Table2_Cytokinetic contractile ring structural progression in an early embryo: positioning of scaffolding proteins, recruitment of α-actinin, and effects of myosin II inhibition.XLSX

Our knowledge of the assembly and dynamics of the cytokinetic contractile ring (CR) in animal cells remains incomplete. We have previously used super-resolution light microscopy and platinum replica electron microscopy to elucidate the ultrastructural organization of the CR in first division sea urchin embryos. To date, our studies indicate that the CR initiates as an equatorial band of clusters containing myosin II, actin, septin and anillin, which then congress over time into patches which coalesce into a linear array characteristic of mature CRs. In the present study, we applied super-resolution interferometric photoactivated localization microscopy to confirm the existence of septin filament-like structures in the developing CR, demonstrate the close associations between septin2, anillin, and myosin II in the CR, as well as to show that septin2 appears consistently submembranous, whereas anillin is more widely distributed in the early CR. We also provide evidence that the major actin cross-linking protein α-actinin only associates with the linearized, late-stage CR and not with the early CR clusters, providing further support to the idea that α-actinin associates with actomyosin structures under tension and can serve as a counterbalance. In addition, we show that inhibition of actomyosin contraction does not stop the assembly of the early CR clusters but does arrest the progression of these structures to the aligned arrays required for functional cytokinesis. Taken together our results reinforce and extend our model for a cluster to patch to linear structural progression of the CR in sea urchin embryos and highlight the evolutionary relationships with cytokinesis in fission yeast.

我们对动物细胞有丝分裂收缩环（CR）的组装与动力学尚不全面。我们先前利用超分辨率光显微镜和铂复制电子显微镜，阐明了第一分裂海胆胚胎中CR的超微结构组织。迄今为止，我们的研究指出，CR起源于含有肌球蛋白II、肌动蛋白、隔膜蛋白和anillin的赤道带簇，随着时间的推移，这些簇逐渐聚合成特征性的线性阵列，构成成熟的CR。在本研究中，我们应用超分辨率干涉光激发定位显微镜，以证实发育中的CR中存在隔膜蛋白纤维状结构，展示隔膜蛋白2、anillin与CR中的肌球蛋白II之间的紧密关联，并显示隔膜蛋白2在早期CR中呈现持续的内质膜下分布，而anillin在早期CR中分布更为广泛。此外，我们提供了主要肌动蛋白交联蛋白α-肌动蛋白仅与线性化、晚期CR而非早期CR簇相结合的证据，这进一步支持了α-肌动蛋白与张力下的肌动蛋白肌球蛋白结构相关联，并能作为平衡力的观点。此外，我们还表明，抑制肌动蛋白肌球蛋白收缩不会阻止早期CR簇的组装，但会阻止这些结构向功能性细胞分裂所需的排列阵列的进展。总之，我们的结果加强并扩展了我们对海胆胚胎中CR从簇到斑到线性结构进化的模型，并突出了与裂殖酵母细胞分裂的进化关系。