September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Dissecting Exosome Biogenesis in Adult Human Retinal Pigment Epithelial Cells (ARPE19):
Specific Interaction of αB-crystallin with Rab GTPase27b
Author Affiliations & Notes
  • Rajendra K Gangalum
    Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Ankur M Bhat
    Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Sirus A Kohan
    Brain Research Insitute, UCLA, Los Angeles, California, United States
  • Suraj P Bhat
    Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
    Molecular Biology Institute and Brain Research Insitute, UCLA, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Rajendra Gangalum, None; Ankur Bhat, None; Sirus Kohan, None; Suraj Bhat, None
  • Footnotes
    Support  NIH Grant to SPB 1R01EY024929
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 245. doi:
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      Rajendra K Gangalum, Ankur M Bhat, Sirus A Kohan, Suraj P Bhat; Dissecting Exosome Biogenesis in Adult Human Retinal Pigment Epithelial Cells (ARPE19):
      Specific Interaction of αB-crystallin with Rab GTPase27b. Invest. Ophthalmol. Vis. Sci. 2016;57(12):245.

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

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Abstract

Purpose : We have previously demonstrated that the small heat shock protein, αB-crystallin (αB) is secreted from ARPE19 cells in culture via exosomes (ARVO 2009; J Biol Chem. 286:3261-9, 2011). Exosomes are 50 -200 nm proteolipid vesicles that carry molecular information (proteins and RNA) from one cell to the other and therefore represent potential vectors of intercellular communication between cells in the RPE; they may also have a role in the maintenance of this epithelial layer as a physiological and physical barrier at the blood/retina interface. As currently understood exosomes are produced from the endocytic pathway that maintains membrane protein (receptor) homeostasis. Here we examine the status of this pathway in ARPE19 cells that express αB-crystallin and those that do not.

Methods : We have employed Flow cytometry, Immunofluorescence and Confocal Microscopy, Western blotting, Co-immunoprecipitation and Double Immunogold labeling and Electron Microscopy to elucidate the status of the endocytic pathway in αB expressing and non-expressing stable ARPE19 αBshRNA transfected) cell lines generated in our laboratory.

Results : In cells not expressing αB, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. However sucrose density gradient analyses of cellular extracts indicate continued exosome synthesis under conditions of impaired exosome secretion. Immunoconfocal microscopy shows increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased labelling of this compartment by Rab7, a Rab GTPase known to be associated with the late endosome maturation. No change is however, seen with Rab5. Interestingly, we find that αB interacts specifically with Rab GTPase27b and not with Rab GTPase 27a in these cells suggesting that this small heat shock protein may have a role in collaboration with other small GTPases in regulating the secretion of exosomes from these cells.

Conclusions : These data point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch-point in the endocytic pathway populated by Rab GTPases known to be involved in multiple phases of secretion from eukaryotic cells.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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