An analysis of the primary structure of the actin-binding protein fesselin revealed it to be the avian homologue of mammalian synaptopodin 2 [Schroeter, Beall, Heid, and Chalovich (2008) Biochem. Biophys. Res. Commun. 371, 582–586]. We isolated two synaptopodin 2 isoforms from rabbit stomach that corresponded to known types of human synaptopodin 2. The purification scheme used was that developed for avian fesselin. These synaptopodin 2 forms shared several key functions with fesselin. Both avian fesselin and mammalian synaptopodin 2 bound to Ca2+–calmodulin, α-actinin and smooth-muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca2+–calmodulin-dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of α-actinin, to polymerize actin in a Ca2+–calmodulin-dependent manner, or to bind to Ca2+–calmodulin or myosin. These properties are consistent with the proposed function of synaptopodin 2 in organizing the cytoskeleton.

INTRODUCTION

Fesselin is an actin-binding protein [1] that is enriched in avian smooth muscle and that is natively unfolded [2]. A key property of fesselin is that it binds to both G-actin and to F-actin [1]. Binding to G-actin nucleates polymerization [3]. Ca2+–calmodulin inhibits this stimulation of nucleation [4] and can further reduce the rate of polymerization below that of pure actin. Binding to F-actin results in the formation of large aggregates. Fesselin also inhibits F-actin activation of myosin S1 ATPase activity [5]. The interaction of fesselin with F-actin is apparently unaffected by calmodulin. Fesselin binds to other muscle proteins, including myosin and α-actinin. The binding to α-actinin and the effects on actin suggest to us that fesselin is important in organizing cellular actin. This hypothesis is supported by the presence of fesselin in smooth-muscle dense bodies [6].

Previously, we have shown that fesselin has a similar primary structure to other synaptopodin family members and that it is an avian homologue of synaptopodin 2 [7]. (Note: human synaptopodin 2 is thought to have at least five splice forms at the mRNA level. Myopodin [8] and genethonin 2 [9] are synonyms of synaptopodin 2. We use the name synaptopodin 2 for the rabbit protein isolated in the present study.) However, unlike other members of the synaptopodin family, avian fesselin is most abundant in smooth muscle. Thus it is possible that avian fesselin has unique properties that give it a special role in smooth muscle. The interaction of fesselin with target proteins has been examined in some detail in solution. However, those same measurements have not been practical with rabbit synaptopodin 2, because of the difficulty in isolating that protein.

We have developed a procedure for preparing milligram amounts of two fesselin homologue proteins from mammalian smooth-muscle tissue. The sequences of these proteins showed that they are identical to isoforms of mammalian synaptopodin 2 and share 60% identity with avian fesselin. The isolated mammalian smooth-muscle synaptopodin 2 isoforms stimulated actin polymerization in a Ca2+–calmodulin-dependent manner. These proteins also bound to α-actinin, myosin and Ca2+–calmodulin, indicating that they are functionally similar to fesselin.

The results of the present study show that synaptopodin 2 has a role in smooth muscle. Both fesselin and mammalian smooth-muscle synaptopodin 2 have properties consistent with a role in organizing actin filaments. This role is consistent with the available data for other members of the synaptopodin family.

Parts of this work were presented the 50th Annual Biophysical Society Meeting held in Salt Lake City, UT, U.S.A., in February 2006, and at the European Muscle Conference held in Heidelberg, Germany, in September 2006.

MATERIALS AND METHODS

Protein preparations

Turkey gizzard fesselin was purified as described by Leinweber et al. [1]. Rabbit muscle actin was purified using a modification [10] of an established method [11]. Calmodulin was prepared from wheat germ as described by Anderson [12], and was coupled to Affi-Gel 10 resin (Bio-Rad) according to the manufacturer's instructions. Recombinant (Arabidopsis) calmodulin (pRZ72; a gift from Dr Ray Zielinski, Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL, U.S.A.) was expressed in Escherichia coli BL21 cells and prepared as described by Pedigo and Shea [13]. Smooth-muscle myosin was prepared as described by Persechini and Hartshorne [14] from turkey gizzards. α-Actinin was purified from turkey gizzards according to the method of Feramisco and Burridge [15].

Purification of a mammalian fesselin homologue

Purification of the fesselin homologue from rabbit stomach (PelFreez, Rogers, AR, U.S.A.) was performed by modifying our standard fesselin preparation [1] in the following ways: the initial ammonium sulfate was brought only to 20% saturation instead of 30% saturation, as described for the avian protein, and for the final purification, gel-filtration chromatography on a Superose 6™ column (GE Healthcare Life Science) was introduced.

Protein sequencing

Sequencing was performed by Edman degradation [16]. Proteins were separated on SDS gels and transferred on to a Sequi-Blot™ PVDF Membrane (Bio-Rad) in a tank blot apparatus [17]. To obtain specific peptide fragments, the protein was digested with trypsin directly on the PVDF membrane [18]. The peptides obtained were separated by HPLC with a Hypersil C18 BDS (3 μm) LC-Packings column (150 mm×1.0 mm; BIA, Bensheim, Germany) and a 130 A HPLC separation system (Applied Biosystems, Weiterstadt, Germany) with 0.1% trifluoroacetic acid as solvent A and 80% acetonitrile in 0.085% trifluoroacetic acid as solvent B. The HPLC-separated fragments were sequenced on polybrene-treated filters, using a Procise 494 A protein sequencer (Applied Biosystems).

MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight MS) fingerprint analysis was performed by Dr Monica Linder (Department of Biochemistry, Medical School, Justus-Liebig University, Giessen, Germany). Proteins were precipitated with 8 vols acetone at −20°C and then washed 3 times with acetone (−20°C) prior to shipping. Peptides were identified using the programs Mascot Search Results (Matrix Science, Boston, MA, U.S.A.) and ProteinProspector v4.0.8 (http://prospector.ucsf.edu/).

Binding of synaptopodin 2 and fesselin to immobilized Ca2+–calmodulin

Protein samples from the penultimate purification step of the rabbit stomach preparation were dialysed against 25 mM Tris/HCl (pH 7.5), 1 mM CaCl2, 100 mM NaCl, 1 mM MgCl2 and 2 mM DTT (dithiothreitol), and loaded on to an Affi-Gel 10 (Bio-Rad) calmodulin-affinity column, which was equilibrated with the same buffer. The column was washed with Ca2+-containing buffer until the absorbance reached baseline and than eluted by replacing Ca2+ by EGTA. Eluted proteins were analysed by SDS gel electrophoresis.

Kinetics of synaptopodin 2 and fesselin binding to Ca2+–calmodulin

Association rates were measured by rapidly mixing MIANS [2-(4′-maleimidoanilino)naphthalene-6-sulfonic acid]-labelled Arabidopsis calmodulin with rabbit synaptopodin 2. Dissociation rates were measured by displacing MIANS–calmodulin with a 20-fold excess of unlabelled calmodulin. In both cases the rapid fluorescence changes were measured using an SX20 stopped-flow spectrofluorimeter (Applied Photophysics, Leatherhead, U.K.). Excitation was set at 320 nm with a monochromator using 1 mm slits. Fluorescence emission was measured using a GG395 long-pass filter (Schott) with a cut on midpoint of 395 nm. Binding was measured at 15°C.

Binding of synaptopodin 2 and fesselin to myosin and α-actinin by sedimentation assays

Smooth-muscle myosin was mixed with various concentrations of fesselin or the homologous rabbit protein in a solution having an ionic strength of 510 mM and a final volume of 0.1 ml. The mixtures were dialysed overnight against 30 mM NaCl, 10 mM Mops (pH 7.0), 5 mM MgCl2, 1 mM EGTA and 1.5 mM DTT. ATP was added to give a final concentration of 1 mM and the samples were immediately centrifuged for 5 min at 19000 rev./min (TLA120.1 rotor, Beckman) at 10°C. Pellets were swollen on ice in 5 μl of 0.5 M NaCl, diluted with the same volume of water and 10 μl of 2× SDS loading buffer. After sonication for 5 min, the samples were heated (95°C) and 40% of the total volume was subjected to SDS/PAGE (8% gel).

For measuring binding to α-actinin, the purified avian and mammalian proteins were mixed with various concentrations of α-actinin in buffer containing 100 mM potassium propionate, 10 mM imidazole (pH 7.0), 2 mM MgCl2 and 1.5 mM DTT in a total volume of 0.2 ml. Following incubation at room temperature (20°C) for 1 h, the samples were centrifuged for 1 h at 35000 rev./min (TLA120.1 rotor, Beckman) at 20°C. Pellets were swollen in 50 μl of 0.1 M NaCl on ice for 1 h, diluted with 50 μl of 2× SDS loading buffer and subjected to SDS/PAGE (8–16% gradient gel).

Binding of synaptopodin 2 and fesselin to F-actin and F-actin cross-linking

Binding was measured by co-sedimentation assays using actin-binding proteins that had been freshly dialysed against 10 mM imidazole (pH 7.0), 100 mM NaCl and 1 mM DTT using a final volume of 0.25 ml. Following sedimentation of actin–ligand complexes, 20% of the pellets were analysed by SDS/PAGE (12% gel). Details of the assay are described elsewhere [4]. Cross-linking assays were run in the same manner, except that centrifugation was carried out at 10000g for 10 min at 20°C, so that only large complexes would sediment.

Actin polymerization assays

Polymerization was measured by the increase in fluorescence of pyrene-labelled actin, as described by Cooper and Pollard [19]. Pyrene-labelled F-actin was depolymerized by dialysis against G-actin buffer [0.2 mM ATP, 2 mM Tris/HCl (pH 7.8), 0.2 mM CaCl2, 0.5 mM 2-mercaptoethanol and 0.005% NaN3], as described by Pardee and Spudich [20]. Residual F-actin was removed by centrifugation at 45000 rev./min (50 Ti rotor, Beckman) for 1 h at 4°C. The G-actin concentration was determined by measuring A290 using a molar absorption coefficient of 0.638 ml/mg per cm and a molecular mass of 42 kDa.

Prior to initiation of polymerization, the G-actin was converted from the Ca2+-bound form into the Mg2+-bound form by incubation with 125 mM EGTA and 50 mM MgCl2 for 5 min [21]. Polymerization was initiated by adding 0.45 ml of 100 mM NaCl, together with other effectors (i.e. fesselin, isolated rabbit proteins and Ca2+–calmodulin) to solutions containing 5 μM G-actin (10% pyrene-labelled), 0.2 mM ATP, 0.12 mM CaCl2, 5.4 mM Tris/HCl (pH 7.8), 4.5 mM imidazole (pH 7.0) and 1.35 mM DTT. Actin polymerization assays were run in 1 ml total volumes. Fluorescence measurements were made on a Fluorolog 2 spectrofluorimeter (Spex) at 25°C with excitation at 365 nm and emission at 407 nm using 0.5 mm slits.

