肌小节和细胞间连接处的 FHOD1 是形成蛋白的组成部分。
Distribution of formins in cardiac muscle: FHOD1 is a component of intercalated discs and costameres.
机构信息
Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany.
Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany; Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Poland.
出版信息
Eur J Cell Biol. 2015 Feb;94(2):101-13. doi: 10.1016/j.ejcb.2014.11.003. Epub 2014 Dec 12.
The formin homology domain-containing protein1 (FHOD1) suppresses actin polymerization by inhibiting nucleation, but bundles actin filaments and caps filament barbed ends. Two polyclonal antibodies against FHOD1 were generated against (i) its N-terminal sequence (residues 1-339) and (ii) a peptide corresponding the sequence from position 358-371, which is unique for FHOD1 and does not occur in its close relative FHOD3. After affinity purification both antibodies specifically stain purified full length FHOD1 and a band of similar molecular mass in homogenates of cardiac muscle. The antibody against the N-terminus of FHOD1 was used for immunostaining cells of established lines, primary neonatal (NRC) and adult (ARC) rat cardiomyocytes and demonstrated the presence of FHOD1 in HeLa and fibroblastic cells along stress fibers and within presumed lamellipodia and actin arcs. In NRCs and ARCs we observed a prominent staining of presumed intercalated discs (ICD). Immunostaining of sections of hearts with both anti-FHOD1 antibodies confirmed the presence of FHOD1 in ICDs and double immunostaining demonstrated its colocalisation with cadherin, plakoglobin and a probably slightly shifted localization to connexin43. Similarly, immunostaining of isolated mouse or pig ICDs corroborated the presence of FHOD1 and its colocalisation with the mentioned cell junctional components. Anti-FHOD1 immunoblots of isolated ICDs demonstrated the presence of an immunoreactive band comigrating with purified FHOD1. Conversely, an anti-peptide antibody specific for FHOD3 with no cross-reactivity against FHOD1 immunostained on sections of cardiac muscle and ARCs the myofibrils in a cross-striated pattern but not the ICDs. In addition, the anti-peptide-FHOD1 antibody stained the lateral sarcolemma of ARCs in a banded pattern. Double immunostaining with anti-cadherin and -integrin-ß1 indicated the additional localization of FHOD1 in costameres. Immunostaining of cardiac muscle sections or ARCs with antibodies against mDia3-FH2-domain showed colocalisation with cadherin along the lateral border of cardiomyocytes suggesting also its presence in costameres.
formin 同源结构域蛋白 1（FHOD1）通过抑制成核来抑制肌动蛋白聚合，但它也能束肌动蛋白丝并覆盖纤维丝的有帽端。我们针对 FHOD1 的（i）N 端序列（残基 1-339）和（ii）一个对应位置 358-371 的肽段，生成了两种 FHOD1 的多克隆抗体，该肽段是 FHOD1 所特有的，在其近亲 FHOD3 中并不存在。经亲和纯化后，两种抗体都能特异性地标记纯化的全长 FHOD1 和心肌匀浆中类似分子量的条带。针对 FHOD1 N 端的抗体被用于对已建立的细胞系、原代新生（NRC）和成年（ARC）大鼠心肌细胞进行免疫染色，并显示 FHOD1 存在于 HeLa 和成纤维细胞中的应力纤维内以及假定的片状伪足和肌动蛋白弧内。在 NRC 和 ARC 中，我们观察到假定的闰盘（ICD）有明显的染色。用两种抗 FHOD1 抗体对心脏切片进行免疫染色，证实了 FHOD1 存在于 ICD 中，双重免疫染色显示其与钙粘蛋白、桥粒斑蛋白和可能略微移位的连接蛋白 43 共定位。同样，对分离的小鼠或猪 ICD 进行免疫染色也证实了 FHOD1 的存在及其与上述细胞连接成分的共定位。分离的 ICD 的抗 FHOD1 免疫印迹显示存在一条与纯化的 FHOD1 共迁移的免疫反应性带。相反，一种针对 FHOD3 的特异性肽抗体，与 FHOD1 无交叉反应，在心肌切片和 ARC 上免疫染色，肌原纤维呈横纹图案，但 ICD 不染色。此外，抗肽-FHOD1 抗体在 ARC 的横向肌膜上呈条带样染色。用抗钙粘蛋白和β-整合素-ß1 进行双重免疫染色表明，FHOD1 还定位于细胞边缘。用针对 mDia3-FH2 结构域的抗体对心脏组织切片或 ARC 进行免疫染色显示，钙粘蛋白沿心肌细胞的侧向边界共定位，这表明它也存在于细胞边缘。
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