©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of a Murine Fascin Homolog from Mouse Brain (*)

Robert A. Edwards (§) , Haydee Herrera-Sosa , Joann Otto (1), Joseph Bryan

From the (1) Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47909

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

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.

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 NaHPO, 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.


RESULTS

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.


DISCUSSION

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,() abalone sperm acrosomal processes (Shiroya and Sakai, 1993), and more specialized structures like mechanosensory bristles in Drosophila (Overton, 1967).

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 -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.

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 -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.

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 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.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-4996; Fax: 713-798-7799.

The abbreviation used is: r-fascin, recombinant fascin.

R. A. Edwards and J. Bryan, unpublished data.


ACKNOWLEDGEMENTS

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.


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