LINC complexes mediate the positioning of cone photoreceptor nuclei in mouse retina

It has long been observed that many neuronal types position their nuclei within restricted cytoplasmic boundaries. A striking example is the apical localization of cone photoreceptors nuclei at the outer edge of the outer nuclear layer of mammalian retinas. Yet, little is known about how such nuclear spatial confinement is achieved and further maintained. Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) consist of evolutionary-conserved macromolecular assemblies that span the nuclear envelope to connect the nucleus with the peripheral cytoskeleton. Here, we applied a new transgenic strategy to disrupt LINC complexes either in cones or rods. In adult cones, we observed a drastic nuclear mislocalization on the basal side of the ONL that affected cone terminals overall architecture. We further provide evidence that this phenotype may stem from the inability of cone precursor nuclei to migrate towards the apical side of the outer nuclear layer during early postnatal retinal development. By contrast, disruption of LINC complexes within rod photoreceptors, whose nuclei are scattered across the outer nuclear layer, had no effect on the positioning of their nuclei thereby emphasizing differential requirements for LINC complexes by different neuronal types. We further show that Sun1, a component of LINC complexes, but not A-type lamins, which interact with LINC complexes at the nuclear envelope, participate in cone nuclei positioning. This study provides key mechanistic aspects underlying the well-known spatia

Synthesis and Characterization of N4 Metal Stablizing Ligands

From the Washington University Undergraduate Research Digest: WUURD, Volume 10, 2014-2015. Published by the Office of Undergraduate Research, Joy Zalis Kiefer Director of Undergraduate Research and Assistant Dean in the College of Arts & Sciences; Stacy Ross, Editor; Kristin Sobotka, Editor; Jennifer Kohl. Mentor: Liviu Miric

A model for the molecular mechanism underlying the baso-apical migration of cone precursor nuclei.

<p>A) Between P4 and P12, cone precursors nuclei initially move towards the basal side of the developing ONL, a movement potentially mediated by microtubules plus-end directed kinesins, before moving back to the apical side. Inset: Depiction of a B-type lamins/Sun1-2/Nesprin2 network of macromolecular complexes that transduce forces generated by dyneins to move cone nuclei precursors back towards the apical side of the developing ONL. B) Disruption of LINC complexes displaces endogenous Nesprin2 (inset) leading to the uncoupling of cone nuclei to dynein. As a result, cone nuclei fail to migrate apically and mislocalize on the inner edge of the ONL. Basalmost localization of these nuclei interferes with the architecture of cone pedicles.</p

Sun1, but not A-type lamins, participates in the positioning of cone photoreceptor nuclei.

<p>A) Immunolocalization of A- and B-type lamins, Sun1, Sun2 and Nesprin2 within the mature ONL of adult retinas (top) or the developing ONL of P8 retinas (bottom). Cartoons: summary of immunolocalization experiments (blue: positive, white: negative). Scale bars: 20 μm. B, C) Immunolocalization of cone nuclei within the ONL of P32 Sun1^−/− (B) and P21 LMNA^−/− (C) retinas in comparison to their respective wild-type littermates. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047180#pone-0047180-g003" target="_blank">Figure 3F and S</a>4 for quantification of cone nuclei positioning in these genotypes. Scale bars: 20 μm.</p

EGFP-KASH2⁺ cone precursor nuclei fail to migrate towards the apical surface of the developing ONL.

<p>A) Cone opsin staining of P8 Tg(CMV-LacZ/EGFP-KASH2) retinas. Note the scattering of wild-type cone nuclei within the apical two thirds of the developing ONL. B) Same experiment on P8 Tg(<i>^HRGP</i>^floxCMV-EGFP-KASH2) littermate retinas. Note the basalmost mislocalization of cone nuclei expressing high levels of EGFP-KASH2. Arrowheads point to pyramid-shaped pedicles. Inset (lower panels): pyramid-shaped pedicles are present beneath wild type or EGFP-KASH2⁺ nuclei that are still confined within the ONL (nucleus 3) but not beneath EGFP-KASH2⁺ cones whose nuclei occupy basalmost locations (nuclei 1 and 2). Scale bars: 20 μm (upper panel) and 10 μm (lower panel).</p

Basalmost localization of EGFP-KASH2+ cone nuclei alters cone terminals morphology in adult Tg(<i>^HRGP</i>^<b>flox</b>CMV-EGFP-KASH2) retina.

