentrators (Amicon, Inc., Beverly, MA) to a final concentration
of 4-6 mg/ml and stored at 4 °C with 1 mM
NaN
. The same procedure can be used to purify the
thioredoxin-fascin fusion protein.
Echinoid Filopodia Preparation
Filopodial cores
and purified echinoid fascin from coelomocytes of S. purpuratus and F-actin from longitudinal body muscle of Parastichopus
californicus were prepared as described (Bryan and Kane, 1982).
Peptide Sequencing
The N-terminal 10 amino acids
of r-fascin and a 39-kDa fragment were determined using an Applied
Biosystems Model 470-A gas-phase protein sequencer equipped with an
in-line Model 120-A phenylthiohydantoin analyzer.
Acid-Urea Gels
Recombinant fascin was subjected to
acid-urea electrophoresis by the method of Allis et al. (1979). Briefly, r-fascin was separated on an 11-cm 6 M
urea, 10% acrylamide gel equilibrated in 5% (v/v) acetic acid for a
total of 1600 V-h. After Coomassie Blue staining, the lane was excised
from the gel and equilibrated in Laemmli (1970) stacking gel buffer
with 10% (w/v) glycerol and 5 mM -mercaptoethanol. The
lane was laid on its side on top of a 10% SDS-polyacrylamide gel and
separated in the second dimension.
Quantitative Cosedimentation Assays
Rabbit
skeletal muscle F-actin was purified as described (Spudich and Watt,
1971) with the addition of a 2.5 100-cm Sephadex G-50 column as
an additional purification step and stored in 100 mM KCl, 2
mM MgCl
, 1 mM ATP, and 1 mM
NaN
. The actin concentration was determined using following
formula: F-actin (mg/ml) = ( A
-
1.34 A
)/0.69, where A
is
the absorbance at 290 nm, 1.34 A
corrects for
light scattering by F-actin, and A
= 0.69 is the extinction coefficient for G-actin. The
concentration of r-fascin was determined using a molar extinction of
54,406 M
cm
at 280 nm
calculated from sequence data.
HPO
, pH 6.8, 100
mM KCl, 2 mM MgCl
, and 1 mM ATP
for 16-18 h at 4 °C. The mixtures were briefly vortexed and
centrifuged for 15 min at 22,000
g. Protein
concentrations were determined on pellets resuspended in 1% SDS using
the bicinchoninic acid assay (Pierce)). Bovine serum albumin was used
as a standard. The data shown are corrected for F-actin sedimentation
in the absence of added fascin and represent the mean of six
experiments.
Bundle Formation and Electron Microscopy
Either
r-fascin or echinoid fascin was mixed with skeletal muscle F-actin at a
molar ratio (fascin/actin) of 1:6 in 10 mM
NaHPO
, pH 7.0, 100 mM KCl, 2
mM MgCl
, and 1 mM ATP and then incubated
for 72 h at 4 °C. Aliquots (5 µl) of the mixture were applied
to Formvar-coated nickel grids (200 mesh), stained with 0.5% aqueous
uranyl acetate, and viewed at
76,000 in a Philips EM-410
transmission electron microscope. The final actin concentration was 5
µM. Electron cryomicroscopy was done on 5-µl aliquots
that were applied to carbon-coated holey grids and then quick-frozen in
liquid ethane. Specimens were imaged in a Jeol 4000 transmission
electron microscope at 400 kV using a low electron dose and nominal
magnification of
30,000.
Murine Fascin Is Enriched in Brain
The tissue
distribution of murine fascin was determined by Western blot analysis
(Fig. 1). A single immunoreactive species with an apparent
M of 55,000 was seen in all of the analyzed
tissues. Mouse brain contained the greatest amount of immunoreactive
fascin/microgram of protein. Significant amounts of fascin were
detected in uterus, small intestine, and spleen. Northern analysis of
whole RNA extracted from the same tissues gave a single band at 2.7
kilobases; fascin mRNA was also most abundant in brain (data not
shown).
Figure 1:
Tissue distribution of murine
fascin: Western blot analysis. 20 µg of protein from the tissues
shown was separated by 10% SDS-polyacrylamide gel electrophoresis,
blotted onto nitrocellulose, and incubated with a monoclonal anti-human
fascin antibody and a goat anti-mouse secondary antibody. The arrow indicates the position of native fascin at 55 kDa. S.
INT, small intestine; L.INT, large
intestine.
Cloning and Expression of Recombinant Murine
Fascin
Based on the abundance of fascin in brain (Fig. 1),
a fetal mouse brain cDNA library (Stratagene) was screened using a
human fascin cDNA probe. Three primary candidate positive clones
isolated from 5 10
-phage yielded two
secondary positive clones. Restriction analysis revealed that these two
clones were identical. Fig. 2gives the sequence and translation
of murine fascin (GenBank
/EMBL accession number L33726).
