Association of Shank 1A Scaffolding Protein with Cone Photoreceptor Terminals in the Mammalian Retina

Photoreceptor terminals contain post-synaptic density (PSD) proteins e.g., PSD-95/PSD-93, but their role at photoreceptor synapses is not known. PSDs are generally restricted to post-synaptic boutons in central neurons and form scaffolding with multiple proteins that have structural and functional roles in neuronal signaling. The Shank family of proteins (Shank 1–3) functions as putative anchoring proteins for PSDs and is involved in the organization of cytoskeletal/signaling complexes in neurons. Specifically, Shank 1 is restricted to neurons and interacts with both receptors and signaling molecules at central neurons to regulate plasticity. However, it is not known whether Shank 1 is expressed at photoreceptor terminals. In this study we have investigated Shank 1A localization in the outer retina at photoreceptor terminals. We find that Shank 1A is expressed presynaptically in cone pedicles, but not rod spherules, and it is absent from mice in which the Shank 1 gene is deleted. Shank 1A co-localizes with PSD-95, peanut agglutinin, a marker of cone terminals, and glycogen phosphorylase, a cone specific marker. These findings provide convincing evidence for Shank 1A expression in both the inner and outer plexiform layers, and indicate a potential role for PSD-95/Shank 1 complexes at cone synapses in the outer retina.National Institutes of Health (U.S.) (K08 Award NS41411)Howard Hughes Medical Institute (Investigator

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin -binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.Peer reviewe

SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes

Shank 1 immunoreactivity is expressed solely within cone terminals of the mouse thy-1.2 YFP 16 line retina.

<p><b>A–D</b>: Shank 1A is co-localized with glycogen phsophorylase (cone photoreceptor marker). <b>A–D</b>: Combined labeling of Shank 1A, glycogen phosphorylase, and YFP in a vertical retinal section. A. Shank 1A labels both the OPL and IPL. The immunoreactive puncta in the OPL are indicative of cone photoreceptor labeling. B. YFP fluorescence is present in bipolar, amacrine, and ganglion cells. C. Glycogen phosphorylase (GP), a cone photoreceptor marker strongly labels the cone terminals with faint labeling of bipolar cell bodies and their axons. D. Combined triple label fluorescence image of Shank 1A (red), YFP (yellow), and glycogen phosphorylase (GP) (blue), and. A box is drawn of a region in the OPL and high magnification images are shown in E–J. <b>E–J</b>: High magnification zoom of a region in the OPL from the inset above in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043463#pone-0043463-g007" target="_blank">Fig. 7D</a>. E. Shank 1A. F. Merge of Shank 1A (red) and YFP (yellow), showing that Shank 1A (red) is expressed above the cone bipolar cell dendrites (yellow). G. Glycogen phosphorylase (GP). H. Merge of Shank 1A (red) and Glycogen phosphorylase (GP), the two immunoreactivities (pink) indicate co-localization of Shank 1a and Glycogenphosphorylase (GP). I. YFP labeled cone bipolar cells and their dendrites. J. Combined image of Shank 1A (red), glycogen phosphorylase (blue), and YFP cone bipolar cell dendrites (yellow). OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, and GCL = ganglion cell layer. Scale bar is 10 µm.</p

Shank 1A immunoreactivity is co-localized with the lectin PNA (peanut agglutinin) in photoreceptor terminals.

<p><b>A–D</b>: Combined labeling of Shank 1A, PNA, and YFP in the mouse thy-1.2 YFP 16 line vertical retinal section. A. Shank 1A labels both the OPL and IPL. The immunoreactive puncta in the OPL are indicative of cone photoreceptor labeling. B. PNA conjugated rhodamine labels the inner and outer segments of cone photoreceptors, and cone photoreceptor terminals in the OPL. C. YFP fluorescence is present in bipolar, amacrine, and ganglion cells. D. Combined triple fluorescence channel image of PNA (red), Shank 1A (blue), and YFP (yellow). A box is drawn of a region in the OPL and high magnification images are shown in E–H. <b>E–H</b>: High magnification zoom of a region in the OPL from D. E. Shank 1A (blue). F. PNA (red). G. YFP labeled cone bipolar cells and their dendrites (yellow). H. A combined triple labeled fluorescent image showing that Shank 1A (blue) is expressed at the same site as PNA (red) above the YFP cone bipolar cell dendrites (yellow). OS = outer segment, IS = inner segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, and GCL = ganglion cell layer. Scale bars is 10 µm.</p

Shank 1A immunoreactivity co-localizes with the lectin WGA at cone photoreceptor terminals.

