Abstract

Mitotic spindle orientation is postulated to be regulated by two mechanisms: (1) Hertwig’s rule in which cells divide perpendicular to their major axis and (2) cortical cues arising from the spatial distribution of extracellular matrix. It was shown that cortical cues override cell geometry in dictating mitotic spindle orientation for cells cultured on 2D surfaces. In this study, we seek to investigate the interplay between cell geometry and cortical cues in determining mitotic spindle orientation for cells cultured on 3D microgratings. Here, cell geometry is manipulated by culturing RPE-1 and HeLa cells on different micrograting widths while cortical cues are modified by ablating focal adhesion contacts using cytochalasin D. We find a significant correlation between cell aspect ratio and spindle angles. Unexpectedly, disruption of cortical cues through focal adhesion inhibition did not lead to spindle misorientation in both RPE-1 and HeLa. Instead, spindle orientation was perturbed by disruption of microtubules (MTs) to a greater degree than abolishment of cortical focal adhesion cues. These results indicate that cell geometry is more important than cortical cues in maintaining proper spindle orientation and that MTs play an important role in spindle orientation for cells grown on 3D microgratings. To incorporate our findings of cell geometry and MTs as important players in spindle orientation, we developed a computational force balance model that relates both cell elongation, astral MTs to spindle angles and found good agreement between the model and experimental data.

摘要

假定有丝分裂纺锤体定向受两种机制调控：（1）Hertwig法则，其中细胞垂直于其主轴分裂；（2）由细胞外基质的空间分布引起的皮质提示。结果表明，在指示二维表面上培养的细胞的有丝分裂纺锤体定向中，皮层提示优先于细胞几何形状。在这项研究中，我们试图调查在确定在3D微光栅上培养的细胞的有丝分裂纺锤体定向中细胞几何结构与皮质提示之间的相互作用。在这里，通过在不同的微光栅宽度上培养RPE-1和HeLa细胞来操纵细胞的几何形状，而通过使用细胞松弛素D消融粘着接触来修饰皮质提示。我们发现细胞长宽比与纺锤体角度之间存在显着相关性。不料，RPE-1和HeLa中通过局灶性粘连抑制破坏皮质线索均不会导致纺锤体方向错误。取而代之的是，与取消皮质粘着斑线索相比，微管（MT）的破坏会更大程度地干扰纺锤体的定向。这些结果表明，在保持适当的纺锤体定向时，细胞几何结构比皮质提示更重要，并且对于在3D微光栅上生长的细胞，MT在纺锤体定向中起着重要作用。为了将我们对单元格几何形状和MTs的发现作为主轴定向中的重要角色进行整合，我们开发了一个计算力平衡模型，该模型将单元格的伸长率，星形MTs与主轴角度相关联，并在模型和实验数据之间找到了很好的一致性。