RESULTS

Isolation and characterization of fesselin homologous protein from rabbit stomach

Using the protocol established for isolating fesselin from turkey gizzard, we isolated two polypeptide bands from rabbit stomach tissue that migrated on an SDS gel with molecular masses of approx. 80 and 70 kDa. Figure 1(A) shows the elution profile of the rabbit protein from a gel-filtration column. Protein of 90–96% purity, as shown in Figure 1(A) (lanes b and c) was used in independent experiments to determine the functions of the fesselin homologous rabbit proteins.

Characterization of the major proteins obtained by applying the turkey fesselin protocol to rabbit stomach tissue

Figure 1
Characterization of the major proteins obtained by applying the turkey fesselin protocol to rabbit stomach tissue

(A) Coomassie blue-stained SDS gel showing the major proteins isolated from a Superose 6™ gel-filtration column. The arrows indicate the fesselin homologous rabbit proteins. Fractions shown in lanes b, c and d were used to characterize rabbit fesselin. To determine the effects of the impurities, we performed independent assays with each of these fractions. (B) Western blot of a two-dimensional gel of the isolated rabbit homologue using affinity-purified anti-fesselin antibody that was raised against gel-excised fesselin purified from turkey. (C) Western blot of a two-dimensional gel of a mixture of the purified turkey and rabbit proteins. Rf values of the proteins were used to identify turkey fesselin (continuous arrows) and the rabbit homologue (broken arrows). Conditions used in (B) and (C) were: first dimension, non-equilibrium isoelectric focusing, pH gradient 3–10, stabilized at pH 8–10; second dimension, SDS gel (12%).

Figure 1
Characterization of the major proteins obtained by applying the turkey fesselin protocol to rabbit stomach tissue

(A) Coomassie blue-stained SDS gel showing the major proteins isolated from a Superose 6™ gel-filtration column. The arrows indicate the fesselin homologous rabbit proteins. Fractions shown in lanes b, c and d were used to characterize rabbit fesselin. To determine the effects of the impurities, we performed independent assays with each of these fractions. (B) Western blot of a two-dimensional gel of the isolated rabbit homologue using affinity-purified anti-fesselin antibody that was raised against gel-excised fesselin purified from turkey. (C) Western blot of a two-dimensional gel of a mixture of the purified turkey and rabbit proteins. Rf values of the proteins were used to identify turkey fesselin (continuous arrows) and the rabbit homologue (broken arrows). Conditions used in (B) and (C) were: first dimension, non-equilibrium isoelectric focusing, pH gradient 3–10, stabilized at pH 8–10; second dimension, SDS gel (12%).

As we have shown previously [1], fesselin has a high isoelectric point. Figure 1(B) shows the Western blot of a two-dimensional gel of the two major isolated rabbit proteins. Both the major 80 kDa and minor 70 kDa bands reacted with an affinity-purified polyclonal anti-fesselin antibody. The relative charges of the rabbit and turkey polypeptides were determined by two-dimensional analyses of mixtures of the two proteins. Figure 1(C) shows that the two rabbit protein chains (broken arrows) had the same migration during isoelectric focusing as fesselin. Therefore both proteins had isoelectric points of approx. 9.3. The rabbit proteins were distinguished from fesselin by their relative mobility on an SDS gel.

Determination of the amino acid sequence of isolated rabbit proteins

Tryptic digests of the purified rabbit proteins produced four peptides that were shown by Edman degradation to be homologous to the predicted sequence of mammalian synaptopodin 2 (Table 1). Because of the high incidence of proline residues, we were unable to utilize the Edman degradation procedure to obtain the complete protein sequence. We performed a MALDI–TOF-MS fingerprint analysis on 25 additional peptides from the upper polypeptide band and 19 peptides from the lower polypeptide band, as shown in Table 2. All sequences obtained by MALDI–TOF-MS matched the predicted human synaptopodin 2 isoforms. All detected fragments were in the sequence boundaries of the synaptopodin 2 splice form (accession no. CAB51856). This analysis covered over 30% of the myopodin sequence (accession no. CAB51856), and verified that the isolated rabbit protein was rabbit synaptopodin 2. We call the purified rabbit stomach protein synaptopodin 2 throughout the remainder of the present study.

Table 1
Peptides of the isolated rabbit stomach protein determined by Edman degradation

Degradation was carried out with the protease trypsin. The numbers correspond to the amino acid residues in human myopodin (accession no. CAB51856); underlined residues are tentative.

Peptide alignmentResidues
RSSTKPMFTFK 428–438 
KIAQPAYP-AR 545–555 
KGPQAAVASQN567–579 
KYVVDSDTVQAH 623–634 
Peptide alignmentResidues
RSSTKPMFTFK 428–438 
KIAQPAYP-AR 545–555 
KGPQAAVASQN567–579 
KYVVDSDTVQAH 623–634 
Table 2
Results of the different polypeptides from the MALDI–TOF MS fingerprint analysis

Degradation was carried out with the protease trypsin.

