Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, United States of America.
Integrated Biomedical Sciences Graduate Program, University of Notre Dame, Notre Dame, IN, United States of America.
PLoS One. 2021 Dec 10;16(12):e0260401. doi: 10.1371/journal.pone.0260401. eCollection 2021.
Proper regulation of microtubule (MT) dynamics is critical for cellular processes including cell division and intracellular transport. Plus-end tracking proteins (+TIPs) dynamically track growing MTs and play a key role in MT regulation. +TIPs participate in a complex web of intra- and inter- molecular interactions known as the +TIP network. Hypotheses addressing the purpose of +TIP:+TIP interactions include relieving +TIP autoinhibition and localizing MT regulators to growing MT ends. In addition, we have proposed that the web of +TIP:+TIP interactions has a physical purpose: creating a dynamic scaffold that constrains the structural fluctuations of the fragile MT tip and thus acts as a polymerization chaperone. Here we examine the possibility that this proposed scaffold is a biomolecular condensate (i.e., liquid droplet). Many animal +TIP network proteins are multivalent and have intrinsically disordered regions, features commonly found in biomolecular condensates. Moreover, previous studies have shown that overexpression of the +TIP CLIP-170 induces large "patch" structures containing CLIP-170 and other +TIPs; we hypothesized that these structures might be biomolecular condensates. To test this hypothesis, we used video microscopy, immunofluorescence staining, and Fluorescence Recovery After Photobleaching (FRAP). Our data show that the CLIP-170-induced patches have hallmarks indicative of a biomolecular condensate, one that contains +TIP proteins and excludes other known condensate markers. Moreover, bioinformatic studies demonstrate that the presence of intrinsically disordered regions is conserved in key +TIPs, implying that these regions are functionally significant. Together, these results indicate that the CLIP-170 induced patches in cells are phase-separated liquid condensates and raise the possibility that the endogenous +TIP network might form a liquid droplet at MT ends or other +TIP locations.
微管（MT）动态的适当调节对于包括细胞分裂和细胞内运输在内的细胞过程至关重要。正端追踪蛋白（+TIPs）动态追踪生长的 MT，并在 MT 调节中发挥关键作用。+TIPs 参与称为+TIP 网络的复杂分子内和分子间相互作用网络。解决+TIP 相互作用目的的假说包括缓解+TIP 自动抑制和将 MT 调节剂定位到生长的 MT 末端。此外，我们提出+TIP:+TIP 相互作用的网络具有物理目的：创建一个动态支架，限制脆弱的 MT 尖端的结构波动，从而充当聚合伴侣。在这里，我们研究了这种拟议的支架是否是生物分子凝聚物（即液滴）的可能性。许多动物+TIP 网络蛋白具有多价性和无规卷曲区域，这些特征常见于生物分子凝聚物中。此外，先前的研究表明，过度表达+TIP CLIP-170 会诱导含有 CLIP-170 和其他+TIP 的大“斑块”结构；我们假设这些结构可能是生物分子凝聚物。为了验证这一假说，我们使用视频显微镜、免疫荧光染色和光漂白后荧光恢复（FRAP）。我们的数据表明，CLIP-170 诱导的斑块具有生物分子凝聚物的特征标志，其中包含+TIP 蛋白并排除其他已知的凝聚物标记物。此外，生物信息学研究表明，无规卷曲区域的存在在关键+TIP 中是保守的，这意味着这些区域具有功能意义。综上所述，这些结果表明细胞中 CLIP-170 诱导的斑块是相分离的液相凝聚物，并提出内源性+TIP 网络可能在 MT 末端或其他+TIP 位置形成液滴的可能性。
