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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

Observations of fluorescently-labeled tubulin and actin. Red fluorescent protein-tubulin and green fluorescent protein-actin were introduced into the A549 cells. The cells were observed using an LCV110 system. Representative images of control A549 cells are shown. The duration of incubation is presented by the horizontal axis (magnification, ×250).
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f3-ol-08-06-2387: Observations of fluorescently-labeled tubulin and actin. Red fluorescent protein-tubulin and green fluorescent protein-actin were introduced into the A549 cells. The cells were observed using an LCV110 system. Representative images of control A549 cells are shown. The duration of incubation is presented by the horizontal axis (magnification, ×250).

Mentions: To obtain time-lapse images of paracrystal formation, we introduced Cellular Lights RFP-Tubulin into the A549 cells and observed the cells using an Olympus LCV110 system. To determine the outline of each cell, GFP-Actin was also introduced. Fig. 3 shows the results of the time-lapse analysis of the cells without vinblastine treatment using the LCV110 system and the progression of the mitotic phases. Green signals were derived from GFP-Actin and red signals were derived from RFP-Tubulin. Microtubule formation during mitotic phases was observed by RFP-Tubulin at the 9 h 00 min time-point, and GFP-Actin was spread through the entire cytoplasm.


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

Nakamura Y, Ishigaki Y - Oncol Lett (2014)

Observations of fluorescently-labeled tubulin and actin. Red fluorescent protein-tubulin and green fluorescent protein-actin were introduced into the A549 cells. The cells were observed using an LCV110 system. Representative images of control A549 cells are shown. The duration of incubation is presented by the horizontal axis (magnification, ×250).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ol-08-06-2387: Observations of fluorescently-labeled tubulin and actin. Red fluorescent protein-tubulin and green fluorescent protein-actin were introduced into the A549 cells. The cells were observed using an LCV110 system. Representative images of control A549 cells are shown. The duration of incubation is presented by the horizontal axis (magnification, ×250).
Mentions: To obtain time-lapse images of paracrystal formation, we introduced Cellular Lights RFP-Tubulin into the A549 cells and observed the cells using an Olympus LCV110 system. To determine the outline of each cell, GFP-Actin was also introduced. Fig. 3 shows the results of the time-lapse analysis of the cells without vinblastine treatment using the LCV110 system and the progression of the mitotic phases. Green signals were derived from GFP-Actin and red signals were derived from RFP-Tubulin. Microtubule formation during mitotic phases was observed by RFP-Tubulin at the 9 h 00 min time-point, and GFP-Actin was spread through the entire cytoplasm.

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