SNAREs Interact with Retinal Degeneration Slow and Rod Outer Segment Membrane Protein-1 during Conventional and Unconventional Outer Segment Targeting

The authors would like to thank Mr. Marc Banworth, Mr. Justin Burnett, and Ms. Jamie Watson for their technical assistance, Drs. Muayyad Al-Ubaidi and David Sherry for their comments on the manuscript, and Drs. Roger Janz, Roderick McInnes, Neeraj Agarwal, Vadim Arshavsky, Robert Molday and Anand Swaroop for the provision of reagents as indicated in the text.Mutations in the photoreceptor protein peripherin-2 (also known as RDS) cause severe retinal degeneration. RDS and its homolog ROM-1 (rod outer segment protein 1) are synthesized in the inner segment and then trafficked into the outer segment where they function in tetramers and covalently linked larger complexes. Our goal is to identify binding partners of RDS and ROM-1 that may be involved in their biosynthetic pathway or in their function in the photoreceptor outer segment (OS). Here we utilize several methods including mass spectrometry after affinity purification, in vitro co-expression followed by pull-down, in vivo pull-down from mouse retinas, and proximity ligation assay to identify and confirm the SNARE proteins Syntaxin 3B and SNAP-25 as novel binding partners of RDS and ROM-1. We show that both covalently linked and non-covalently linked RDS complexes interact with Syntaxin 3B. RDS in the mouse is trafficked from the inner segment to the outer segment by both conventional (i.e., Golgi dependent) and unconventional secretory pathways, and RDS from both pathways interacts with Syntaxin3B. Syntaxin 3B and SNAP-25 are enriched in the inner segment (compared to the outer segment) suggesting that the interaction with RDS/ROM-1 occurs in the inner segment. Syntaxin 3B and SNAP-25 are involved in mediating fusion of vesicles carrying other outer segment proteins during outer segment targeting, so could be involved in the trafficking of RDS/ROM-1.Yeshttp://www.plosone.org/static/editorial#pee

Expression of the human usherin c.2299delG mutation leads to early-onset auditory loss and stereocilia disorganization

Abstract Usher syndrome (USH) is the leading cause of combined deafness and blindness, with USH2A being the most prevalent form. The mechanisms responsible for this debilitating sensory impairment remain unclear. This study focuses on characterizing the auditory phenotype in a mouse model expressing the c.2290delG mutation in usherin equivalent to human frameshift mutation c.2299delG. Previously we described how this model reproduces patient’s retinal phenotypes. Here, we present the cochlear phenotype, showing that the mutant usherin, is expressed during early postnatal stages. The c.2290delG mutation results in a truncated protein that is mislocalized within the cell body of the hair cells. The knock-in model also exhibits congenital hearing loss that remains consistent throughout the animal’s lifespan. Structurally, the stereocilia bundles, particularly in regions associated with functional hearing loss, are disorganized. Our findings shed light on the role of usherin in maintaining structural support, specifically in longer inner hair cell stereocilia, during development, which is crucial for proper bundle organization and hair cell function. Overall, we present a genetic mouse model with cochlear defects associated with the c.2290delG mutation, providing insights into the etiology of hearing loss and offering potential avenues for the development of effective therapeutic treatments for USH2A patients

Syn3B associates with multiple types of RDS complexes.

<p>Protein extract from WT (<b>A</b>) or <i>rds</i>^{<i>C150S/C150S</i>} (<b>B</b>) mouse retinas was IPed with anti-Syn3B-478. <b>A.</b> Elution/SDS-PAGE was performed under non-reducing conditions. RDS (mAB 2B7) and ROM-1 (mAB 2H5) antibodies show two bands on non-reducing western blot, monomers at around 37 kDa and dimers at around 75 kDa. <b>B.</b> Reducing SDS/PAGE after IP for RDS from <i>rds</i>^{<i>C150S/C150S</i>} retinal extracts shows that oligomerization-incompetent RDS retains the ability to bind Syn3B. <b>C</b>. Extracts from HEK293 cells transfected with WT or C150S RDS and Syn3B underwent IP for RDS (using RDS mAB 2E7 or RDS-CT) antibodies. Blots were subsequently probed for RDS (RDS mAB 2B7) or Syn3B (mAB 12E5) antibodies. <b>D-G.</b> Total retinal extracts (<b>D</b>, WT N = 10, <i>rom1</i>^<i>-/-</i> N = 6, <i>rds</i>^{<i>C150S/C150S</i>} N = 3), fractions from non-reducing sucrose gradient velocity sedimentations (<b>E-F</b>), or retinal extracts from Syn3B IP (<b>G</b>) underwent digestion with EndoH (H), PNGase F (F), or were undigested (mock, M). Results from <b>F</b> are summarized graphically in <b>E</b>.</p

RDS/ROM-1/Syn3B interactions are visualized via PLA.

