July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Peripherin-2/rds (P/rds) self-assembly drives membrane curvature generation
Author Affiliations & Notes
  • Michelle Lynn Milstein
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Breyanna Lynn Cavanaugh
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Andrew F X Goldberg
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Footnotes
    Commercial Relationships   Michelle Milstein, None; Breyanna Cavanaugh, None; Andrew Goldberg, None
  • Footnotes
    Support  R01EY025291
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 6042. doi:
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      Michelle Lynn Milstein, Breyanna Lynn Cavanaugh, Andrew F X Goldberg; Peripherin-2/rds (P/rds) self-assembly drives membrane curvature generation. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6042.

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

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Abstract

Purpose : We are investigating the molecular mechanisms by which peripherin-2/rds (P/rds) supports rod and cone photoreceptor outer segment (OS) structure, and how inherited defects in this protein can lead to progressive retinal degeneration. Defects in disk and OS structure cause a broad variety of blinding diseases; however, disease etiology is not understood, in part, because P/rds protein molecular function remains undefined. Here, we answer the question about the role of subunit assembly in shaping membranes using our previously developed assay for P/rds curvature generating activity.

Methods : Biochemical, biophysical, and imaging techniques were used to investigate the importance of P/rds self-assembly in generating membrane curvature. All assays of curvature generation utilized variants of PΔAH, an activated form of P/rds. Double mutants that blocked defined stages of subunit assembly included C150S-PΔAH and L185P-PΔAH. Protein biosynthesis, trafficking, subcellular localization, and impact on membrane ultrastructure were analyzed by velocity sedimentation, ICC/IHC, and transmission electron microscopy (TEM), respectively.

Results : PΔAH variants expressed robustly in HEK293 cells, without measurable effect on biosysnthesis or subcellular localization. Sedimentation analyses demonstrated that the C150S mutation blocked disulfide-mediated polymerization; and the L185P mutation, which underlies digenic retinitis pigmentosa, blocked tetrameric subunit assembly. Each mutation affected curvature generating activity in a distinctive manner, as observed by TEM.

Conclusions : This study demonstrates that P/rds is necessary and sufficient to generate the membrane curvature underlying OS disk rim structure. Moreover, the multiple steps of self-assembly previously documented for this protein are required for its membrane-shaping activity. Inhibiting this mechanism interferes with curvature generation, which explains how mutations in P/rds that affect protein self-assembly can lead to defects in OS structure and retinal dysfunction and disease. Together, these results provide clear evidence that the primary function of this protein is to generate membrane curvature, and that self-assembly is the mechanistic driver of this activity.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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