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Immunostaining and time-lapse analysis of vinblastine-induced paracrystal formation in human A549 cells.

Nakamura Y, Ishigaki Y - Oncol Lett (2014)

Bottom Line: Vinblastine treatment results in the formation of paracrystalline aggregates in the cells, which are formed from tightly packed tubulin molecules.This was achieved using exogenous expression of fluorescent proteins conjugated with tubulin and time-lapse microscopy.It may be concluded that the indicated method was successful for the real-time analysis of paracrystal formation in human cells.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Institute, Kanazawa Medical University, Ishikawa 920-0293, Japan.

ABSTRACT
Vinblastine is a vinca alkaloid that binds to tubulin and inhibits microtubule formation in cells. Vinblastine treatment results in the formation of paracrystalline aggregates in the cells, which are formed from tightly packed tubulin molecules. Mitotic spindle assemblies in treated cells are disrupted and cell cycle progression is arrested at the mitosis phase. Vinblastine is therefore widely used for cancer treatment. However, the mechanism underlying paracrystal formation has not been fully elucidated. The present study attempted to observe paracrystal formation in human A549 cells. Initally, paracrystal formation was detected using the anti-tubulin antibody. Secondly, the exogenousuly expressed RFP-conjugated tubulin also formed paracrystals. Additionally, immunostaining with the anti-RBM8A antibody overlapped with paracrystal images obtained from RFP conjugated tubulin. This suggested that the localization of the RBM8A proteins was adjacent to the tubulin molecules prior to vinblastine treatment. Furthermore, a time-lapse analysis was developed for paracrystal formation in viable human A549 cells. This was achieved using exogenous expression of fluorescent proteins conjugated with tubulin and time-lapse microscopy. It may be concluded that the indicated method was successful for the real-time analysis of paracrystal formation in human cells.

No MeSH data available.


Related in: MedlinePlus

Time-lapse observations of paracrystal formation in viblastine-treated A549 cells. Cells with fluorescently-labeled tubulin and actin were observed using an LCV110 system. Incubation times are shown on the left of the images. Duration of incubation is presented by the horizontal axis (magnification, ×140).
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f4-ol-08-06-2387: Time-lapse observations of paracrystal formation in viblastine-treated A549 cells. Cells with fluorescently-labeled tubulin and actin were observed using an LCV110 system. Incubation times are shown on the left of the images. Duration of incubation is presented by the horizontal axis (magnification, ×140).

Mentions: In comparison, the time-lapse results for the vinblastine-treated cells are shown in Fig. 4 (images obtained on different days). Tube-like structures were clearly demonstrated by the red signals derived from RFP at the 7 h 11 min time-point. These paracrystals gradually grew until the 12 h 11 min time-point. During paracrystal formation, cell cycle progression was arrested around mitosis, as the cell shapes were round. At the same time, the green signals derived from GFP spread as shown in the negative control cells. Subsequent to the 15 h 41 min time-point, rounded cells predominantly appeared and cell cycle progression was inhibited. These cells retained paracrystals and appeared to be arrested at the mitotic phase.


Immunostaining and time-lapse analysis of vinblastine-induced paracrystal formation in human A549 cells.

Nakamura Y, Ishigaki Y - Oncol Lett (2014)

Time-lapse observations of paracrystal formation in viblastine-treated A549 cells. Cells with fluorescently-labeled tubulin and actin were observed using an LCV110 system. Incubation times are shown on the left of the images. Duration of incubation is presented by the horizontal axis (magnification, ×140).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214480&req=5

f4-ol-08-06-2387: Time-lapse observations of paracrystal formation in viblastine-treated A549 cells. Cells with fluorescently-labeled tubulin and actin were observed using an LCV110 system. Incubation times are shown on the left of the images. Duration of incubation is presented by the horizontal axis (magnification, ×140).
Mentions: In comparison, the time-lapse results for the vinblastine-treated cells are shown in Fig. 4 (images obtained on different days). Tube-like structures were clearly demonstrated by the red signals derived from RFP at the 7 h 11 min time-point. These paracrystals gradually grew until the 12 h 11 min time-point. During paracrystal formation, cell cycle progression was arrested around mitosis, as the cell shapes were round. At the same time, the green signals derived from GFP spread as shown in the negative control cells. Subsequent to the 15 h 41 min time-point, rounded cells predominantly appeared and cell cycle progression was inhibited. These cells retained paracrystals and appeared to be arrested at the mitotic phase.

Bottom Line: Vinblastine treatment results in the formation of paracrystalline aggregates in the cells, which are formed from tightly packed tubulin molecules.This was achieved using exogenous expression of fluorescent proteins conjugated with tubulin and time-lapse microscopy.It may be concluded that the indicated method was successful for the real-time analysis of paracrystal formation in human cells.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Institute, Kanazawa Medical University, Ishikawa 920-0293, Japan.

ABSTRACT
Vinblastine is a vinca alkaloid that binds to tubulin and inhibits microtubule formation in cells. Vinblastine treatment results in the formation of paracrystalline aggregates in the cells, which are formed from tightly packed tubulin molecules. Mitotic spindle assemblies in treated cells are disrupted and cell cycle progression is arrested at the mitosis phase. Vinblastine is therefore widely used for cancer treatment. However, the mechanism underlying paracrystal formation has not been fully elucidated. The present study attempted to observe paracrystal formation in human A549 cells. Initally, paracrystal formation was detected using the anti-tubulin antibody. Secondly, the exogenousuly expressed RFP-conjugated tubulin also formed paracrystals. Additionally, immunostaining with the anti-RBM8A antibody overlapped with paracrystal images obtained from RFP conjugated tubulin. This suggested that the localization of the RBM8A proteins was adjacent to the tubulin molecules prior to vinblastine treatment. Furthermore, a time-lapse analysis was developed for paracrystal formation in viable human A549 cells. This was achieved using exogenous expression of fluorescent proteins conjugated with tubulin and time-lapse microscopy. It may be concluded that the indicated method was successful for the real-time analysis of paracrystal formation in human cells.

No MeSH data available.


Related in: MedlinePlus