Characterization of α-actinin from Neurospora crassa

Over-expression of α-actinin and α-actinin domains in a GST system and antibody production The GST fusion proteins constructed were found to vary in their level of expression and solubility. In general, both expression and solubility increased when the culture time was prolonged at low temperature (25°C). The full length α-actinin-GST protein, containing the N-terminal extension and characteristic domains (Figure 6B, Material and Methods), was used as an immunogen to produce specific antibodies against N. crassa α-actinin. The resulting polyclonal antibody was named anti-Neurospora α-actinin and has been used for several biochemical analyses described in Publications II and IV and in this part of this work.

Immunochemical detection of proteins reacting with anti-Neurospora α-actinin antibody

The anti-Neurospora α-actinin antibody reacted against α-actinin from different ascomicetous fungi.

In N. crassa the antibody detected an 80 kDa peptide corresponding in molecular weight to the protein containing the two N-terminal CH-domains, the rod domain and C-terminal EF-hand motifs (Figure 13; Fig. 1B, lane d in Publication II). In Magnapothe grisea the antibody detected a 72 kDa peptide also corresponding to a protein with the specific α-actinin domains (Figure 13; Fig. 1B, lane e in Publication II). The antibody was highly reactive against the 80 kDa α-actinin protein from a Botrytis cinerea crude extract (Figure 13). In Phytophthora infestans an immunoreacting band with anti-Neurospora α-actinin antibody was not obtained under the test conditions used (Fig. 1B, lane f in Publication II).

We also tested the antibody against the three over-expressed fragments of the N. crassa α-actinin, corresponding to the Actin-Binding Domain (ABD), the rod domain and the Ca²⁺-binding domain. The antibody showed poor affinity for ABD (approaching the background signal). The principal epitopes for the anti-Neurospora α-actinin antibody seem to be in the rod and Ca²⁺-binding domains (Figure 14).

Characterization of Neurospora α-actinin properties

The actin- and calcium-binding properties of Neurospora α-actinin have been described in detail in Publication IV. The results show that calcium binds to α-actinin from N. crassa with the same affinity as the α-actinin from chicken gizzard. Since we used recombinant GST-α-actinin for this binding assay, the GST protein was used as a negative control and shows that this tag does not have affinity for calcium (Fig. 4 in Publication IV). A co-sedimentation assay and electron microscopy were used to analyze the actin-binding properties of Neurospora α-actinin. This protein binds actin in a

calcium-dependent manner (Fig. 2 in Publication IV) and cross-links the actin microfilaments to organize them in parallel structures as bundles (Fig. 3B in Publication IV).

Localization of α-actinin in N. crassa

Neurospora α-actinin clearly localized within the septum (Fig. 5C-D and Fig. 7A-B in Publication IV;

Figure 15). Results obtained from the in vivo localization revealed that the α-actinin is only present at this location during septum formation (Figure 15; Fig. 7 in Publication IV). Immunofluorescence and GFP signal were found at the germination site in the conidia and in the tip of the emerging tube (Fig.

5A and Fig. 6A–C in Publication IV). α-Actinin immunolocalizes at the peripheral region of the cell in growing hyphae (Fig. 5B in Publication IV). This localization was not confirmed by the α-actinin GFP fluorescence.

Phenotype of the heterokaryon α-actinin knock-out strain of Neurospora

We approached the Neurospora Genome project (Colot et al., 2006) to create an α-actinin knock-out strain. The heterokaryon knock-out strain was deposited in the FGSC. The homokaryon strain was defined as lethal. The heterokaryon mutant in Davis and De Serres medium (1970) showed a shortening of aerial hyphae (Fig. 8A in Publication IV) and a delay in colony expansion in Vogel 2%

saccharose plates in comparison to the wild type (Figure 16). The morphology of the hyphae was different with respect to their branching pattern, showing a predominantly dichotomous phenotype compared to sympodial branching in the wild type (Fig. 8B in Publication IV).

Immunochemical characterization of “α-actinin-GFP-expressing” and “heterokaryon α-actinin knock-out” strains of N. crassa

We examined the crude extract of the α-actinin-GFP-expressing and wild type (wt) Neurospora strains with the anti-Neurospora α-actinin antibody. This antibody reacted with the 80 kDa peptide both in the wild type and the transformed strains (Figure 17, lanes a', b'), but additional bands appear at 110 and 140 kDa in the transformed strain (Figure 17, lanes b'). In an attempt to clarify the identity of these two bands we used a commercial anti-GFP antibody, which reacted in this same crude extract with the 110 and 140 kDa bands (Figure 17, lane d’). In order to confirm the identity of these two bands as the recombinant GFP-fusion proteins proposed in Figure 17, it will be necessary to perform sequence analysis.

In a crude extract from the heterokaryon α-actinin knock-out strain of N. crassa, the anti-Neurospora α-actinin antibody gives a faint signal that could reflect its reduced gene copy number when compared with wild type (Figure 17, lane c').

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Figure 13. Immunoblot of α-actinin from different ascomycetous fungi. (Lane a) N. crassa;

(lane b) M. grisea; (lane c) B. cinerea.

Figure 14. Immunoreactivity of anti-Neurospora α-actinin antibody with the different domains of α-actinin. (Lane a) full-length GST-α-actinin; (lane b) GST-Actin-binding domain;

(lane c) GST-rod domain; (lane d) GST-Calcium-binding domain. (Arrowhead shows the position of the GST-Actin- binding domain).

Figure 15. Confocal images of germinating conidia expressing α-actininGFP showing its localization during the septa formation. The GFP signal appears at the site of septum formation and disappear following completion of the septum after 10 min. (Arrow: septum). Bar 5 μm.

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Figure 16. Growth on solid medium of the Neurospora heterokaryon α-actinin knock-out strain compared to the wild type.

80 kDa 110 kDa GFP

140 kDa GFP

a b c a’ b’ c’ d d' 170

kDa

116

76

Figure 17. Anti-Neurospora α-actinin immunoreacting peptides in the heterokaryon α-actinin knock-out and α-α-actinin-GFP containing strains compared to the wild type.

Immunoblot of the protein revealed with anti-Neurosporaα-actinin (left panel) and anti-GFP ( right panel, lane d’) antibodies. Lanes a-a’ corresponds to the wild type strain; lanes b-b’, to the α-actinin-GFP containing strain and c-c’ to the heterokaryonα-actinin knock-out strain; lane d-d’ is the same as b-b’.

Immunolocalization of α-actinin and actin in Botrytis cinerea

N. crassa was the principal model in this work for the characterization of α-actinin. The study of theis protein in other fungi could give us more insights into the role of this actin-binding protein in fungi.

As the Western analysis of the whole crude extract proteins from B. cinerea resulted in a strong reacting band with the anti-Neurospora α-actinin antibody, we have performed the in situ localization of the α-actinin in this ascomycetous fungus. After 12 h of growth the cultures were in the exponential growth phase. In this phase α-actinin was localized throughout the cytoplasm and concentrated in the septum and tip region (Figure 18A–D). Actin principally localized as cortical patches and occasionally an immuno-fluorescence signal was detected in the septum (Figure 18E–F).

Figure 18. Immunolocalization of Botrytis cinerea α-actinin (A-B) and actin (C-D). α-Actinin localization in the tip region (A) and in the septum of the hyphae (B). Actin was mostly concentrated in the septum and as dots in the apical region (C-D). Bar 5 μm.

5. DISCUSSION, CONCLUSIONS AND PERSPECTIVES