Condensed desmin and actin cytoskeletal communication in lipid droplets
Condensed desmin and actin cytoskeletal communication in lipid droplets
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脂滴中浓缩结蛋白和肌动蛋白细胞骨架通讯
DOI：
10.1002/cm.21573
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
K.
中科院分区：
文献类型：
--
作者：
Miyasaka;Y.;Murakami;K.;Ito;K.;Kumaki;J.;Makabe;K.;Hatori;K.
中间丝（if）和其他细胞骨架成分之间的相互作用对细胞的完整性和运动性很重要。中频组装对其他成分和细胞形态的影响尚不完全清楚。因此，我们研究了聚乳酸蛋白和肌动蛋白组合对人工细胞大小液滴中细胞骨架网络排列的影响。荧光标记的去精蛋白，有或没有肌动蛋白，被包裹在用1,2 -二油基- sn -甘油- 3 -磷酸乙醇胺（DOPE）用水-油-油法制备的液滴中。在150 mM KCl， 20 mM咪唑- hcl (pH 7.4)， 2 mM MgCl2和1 mM 5' -三磷酸腺苷的存在下，用荧光显微镜观察蛋白网络，用于desmin和actin的组装。由于聚醚蛋白可以在几秒钟内组装成细丝，因此在组装开始后的10分钟内观察到聚醚蛋白网络主要集中在液滴的内缘。随后，液滴出现变形。此外，部分液滴形成了几微米的富含聚二胺的突起。值得注意的是，在相同的条件下，单独的肌动蛋白很少形成突起。当使用1,2 -二油基- sn -甘油- 3 -磷脂胆碱代替DOPE时，突出变得不那么频繁。聚丝蛋白和肌动蛋白的结合增加了变形液滴的数量，这些液滴中的蛋白质在很大程度上是共定位的。desmin的组装过程促进了本地化。原子力显微镜未能揭示两种灯丝类型之间的相互作用。这些结果表明，聚乳酸蛋白网络的力学特性可能影响肌动蛋白网络的行为以及膜形态，可能反映了肌细胞中聚乳酸蛋白丝的力学功能。
The interplay between intermediate filaments (IFs) and other cytoskeletal components is important for the integrity and motility of cells. The impact of IF assembly on other components and cell morphology is not yet fully understood. Therefore, we examined the effects of combined desmin and actin assembly on cytoskeletal network arrangement in artificial cell‐sized droplets. Fluorescently labeled desmin, with or without actin, was enclosed in droplets prepared with 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE) using the water‐in‐oil method. Protein networks were observed using fluorescence microscopy in the presence of 150 mM KCl, 20 mM imidazole–HCl (pH 7.4), 2 mM MgCl2, and 1 mM adenosine 5'‐triphosphate for both desmin and actin assembly. As desmin alone can assemble into filaments within seconds, desmin networks mainly localizing at the inner margins of the droplets were observed within 10 min after assembly initiation. Subsequently, deformations of droplets appeared. Furthermore, a portion of droplets formed desmin‐rich protrusions of several micrometers. Notably, actin alone rarely formed protrusions under the same conditions. When 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine was used instead of DOPE, protrusions became less frequent. The combination of desmin and actin increased the number of deformed droplets in which the proteins were considerably colocalized. The assembly process of desmin facilitated colocalization. Atomic force microscopy failed to reveal interactions between the two filament types. These results suggest that the mechanical properties of desmin networks may influence the behavior of actin networks, as well as membrane morphology, possibly reflecting the mechanical function of desmin filaments in muscle cells.