Calcium binding is essential for plastin 3 function in Smn-deficient motoneurons

Abstract

The actin-binding and bundling protein, plastin 3 (PLS3), was identified as a protective modifier of spinal muscular atrophy (SMA) in some patient populations and as a disease modifier in animal models of SMA. How it functions in this process, however, is not known. Because PLS3 is an actin-binding/bundling protein, we hypothesized it would likely act via modification of the actin cytoskeleton in axons and neuromuscular junctions to protect motoneurons in SMA. To test this, we examined the ability of other known actin cytoskeleton organizing proteins to modify motor axon outgrowth phenotypes in an smn morphant zebrafish model of SMA. While PLS3 can fully compensate for low levels of smn, cofilin 1, profilin 2 and α-actinin 1 did not affect smn morphant motor axon outgrowth. To determine how PLS3 functions in SMA, we generated deletion constructs of conserved PLS3 structural domains. The EF hands were essential for PLS3 rescue of smn morphant phenotypes, and mutation of the Ca(2+)-binding residues within the EF hands resulted in a complete loss of PLS3 rescue. These results indicate that Ca(2+) regulation is essential for the function of PLS3 in motor axons. Remarkably, PLS3 mutants lacking both actin-binding domains were still able to rescue motor axons in smn morphants, although not as well as full-length PLS3. Therefore, PLS3 function in this process may have an actin-independent component.

Figure 1.

Expression of PLS3 rescues motor axon defects in smn morphants. Representative images of ventral axons in 28 hpf Tg(mnx1:0.6hsp70:EGFP) embryos injected with (A) control MO, (B) smn MO and (C) smn MO with 250 pg PLS3 mRNA. (D) Quantitation and statistical analysis of the motor axon defects using a Mann–Whitney non-parametric rank test. The arrows in (A)–(C) indicate individual motor axons. ns, non-significant; ***P ≤ 0.001.

Figure 2.

Actin-binding proteins are unable to rescue motor axon defects in smn morphants. (A) Structural motifs in actin-binding protein constructs. PLS3 contains N-terminal EF hand motifs, and two C-terminal actin-binding domains, each composed of two calponin homology domains. αACTN1 is structurally similar to PLS3, with an N-terminal ABD, composed of two calponin homology domains, and two C-terminal EF hand motifs. αACTN1 also has central spectrin (S) repeat domains. CFL1 is composed of one actin depolymerization factor domain, and PFN2 is composed of a profilin domain. All constructs had V5 and 6xHis C-terminal tags. Constructs are drawn to scale. (B) Co-injection of smn MO with RNA encoding actin-binding proteins. (C) Table depicting statistical significance determined by Mann–Whitney analysis of distribution between injection conditions. Significance is set at *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ns indicates not significant.

Figure 3.

PLS2, but not PLS1, rescues motor axon defects in smn morphants. (A) Co-injection of smn MO with plastin family members. PLS2 and PLS1 rescue was tested at 250 and 500 pg (B) Table showing statistical significance between injection conditions as determined by Mann–Whitney analysis. PLS2 and PLS1 statistics are both shown at 500 pg doses.

Figure 4.

Schematic diagram of human PLS3 deletion constructs. PLS3 is a 630 amino acid protein with several evolutionarily conserved structural motifs. The full-length (FL) construct contains a nuclear export signal that overlaps with an N-terminal EF hand motif. The C-terminus contains a pair of actin-binding domains, ABD1 and ABD2, each composed of two calponin homology (CH) domains. All constructs have V5 and 6xHis tags at the C-terminus.

Figure 5.

EF hands are required for PLS3 function in smn morphant motor axons. (A) Motor axon defects observed in smn morphants with 250 pg injection of PLS3 C-terminal deletions: Δvim, ΔABD2, ΔCH2/ABD2 and ΔABD1/2 mRNA (B) Motor axon defects observed in smn morphants with 250 pg injection of PLS3 N-terminal deletions: ΔNES and ΔEF hands mRNA (C) Table showing statistical significance between injection conditions as determined by Mann–Whitney analysis. (D) Western blots from 10 and 24 hpf embryos injected with RNA from PLS3 constructs at the one-cell stage as detected by V5 expression. β-Actin is used as a loading control. Owing to its small size, ABD1/2 was run separately on a 15% polyacrylamide gel, whereas all other constructs were run on a 7.5% gel.

