Mutations changing tropomodulin affinity for tropomyosin alter neurite formation and extension

Plasmid construction and mutagenesis

For transfection experiments and expression in Escherichia coli, mouse Tmod2 (accession No. NM_016711) was subcloned into pReceiver-M55 with mCherry tag and into pReceiver-B01 with His-tag (Genecopoeia, Rockville, MD), respectively. The plasmid for mouse Tmod1 (accession No. NM_21883) subcloned into pEGFP-C1 was a generous gift from Dr. Carol Gregorio (Arizona State University) previously used in (Tsukada et al., 2011). The GFP-Tmod1 plasmid was used both for PC12 transfection and as a template for mutagenesis. The pET(His)Tmod1 plasmid (Kostyukova et al., 2000) was used for Tmod1 expression in E. coli and as a template for mutagenesis. Site-directed mutagenesis was performed using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The plasmids were amplified by PCR according to the manufacturer’s instructions with the modification described in (Tsukada et al., 2011) using Pfu Ultra Hotstart DNA polymerase (Agilent) and two complementary sets of oligonucleotides, which contain changed triplets.

To change Ala 21 to Lys in chicken or mouse Tmod1, the sets of oligonucleotides were 5′-GAA GAC AAG ATC CTC GGA AAG CTG ACG GAG GAA GAG CTC-3′ and 5′-GAG CTC TTC CTC CGT CAG CTT TCC GAG GAT CTT GTC TTC-3′; or 5′-GAG GAT GAA ATC CTG GGG AAG CTC ACA GAG GAG GAG C-3′ and 5′-GCT CCT CCT CTG TGA GCT TCC CCA GGA TTT CAT CCT C-3′, respectively.

Then once a single mutation was introduced and confirmed for each plasmid, the singly-mutated plasmid was then used as a template to introduce the second mutation.

To change Glu 33 to Val in chicken or mouse Tmod1, the sets of oligonucleotides were: 5′-CTC AGG AAG TTG GAG AAC GTG CTG GAA GAG CTG GAC-3′ and 5′-GTC CAG CTC TTC CAG CAC GTT CTC CAA CTT CCT GAG-3′, or 5′-CTG AGG ACG CTG GAA AAT GTG CTA GAT GAA CTA GAC-3′ and 5′-GTC TAG TTC A TCT AGC ACA TTT TCC AGC GTC CTC AG-3′, respectively.

After PCR, the original plasmid was digested using DpnI. The digest was used to transform E. coli maximum-efficiency DH5α (Invitrogen). Cells were grown in the presence of 100 mg/L carbenicillin for all plasmids except the one for GFP-Tmod1, which was grown in the presence of 50 mg/L kanamycin. After plasmid purification (using Qiagen mini-prep kit), the presence of mutations was confirmed by DNA sequencing. Synthesis of all oligonucleotides was done by Integrated DNA Technologies Inc. (Coralville, Iowa), and DNA sequencing was done by Genewiz (South Plainfield, NJ).

Tmod purification

Tmod1 (both wild type and mutant) was overexpressed in E. coli BL21 (DE3) pLysE while Tmod2 was overexpressed in E. coli BL21 (DE3). Auto-inducible ZYP-5052 media (Studier, 2005) containing 100 mg/L carbenicillin (plus 50 mg/L chloramphenicol for E. coli BL21 (DE3) pLysE) was inoculated and cells were grown for 12–15 hours at 37 °C. Cells were harvested by centrifugation at 8,000 rpm (Sorvall SLA-3000 Rotor), 4 °C, for 10 min. Pellets were re-suspended in 20 mM Tris-HCL, pH 7.0, 100 mM NaCl, containing a protease inhibitor cocktail (Roche), containing 1 mM pefabloc, and 1 mM tosyl-L-lysine chloromethyl ketone (TLCK). Re-suspended pellets were sonicated for 10 min on ice. The homogenized solution was then centrifuged at 20,000 rpm (Sorvall SA-300 Rotor), 4 °C, for 20 minutes and the supernatant was loaded onto a Superflow Ni-NTA agarose column (Qiagen) equilibrated with 50 mM Na-phosphate, pH 6.8, containing 10 mM imidazole. Once the protein was loaded, the column was washed with 50 mM Na-phosphate, pH 6.8, containing 10 mM imidazole and 5 mM β-mercaptoethanol. Proteins were eluted by a 50–250 mM imidazole gradient in the same buffer. Fractions containing Tmod were combined, dialyzed overnight against 20 mM Tris-HCl, pH 8, containing 1 mM EDTA and 1 mM DTT, and loaded on an anion-exchange column Poros HQ/L (PerSeptive Biosystems) using an FPLC system (Pharmacia). Proteins were eluted by a 12–25% (for Tmod1) or 10-30% (for Tmod2) NaCl gradient. Fractions containing Tmod were combined and dialyzed against 20 mM Tris-HCl, pH 8, containing 1mM EDTA and 1mM DTT. Protein purity was evaluated using SDS-PAGE.

