June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
βA3-Crystallin Reduces the Aggresome Formation of αA- and αB-Crystallins and their Deamidated Mutants in HeLa Cells
Author Affiliations & Notes
  • Ekta Tiwary
    VisionSciences, University of Alabama at Birmingham, Birmingham, AL
  • Shylaja Hegde
    VisionSciences, University of Alabama at Birmingham, Birmingham, AL
  • Om P Srivastava
    VisionSciences, University of Alabama at Birmingham, Birmingham, AL
  • Footnotes
    Commercial Relationships Ekta Tiwary, None; Shylaja Hegde, None; Om Srivastava, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5590. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Ekta Tiwary, Shylaja Hegde, Om P Srivastava; βA3-Crystallin Reduces the Aggresome Formation of αA- and αB-Crystallins and their Deamidated Mutants in HeLa Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5590.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: The objective of the study was to determine the aggresome formation of αA- and αB- crystallins and their deamidated mutants (αA N101D, αA N123D, αB N78D and αB N146D) in the presence of βA3-crystallin in HeLa cells.

Methods: WT αA- and αB- crystallins and their deamidated mutants were cloned in pZs Yellow N1 vector and βA3-crystallin was cloned in pAm Cyan N1 vector. The recombinant plasmids were transfected in HeLa cells using lipofectamine, and the expression of CFP-tagged βA3-crystallin and YFP-tagged αA- and αB-crystallins were determined after 24 h. Further, cells were fixed with 4% paraformaldehyde and aggresome staining was performed using a Proteostat staining kit (Enzo Life Sciences). Expression of crystallins and their aggresome formation were analysed under a fluorescence microscope using CFP-, YFP- and red filters.

Results: βA3-crystallin did not show aggresome formation while αA- and αB-crystallins and their deamidated mutants formed aggresomes. A greater than 90% HeLa cells were positive for aggresome staining when they expressed WT αA- or WT αB-crystallins. Similarly, >90% HeLa cells, transfected with deamidated mutants of αA- and αB-crystallins, showed aggresome formation. A significant decrease (about 70-80% reduction) in aggresome formation was observed when either WT αA- or WT αB-crystallin was co-expressed with βA3-crystallin. Similar results were observed on co-expression of deamidated mutants of αA-or αB-crystallins with βA3-crystallin and aggresome formation was reduced to 40-60%.

Conclusions: WT αA- and WT αB-crystallins and their deamidated mutants formed aggresomes on their expression in HeLa cells. The co-expression of βA3-crystallin with them significantly reduces the aggresome formation. The results are consistent with the earlier reports, suggesting that βA3-crystallin plays a role in the clearance of cellular debris. A study of βA3-crystallin knockout mouse model in our laboratory also showed impaired autophagy, which resulted in accumulation of aggregates during cataract development.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×