Polypeptide formPeptide alignment with human myopodin (accession no. CAB51856) (residues)
80 kDa DHSRPHK (82–88)*; MEMLPDTTGKGALMKAK (219–235); ERMDQITAQK (238–247); MDQITAQK (240–247); TTTSYQR (270–276); KEEESVR (277–283); TAKPFPGSVNQPATPFSPTR (326–345); IASRDER (405–411); TGILQEAK (419–426); TGILQEAKR (419–427); STTKPMFTFK (429–438); STTKPMFTFKEPK (429–441); VSPNPELLSLLQNSEGK(442–458); VSPNPELLSLLQNSEGKR (442–459); TPPPVAPKPAVK (493–504); IAQPSYPPARPASTLNVAGPFK (546–567); YVVDSDTVQAHAAR (624–637); AQSPTPSLPASWK (638–650); SQPSAAQPSK (678–687); GKKPLNALDVMK (693–704); KPLNALDVMK (695–704); HQPYQLNASLFTFQPPDAK (705–723); DGLPQKSSVK (724–733); KSGVTIQVWKPSVVEE (816-831); SGVTIQVWKPSVVEE (817–831) 
70 kDa MEMLPDTTGKGALMKAK (219–235); TTTSYQR (270–276); TAKPFPGSVNQPATPFSPTR (326–345); TGILQEAK (419–426); TGILQEAKR (419–427); STTKPMFTFK (429–438); STTKPMFTFKEPK (429–441); VSPNPELLSLLQNSEGK (442–458); VSPNPELLSLLQNSEGKR (442–459); TPPPVAPKPAVK (493–504); YVVDSDTVQAHAAR (624–637); AQSPTPSLPASWK (638–650); SQPSAAQPSK (678–687); GKKPLNALDVMK (693–704); KPLNALDVMK (695–704); HQPYQLNASLFTFQPPDAK (705–723); DGLPQKSSVK (724–733); KSGVTIQVWKPSVVEE (816–831); SGVTIQVWKPSVVEE (817–831) 
Polypeptide formPeptide alignment with human myopodin (accession no. CAB51856) (residues)
80 kDa DHSRPHK (82–88)*; MEMLPDTTGKGALMKAK (219–235); ERMDQITAQK (238–247); MDQITAQK (240–247); TTTSYQR (270–276); KEEESVR (277–283); TAKPFPGSVNQPATPFSPTR (326–345); IASRDER (405–411); TGILQEAK (419–426); TGILQEAKR (419–427); STTKPMFTFK (429–438); STTKPMFTFKEPK (429–441); VSPNPELLSLLQNSEGK(442–458); VSPNPELLSLLQNSEGKR (442–459); TPPPVAPKPAVK (493–504); IAQPSYPPARPASTLNVAGPFK (546–567); YVVDSDTVQAHAAR (624–637); AQSPTPSLPASWK (638–650); SQPSAAQPSK (678–687); GKKPLNALDVMK (693–704); KPLNALDVMK (695–704); HQPYQLNASLFTFQPPDAK (705–723); DGLPQKSSVK (724–733); KSGVTIQVWKPSVVEE (816-831); SGVTIQVWKPSVVEE (817–831) 
70 kDa MEMLPDTTGKGALMKAK (219–235); TTTSYQR (270–276); TAKPFPGSVNQPATPFSPTR (326–345); TGILQEAK (419–426); TGILQEAKR (419–427); STTKPMFTFK (429–438); STTKPMFTFKEPK (429–441); VSPNPELLSLLQNSEGK (442–458); VSPNPELLSLLQNSEGKR (442–459); TPPPVAPKPAVK (493–504); YVVDSDTVQAHAAR (624–637); AQSPTPSLPASWK (638–650); SQPSAAQPSK (678–687); GKKPLNALDVMK (693–704); KPLNALDVMK (695–704); HQPYQLNASLFTFQPPDAK (705–723); DGLPQKSSVK (724–733); KSGVTIQVWKPSVVEE (816–831); SGVTIQVWKPSVVEE (817–831) 
*

Unique for the higher molecular-mass polypeptide.

Binding of synaptopodin 2 and fesselin to Ca2+–calmodulin, α-actinin and smooth-muscle myosin

Ca2+–calmodulin binds tightly to avian fesselin and alters the interaction of fesselin with G-actin [4]. Figure 2(A) shows that rabbit synaptopodin 2 bound to a calmodulin-affinity column in the presence of Ca2+ and was eluted by chelating the free Ca2+ with EGTA.

Binding of synaptopodin 2 to Ca2+–calmodulin

Figure 2
Binding of synaptopodin 2 to Ca2+–calmodulin

(A) SDS gel showing binding of proteins from an intermediate step in the purification to a calmodulin-affinity column. The wash fraction of a Ca2+ buffer at 118 mM ionic strength (lane a) contains no fesselin-like proteins. Proteins migrating in a similar manner to fesselin were eluted by the same buffer containing EGTA (lanes b and c). (B and C) Kinetics of rabbit synaptopodin 2 binding to MIANS-labelled Arabidopsis calmodulin. Binding was measured on a stopped-flow fluorimeter at 15°C. The buffer conditions were 200 mM KCl, 10 mM Mops (pH 7.0), 2 mM MgCl2 and 0.5 mM CaCl2. Data are: (B) association and (C) dissociation. In order to measure binding we used 0.164 μM rabbit synaptopodin 2 and 0.05 μM MIANS-labelled calmodulin (final concentration). Dissociation was measured by displacing MIANS–calmodulin with unlabelled calmodulin from the rabbit protein. The concentrations used were 0.324 μM rabbit synaptopodin 2, 0.1 μM MIANS-labelled calmodulin, and 10 μM unlabelled calmodulin (concentrations before mixing).