Peptides

N-acetylated TM peptides (αTm1bzip, γTm1bzip, and δTm1bzip) were synthesized by the Tufts University Core Facility (Boston, MA). Chimeric TM peptides designed for structural and functional studies (Greenfield et al., 2001; Kostyukova, 2007; Meshcheryakov et al., 2011) contain 19 N-terminal residues of short α-, γ-, or δ-TM respectively, encoded by exon 1b. To stabilize the coiled-coil structure, each peptide contained an additional 18 C-terminal residues of the GCN4 leucine Zipper domain. Quality of the synthetic peptides was confirmed using mass spectroscopy; molecular weights of the peptides were the same as the predicted ones.

Isoelectric points of TM peptides were calculated using protparam http://web.expasy.org/protparam/. Concentrations of proteins and peptides were determined both by using the BCA protein assay kit (Pierce) and by measuring their difference spectra in 6 M guanidine-HCl between pH 12.5 and 7.0, using the extinction coefficients of 2357 per tyrosine and 830 per tryptophan at 294 nm as in (Kostyukova, Hitchcock-Degregori & Greenfield, 2007). Standard deviations of concentrations determined by the two methods were 20% for Tmod2 and between 4 and 16% for TM peptides.

Binding experiments

Binding was detected using native gel electrophoresis in 9% polyacrylamide gels that were polymerized in the presence of 10% glycerol without SDS. To prepare complexes for loading onto gels, stock solutions of 12 µM Tmods were mixed with TM peptides in a 1:2 molar ratio in 20 mM Tris-HCl, pH 8.0, containing 1 mM DTT and 1 mM EDTA. Presence ( ≤ 100 mM) or absence of NaCl did not affect binding results. Sample loading buffer (125 mM Tris-HCl, pH 6.8, 40% glycerol, 0.01% bromophenol blue) was added to the samples to a Tmod final concentration of 7.5 µM. Running buffer for electrophoresis contained 25 mM Tris-HCl and 192 mM glycine. For titration, TM peptides were added at different ratios (up to 3:1) to the stock solution of Tmod2. Samples were analyzed by staining native polyacrylamide gels with Coomassie R-250 and quantified using a ChemiDoc XRS+ with Image Lab Software (BioRad). Density of the complex bands was normalized in order to average results of several titrations.

Cell culture and imaging

Undifferentiated PC12 cells were grown in DMEM (Invitrogen) supplemented with 5% fetal bovine serum (FBS, Invitrogen) and 10% horse serum (HS, Invitrogen), and were sub-cultured every three days. For neuronal differentiation, PC12 cells were grown on 100 µg/ml poly-D-lysine–coated 8-well plates or 35-mm culture dishes (ibidi LLC) in DMEM supplemented with 0.5% FBS, 1% HS, and 100 ng/ml nerve growth factor (NGF, invitrogen). PC12 cells were transfected with GFP-Tmod1 WT, GFP-Tmod1 A21K/E33V, mChFP-Tmod2, or GFP-actin using lipofectamin 2000 (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours after transfection, neuronal differentiation was induced and neurite outgrowth was monitored on day 3 and day 6 on a laser scanning confocal microscope LSM710 (Zeiss) with a 63x oil immersion lens (Zeiss). Images were acquired with Zen software (Zeiss) and the number and length of neurites were analyzed in Imaris software (Bitplane). Statistical analysis was performed by One-way ANOVA.

