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De novo actin polymerization is required for model Hirano body formation in Dictyostelium.

Dong Y, Shahid-Salles S, Sherling D, Fechheimer N, Iyer N, Wells L, Fechheimer M, Furukawa R - Biol Open (2016)

Bottom Line: Whereas model Hirano bodies could form in WASH-deficient cells, they failed to form in cells lacking HSPC300, a member of the WAVE complex.In the case of VASP, both its G- and F-actin binding domains were required for model Hirano body formation.Collectively, our results indicate that de novo actin polymerization is required to form model Hirano bodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology, University of Georgia, Athens, GA, USA 30602.

No MeSH data available.


Related in: MedlinePlus

Localization of Profilin I and F-actin in Dictyostelium cells expressing model Hirano bodies. (A-H) Cells expressing CT or profilin knockdown strain [AS]proA transformed with CT were observed by DIC microscopy (A,E), and by fluorescence microscopy using either TRITC-phalloidin to stain F-actin (C,G) or antibody against profilin I (D) or B2C that recognizes CT (H), followed by FITC-conjugated secondary antibody. Profilin I is enriched in model Hirano bodies (B) and in cells with reduced levels of profilin (F). Scale bar=10 µm. (I) Profilin I affects the size of model Hirano bodies. The size of model Hirano bodies were measured in profilin knockdown strain [AS]proA-CT and CT cells in three independent experiments. A representative plot is shown. The size of model Hirano bodies significantly decreased when profilin I was knocked down (P<0.001 for all three experiments).
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BIO014944F1: Localization of Profilin I and F-actin in Dictyostelium cells expressing model Hirano bodies. (A-H) Cells expressing CT or profilin knockdown strain [AS]proA transformed with CT were observed by DIC microscopy (A,E), and by fluorescence microscopy using either TRITC-phalloidin to stain F-actin (C,G) or antibody against profilin I (D) or B2C that recognizes CT (H), followed by FITC-conjugated secondary antibody. Profilin I is enriched in model Hirano bodies (B) and in cells with reduced levels of profilin (F). Scale bar=10 µm. (I) Profilin I affects the size of model Hirano bodies. The size of model Hirano bodies were measured in profilin knockdown strain [AS]proA-CT and CT cells in three independent experiments. A representative plot is shown. The size of model Hirano bodies significantly decreased when profilin I was knocked down (P<0.001 for all three experiments).

Mentions: Using inducible promoters, it has been observed that model Hirano bodies begin as small actin foci that coalesce to form a large inclusion (Griffin et al., 2014; Reyes et al., 2009). It is not known whether the foci disassemble and reassemble to form the large inclusion or whether the foci coalesce into one inclusion. Upon examination of the mass spectrometry analysis, we observed that profilin and Arp2/3 were putatively present in model Hirano bodies. We further examined these proteins since they are important in the regulation of actin filament formation. We examined the colocalization of profilin I with model Hirano bodies in Dictyostelium expressing CT (c-terminal portion of 34 kDa protein, amino acids 129-295, see Table S1) by immunofluorescence. Profilin I was enriched and colocalized well with the model Hirano bodies (Fig. 1A-D). This result verified the presence of profilin I in model Hirano bodies. To determine if profilin I affects model Hirano body formation, profilin I knockdown strain ([AS]proA) (Haugwitz et al., 1994) was transformed with plasmid encoding CT under a constitutive promoter (Maselli et al., 2002). Transformed cells ([AS]proA-CT), [AS]proA and wild-type cells expressing CT were immunoblotted to confirm that expression of profilin I was reduced (data not shown). Model Hirano bodies still formed in transformed cells (Fig. 1E-H); however, compared with wild-type cells that expressed CT, model Hirano bodies that were formed when profilin I was knocked down were significantly smaller (I, P<0.001). This result suggests that profilin I promotes model Hirano body formation.Fig. 1.


De novo actin polymerization is required for model Hirano body formation in Dictyostelium.

Dong Y, Shahid-Salles S, Sherling D, Fechheimer N, Iyer N, Wells L, Fechheimer M, Furukawa R - Biol Open (2016)

Localization of Profilin I and F-actin in Dictyostelium cells expressing model Hirano bodies. (A-H) Cells expressing CT or profilin knockdown strain [AS]proA transformed with CT were observed by DIC microscopy (A,E), and by fluorescence microscopy using either TRITC-phalloidin to stain F-actin (C,G) or antibody against profilin I (D) or B2C that recognizes CT (H), followed by FITC-conjugated secondary antibody. Profilin I is enriched in model Hirano bodies (B) and in cells with reduced levels of profilin (F). Scale bar=10 µm. (I) Profilin I affects the size of model Hirano bodies. The size of model Hirano bodies were measured in profilin knockdown strain [AS]proA-CT and CT cells in three independent experiments. A representative plot is shown. The size of model Hirano bodies significantly decreased when profilin I was knocked down (P<0.001 for all three experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920178&req=5

BIO014944F1: Localization of Profilin I and F-actin in Dictyostelium cells expressing model Hirano bodies. (A-H) Cells expressing CT or profilin knockdown strain [AS]proA transformed with CT were observed by DIC microscopy (A,E), and by fluorescence microscopy using either TRITC-phalloidin to stain F-actin (C,G) or antibody against profilin I (D) or B2C that recognizes CT (H), followed by FITC-conjugated secondary antibody. Profilin I is enriched in model Hirano bodies (B) and in cells with reduced levels of profilin (F). Scale bar=10 µm. (I) Profilin I affects the size of model Hirano bodies. The size of model Hirano bodies were measured in profilin knockdown strain [AS]proA-CT and CT cells in three independent experiments. A representative plot is shown. The size of model Hirano bodies significantly decreased when profilin I was knocked down (P<0.001 for all three experiments).
Mentions: Using inducible promoters, it has been observed that model Hirano bodies begin as small actin foci that coalesce to form a large inclusion (Griffin et al., 2014; Reyes et al., 2009). It is not known whether the foci disassemble and reassemble to form the large inclusion or whether the foci coalesce into one inclusion. Upon examination of the mass spectrometry analysis, we observed that profilin and Arp2/3 were putatively present in model Hirano bodies. We further examined these proteins since they are important in the regulation of actin filament formation. We examined the colocalization of profilin I with model Hirano bodies in Dictyostelium expressing CT (c-terminal portion of 34 kDa protein, amino acids 129-295, see Table S1) by immunofluorescence. Profilin I was enriched and colocalized well with the model Hirano bodies (Fig. 1A-D). This result verified the presence of profilin I in model Hirano bodies. To determine if profilin I affects model Hirano body formation, profilin I knockdown strain ([AS]proA) (Haugwitz et al., 1994) was transformed with plasmid encoding CT under a constitutive promoter (Maselli et al., 2002). Transformed cells ([AS]proA-CT), [AS]proA and wild-type cells expressing CT were immunoblotted to confirm that expression of profilin I was reduced (data not shown). Model Hirano bodies still formed in transformed cells (Fig. 1E-H); however, compared with wild-type cells that expressed CT, model Hirano bodies that were formed when profilin I was knocked down were significantly smaller (I, P<0.001). This result suggests that profilin I promotes model Hirano body formation.Fig. 1.

Bottom Line: Whereas model Hirano bodies could form in WASH-deficient cells, they failed to form in cells lacking HSPC300, a member of the WAVE complex.In the case of VASP, both its G- and F-actin binding domains were required for model Hirano body formation.Collectively, our results indicate that de novo actin polymerization is required to form model Hirano bodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology, University of Georgia, Athens, GA, USA 30602.

No MeSH data available.


Related in: MedlinePlus