Figure 2
Binding of synaptopodin 2 to Ca2+–calmodulin

(A) SDS gel showing binding of proteins from an intermediate step in the purification to a calmodulin-affinity column. The wash fraction of a Ca2+ buffer at 118 mM ionic strength (lane a) contains no fesselin-like proteins. Proteins migrating in a similar manner to fesselin were eluted by the same buffer containing EGTA (lanes b and c). (B and C) Kinetics of rabbit synaptopodin 2 binding to MIANS-labelled Arabidopsis calmodulin. Binding was measured on a stopped-flow fluorimeter at 15°C. The buffer conditions were 200 mM KCl, 10 mM Mops (pH 7.0), 2 mM MgCl2 and 0.5 mM CaCl2. Data are: (B) association and (C) dissociation. In order to measure binding we used 0.164 μM rabbit synaptopodin 2 and 0.05 μM MIANS-labelled calmodulin (final concentration). Dissociation was measured by displacing MIANS–calmodulin with unlabelled calmodulin from the rabbit protein. The concentrations used were 0.324 μM rabbit synaptopodin 2, 0.1 μM MIANS-labelled calmodulin, and 10 μM unlabelled calmodulin (concentrations before mixing).

Because many proteins bind to Ca2+–calmodulin, we measured the kinetics of binding and dissociation of synaptopodin 2 as another measure of the functional similarity to fesselin. Figure 2(B) shows the rate of displacement of bound MIANS–calmodulin following rapid mixing with an excess of unlabelled calmodulin. Mammalian synaptopodin 2 dissociated from MIANS–calmodulin with a t1/2 of 3.8 ms, which is the same as that observed previously for turkey gizzard fesselin [4].

Fesselin binds to α-actinin and forms complexes that can be isolated by sedimentation [22]. Figure 3(A) shows a comparison of synaptopodin 2 with fesselin in sedimentation assays to access their relative abilities to bind to α-actinin. Neither fesselin (lane 1) nor synaptopodin 2 (lane 5) sedimented appreciably by themselves under the conditions used. However, both fesselin (lanes 2–4) and synaptopodin 2 (lanes 6–8) formed sediments in the presence of α-actinin. In the case of turkey fesselin, neither fesselin nor α-actinin were seen in the supernatant with 0.5 μM α-actinin (Western blot; results not shown). That is, complete binding occurred at a free α-actinin concentration of approx. 0.1 μM. This is consistent with high affinity (approx. 4×107 M−1), as reported previously for this complex [22]. It was possible to detect α-actinin in the supernatants at concentrations of 1 and 1.5 μM α-actinin, indicating that the fesselin sites were saturated by 0.5 μM α-actinin. No rabbit synaptopodin 2 was detected by anti-fesselin antibodies in any of the supernatants of the synaptopodin 2/α-actinin mixtures; this is consistent with an affinity >107 M−1.

Binding of synaptopodin 2 and fesselin to α-actinin and smooth-muscle myosin

Figure 3
Binding of synaptopodin 2 and fesselin to α-actinin and smooth-muscle myosin

(A) Co-sedimentation assay of smooth-muscle α-actinin with constant amounts of fesselin (1 μM) or rabbit synaptopodin 2 (0.4 μM). Protein pellets after a sedimentation assay of both proteins were separated on an 8–16% polyacrylamide-gradient SDS gel. The samples were ultracentrifuged for 1 h at 35000 rev./min (TLA120.1 rotor, Beckman). Lanes 1–4: fesselin with 0, 0.5, 1 and 1.5 μM α-actinin respectively. Lanes 5–8: rabbit synaptopodin 2 with 0, 0.5, 1 and 1.5 μM α-actinin. Lanes 9–11: sedimentation of 0.5, 1 and 1.5 μM α-actinin by itself. α-Actinin migrates with an apparent molecular mass of 100 kDa and is indistinguishable from the upper fesselin polypeptide band (103 kDa), but it is readily distinguished from rabbit synaptopodin 2. (B) Electrophoretic analysis of protein pellets (8% polyacrylamide SDS gel) from turkey fesselin and synaptopodin 2 after co-sedimentation with smooth-muscle myosin. Broken arrow, synaptopodin 2; continuous arrows, fesselin. Lanes 1–3: 1 μM myosin and 1 μM, 2 μM or no avian fesselin respectively. Lanes 4–6: 1 μM myosin and 0.4 μM, 0.8 μM or no rabbit synaptopodin 2 respectively. Lane 7: 0.4 μM synaptopodin 2 without myosin.

Figure 3
Binding of synaptopodin 2 and fesselin to α-actinin and smooth-muscle myosin

(A) Co-sedimentation assay of smooth-muscle α-actinin with constant amounts of fesselin (1 μM) or rabbit synaptopodin 2 (0.4 μM). Protein pellets after a sedimentation assay of both proteins were separated on an 8–16% polyacrylamide-gradient SDS gel. The samples were ultracentrifuged for 1 h at 35000 rev./min (TLA120.1 rotor, Beckman). Lanes 1–4: fesselin with 0, 0.5, 1 and 1.5 μM α-actinin respectively. Lanes 5–8: rabbit synaptopodin 2 with 0, 0.5, 1 and 1.5 μM α-actinin. Lanes 9–11: sedimentation of 0.5, 1 and 1.5 μM α-actinin by itself. α-Actinin migrates with an apparent molecular mass of 100 kDa and is indistinguishable from the upper fesselin polypeptide band (103 kDa), but it is readily distinguished from rabbit synaptopodin 2. (B) Electrophoretic analysis of protein pellets (8% polyacrylamide SDS gel) from turkey fesselin and synaptopodin 2 after co-sedimentation with smooth-muscle myosin. Broken arrow, synaptopodin 2; continuous arrows, fesselin. Lanes 1–3: 1 μM myosin and 1 μM, 2 μM or no avian fesselin respectively. Lanes 4–6: 1 μM myosin and 0.4 μM, 0.8 μM or no rabbit synaptopodin 2 respectively. Lane 7: 0.4 μM synaptopodin 2 without myosin.