<p>Paired WT retinal cryosections underwent IF (left picture in each pair) or PLA (right picture in each pair) for the indicated proteins. Label colors correspond with colors in IF. Red labeling in PLA indicates interaction between the two proteins. Nuclei were stained with DAPI (blue). <b>A-B</b>. RDS/Syn3B (<b>A</b>) and ROM-1/Syn3B (<b>B</b>) interactions were assessed using RDS mAB 2E7, ROM-1 mAB 2H5, and Syn3B-478 antibodies. Insets show magnified regions. <b>C-E</b>. RDS/Cytochrome C (RDS mAB 2E7, Cyto-C polyclonal) served as a negative control (<b>C</b>) while positive controls included RDS/ROM-1 [RDS mAB 2E7, ROM1-CT antibodies, (<b>D</b>)], and Syn3B/SNAP-25 [(Syn3B-478, SNAP-25 antibodies, (<b>E</b>)]. OS: outer segments, IS: inner segments, ONL: outer nuclear layer. Scale bars, 10 μm.</p

RDS/ROM-1 share interacting partners with each other and Syn3B.

<p>Euler diagrams depicting results from LC-MS studies to identify RDS/ROM-1 interacting partners. Numbers in each diagram represent proteins identified. <b>A-B.</b> WT and <i>rds</i>^<i>-/-</i> retinal extracts were IPed using RDS mAB 2E7 (<b>A</b>) or ROM1 mAB 2H5 (<b>B</b>), cross-linked to sepharose followed by LC-MS. <b>C.</b> This diagram depicts overlap between proteins identified in RDS pull-down (<b>A</b>) and ROM-1 pull down (<b>B</b>) after subtraction of negative controls (blue circles <b>A-B</b>). <b>D.</b> WT retinal extracts were IPed with anti-Syn3B-SySy or IgG coupled to protein A beads followed by LC-MS. <b>E-F.</b> These diagrams depict overlap between proteins in the RDS (<b>E-left</b>) or ROM-1 (<b>E-right</b>) IP and the Syn3B IP, or overlap between all three (<b>F</b>).</p

Mutant RDS that retains the ability to target to the OS interacts with Syn3B.

<p><b>A-B.</b> Retinal extracts from WT and <i>nrl</i>^<i>-/-</i> (<b>A</b>) or R172W transgenic and Y141C knockin (<b>B</b>) underwent IP for RDS (using RDS mAB 2E7 or RDS-CT) antibodies. Blots were subsequently probed for RDS (RDS mAB 2B7) or Syn3B (mAB 12E5) antibodies.</p

RDS and ROM-1 interact <i>in vitro</i> with Syn3B.

<p><b>A-B</b>. HEK293 cells were double transfected with RDS/Syn3B and ROM-1/Syn3B. <b>A.</b> IF of transfected HEK293 cells demonstrating double transfection; RDS (RDS-CT) and ROM-1 (ROM1-CT) antibodies are shown in green, with Syn3B (mAB 12E5) antibody in red. <b>B.</b> Transfected cell extracts underwent reciprocal co-IP with RDS (RDS-CT), ROM-1 (mAB 2H5), or Syn3B (Syn3B-478) antibodies, and resulting WBs were probed for RDS (mAB 2B7), ROM-1 (mAB 2H5), or Syn3B (mAB 12E5) antibodies. <b>C.</b> HEK293 cells were single transfected with the indicated plasmids and cell extracts were subsequently mixed in a 1:1 ratio prior to IP (same antibodies as in <b>B</b>). FT: flow-through.</p

Antibodies Used.

<p>Antibodies Used.</p

VAMP2 shows a weak interaction with ROM-1 and RDS <i>in vitro</i> but not <i>in vivo</i>.

<p><b>A</b>. HEK293 cells were co-transfected with vectors containing RDS, ROM-1, or Syn3B and flag-tagged VAMP2 cDNA and fluorescently labeled for RDS (RDS-CT, red) ROM-1 (ROM1-CT, red), Syn3B (Syn3B-529, red), or VAMP2 (anti-DDK, green) antibodies. <b>B.</b> CHAPS-solubilized cell extracts were used for IP with RDS (RDS-CT), ROM-1 (mAB 2H5), and Syn3B (mAB 12E5) antibdoies, and resultant WBs were probed with RDS (mAB 2B7), ROM-1 (mAB 2H5), Syn3B (mAB 12E5), or VAMP2 (anti-DDK) antibodies. <b>C.</b> IP was performed on CHAPS-solubilized WT retinal extracts with the same antibodies as in <b>B</b>, and blots were probed for RDS (mAB 2B7), ROM-1 (mAB 2H5), Syn3B (mAB 12E5), and VAMP2 (VAMP2-SySy) antibodies. <b>D.</b> WT retinal sections underwent IF for VAMP2 (VAMP2-SySy, green), and RDS (RDS-CT, red), ROM-1 (ROM1-CT, red), or Syn3B (Syn3B-529, red) antibodies. Scale bars: 10 μm. OS: outer segment, IS: inner segment, ONL: outer nuclear layer. FT: flow through.</p

SNARE and Trafficking Telated Proteins Identified in MS Analysis.

<p>SNARE and Trafficking Telated Proteins Identified in MS Analysis.</p