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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 VASP and mutant forms of VASP and F-actin in Dictyostelium cells transformed with CT to induce model Hirano bodies. vasP− were transformed with plasmid to express GFP-VASP, GFP-ΔGAB, GFP-ΔFAB, GFP-ΔGAB/FAB and in the presence or absence of CT to induce model Hirano bodies. The cells were observed by fluorescence microscopy using either TRITC-phalloidin or GFP. Except for CT/vasP–, the merged TRITC-phalloidin and GFP images are shown. When CT is expressed in vasP−, punctate small actin foci were formed. Model Hirano bodies were formed when CT/vasP− was rescued with full length GFP-VASP. Small foci containing either actin, VASP, or both were observed when either GFP-ΔGAB or GFP-ΔFAB VASP was expressed in vasP−. In the presence of CT and either GFP-ΔGAB or GFP-ΔFAB VASP, no large model Hirano bodies were formed, only small foci. When CT is expressed in GFPΔGAB/FAB vasP−, punctate actin foci that resembled those formed in vasP– cells were observed. VASP and its ability to bind actin is required either for F-actin foci to coalesce or elongate from F-actin foci to form model Hirano bodies. Arrowheads indicate either actin foci or model Hirano bodies. Scale bar=5 µm.
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BIO014944F8: Localization of VASP and mutant forms of VASP and F-actin in Dictyostelium cells transformed with CT to induce model Hirano bodies. vasP− were transformed with plasmid to express GFP-VASP, GFP-ΔGAB, GFP-ΔFAB, GFP-ΔGAB/FAB and in the presence or absence of CT to induce model Hirano bodies. The cells were observed by fluorescence microscopy using either TRITC-phalloidin or GFP. Except for CT/vasP–, the merged TRITC-phalloidin and GFP images are shown. When CT is expressed in vasP−, punctate small actin foci were formed. Model Hirano bodies were formed when CT/vasP− was rescued with full length GFP-VASP. Small foci containing either actin, VASP, or both were observed when either GFP-ΔGAB or GFP-ΔFAB VASP was expressed in vasP−. In the presence of CT and either GFP-ΔGAB or GFP-ΔFAB VASP, no large model Hirano bodies were formed, only small foci. When CT is expressed in GFPΔGAB/FAB vasP−, punctate actin foci that resembled those formed in vasP– cells were observed. VASP and its ability to bind actin is required either for F-actin foci to coalesce or elongate from F-actin foci to form model Hirano bodies. Arrowheads indicate either actin foci or model Hirano bodies. Scale bar=5 µm.

Mentions: Since profilin and the actin nucleation factor Arp2/3 have been shown to be important for model Hirano body formation, we also studied the effect of VASP, an actin elongation factor. To determine whether model Hirano bodies require VASP to form, Dictyostelium lacking VASP (vasP−) (Han et al., 2002) were transformed with CT to induce model Hirano bodies in the presence or absence of full length VASP, VASP lacking either the G- (ΔGAB) or F-actin binding domains (ΔFAB), or both the G- and F-actin binding domains (ΔGAB/FAB) (Breitsprecher et al., 2008; Schirenbeck et al., 2006). Transient expression in cells was determined with TRITC-labeled phalloidin to localize actin and GFP fluorescence was used to localize VASP and its mutants as shown in Fig. 8. No model Hirano bodies formed in CT/vasP− cells, only numerous punctate F-actin foci were observed. In CT/VASP/vasP− cells, model Hirano bodies formed and VASP did not colocalize to the model Hirano body. VasP− cells transformed with VASP lacking either the ΔGAB or ΔFAB formed small punctuate foci that were enriched in either actin, VASP, or both (Fig. 8). When CT is expressed in these cells, no model Hirano bodies form, only small foci formed as seen in the control cells. The phenotype of numerous, small punctate actin foci in the CT/vasP− cells was recovered when ΔGAB/FAB VASP was expressed. While VASP does not colocalize to model Hirano bodies, it is required for model Hirano body formation.Fig. 8.