The longest open reading frame begins at base pair 90 with a good Kozak
consensus initiation sequence (Kozak, 1987) and terminates at base pair
1569, encoding a peptide of 493 residues. A consensus polyadenylation
sequence (AATAAA) begins at residue 2636. The molecular mass of the
encoded protein is 54,412 Da, with a calculated pI of 6.63. The peptide
sequence of murine fascin was compared with those of the echinoid,
Drosophila, human, murine, and Xenopus fascins
obtained from GenBank
. The overall sequence identity is
17%. 15% of the substitutions are conservative. The murine and human
sequences differ at 24 out of 493 residues.
Figure 2:
Nucleotide and protein sequences of murine
fascin. The cloned cDNA was sequenced using M13mp18 and M13mp19 in both
the 5`- and 3`-directions. The longest open reading frame of 1479
nucleotides runs from positions 90 to 1568 and encodes a predicted
polypeptide of 493 residues. The consensus poly(A) sequence (shown in
boldface) begins at nucleotide 2624. Nucleotides are numbered
in the leftmargin; amino acid residues are numbered
in the rightmargin.
To demonstrate that the
cloned sequence encodes a functional fascin, we expressed mouse fascin
cDNA in E. coli. Early attempts at expression of either fascin
itself or as a glutathione S-transferase fusion protein using
T7-based plasmids produced insoluble material. Expression of a soluble
functional peptide was achieved by cloning the coding region of fascin
in frame behind the E. coli thioredoxin A gene in plasmid
pTRXFUS (LaVallee et al., 1993). The original vector produced
small amounts of soluble material. The level of synthesis was improved
by putting the cDNA encoding the fusion protein under control of a T7
promoter and inducing expression in E. coli BL21 (DE3) cells.
60% of the thioredoxin-fascin fusion protein can be recovered by
hypotonic shock. We have taken advantage of the relative insensitivity
of fascin to chymotrypsin to cleave the fusion protein and to purify
r-fascin. This was done by treating the shockate with
-chymotrypsin at an enzyme/substrate ratio of 1:30 to effect
cleavage. The hydrolysate was then fractionated over DEAE-Sephacryl,
hydroxylapatite, and DEAE-Sephacel to purify r-fascin. Essentially the
same procedure can be used to purify the fusion protein. The results
for the cleaved protein are shown in Fig. 3. Comparison of
lanes1 and 2 in Fig. 3and Western
blot data (not shown) indicate that induction was modest and that
expression from this plasmid is leaky, although no deleterious effects
have been noted on bacterial physiology. Lane3 shows
the protein profile of the shockate. 4 liters of induced culture were
pelleted and used to produce 100 ml of shockate. The degree of
enrichment of several proteins is apparent. The prominent 67-kDa band
is the thioredoxin-fascin fusion protein; the band at 43 kDa is the
E. coli elongation factor Tu protein. Lane4 indicates the effect of treatment with chymotrypsin and
purification on a DEAE-Sephacryl column, while lanes5 and 6 show the extent of purification after
chromatography on hydroxylapatite and DEAE-Sephacel, respectively. The
39,000-Da band in lane6 is a proteolytic
fragment of fascin that begins at Ser
and presumably
extends to the C terminus.
Figure 3:
Purification of recombinant murine fascin.
E. coli BL21 cells were transformed with pMFAST7, grown, and
harvested as described under ``Experimental Procedures.''
Samples of protein from different stages in the purification were
separated on 10% SDS-polyacrylamide gels and stained with Coomassie
Blue. The major band at M 67,000 in lane3 is the thioredoxin-fascin fusion protein. The
M
55,000 band in lanes 4-6 is
cleaved murine fascin. Lane 1, uninduced culture; lane
2, induced culture; lane 3, crude shockate; lane
4, chymotrypsin digest/DEAE-Sephacryl-pooled fractions; lane
5, hydroxylapatite-pooled fractions; lane 6,
sulfopropyl-Sephadex-pooled fractions. The large increase in abundance
of the fusion protein in the shockate ( lane 3) reflects the
selective release of the fusion protein by osmotic
shock.
Purified r-fascin has an apparent
molecular mass of 55 kDa on SDS gels, identical to that observed for
the brain protein. Recombinant fascin was used for N-terminal
microsequencing to define the site of chymotryptic cleavage. The first
10 residues were TANGTAEAVQ, identical to the deduced peptide sequence
if the N-terminal methionine has been cleaved. Echinoid fascin is also
missing its N-terminal Met (Bryan et al.,1993). We
have not attempted to sequence native mouse fascin, but conclude that
purified r-fascin probably corresponds to the native cytoplasmic form
of the molecule with the exception of the changed ratios of isoforms.