Fesselin also binds to myosin-forming complexes that can be isolated by sedimentation [5]. We compared synaptopodin 2 with fesselin for binding to turkey gizzard myosin using a sedimentation assay. Figure 3(B) shows an SDS gel of the pellets of one such assay. Both avian fesselin and rabbit synaptopodin 2 sedimented in the presence, but not in the absence, of myosin. We probed the supernatant lanes of the gels with antibodies directed against fesselin (results not shown). No immunoreaction was observed in the supernatant following sedimentation of a solution composed of 1 μM myosin and either 1 μM fesselin or 0.4 μM synaptopodin 2. However, both ligands were observed in supernatants when fesselin was increased to 2 μM or mammalian synaptopodin 2 was increased to 0.8 μM. The fact that free synaptopodin 2 was observed at a total concentration of 0.8 μM, whereas no free fesselin was observed at a total fesselin concentration of 1 μM, indicates that synaptopodin 2 binds less tightly to myosin than does fesselin. Assuming that we could detect 10% of the proteins in the supernatants with the anti-fesselin antibodies, the affinity of myosin for synaptopodin 2 is approx. 5×106 M−1.

F-actin cross-linking and binding

Upon mixing either fesselin or synaptopodin 2 with actin, the solutions became turbid, as expected in the case of cross-linking. Low-speed sedimentation of the mixtures produced pellets that were analysed by SDS gel electrophoresis (Figure 4A). The pellets contained actin and either fesselin or synaptopodin 2. Western blotting showed that rabbit synaptopodin 2, which had co-sedimented with actin, cross-reacted with affinity-purified anti-fesselin polyclonal antibodies. We could not detect free synaptopodin 2 following low-speed centrifugation (Figure 4A, lanes 8 and 9) by Western blot analysis. Because synaptopodin 2 induces actin polymerization at sub-stoichiometric concentrations (see below), we could not estimate the free actin concentration nor the affinity of synaptopodin 2.

Cross-linking and binding of F-actin by synaptopodin 2 and fesselin

Figure 4
Cross-linking and binding of F-actin by synaptopodin 2 and fesselin

(A) Avian fesselin and mammalian synaptopodin 2 cross-link F-actin independently of Ca2+–calmodulin (CaM). 5 μM G-actin, 0.2 μM avian fesselin or rabbit synaptopodin 2, and either 0 or 1 μM calmodulin, were mixed in a buffer containing 5.4 mM Tris/HCl (pH 7.8), 4.5 mM imidazole (pH 7.0), 0.2 mM ATP, 45 mM NaCl, 0.12 mM CaCl2 and 1.35 mM DTT. After a 60 min incubation, samples were centrifuged 10 min at low speed (10000 g; L). The resulting supernatants were afterwards subjected to high-speed sedimentation for 60 min at 100000 g (H). The pellets (P) from the low-speed and high-speed sedimentation, as well as the remaining supernatants (SN), were subjected to electrophoresis on 12% polyacrylamide SDS gels, transferred on to nitrocellulose and probed with an affinity-purified anti-fesselin antibody. (B) Binding of turkey fesselin and rabbit synaptopodin 2 to F-actin in the absence and presence of Ca2+–calmodulin. Analysis of the pellets from a high-speed sedimentation assay (60 min at 100000 g) on a 12% polyacrylamide SDS gel. Protein concentrations were: F-actin, 5 μM; avian fesselin, 0.5 μM; rabbit synaptopodin 2, 0.5 μM; Ca2+–calmodulin, 5 μM. The final buffer conditions were 20 mM NaCl, 80 mM potassium propionate, 10 mM imidazole, 1 mM DTT and 0.2 mM CaCl2.

Figure 4
Cross-linking and binding of F-actin by synaptopodin 2 and fesselin

(A) Avian fesselin and mammalian synaptopodin 2 cross-link F-actin independently of Ca2+–calmodulin (CaM). 5 μM G-actin, 0.2 μM avian fesselin or rabbit synaptopodin 2, and either 0 or 1 μM calmodulin, were mixed in a buffer containing 5.4 mM Tris/HCl (pH 7.8), 4.5 mM imidazole (pH 7.0), 0.2 mM ATP, 45 mM NaCl, 0.12 mM CaCl2 and 1.35 mM DTT. After a 60 min incubation, samples were centrifuged 10 min at low speed (10000 g; L). The resulting supernatants were afterwards subjected to high-speed sedimentation for 60 min at 100000 g (H). The pellets (P) from the low-speed and high-speed sedimentation, as well as the remaining supernatants (SN), were subjected to electrophoresis on 12% polyacrylamide SDS gels, transferred on to nitrocellulose and probed with an affinity-purified anti-fesselin antibody. (B) Binding of turkey fesselin and rabbit synaptopodin 2 to F-actin in the absence and presence of Ca2+–calmodulin. Analysis of the pellets from a high-speed sedimentation assay (60 min at 100000 g) on a 12% polyacrylamide SDS gel. Protein concentrations were: F-actin, 5 μM; avian fesselin, 0.5 μM; rabbit synaptopodin 2, 0.5 μM; Ca2+–calmodulin, 5 μM. The final buffer conditions were 20 mM NaCl, 80 mM potassium propionate, 10 mM imidazole, 1 mM DTT and 0.2 mM CaCl2.