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 VASP and mutant forms of VASP and F-actin in Dictyostelium cells transformed with CT to induce model Hirano bodies. vasP− were transformed with plasmid to express GFP-VASP, GFP-ΔGAB, GFP-ΔFAB, GFP-ΔGAB/FAB and in the presence or absence of CT to induce model Hirano bodies. The cells were observed by fluorescence microscopy using either TRITC-phalloidin or GFP. Except for CT/vasP–, the merged TRITC-phalloidin and GFP images are shown. When CT is expressed in vasP−, punctate small actin foci were formed. Model Hirano bodies were formed when CT/vasP− was rescued with full length GFP-VASP. Small foci containing either actin, VASP, or both were observed when either GFP-ΔGAB or GFP-ΔFAB VASP was expressed in vasP−. In the presence of CT and either GFP-ΔGAB or GFP-ΔFAB VASP, no large model Hirano bodies were formed, only small foci. When CT is expressed in GFPΔGAB/FAB vasP−, punctate actin foci that resembled those formed in vasP– cells were observed. VASP and its ability to bind actin is required either for F-actin foci to coalesce or elongate from F-actin foci to form model Hirano bodies. Arrowheads indicate either actin foci or model Hirano bodies. Scale bar=5 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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BIO014944F8: Localization of VASP and mutant forms of VASP and F-actin in Dictyostelium cells transformed with CT to induce model Hirano bodies. vasP− were transformed with plasmid to express GFP-VASP, GFP-ΔGAB, GFP-ΔFAB, GFP-ΔGAB/FAB and in the presence or absence of CT to induce model Hirano bodies. The cells were observed by fluorescence microscopy using either TRITC-phalloidin or GFP. Except for CT/vasP–, the merged TRITC-phalloidin and GFP images are shown. When CT is expressed in vasP−, punctate small actin foci were formed. Model Hirano bodies were formed when CT/vasP− was rescued with full length GFP-VASP. Small foci containing either actin, VASP, or both were observed when either GFP-ΔGAB or GFP-ΔFAB VASP was expressed in vasP−. In the presence of CT and either GFP-ΔGAB or GFP-ΔFAB VASP, no large model Hirano bodies were formed, only small foci. When CT is expressed in GFPΔGAB/FAB vasP−, punctate actin foci that resembled those formed in vasP– cells were observed. VASP and its ability to bind actin is required either for F-actin foci to coalesce or elongate from F-actin foci to form model Hirano bodies. Arrowheads indicate either actin foci or model Hirano bodies. Scale bar=5 µm.
Mentions: Since profilin and the actin nucleation factor Arp2/3 have been shown to be important for model Hirano body formation, we also studied the effect of VASP, an actin elongation factor. To determine whether model Hirano bodies require VASP to form, Dictyostelium lacking VASP (vasP−) (Han et al., 2002) were transformed with CT to induce model Hirano bodies in the presence or absence of full length VASP, VASP lacking either the G- (ΔGAB) or F-actin binding domains (ΔFAB), or both the G- and F-actin binding domains (ΔGAB/FAB) (Breitsprecher et al., 2008; Schirenbeck et al., 2006). Transient expression in cells was determined with TRITC-labeled phalloidin to localize actin and GFP fluorescence was used to localize VASP and its mutants as shown in Fig. 8. No model Hirano bodies formed in CT/vasP− cells, only numerous punctate F-actin foci were observed. In CT/VASP/vasP− cells, model Hirano bodies formed and VASP did not colocalize to the model Hirano body. VasP− cells transformed with VASP lacking either the ΔGAB or ΔFAB formed small punctuate foci that were enriched in either actin, VASP, or both (Fig. 8). When CT is expressed in these cells, no model Hirano bodies form, only small foci formed as seen in the control cells. The phenotype of numerous, small punctate actin foci in the CT/vasP− cells was recovered when ΔGAB/FAB VASP was expressed. While VASP does not colocalize to model Hirano bodies, it is required for model Hirano body formation.Fig. 8.

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