School of Medical Sciences, UNSW Sydney, NSW 2052, Australia.
Membrane Dynamics, Department of Biology, Technische Universität Darmstadt, Schnittspahnstrasse 3, 64287 Darmstadt, Germany.
J Cell Sci. 2018 Mar 19;131(6):jcs212654. doi: 10.1242/jcs.212654.
Many actin filaments in animal cells are co-polymers of actin and tropomyosin. In many cases, non-muscle myosin II associates with these co-polymers to establish a contractile network. However, the temporal relationship of these three proteins in the assembly of actin filaments is not known. Intravital subcellular microscopy of secretory granule exocytosis allows the visualisation and quantification of the formation of an actin scaffold in real time, with the added advantage that it occurs in a living mammal under physiological conditions. We used this model system to investigate the assembly of actin, tropomyosin Tpm3.1 (a short isoform of TPM3) and myosin IIA (the form of non-muscle myosin II with its heavy chain encoded by ) on secretory granules in mouse salivary glands. Blocking actin polymerization with cytochalasin D revealed that Tpm3.1 assembly is dependent on actin assembly. We used time-lapse imaging to determine the timing of the appearance of the actin filament reporter LifeAct-RFP and of Tpm3.1-mNeonGreen on secretory granules in LifeAct-RFP transgenic, Tpm3.1-mNeonGreen and myosin IIA-GFP (GFP-tagged MYH9) knock-in mice. Our findings are consistent with the addition of tropomyosin to actin filaments shortly after the initiation of actin filament nucleation, followed by myosin IIA recruitment.
许多动物细胞中的肌动蛋白丝是肌动蛋白和原肌球蛋白的共聚物。在许多情况下，非肌肉肌球蛋白 II 与这些共聚物结合，建立一个收缩网络。然而，这三种蛋白质在肌动蛋白丝组装中的时间关系尚不清楚。分泌颗粒胞吐的活体亚细胞显微镜允许实时可视化和定量组装肌动蛋白支架，其优点是它在生理条件下的活体哺乳动物中发生。我们使用这个模型系统来研究肌动蛋白、原肌球蛋白 Tpm3.1（TPM3 的短同工型）和非肌肉肌球蛋白 IIA（由编码的非肌肉肌球蛋白 II 形式）在小鼠唾液腺分泌颗粒上的组装。用细胞松弛素 D 阻断肌动蛋白聚合表明 Tpm3.1 组装依赖于肌动蛋白组装。我们使用延时成像来确定肌动蛋白丝报告蛋白 LifeAct-RFP 和 Tpm3.1-mNeonGreen 在 LifeAct-RFP 转基因、Tpm3.1-mNeonGreen 和肌球蛋白 IIA-GFP（GFP 标记的 MYH9）敲入小鼠中在分泌颗粒上出现的时间。我们的发现与肌动蛋白丝核形成后不久添加原肌球蛋白到肌动蛋白丝，随后招募肌球蛋白 IIA 一致。