Ca2+–calmodulin did not affect actin cross-linking by synaptopodin 2. Again, neither fesselin (results not shown) nor rabbit synaptopodin 2 were detected in the supernatants. This places a lower limit on the affinity of the calmodulin-Ca2+-bound proteins to actin of 106 M−1. We observed that cross-linking could occur when either fesselin or synaptopodin 2 were added to either G- or F-actin. This is a positive indication that mammalian synaptopodin 2 shares another important feature with fesselin, the ability to polymerize actin.

In a separate experiment, the actin-binding proteins were mixed directly with F-actin and sedimented at high-centrifugal force. Figure 4(B) shows an SDS gel of pellets following centrifugation. Neither protein sedimented in the absence of actin. Similar amounts of both fesselin and synaptopodin 2 bound to F-actin in the absence and presence of Ca2+–calmodulin. The affinity of synaptopodin 2 for actin must be similar to that of fesselin, or approx. 3×106 M−1 [1].

Fesselin and rabbit synaptopodin 2 induced actin polymerization under the regulation of Ca2+–calmodulin

Another characteristic feature of fesselin is its ability to polymerize actin [3] in a Ca2+–calmodulin-dependent manner [4]. Figure 5 shows the polymerization of 5 μM actin under three different conditions. In the absence of other proteins, actin polymerization is characterized by a lag phase, followed by a slow elongation phase (Figure 5, curve 3). The addition of 80 nM synaptopodin 2 eliminated the lag phase and produced a significantly accelerated polymerization (Figure 5, curve 1). Ca2+–calmodulin reduced the acceleration of polymerization caused by synaptopodin 2. This is the same pattern that we observed previously for fesselin [4], as shown in Figure 5 (inset).

Actin polymerization induced by synaptopodin 2 under the regulation of Ca2+–calmodulin

Figure 5
Actin polymerization induced by synaptopodin 2 under the regulation of Ca2+–calmodulin

G-actin (5 μM; 10% pyrene-labelled) was polymerized by 0.08 μM rabbit synaptopodin 2. Reactions were initiated by adding NaCl (45 mM final concentration), together with the purified turkey or rabbit proteins in the presence or absence of 1 μM Ca2+–calmodulin. Synaptopodin-2-induced actin polymerization (1), synaptopodin-2-induced actin polymerization under Ca2+–calmodulin regulation (2) and salt-induced actin polymerization (control) (3). Inset: actin polymerization induced by 0.2 μM fesselin (4), in presence of Ca2+–calmodulin (5) and salt-induced actin polymerization (control) (6). The axes scales of the inset are identical to that of the main Figure.

Figure 5
Actin polymerization induced by synaptopodin 2 under the regulation of Ca2+–calmodulin

G-actin (5 μM; 10% pyrene-labelled) was polymerized by 0.08 μM rabbit synaptopodin 2. Reactions were initiated by adding NaCl (45 mM final concentration), together with the purified turkey or rabbit proteins in the presence or absence of 1 μM Ca2+–calmodulin. Synaptopodin-2-induced actin polymerization (1), synaptopodin-2-induced actin polymerization under Ca2+–calmodulin regulation (2) and salt-induced actin polymerization (control) (3). Inset: actin polymerization induced by 0.2 μM fesselin (4), in presence of Ca2+–calmodulin (5) and salt-induced actin polymerization (control) (6). The axes scales of the inset are identical to that of the main Figure.

DISCUSSION

The synaptopodin family of proteins is interesting because of its association with human disorders. Genethonin 2 was first observed as a down-regulated gene in Duchenne muscular dystrophy patients [9]. The function of the gene product was unknown. Shortly afterwards, Weins et al. [8] described a novel actin-binding protein, myopodin, that was discovered to be the gene expression product of genethonin 2 and a member of a subgroup of the synaptopodin family [8]. The distribution of myopodin is dependent on stress and differentiation. In undifferentiated muscle cells myopodin is located in the nucleus, where it induces the formation of actin loops. During myotube differentiation myopodin binds to stress fibres. In differentiated myocytes myopodin is incorporated into Z-lines [8]. These observations show that myopodin is closely associated with cellular actin in various structural configurations.

We have recently demonstrated that fesselin is the avian form of synaptopodin 2 [7]. Fesselin differs from mammalian synaptopodin 2 in that it is most heavily expressed in smooth muscle. This difference raised the possibility that avian fesselin and mammalian synaptopodin 2 could differ in some key properties. Synaptopodin 2 has not been studied extensively in solution, probably because of difficulties in extracting and purifying this protein. We purified two proteins from rabbit smooth muscle and showed them to be homologous to synaptopodin 2. We further showed that the rabbit synaptopodin 2 has properties that were very similar to those reported previously [1,35,22] for avian fesselin.

The purification of mammalian synaptopodin 2 was accomplished with a minor modification to our procedure for fesselin purification. By reducing the ammonium sulfate saturation from 30% to 20%, we were able to eliminate several proteins that were difficult to remove by other methods. The higher purity of synaptopodin came at the cost of a decreased yield. The purified synaptopodin 2 polypeptides migrated as a doublet in SDS gel electrophoresis. The synaptopodin 2 polypeptides had similar isoelectric points to fesselin polypeptides. In Western blots, affinity-purified polyclonal anti-fesselin antibodies cross-reacted with the isolated proteins.