Multiple Fascin Isoforms Exist in Vivo and in
Vitro
Two-dimensional gel electrophoresis of mouse brain
extracts revealed the presence of at least four isoelectric variants,
two major and two minor species (Fig. 4, panel 1).
Multiple variants were also seen in r-fascin ( panel 2).
Panel 3 shows the coelectrophoresis of brain and recombinant
fascins. The four variants comigrate, and the results show that the
most alkaline variant is most prominent in r-fascin. The minor spots in
the upper left and lower right corners of panel 3 are the
undigested fusion protein and the 39-kDa proteolytic fragment,
respectively. The results suggest that fascin has been
post-translationally modified to decrease its isoelectric point. This
could occur either by phosphorylation or by modification of amino
groups, e.g. methylation or acetylation of lysine. The results
are consistent with r-fascin being incompletely modified, while most of
the brain protein is altered. All of our attempts to provide support
for the idea that fascin is phosphorylated have been negative. These
efforts included: (i) treatment with acid or alkaline phosphatase,
which did not change the two-dimensional gel pattern; (ii) metabolic
labeling of 3T3 fibroblasts with PO
, which
gave no incorporation of label into fascin; (iii) phosphorylation
trials using purified rat brain protein kinase C (Huang et
al., 1986), which failed to label r-fascin; and (iv) amino acid
analysis of r-fascin, which indicated that only trace amounts of
phosphoamino acids were present. The results of these experiments (data
not shown) suggest that fascin isoforms are not the result of
phosphorylation.
Figure 4:
Two-dimensional electrophoresis of native
and recombinant fascins. 100 µg of murine brain extract ( panel
1) was separated by isoelectric focusing and 10%
SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose,
and developed with anti-fascin antibodies as described in the legend to
Fig. 1. At least four isoelectric variants are present. Similar
patterns of isoelectric variants are seen with 500 ng of recombinant
mouse fascin ( panel 2) and a mixture of 50 µg of brain
extract and 250 ng of recombinant fascin ( panel 3). Note also
the more basic form in panels2 and
3.
Fascin Isoforms Appear to be Generated by
Acetylation
Our inability to demonstrate phosphorylation of
r-fascin in vitro or of native fascin in vivo suggested that fascin might have modified amino groups. To address
this possibility, we subjected r-fascin to acid-urea electrophoresis
(Allis et al., 1979), which separates peptides under acid pH
conditions expected to resolve differences due to peptide acetylation,
but not phosphorylation. As seen in Fig. 5, this two-dimensional
gel system resolves r-fascin into at least three spots, suggesting
their generation by acetylation.
Figure 5:
Acid-urea electrophoresis of r-fascin.
Recombinant fascin was separated in the first dimension on an acid-urea
gel system, which separates acetylation (but not phosphorylation)
isoforms. The lane containing r-fascin was then applied to an
SDS-polyacrylamide gel and separated in the second dimension. The gel
was stained with Coomassie Blue. At least three isoforms are resolved,
suggesting that they vary in the degree of acetylation. PAGE,
polyacrylamide gel electrophoresis.
Recombinant Fascin Produces Highly Ordered Bundles in
Vitro
Recombinant fascin and the thioredoxin-fascin fusion
protein actively bundle F-actin. Bundles were generated at various
actin/fascin ratios by mixing fascin and 3.5 or 7 µM
F-actin and incubating at 4 °C. Bundles were recovered by low speed
centrifugation (22,000 g for 15 min) to separate them
from single filaments. The pelleted material was resuspended in 1% SDS,
and the protein concentration was determined. At low levels of added
fascin, the amount of protein recovered by low speed centrifugation
increased nearly linearly with the amount of added fascin
(Fig. 6). The values are corrected for F-actin pelleted in the
absence of added fascin. The points in the linear range were used to
calculate a regression line. The slope of this line,
4.1, gives
the weight ratio of bundles formed (defined by low speed sedimentation)
per weight of added fascin. Assuming that all of the added fascin is
incorporated into bundles, the slope gives a minimum estimate of the
actin/fascin ratio of 4.1:1. A similar value is observed if the
thioredoxin-fascin fusion protein is used in place of r-fascin.
Figure 6:
Quantitative fascin-actin cosedimentation
analysis. Bundles were produced from various mixtures of actin and
r-fascin and sedimented, and the amount of protein in the bundles was
determined. The results, at two actin concentrations (, 3.5
µM actin;
, 7 µM actin), are plotted
versus the concentration of added fascin. The solidline shows the least-squares linear regression line for
the two sets of data. The slope of the line is 4.1. The dottedlines are the estimated plateaus. The minimum
actin/fascin stoichiometry is estimated at 4.1:1 from the slope of the
regression line assuming that all of the added fascin is incorporated
into bundles. The twoarrows give the estimated
values at saturation determined from the intercepts of the regression
line with the estimated plateau values.