<p><b>A–C</b>: A. Shank 1A B. WGA labeling at the OPL. Arrowheads indicate WGA rod spherule labeling, and arrows indicate the location of WGA labeling of cone pedicles. C. YFP cone bipolar cell dendrites. <b>D–F</b>: Shank 1A (blue) and WGA (red) co-localize at cone terminals in the OPL. G–I: YFP cone bipolar cell dendrites (yellow) synapse with WGA (red) labeled cone terminals, which cradle Shank 1A immunoreactive puncta (blue). Scale bar is 10 µm.</p

Shank 1A is expressed with PSD-95 labeled photoreceptor terminals in the mouse thy-1.2 YFP 16 line retina.

<p><b>A–D</b>: Zoomed confocal region of the OPL immunostained with rabbit anti-Shank1A and mouse anti-PSD-95 antibodies. A. Shank 1A puncta labeling in the OPL in the mouse retina. B. PSD-95 immunolabels rod and cone photoreceptor terminals in the OPL. C. YFP labeled dendrites from cone bipolar cells in the OPL. D. Merged confocal image showing Shank 1A (red), PSD-95 (blue), and YFP (yellow). <b>E–J</b>: High magnification single plane confocal images of Shank 1A and PSD-95 in the mouse thy 1.2 YFP 16 line retina (100× objective, N.A. 1.3). E. Shank1 A (red). F. PSD-95 (blue). G. YFP (yellow). H. Merged image of YFP and Shank 1A, YFP and Shank 1A puncta are not co-localized at cone bipolar cell dendrites (see arrowheads, shank fluorescence above YFP dendrite. I. Merged image of Shank 1A and PSD-95, the two immunoreactivities (pink) indicate co-localization of Shank 1a and PSD-95 (see arrows which indicate that Shank 1A is co-localized with PSD-95). Arrows indicate location of Shank 1A immunoractive puncta co-localized with PSD-95. J. Merged image of inset Shank 1A (red), PSD-95 (blue), and YFP (yellow). Primary antibodies were detected using a secondary goat anti-rabbit Alexa 568 IgG for Shank 1A, and a goat anti-mouse Alexa 633 IgG to PSD-95. Scale bar is 5 µm.</p

Shank 1A immunoreactivity is absent when the Shank 1 gene has been deleted in mouse retina.

<p><b>A</b>: Shank 1A immunoreactivity in wild type mouse retina, punctate antibody labeling in the OPL in the outer retina and throughout the IPL. <b>B</b>: No Shank 1A immunoreactivity was found in retinal sections obtained from animals where the Shank 1 gene was deleted. (OS = outer segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, and GCL = ganglion cell layer). Scale bar is 10 µm.</p

Shank 1A immunoreactivity is in both the inner plexiform layer (IPL) and outer plexiform layer (OPL) of the mouse YFP-16 line retina.

<p><b>A–C</b>: A. Image of a retinal section immunostained for Shank 1A. B. Mouse YFP-16 line vertical retinal section. C. Shank 1A (red) immunolabeling and YFP (yellow). Shank1A expression is restricted to the OPL and IPL. A regular pattern of Shank 1A immunolabeling appears in the OPL, which is indicative of cone photoreceptor terminals. <b>D–E</b>: High magnification zoom of the OPL demonstrates that Shank 1A puncta (red) are distal to the dendrite tips (yellow) of YFP labeled cone bipolar cells, suggesting that Shank 1A is expressed presynaptic to the YFP cone bipolar cell dendrite. <b>G–L</b>: High magnification zoom of the IPL demonstrates that Shank 1A puncta are likely expressed postsynaptically to bipolar cell terminals. G. Shank 1A immunoreactive puncta. H. YFP labeled neurons and processes within the IPL region. I. PKCα labeled rod bipolar cell axons and terminals. J. Combined Shank 1A (red) and PKCα (blue) immunolabeling illustrate that shank 1A puncta are postsynaptic to rod bipolar cell terminals in the IPL. K. Combined Shank 1A (red) immunolabeling and YFP (yellow) in the IPL demonstrate that Shank 1A puncta are postsynaptic to cone bipolar cell terminals in the IPL. L. Combined triple fluorescent image of Shank 1A (red), PKCα (blue), and YFP (yellow) in the IPL. OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, and GCL = ganglion cell layer. Scale bars = 10 µm.</p

Antibodies and Lectins.

<p>Antibodies and Lectins.</p