Figure 6.

Immunofluorescence of PLS3 colocalization with filamentous actin in HEK293 cells. PLS3 constructs are stained with antibody to C-terminal V5 tag (red). Filamentous actin is labeled with AlexaFluor488-phalloidin conjugate (green). DAPI (blue) labels the nucleus, (A and B) empty pDEST40 vector, (C and D) FL PLS3, (E and F) FL PLS3 pDEST40 (labeled only with secondary antibody, control for staining specificity), (G and H) ΔNES, (I and J) ΔEF hands, (K and L) Δvimentin interact, (M and N) ΔABD2, and (O and P) ΔABD1/2. All imaging was performed with the same laser intensity.

Figure 7.

Ca²⁺ binding to EF hands is required for PLS3 function in smn-depleted motor axons. (A) Schematic depicting PLS3 constructs containing point mutations for Ca²⁺ binding residues in the EF hands of PLS3. EF mut 1 contains the following point mutations: D25A, N27A, N29A, F31A, C33A, E36A. EF mut 2 contains D65A, N67A, D69A, K71A, S73A, E76A. EF mut 1 + 2 contains all of the mutations mentioned. (B) Motor axon defects observed in smn morphants with 250 pg mRNA injection of EF hand point mutants: EF mut 1, EF mut 2 and EF mut 1 + 2. (C) Statistical table depicting significance between injection groups as determined by Mann–Whitney analysis.

Figure 8.

Ca²⁺ inhibits actin-bundling activity of PLS3, but has no effect on actin binding. (A) Binding of 2 μm of PLS3, EF mut 1 + 2, ΔEF hands and 30 μm of ΔABD1/2 to 5 μm of F-actin in the presence of different concentrations of free Ca²⁺ was assessed by the high speed co-sedimentation assay (200 000g for 30 min) followed by SDS–PAGE analysis of pellet and supernatant fractions. Pellets (p) contain F-actin and bound PLS proteins; supernatants (s) contain unbound PLS. Sedimentation of PLS3 and its mutants in the absence of actin is shown as a control on the left. The positions of wild-type PLS3, EF mut 1 + 2, and ΔEF hands and actin are marked on the right. The experiment was conducted under different pCa²⁺ 8.5, 7.7, 6.4, 5.3, 5.0, 4.6 and 3.8; but only pCa²⁺ 6.4–4.6 range is presented in (A). (B) The amounts of PLS3 proteins bound to actin from three independent experiments were expressed as the per cent of the corresponding total PLS3 protein (combined PLS3 in the supernatant and pellet) and shown on the graph. Error bars, SEM. (C) Time course of change in light scattering intensity of 5 μm F-actin solution was recorded by spectrofluorometer with emission and excitation wavelengths set at 330 nm. Addition of PLS3 (2 μm) to F-actin increased light scattering of the sample reflecting bundling of actin filaments by PLS3. Subsequent addition of CaCl₂ (0.1 mm free Ca²⁺, pCa²⁺ 4.0) reduced the signal to a nearly initial level. (D) Bundling of F-actin (5 μm) by PLS3, EF mut 1 + 2, ΔEF hands, and ΔABD (2, 2, 2, and 30 μm, respectively) in buffers with various concentrations of Ca²⁺ was assessed by low-speed sedimentation (17 000g for 15 min) followed by the SDS–PAGE analysis. Under these sedimentation conditions, F-actin bundles were found in the pellet fraction (p), whereas unbundled actin filaments remained in the supernatant (s). Sedimentation of F-actin alone is shown on the left as a control for unbundled actin. (E) Amount of bundled F-actin in pellet was expressed as a per cent of total actin (combined supernatant and pellet) and shown on the graph. Error bars, SEM (n = 3).

Figure 9.

Summary of PLS3 functional activity. Full-length PLS3, ΔABD1/2, ΔEF hands, and EF mut 1 + 2 are described in terms of their ability to rescue of smn morphant motor axon defects, bind and bundle actin, bind Ca²⁺, and whether actin-bundling activity is Ca²⁺ regulated.