The MS and Edman sequence determinations of the isolated proteins verified that they were mammalian synaptopodin 2. The smaller polypeptide chain was most similar to the myopodin isoform, containing 698 amino acid residues, as described by Lin et al. [23]. The predicted molecular mass of this smaller myopodin is 74.7 kDa. This is in good agreement with the molecular mass of 70 kDa observed on SDS gels. Furthermore, the MALDI–TOF-MS fingerprint analysis of the smaller polypeptide gave sequences that were all in the boundaries of the ORF (open reading frame) of the 74.7 kDa myopodin (accession no. EAW73659).

The larger polypeptide was likely to be identical with the predicted ORF of 831 residues for human myopodin (accession no. CAB51856), as described by Weins et al. [8]. The prediction of the larger myopodin gives a molecular mass of 89.3 kDa. On SDS gels the mobility of the higher molecular polypeptide was consistent with a molecular mass of 80 kDa. This is the same electrophoretic mobility that was observed by Weins et al. [8]. Furthermore we detected a small N-terminal fragment (82–88 residues) (accession no. CAB51856) that was unique for the larger polypeptide. The slight discrepancies between predicted molecular masses and observed migration on SDS gels for both isolated polyeptides may be due to species differences, post-translational modifications or proteolytic degradation.

Our results indicate that at least two different synaptopodin 2 isoforms are expressed in stomach smooth-muscle tissue. Both detected isoforms are supported by cDNAs for human myopodin as observed previously [8,23].

In addition to the two isoforms that are described above, three mRNAs have been isolated, having ORFs coding for larger myopodin isoforms (accession nos. Q9UMS6, EU481975 and NP597734). More recently, De Ganck et al. [24] described the in vitro amplification and heterologous expression of these myopodins. The extended N-termini of these recombinant proteins contain PDZ domains with unknown functions. To date, no natural myopodins, including this domain, have been detected. The major differences among theses three PDZ-containing myopodin isoforms are the exons used to code for the C-termini [24]. The importance of these differences is unknown.

Properties of synaptopodin 2

Having confirmed that we had isolated mammalian synaptopodin 2, we proceeded to examine its biochemical properties. Mammalian synaptopodin 2 bound to F-actin, with an affinity similar to that reported for fesselin [1]. The binding of synaptopodin 2 to F-actin resulted in the formation of large complexes or bundles that were sedimented at a low speed. Similar to fesselin [4], synaptopodin 2 stimulated the rate of actin polymerization, and that rate enhancement was inhibited by Ca2+–calmodulin. We verified that synaptopodin 2 bound directly to calmodulin. Because of the regulatory significance of calmodulin binding, we measured the rate of dissociation of Ca2+–calmodulin from synaptopodin 2. The displacement of MIANS-labelled Ca2+–calmodulin from mammalian synaptopodin 2 occurred with the same rate as observed previously for fesselin [4].

The binding of synaptopodin 2 to cellular actin is a key feature of synaptopodin 2 function. Although we have established some key properties of purified native synaptopodin 2, some of these same properties have been shown in cellular systems. Myopodin was localized to the actin filaments and to Z-disks of myocytes [8]. Overexpression of myopodin or microinjection of recombinant myopodin resulted in cross-linking of actin filaments in myocytes [8]. The founding member of the family, synaptopodin, stimulates α-actinin-induced polymerization of actin in podocytes [25]. We now found that α-actinin was not required for synaptopodin-2-stimulated actin polymerization in solution. There are no previous reports of synaptopodin 2 or myopodin binding to Ca2+–calmodulin, although such an activity was reported for synaptopodin [26,27]. No other reports of Ca2+–calmodulin regulation of a synaptopodin family member exist. The only report of regulation of synaptopodin 2 function is the regulation of nuclear shuttling by phosphorylation synaptopodin 2 [28,29].

We show here that rabbit smooth-muscle synaptopodin 2 binds directly to α-actinin, with an affinity similar to that of fesselin [22]. As we described previously for fesselin [22], the binding of synaptopodin 2 to α-actinin results in the formation of a large complex that can be isolated by centrifugation. Myopodin was shown previously to co-localize with α-actinin and immunoprecipitate with α-actinin [8,28]. Furthermore, synaptopodin binds to brain and kidney isotype-specific forms of α-actinin [25].

Although no member of the synaptopodin family has been shown to bind to myosin, we observed binding of rabbit synaptopodin 2 to gizzard smooth-muscle myosin. Binding appears to be weaker than that observed for avian fesselin, but more detailed binding studies are required to confirm this.

The establishment of fesselin as a synaptopodin family member, and the ability to purify cellular fesselin and synaptopodin 2, create opportunities for understanding the cellular function of these proteins. Of particular importance is the tumour-suppressive function of myopodin [30,31], which appears to be a deterrent to tumour growth and invasion [31]. Reduced myopodin expression [30], reduced nuclear myopodin concentrations [30] and deletion of the myopodin gene [23] have been found in diverse cancers.

Abbreviations

     
  • DTT

    dithiothreitol

  •  
  • MALDI–TOF-MS

    matrix-assisted laser-desorption ionization–time-of-flight MS

  •  
  • MIANS

    2-(4′-maleimidoanilino)naphthalene-6-sulfonic acid

  •  
  • ORF

    open reading frame

We thank Dr Ray Zielinski (Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL, U.S.A.) for providing the Arabidopsis calmodulin clone. This work was supported by grant AR035216 from the National Institutes of Health to J.M.C.

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