The
structure of the bundles was examined by negative staining and
quick-freezing. Highly ordered bundles were formed over the pH range
6.0-8.5 in 100 mM KCl at 4 °C using a 6:1
actin/fascin ratio (5 µM actin, 0.83 µM
fascin). A negatively stained bundle, formed at pH 7.0 from r-fascin
and rabbit skeletal muscle F-actin, is shown in Fig. 7 a.
A transverse periodicity spaced every 36 nm at an angle of 61° to
the bundle axis is marked (Fig. 7 a,
arrowheads). This periodicity is clearly visible in a
reconstituted vitreous ice-embedded bundle (Fig. 7 b). To
compare the fine structure of bundles formed from mammalian and
invertebrate fascins, sea urchin filopodial cores, which consist almost
entirely of actin and echinoid fascin
(5) , were also negatively
stained (Fig. 7 c) and ice-embedded
(Fig. 7 d). Due to the inherent axial twist in these
bundles, one of two predominant banding patterns is seen, depending on
which orientation of the bundle is observed. Near the right edge of the
filopodial core, a constant 12-nm spacing is visible
(Fig. 7 c, arrowheads). The frozen echinoid
bundle shows pairs of bands 12 nm apart, separated by a spacing of 24
nm (Fig. 7 d, arrowheads). A similar spacing
pattern can be discerned in Fig. 7 c ( arrows).
Figure 7:
Structure of reconstituted fascin-actin
bundles. a, rabbit skeletal muscle F-actin (5 µM)
was incubated with 0.33 µM cleaved r-fascin for 72 h at 4
°C. Aliquots were blotted onto Formvar-coated nickel grids and
stained for 60 s with 0.5% aqueous uranyl acetate The characteristic
36-nm transverse repeat is visible ( arrowheads). b,
alternatively, r-fascin bundles were quick-frozen on carbon-coated
holey grids and viewed without staining or fixation; the same
periodicity is marked ( arrowheads). c, for
comparison, echinoid filopodial cores were negatively stained,
revealing a different transverse repeat of alternating 12- and 24-nm
spacing ( arrows) or of constant 12-nm spacing
( arrowheads). d, filopodial cores were quick-frozen,
showing the same alternating repeats ( arrowheads) as in
c. Bars = 100 nm.
Reconstituted bundles produced with Drosophila fascin and
chicken skeletal muscle F-actin (Cant et al., 1994) appear to
be similar in structure to the fiber bundles found in Drosophila bristles (Overton, 1967). Similarly, early experiments in which
echinoid fascin was mixed with rabbit skeletal muscle actin produced
bundles whose morphology was identical to the echinoid bundles shown in
Fig. 7
( c and d). This suggests that fascin,
not the actin filaments, determines the final bundle structure. To
reinforce this conclusion, we determined the structure of bundles
reconstituted from murine fascin and echinoid F-actin isolated from
P. californicus. The resulting bundles were indistinguishable
from the murine fascin-rabbit actin bundles shown in
Fig. 7
( a and b). From these results, we
conclude that the phenotype of a fascin-actin bundle is determined by
the species of fascin, not actin, utilized.
(
)
abalone sperm acrosomal processes (Shiroya and Sakai,
1993), and more specialized structures like mechanosensory bristles in
Drosophila (Overton, 1967).
-chymotrypsin to cleave the
fusion protein. A chromatographic procedure has been developed to
purify the cleaved fascin, here termed r-fascin. The fusion protein and
purified r-fascin both bundle filaments. Two-dimensional gel
electrophoresis shows that native brain fascin, detected by Western
blotting, has multiple charge isoforms. These same isoforms are found
in r-fascin. The relative abundances indicate that the precursor of the
other isoforms is the most alkaline variant. The present data support
the idea that the isoforms are generated by modification of amino
groups. While is it widely appreciated that eukaryotes modify amino
groups via acetylation (Allis et al., 1979) and glutamylation
(Eddé et al., 1990), it has only recently been
demonstrated that peptides expressed in E. coli may be
acetylated (Violand et al., 1994). The significance of fascin
isoforms is unclear since our preliminary data indicate that the two
major brain isoforms are equally incorporated into bundles.
-chymotryptic digestion (Fig. 3, lane 6) is not
incorporated into bundles with r-fascin and will not produce bundles
when tested alone. Similarly, this fragment does not bind to F-actin.
The importance of the other conserved regions remains to be tested.
7-8% from those given by the Holmes model. It will be
necessary to alter the bundle model to accommodate these newer
parameters, but it is not clear if inclusion of the new parameters will
alter the predicted cross-link/actin ratio or if the model can
accommodate the vertebrate structure.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.