<p>A) Maximum intensity projection of a Z-stack series acquired from Tg(<i>^HRGP</i>^floxCMV-EGFP-KASH2) retina stained with either anti-CAR (A) or Alexa594-PNA (B). Arrows in A) denote the staining pattern of CAR underneath EGFP-KASH2⁺ cones nuclei located within the OPL. As shown in B), these nuclei also displayed weaker or no basal PNA signal. Sale bars: 10 μm. C) PNA signal underneath EGFP-KASH2⁺ cone nuclei located within the OPL (EGFP-KASH2⁺) is significantly weaker (p<0.01) by comparison to PNA signals measured from regions devoid of EGFP-KASH2⁺ nuclei (EGFP-KASH2⁻).</p

LINC complexes mediate the positioning of cone photoreceptor nuclei.

<p>A) Genetic strategy used to derive Tg(<i>^HRGP</i>^floxCMV-EGFP-KASH2) mice expressing EGFP-KASH2 specifically in cone photoreceptors. B) CAR immunostaining of P26 Tg(CMV-LacZ/EGFP-KASH2) and Tg(<i>^HRGP</i>^floxCMV-EGFP-KASH2) littermates retinas. Lower panel: Zoomed view of the basal side of the ONL showing CAR⁺/EGFP-KASH2⁺ nuclei in the outer plexiform layer. Yellow arrows in merged image point to OS atop IS of cone nuclei expressing high levels of EGFP-KASH2. Scale bars: 50 μm and 20 μm (lower panel). C) Basalmost EGFP-KASH2⁺ cone nuclei express a significantly higher level of EGFP-KASH2 recombinant protein in comparison to their apical counterparts (p<0.001, Student's t-test). Error bars represent ±SEM from measurement of EGFP intensities of basal and apical nuclei from three random fields within ONL two Tg(<i>^HRGP</i>^floxCMV-EGFP-KASH2) littermate retinas. D) Mispositioned EGFP-KASH2⁺ nuclei are significantly less elongated. Maximal Feret diameters were significantly smaller in basal (n = 85) vs. apical (n = 68) EGFP-KASH2+ nuclei (p<0.01, Student's t-test). Error bars represent ±SEM from measurements of two random fields within two Tg(<i>^HRGP</i>^floxCMV-EGFP-KASH2) littermate retinas. E) Depiction and measurement of inclusion zones for the indicated genotypes (see text for details). Red nuclei are ectopic (centroids outside the inclusion zone) while green nuclei are correctly positioned (centroids within the inclusion zone). F) Percentages of ectopic (red) and of correctly positioned (green) nuclei of populations of n cone nuclei of the indicated genotypes. G) The size of cone populations estimated by the number of cone outer segments labeled with CAR in 4 month-old Tg(CMV-LacZ/EGFP-KASH2) and Tg(<i>^HRGP</i>^floxCMV-EGFP-KASH2) littermates retinas are not significantly different (p>0.05, Student's t-test). Error bars represent ±SEM from the counting of 5 random fields within littermate retinas of each genotype.</p

Transgenic expression pattern of Tg(CMV-LacZ/EGFP-KASH2) retinas.

<p>A) Depiction of the organization of LINC complexes that physically couple the nuclear lamina to peripheral cytoskeletal networks and molecular motors. INM, ONM: Inner and outer nuclear membrane, respectively. PNS: perinuclear space. Nesprin α, β and γ depict shorter isoforms originating from the alternative splicing of Nesprin 1 and 2 genes. B) Top: depiction of the CMV-LacZ/EGFP-KASH2 genetic construct (see text for details). Left panel: Transgenic expression pattern detected by X-gal staining of Tg(CMV-LacZ/EGFP-KASH2) retinal flat mount. Note the preferential transgenic expression on the dorsal side of the retina. Right panel: X-gal staining of vertical slices. Note the restriction of transgenic expression to the outer nuclear layer. ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. C) LacZ/V5 is mostly expressed in rods and a few cones. Vertical sections of P32 Tg(CMV-LacZ/EGFP-KASH2) were immunostained with anti-Cone arrestin (CAR) and anti-V5 antibodies. The arrow points to a CAR+/V5+ transgenic cone. Scale bars: 50 μm.</p