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Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells
Published on: December 12, 2014
Diurnal Photoreceptor Outer Segment Renewal in Mice Is Independent of Galectin-3
Nicholas J Esposito1, Francesca Mazzoni1, Jade A Vargas1
1Center for Cancer, Genetic Diseases and Gene Regulation, Department of Biological Sciences, Fordham University, Bronx, New York, United States.
Purpose:
Galectin-3 (gal-3) is a soluble glycoprotein that has been associated with diverse forms of phagocytosis, including some mediated by the engulfment receptor MerTK. Retinal pigment epithelium (RPE) in vivo uses MerTK (or the related Tyro3) for phagocytosis of shed outer segment fragments during diurnal outer segment renewal. Here, we test if gal-3 plays a role in outer segment renewal in mice and if exogenous gal-3 can promote MerTK-dependent engulfment of isolated outer segment fragments by primary RPE cells in culture.
Methods:
We explored age- and strain-matched wild-type (wt), lgals3-/- and mertk-/- mice. Immunofluorescence and immunoblotting characterized gal-3 and RPE/retina protein expression, respectively. Outer segment renewal was investigated by live imaging of phosphatidylserine (PS) exposure on photoreceptor outer segment distal tips and by microscopy of rhodopsin-labeled RPE phagosomes in tissue sections. Retinal function was assessed by recording electroretinograms (ERGs). Phagocytosis assays feeding purified outer segment fragments (POS) were conducted with added recombinant proteins testing unpassaged primary mouse RPE.
Results:
Gal-3 localizes to neural retina and RPE in wt mice. The lgals3-/- photoreceptor outer segments display normal diurnal PS exposure at distal tips. The number of rhodopsin-positive phagosomes in wt and lgals3-/- RPE does not differ at peak or trough of diurnal phagocytosis activity. lgals3-/- mice show light responses like wt, and their eyes contain wt levels of retinal and RPE proteins. Unlike purified protein S, recombinant gal-3 fails to promote POS engulfment by mouse primary RPE in culture.
Conclusions:
Gal-3 has no essential role in MerTK-dependent outer segment renewal in mice.
Insights
Galectin-3 (gal-3) does not play a role in retinal pigment epithelium (RPE) phagocytosis of outer segments in mice. Studies show gal-3 is not essential for MerTK-dependent outer segment renewal.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Galectin-3 (gal-3) is a glycoprotein linked to phagocytosis, including MerTK-mediated processes.
- Retinal pigment epithelium (RPE) utilizes MerTK for clearing shed photoreceptor outer segments during daily renewal.
Purpose of the Study:
- To investigate the role of gal-3 in mouse outer segment renewal.
- To determine if gal-3 enhances MerTK-dependent engulfment of outer segments by RPE cells.
Main Methods:
- Exploration of wild-type, lgals3-/-, and mertk-/- mice.
- Analysis of gal-3 and RPE/retina protein expression via immunofluorescence and immunoblotting.
- Assessment of outer segment renewal through live imaging of phosphatidylserine exposure and microscopy of rhodopsin-labeled phagosomes.
- Evaluation of retinal function using electroretinograms (ERGs).
- In vitro phagocytosis assays with primary mouse RPE and purified outer segments.
Main Results:
- Gal-3 is present in neural retina and RPE of wild-type mice.
- Outer segment renewal, marked by phosphatidylserine exposure and phagosome activity, proceeds normally in lgals3-/- mice.
- Rhodopsin-positive phagosome counts in RPE do not differ between wild-type and lgals3-/- mice.
- Retinal function and protein levels are comparable between wild-type and lgals3-/- mice.
- Recombinant gal-3 does not promote outer segment fragment engulfment by primary RPE cells in culture.
Conclusions:
- Galectin-3 is not essential for MerTK-dependent outer segment renewal in mice.
- The study clarifies gal-3's non-involvement in this specific RPE phagocytic process.
