Limits...
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

Paracrystal formation in human A549 cells. A549 cells were treated with a vinblastine solution and fixed. The cells were stained with an anti-tubulin antibody followed by an appropriate secondary antibody. Nuclei were stained with DAPI. Images were acquired using an Axiovert 200M microscope. Bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214480&req=5

f1-ol-08-06-2387: Paracrystal formation in human A549 cells. A549 cells were treated with a vinblastine solution and fixed. The cells were stained with an anti-tubulin antibody followed by an appropriate secondary antibody. Nuclei were stained with DAPI. Images were acquired using an Axiovert 200M microscope. Bar = 10 μm.

Mentions: To confirm paracrystal formation in the A549 cells, the cells were treated with vinblastine for 12 h and the paracrystals that formed were stained with an anti-tubulin antibody, as described in previous studies (26,27). As shown in Fig. 1A–C, tubulin cytoplasmic localization was verified in the cells without vinblastine treatment. In the vinblastine-treated cells, characteristic paracrystal tube-like structure formation could clearly be detected (Fig. 1G–I). Thus, vinblastine treatment caused paracrystal formation in the A549 cells.


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

Nakamura Y, Ishigaki Y - Oncol Lett (2014)

Paracrystal formation in human A549 cells. A549 cells were treated with a vinblastine solution and fixed. The cells were stained with an anti-tubulin antibody followed by an appropriate secondary antibody. Nuclei were stained with DAPI. Images were acquired using an Axiovert 200M microscope. Bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-ol-08-06-2387: Paracrystal formation in human A549 cells. A549 cells were treated with a vinblastine solution and fixed. The cells were stained with an anti-tubulin antibody followed by an appropriate secondary antibody. Nuclei were stained with DAPI. Images were acquired using an Axiovert 200M microscope. Bar = 10 μm.
Mentions: To confirm paracrystal formation in the A549 cells, the cells were treated with vinblastine for 12 h and the paracrystals that formed were stained with an anti-tubulin antibody, as described in previous studies (26,27). As shown in Fig. 1A–C, tubulin cytoplasmic localization was verified in the cells without vinblastine treatment. In the vinblastine-treated cells, characteristic paracrystal tube-like structure formation could clearly be detected (Fig. 1G–I). Thus, vinblastine treatment caused paracrystal formation in the A549 cells.

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