A21K and E33V mutations in Tmod1 cause decrease in neurite length

Mutagenesis of Ala21 to Lys and Glu33 to Val in the Tmod1 fragment, Tmod1s1, increased the affinity of this fragment for TM peptides (Uversky et al., 2011). Tmod1s1 corresponded to the TM-binding site 1 of Tmod1 (res. 1-38). TM chimeric peptides, γTM1bzip and δTM1bzip, contained the N-terminal sequences of short TM isoforms TM5NM1 and TM4, respectively. To study the effects of this changed affinity on neurite formation, these mutations were introduced in full-length GFP-Tmod1. The mutant was expressed in PC12 cells, a model system for neuronal differentiation (Greene & Tischler, 1976) and functional assessment of neurite outgrowth (Guillaud et al., 1998), to test its effect on neurite formation and extension.

We first checked expression of GFP-Tmod1 and mChFP-Tmod2 in PC12 cells. Both GFP-Tmod1 and mChFP-Tmod2 were successfully expressed and localized in both the cell body and neuronal extensions in differentiated PC12 cells (Fig. 1A); however, mChFP-Tmod2 appeared be more restricted to the shaft of the neurites, while GFP-Tmod1 localized in the shaft and at the very end of the growth cones (Fig. 2A).

We next observed the effect of GFP-Tmod1 and mChFP-Tmod2 overexpression on neurite formation and outgrowth of PC12 cells, three and six days after NGF induction. Overexpression of GFP-Tmod1 did not affect neuronal differentiation when compared with overexpression of GFP alone (data not shown), but overexpression of mChFP-Tmod2 impaired neuronal differentiation of PC12 cells (Figs. 1A and 1B). PC12 cells expressing GFP-Tmod1 showed proper neurite outgrowth after three and six days of NGF-induced neuronal differentiation. On the other hand, PC12 cells expressing mChFP-Tmod2 showed a significant reduction in the number (4-fold) and the length (3 to 10-fold) of neurites (Figs. 1C and 1D).

Additionally, we co-expressed GFP-Tmod1 and mChFP-Tmod1 in PC12 cells and checked their effect on neuronal differentiation. Interestingly, co-expression of Tmod1 and Tmod2 did not impair NGF-induced neuronal differentiation after three days (Fig. 2A). The number (3.1 ± 0.6, n = 20) and length (61.3 ± 8.3 µm, n = 20) of neurites were similar to those observed in Tmod1 over-expressing cells (Figs. 1C and 1D). Tmod2 did not localize with actin filaments in the tips of neurites of PC12 cells over-expressing mChFP-Tmod2 and GFP-actin (Fig. 2B).

We further checked the effect of A21K and E33V mutations in GFP-Tmod1 on PC12 cell’s neuronal differentiation. PC12 cells overexpressing GFP-Tmod1[A21K/E33V] did not exhibit a significant decrease in the number of neurites per cell; however, the average length of neurites decreased approximately 2 to 3-fold (Figs. 1A–1C). Interestingly, GFP-Tmod1[A21K/E33V] shared a similar localization pattern with mChFP-Tmod2 (data not shown).

Effect of the mutations in full-length Tmod1 on binding TM isoforms

Experiments that demonstrated increased Tmod1 binding to short non-muscle TM isoforms were performed using synthetic TM and Tmod peptides (Uversky et al., 2011). Residues 7–14 of short TMs form the binding site for Tmod (Vera et al., 2000). Therefore the data obtained for TM peptides, which contain 19 N-terminal residues, may be extrapolated to full-length TMs. Tmods contain two TM-binding sites; changes in one site may or may not affect TM-binding properties of the full-length molecule.

To explore the effect of the changes on full-length Tmod1, A21K and E33V mutations were introduced into full-length, His-tagged Tmod1. TM-binding properties of purified mutant Tmod1[A21K/E33V] and wild-type Tmod1 and Tmod2 were analyzed using native gel-electrophoresis (Fig. 3). Three TM peptides, αTM1bzip, γTM1bzip and δTM1bzip, were used in this experiment. All of them contained TM N-terminal sequences encoded by exon 1b of α, γ, and δ-genes, respectively. Tmods alone and mixed with TM peptides were loaded on native gels. TM peptides cannot be seen on native gels; they are positively charged and do not enter a gel unless they are in a complex with Tmod (isoelectric points of αTM1bzip, γTM1bzip and δTM1bzip are 9.98, 9.82, and 9.82, respectively). Binding positively charged TM peptides decreased Tmod mobility in gels; therefore, a shift of bands corresponding to the formed complexes was observed.

