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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

CK666 affects the distribution of model Hirano body size and number of cells that generate model Hirano bodies. The size distribution of model Hirano bodies was measured after 6 h (A), 12 h (B) or 18 h (C) of incubation with CK666 in media (green) after removal of folic acid to induce expression of E60K-GFP, compared with cells incubated with DMSO plus media (red) or media only (blue) under the same condition to induce model Hirano bodies. The distributions of the model Hirano body size were significantly different (P<0.01 at 6 h, P<0.001 at 12 h and at 18 h). The proportions of cells that generated model Hirano bodies with or without CK666 were also counted, and compared with the control (D). The number of cells that produced model Hirano bodies were significantly reduced when incubated in the presence CK666 (P<0.001 for all three time points, data represented as means±S.D.).
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BIO014944F4: CK666 affects the distribution of model Hirano body size and number of cells that generate model Hirano bodies. The size distribution of model Hirano bodies was measured after 6 h (A), 12 h (B) or 18 h (C) of incubation with CK666 in media (green) after removal of folic acid to induce expression of E60K-GFP, compared with cells incubated with DMSO plus media (red) or media only (blue) under the same condition to induce model Hirano bodies. The distributions of the model Hirano body size were significantly different (P<0.01 at 6 h, P<0.001 at 12 h and at 18 h). The proportions of cells that generated model Hirano bodies with or without CK666 were also counted, and compared with the control (D). The number of cells that produced model Hirano bodies were significantly reduced when incubated in the presence CK666 (P<0.001 for all three time points, data represented as means±S.D.).

Mentions: Subsequently, E60K-GFP cells were induced in the presence or absence of 100 µM CK666. Cells were fixed and stained with TRITC-phalloidin to visualize the F-actin at 6 h, 12 h and 18 h after induction of model Hirano bodies (12 h shown in Fig. 3M-U). The cells in the presence of CK666 formed fewer model Hirano bodies than control cells, consistent with the data that Arp2/3 colocalized with model Hirano bodies. Some of the model Hirano bodies that formed in the presence of CK666 showed abnormal morphology compared to the controls. To quantify these observations, the proportion of cells with model Hirano bodies, and the size of model Hirano bodies was counted in each of the samples. At each of the time points, compared with the controls, the proportion of cells that formed model Hirano bodies was dramatically lowered by 90% (Fig. 4, P<0.001 for all three time points). Moreover, the distribution of area of model Hirano bodies was significantly different compared to solvent control, (P<0.01 at 6 h, P<0.001 at 12 h and at 18 h; Fig. 4). These findings were repeated in three independent experiments.Fig. 4.


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)

CK666 affects the distribution of model Hirano body size and number of cells that generate model Hirano bodies. The size distribution of model Hirano bodies was measured after 6 h (A), 12 h (B) or 18 h (C) of incubation with CK666 in media (green) after removal of folic acid to induce expression of E60K-GFP, compared with cells incubated with DMSO plus media (red) or media only (blue) under the same condition to induce model Hirano bodies. The distributions of the model Hirano body size were significantly different (P<0.01 at 6 h, P<0.001 at 12 h and at 18 h). The proportions of cells that generated model Hirano bodies with or without CK666 were also counted, and compared with the control (D). The number of cells that produced model Hirano bodies were significantly reduced when incubated in the presence CK666 (P<0.001 for all three time points, data represented as means±S.D.).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

BIO014944F4: CK666 affects the distribution of model Hirano body size and number of cells that generate model Hirano bodies. The size distribution of model Hirano bodies was measured after 6 h (A), 12 h (B) or 18 h (C) of incubation with CK666 in media (green) after removal of folic acid to induce expression of E60K-GFP, compared with cells incubated with DMSO plus media (red) or media only (blue) under the same condition to induce model Hirano bodies. The distributions of the model Hirano body size were significantly different (P<0.01 at 6 h, P<0.001 at 12 h and at 18 h). The proportions of cells that generated model Hirano bodies with or without CK666 were also counted, and compared with the control (D). The number of cells that produced model Hirano bodies were significantly reduced when incubated in the presence CK666 (P<0.001 for all three time points, data represented as means±S.D.).
Mentions: Subsequently, E60K-GFP cells were induced in the presence or absence of 100 µM CK666. Cells were fixed and stained with TRITC-phalloidin to visualize the F-actin at 6 h, 12 h and 18 h after induction of model Hirano bodies (12 h shown in Fig. 3M-U). The cells in the presence of CK666 formed fewer model Hirano bodies than control cells, consistent with the data that Arp2/3 colocalized with model Hirano bodies. Some of the model Hirano bodies that formed in the presence of CK666 showed abnormal morphology compared to the controls. To quantify these observations, the proportion of cells with model Hirano bodies, and the size of model Hirano bodies was counted in each of the samples. At each of the time points, compared with the controls, the proportion of cells that formed model Hirano bodies was dramatically lowered by 90% (Fig. 4, P<0.001 for all three time points). Moreover, the distribution of area of model Hirano bodies was significantly different compared to solvent control, (P<0.01 at 6 h, P<0.001 at 12 h and at 18 h; Fig. 4). These findings were repeated in three independent experiments.Fig. 4.

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