Sonic hedgehog (Shh) is a morphogen active during vertebrate development and tissue homeostasis in adulthood. Dysregulation of the Shh signalling pathway is known to incite carcinogenesis. Due to the highly lipophilic nature of this protein imparted by two post-translational modifications, Shh’s method of transit through the aqueous extracellular milieu has been a long-standing conundrum, prompting the proposition of numerous hypotheses to explain the manner of its displacement from the surface of the producing cell. Detection of high molecular-weight complexes of Shh in the intercellular environment has indicated that the protein achieves this by accumulating into multimeric structures prior to release from producing cells. The mechanism of assembly of the multimers, however, has hitherto remained mysterious and contentious. Here, with the aid of high-resolution optical imaging and post-translational modification mutants of Shh, we show that the C-terminal cholesterol and the N-terminal palmitate adducts contribute to the assembly of large multimers and regulate their shape. Moreover, we show that small Shh multimers are produced in the absence of any lipid modifications. Based on an assessment of the distribution of various dimensional characteristics of individual Shh clusters, in parallel with deductions about the kinetics of release of the protein from the producing cells, we conclude that multimerization is driven by self-assembly underpinned by the law of mass action. We speculate that the lipid modifications augment the size of the multimolecular complexes through prolonging their association with the exoplasmic membrane.

声波刺猬 (Shh) 是一种在脊椎动物发育和成年组织稳态过程中活跃的形态发生素。众所周知，Shh 信号通路的失调会引发癌变。由于这种蛋白质由两次翻译后修饰赋予的高度亲脂性，Shh穿过水性细胞外环境的方法一直是一个长期存在的难题，促使提出许多假说来解释其从表面移位的方式的生产细胞。对细胞间环境中Shh高分子量复合物的检测表明，该蛋白质通过在从生产细胞中释放之前积累成多聚体结构来实现这一点。然而，多聚体的组装机制迄今为止仍然是神秘且有争议的。在这里，借助高分辨率光学成像和Shh的翻译后修饰突变体，我们发现C端胆固醇和N端棕榈酸酯加合物有助于大多聚体的组装并调节其形状。此外，我们还表明，小Shh多聚体是在没有任何脂质修饰的情况下产生的。基于对各个 Shh 簇的各种维度特征的分布的评估，同时推论蛋白质从生产细胞中释放的动力学，我们得出结论，多聚化是由质量作用定律支撑的自组装驱动的。我们推测脂质修饰通过延长多分子复合物与外质膜的结合来增大多分子复合物的尺寸。