Compact bands were formed for the mixtures of both Tmod1 and Tmod2 with αTM1bzip, (Fig. 3, lane 2) indicating formation of a stable complex. Tmod1 has been shown to bind an αTM1bzip peptide at both sites (Kostyukova, Choy & Rapp, 2006). Although Tmod1 also has two binding sites for γTM1bzip, formation of a complex with only one γTM1bzip peptide bound to site 2 can be detected using native gel-electrophoresis (Kostyukova, Hitchcock-Degregori & Greenfield, 2007). Binding one peptide instead of two resulted in higher electrophoretic mobility of the Tmod1/γTM1bzip band on the gel, and resulted from the weak affinity of γTM1bzip to site 1. The similar position of the band for the Tmod1/δTM1bzip complex, indicated that only one δTM1bzip peptide was bound to Tmod1 under these conditions (Fig. 3A, lane 4). A smear, which appeared under the Tmod1/γTM1bzip and Tmod1/δTM1bzip bands, can be explained by complex dissociation during electrophoresis, indicating weaker affinities of Tmod1 with these TM isoforms (Fig. 4A, lanes 3–4).

Mutations A21K and E33V were designed based on previous data (Uversky et al., 2011) to enhance interaction of Tmod1 with short γ- and δ-TMs in site 1. With these mutations, we expected to see a band with the mobility of the band formed by Tmod1 and αTM1bzip. However, we saw no change of mobility for the complexes of Tmod1[A21K/E33V] with γTM1bzip and δTM1bzip (Fig. 4B, lanes 3, 4). Unexpectedly, rather than a single band, for the complex of Tmod1[A21K/E33V] with αTM1bzip, an additional lower band appeared (Fig. 3B, lane 2). The usual band representing a complex of Tmod1[A21K/E33V] with two αTM1bzip was also observed. The increased mobility of the additional band indicated formation of a complex with one TM peptide. Therefore, these mutations decreased the affinity of Tmod1 for the αTM1bzip in site 1, but did not affect the formation of the complex of Tmod1 with the two other TM peptides at least that can be detected by native gel-electrophoresis.

The mobilities of the Tmod2/γTM1bzip and Tmod2/δTM1bzip bands were slightly greater than the mobility of Tmod2/αTM1bzip (Fig. 3C). The difference may be explained either by the slightly higher isoelectric point of αTM1bzip or by binding only one peptide.

Titration of Tmod2 by TM peptides

To find out how many TM molecules bind to Tmod2, Tmod2 was titrated with αTM1bzip, γTM1bzip, and δTM1bzip (Fig. 4). In all cases, the Tmod2 band disappeared completely at a 1:1 molar TM/Tmod ratio, indicating that at this ratio all Tmod2 molecules were in a complex with one TM peptide. Complex formation between Tmod2 and αTM1bzip was different than with the two other peptides. The intensity of the Tmod2/αTM1bzip band continued to increase gradually, reaching maximum at a 2:1 ratio. It was more difficult to understand if the intensity of complex bands for Tmod2/γTM1bzip and Tmod2/δTM1bzip also continued to increase after disappearance of Tmod2 band. To clarify this, the change of the complexes in the mixture was monitored by scanning and quantifying the complex bands in native gels. The normalized density of the complexes is shown in Fig. 5 as a function of the TM/Tmod molar ratio. All curves demonstrate saturation at a 2:1 ratio. This result may be explained if binding of Tmod2 to all peptides was not cooperative and all Tmod2 molecules bind one TM peptide first, in a site that has a higher affinity; then a second peptide starts to bind. The change in the position of the complex band also confirms this explanation. Mobility of the complex band at a 1:1 ratio is slightly higher than at a 2:1 ratio (Fig. 4).