Collective epithelial cell sheet adhesion and migration on polyelectrolyte multilayers with uniform and gradients of compliance.
Collective epithelial cell sheet adhesion and migration on polyelectrolyte multilayers with uniform and gradients of compliance.
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DOI：
10.1016/j.yexcr.2016.06.002
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发表时间：
2016-08-01
影响因子：
3.7
通讯作者：
Keller TC 3rd
中科院分区：
文献类型：
--
作者：
Martinez JS;Schlenoff JB;Keller TC 3rd
聚电解质多层膜(PEMU)是一种可调薄膜，可用作生物医学植入物的涂层。用光敏的4-(2-羟乙氧基)二苯甲酮(PAABp)修饰的聚阴离子聚丙烯酸(PAA)和聚阳离子聚烯丙胺盐酸盐(PAH)逐层构建的PEMU可以通过紫外光照射机械调节，形成层间的共价键，增加PEMU的硬度。PAH-PEMU(PAH-PEMU)是未交联体、紫外交联体或通过中性密度光学梯度滤膜形成连续顺应性梯度的、未交联体、紫外光交联体或通过中性密度光学梯度滤膜形成连续顺应性梯度的PEMU，用来研究PEMU硬度的差异如何影响凤尾鱼(Poecilia Sphenops)和鲫鱼(Carassius Auratus)鳞片上皮细胞片的黏附和迁移。在渐进的集体细胞迁移过程中，与在较硬的PEMU或玻璃上形成的薄片细胞相比，在较软的未交联化的PEMU和较少交联区的梯度上的薄片中的边缘细胞(也称为领导细胞)形成更多的肌动蛋白细丝和含纽蛋白的粘连连接和焦点粘连。在薄片迁移过程中，边缘细胞与内部细胞(也称为“跟随者”细胞)在较软的PEMU上的运动比在较硬的PEMU或玻璃上更大，导致整个薄片上产生张力和回缩周期，在此期间，边缘细胞失去与衬底的粘附性，并且薄片的区域向更粘连的内部细胞区域收缩。这些回缩事件被肌球蛋白II抑制剂Blebbistatin抑制，后者降低了运动速度比，使其与较坚硬的PEMU上的运动速度比。Blebbistatin还导致肌动蛋白细丝解体，局灶性粘连重组，前沿细胞扩散增加，以及所有表面上皮细胞片中边缘细胞与细胞连接的丢失。有趣的是，在未交联化的PEMU上，整个膜的内部区域的细胞在用Blebbistatin处理后，在粘连的连接处保留了它们的肌动蛋白和纽蛋白组织。与Blebbistatin类似，Rho-Kinase(ROCK)抑制剂Y27632促进边缘细胞之间细胞间连接的丢失，而rac1抑制剂NSC23766主要改变边缘细胞中的片脂突起。顺应性梯度PAH-PEMU可促进细胞膜的趋性，但不能促进单个角质形成细胞的趋性，说明趋性和趋性一样，是细胞膜组织的一种新出现的特性。
Polyelectrolyte multilayers (PEMUs) are tunable thin films that could serve as coatings for biomedical implants. PEMUs built layer by layer with the polyanion poly(acrylic acid) (PAA) modified with a photosensitive 4-(2-hydroxyethoxy) benzophenone (PAABp) group and the polycation poly(allylamine hydrochloride) (PAH) are mechanically tunable by UV irradiation, which forms covalent bonds between the layers and increases PEMU stiffness. PAH-terminated PEMUs (PAH-PEMUs) that were uncrosslinked, UV-crosslinked to a uniform stiffness, or UV-crosslinked with an edge mask or through a neutral density optical gradient filter to form continuous compliance gradients were used to investigate how differences in PEMU stiffness affect the adhesion and migration of epithelial cell sheets from scales of the fish Poecilia sphenops (Black Molly) and Carassius auratus (Comet Goldfish). During the progressive collective cell migration, the edge cells (also known as ‘leader’ cells) in the sheets on softer uncrosslinked PEMUs and less crosslinked regions of the gradient formed more actin filaments and vinculin-containing adherens junctions and focal adhesions than formed in the sheet cells on stiffer PEMUs or glass. During sheet migration, the ratio of edge cell to internal cell (also known as ‘follower’ cells) motilities were greater on the softer PEMUs than on the stiffer PEMUs or glass, causing tension to develop across the sheet and periods of retraction, during which the edge cells lost adhesion to the substrate and regions of the sheet retracted toward the more adherent internal cell region. These retraction events were inhibited by the myosin II inhibitor Blebbistatin, which reduced the motility velocity ratios to those for sheets on the stiffer PEMUs. Blebbistatin also caused disassembly of actin filaments, reorganization of focal adhesions, increased cell spreading at the leading edge, as well as loss of edge cell-cell connections in epithelial cell sheets on all surfaces. Interestingly, cells throughout the interior region of the sheets on uncrosslinked PEMUs retained their actin and vinculin organization at adherens junctions after treatment with Blebbistatin. Like Blebbistatin, a Rho-kinase (ROCK) inhibitor, Y27632, promoted loss of cell-cell connections between edge cells, whereas a Rac1 inhibitor, NSC23766, primarily altered the lamellipodial protrusion in edge cells. Compliance gradient PAH-PEMUs promoted durotaxis of the cell sheets but not of individual keratocytes, demonstrating durotaxis, like plithotaxis, is an emergent property of cell sheet organization.