Correction: Obscurins: Goliaths and Davids Take over Non-Muscle Tissues.

[This corrects the article DOI: 10.1371/journal.pone.0088162.]

Obscurins: Goliaths and Davids Take over Non-Muscle Tissues

<div><p>Obscurins comprise a family of proteins originally identified in striated muscles, where they play essential roles in myofibrillogenesis, cytoskeletal organization, and Ca²⁺ homeostasis. They are encoded by the single <i>OBSCN</i> gene, and are composed of tandem adhesion domains and signaling motifs. To date, two giant obscurin isoforms have been described in detail that differ only at the extreme COOH-terminus; while obscurin-A (∼720 kDa) contains a non-modular COOH-terminus that harbors binding sites for the adaptor proteins ankyrins, obscurin-B (∼870 kDa) contains two COOH-terminal serine-threonine kinase domains preceded by adhesion motifs. Besides the two known giant obscurins, a thorough search of transcript databases suggests that complex alternative splicing of the obscurin transcript results in the generation of additional giant as well as small isoforms with molecular masses ranging between ∼50–970 kDa. These novel isoforms share common domains with the characterized isoforms, but also contain unique regions. Using a panel of highly specific antibodies directed against epitopes spanning the entire length of giant obscurins, we employed western blotting and immunohistochemistry to perform a systematic and comprehensive characterization of the expression profile of obscurins in muscle and non-muscle tissues. Our studies demonstrate for the first time that obscurins are not restricted to striated muscles, but are abundantly expressed in several tissues and organs including brain, skin, kidney, liver, spleen, and lung. While some obscurin isoforms are ubiquitously expressed, others are preferentially present in specific tissues and organs. Moreover, obscurins are present in select structures and cell types where they assume nuclear, cytosolic, and membrane distributions. Given the ubiquitous expression of some obscurins, along with the preferential expression of others, it becomes apparent that obscurins may play common and unique roles, respectively, in the regulation and maintenance of cell homeostasis in various tissues and organs throughout the body.</p></div

Distribution of obscurins in mouse and rat skin.

<p>Obscurins localize to the epidermis (black arrows) of mouse (A-A1) and rat (C-C1) skin. They are also found in the cytoplasm (pink arrows) and nuclei (white asterisks) of epithelial cells composing the root sheath of the hair follicle as well as the cytoplasm (green arrows) and nuclei (white asterisks) of the cells within the sebaceous glands in both mouse (B-B2) and rat (D-D2) tissue. In addition, obscurins are present in the connective tissue (purple arrows) and cells within the connective tissue (yellow arrows) in mouse (B and B3) and rat (D and D3) skin. Similar to the vasculature of striated muscles, obscurins reside within the vasculature of mouse and rat skin (B and B4, and D and D4, respectively; dark blue arrows). Images are shown at multiple magnifications to highlight the various immunopositive structures. Scale bars are included in each panel for reference.</p

Epitopes present in small obscurins.

<p>The ability of each of the four obscurin antibodies (α-NH₂ in red, α-COOH in blue, α-ABD in green, and α-Kinase in yellow) to recognize small obscurins (∼40–260 kDa) is depicted for each murine tissue and organ.</p

Expression of intermediate obscurins in rodent tissues and organs.

<p>Western blot analysis of 70 µg of protein homogenates prepared from various adult mouse (A) and rat (B) tissues were probed with antibodies specific to obscurins. As before, probing for GAPDH ensured equal loading. The blots have been cut to include intermediate obscurins, ranging in size between ∼260–600 kDa. Each lane is a representative image from multiple replicates.</p

Localization of obscurins in the rodent liver.

<p>Obscurins localize to the outer surface of the liver, namely Gilsson’s capsule (black arrows) in both mouse (A-A1) and rat (F-F1) tissues. Interestingly, only epitopes at the NH₂-terminus of obscurins are found within the connective tissue (yellow arrows) in both mouse (B) and rat (G). Obscurins are also found lining the sinusoids (C-C1, D, and H-H1, mouse and rat tissues, respectively; pink arrows) as well as within the cytoplasm of Kuppfer cells (C, C2, D and E, and H, I, and I2, mouse and rat, respectively; purple arrows) and hepatocytes (C, D, D2, E, and E2 and H, I-I1, J, and J2, mouse and rat, respectively; light blue arrows). Moreover, they are localized to the cell-cell contacts of hepatocytes (green arrows) within both mouse (D-D1) and rat (H and H2) tissue and hepatocyte nuclei (E and E2, and I-I1, mouse and rat respectively; white asterisks). Similar to other tissues and organs, obscurins reside within the VECs (dark blue arrows) of the liver vasculature in both mouse (E-E1) and rat (J-J1). Images are shown at multiple magnifications to highlight the various immunopositive structures. Scale bars are included in each panel for reference.</p

Expression of small obscurins in rodent tissues and organs.

<p>Western blot analysis of 70 µg of protein homogenates prepared from various adult mouse (A) and rat (B) tissues were probed with antibodies specific to obscurins and a GAPDH loading control. The blots have been cut to show small obscurins with molecular weights of ∼40–260 kDa. A representative blot for each tissue is shown in every lane.</p

Localization of obscurins in rodent striated muscles.

<p>Adult mouse and rat heart (A-H2) and tibialis anterior (I-N1) muscle sections were analyzed by immunohistochemistry using antibodies specific for obscurins. In accordance with previous studies, obscurins exhibited a striated pattern in both cardiac and skeletal muscles of mouse and rat origin. <b>Cardiac </b><b>Tissue</b>: Obscurins reside in the mouse and rat epicardium (A-A1 and E-E1, respectively; black arrows). In the myocardial layer, obscurins are found at the sarcolemma (B-B1 and G-G1, mouse and rat tissues, respectively; green arrows) and in sarcomeric striations (C-C1 and F-F1, mouse and rat tissues, respectively; light blue arrows). Interestingly, intercalated disks (pink arrows) and the nuclei of cardiomyocytes (black asterisk) are also labeled in both mouse (D-D1) and rat (H-H2) tissues. In addition, obscurins are found within the cells lining the vasculature throughout the heart (B and B2, and F and F2, mouse and rat tissues, respectively; dark blue arrows). <b>Skeletal Muscle </b><b>Tissue:</b> Similar to earlier observations, obscurins localize to myofibrillar striations (light blue arrows) of both mouse (I-I1) and rat (L-L1) tissues. Obscurins are also present at the sarcolemma (J-J1 and M-M1, mouse and rat tissues, respectively; green arrows) and the nuclei of mouse, but not rat, skeletal muscle using the α-ABD antibody (K and K2, black asterisk). Moreover, obscurins are found within the cells lining the walls of the vasculature (dark blue arrow) in both mouse (K-K1) and rat (N-N1) tissue. Images are shown at multiple magnifications to highlight the various immunopositive structures. Scale bars are included in each panel for reference; for details please refer to the relevant section in the Materials and Methods.</p

Epitopes present in giant obscurins.

<p>The ability of each of the four obscurin antibodies (α-NH₂ in red, α-COOH in blue, α-ABD in green, and α-Kinase in yellow) to recognize giant obscurins (>60 kDa) is depicted for each murine tissue and organ.</p