Integration of Actin Dynamics and Adhesion in Cell Migration

纽蛋白稳定迁移细胞的新生粘连并建立板层-板层边界 Ingo Thievessen，R.Ross 位于迁移细胞前沿的肌动蛋白细胞骨架由两个肌动蛋白网络组成，一个是以快速聚合驱动的肌动蛋白逆行流动为特征的片层(LP)，另一个是以慢速肌球蛋白II(MyoII)为特征的肌动蛋白网络(LM)。通过新生的整合素介导局灶性粘连(FA)，LP肌动蛋白与ECM的结合建立了LP和LM之间的流速梯度。新生的粘连随后通过myoII LM肌动蛋白流动而延长和成熟。整合素是如何连接到逆行肌动蛋白流的尚不清楚。利用原代缺乏纽蛋白基因(VCL)的小鼠胚胎成纤维细胞(MEF)，我们试图验证纽蛋白在FA中介导肌动蛋白逆行向ECM偶联的假说。单个VCL-/-MEF的迁移速度快于对照(Vclf1/f1)MEF，并显示各向异性扩散受损。为了确定纽蛋白缺失是否会影响LP/LM的组织结构，我们分析了磷酸化肌球蛋白轻链(PMLC)、皮质酮和帕西林的分布。这表明pMLC的分布向细胞边缘移动，LP paxlin强度降低，前沿的接触蛋白条带变宽。这表明LP和LM之间失去了界限。为了测试这一点，我们对含有荧光小黄素和肌动蛋白的MEF进行了旋转圆盘共聚焦(SDC)显微镜观察。这表明，在VCL-/-MEF的LP中，短暂的、衍射受限的FA的形成速度降低，表明新生LP FA的稳定性受到损害。肌动蛋白的高分辨DIC和定量荧光散斑SDC显微镜分析表明，在VCL-/-MEF的LM区，在前沿附近没有两个明显的f-肌动蛋白逆行速度区，而逆行速度区的速度增加。我们认为，纽蛋白通过偶联板脂肌动蛋白流来稳定新生FA，从而建立LP和LM之间的流速梯度，促进新生粘连的成熟。这意味着在细胞迁移过程中，纽蛋白是连接动态肌动蛋白细胞骨架和细胞外基质的重要组成部分。 这项工作是与Bob Ross合作完成的，并已在细胞生物学学会会议和Gordon会议上发表。 项目2：基于整合素的细胞黏附的纳米结构 帕科恩·坎查那旺 焦点粘连(FA)介导细胞与其细胞外基质的相互作用 (ECM)，由整合素ECM受体通过肌动蛋白细胞骨架连接 质膜相关蛋白斑块。尽管它们在 多细胞生物，FAs内蛋白质的三维组织是 未知。在这里，我们使用3D超分辨率来确定FA的分子结构 显微镜(干涉光激活定位显微镜)绘制纳米级地图 蛋白质组织。我们发现FA由部分重叠的特定蛋白组成 15-50 nm厚的垂直层，整合素和肌动蛋白由30- 50 nm FA芯，由塔林系绳跨越。这揭示了FA的结构基础 通过多层核心架构来调节相互依赖的细胞的功能 黏附、信号、力传递和肌动蛋白细胞骨架调节的过程。 这项研究是与Mike Davidson、Gleb Stengle、Harald Hess合作完成的，已被修订并重新提交给《自然》杂志。 项目3：肌动蛋白动力学与局部粘连的力传递相关 克莱尔·沃特曼 焦点粘连(FA)如何将逆行肌动蛋白细丝(F-Actin)流转化为细胞外基质上的牵引力，从而驱动细胞迁移，目前尚不清楚。结合牵引力和荧光散斑显微镜，我们观察到F-肌动蛋白速度和牵引力之间有很强的双相关系。F-肌动蛋白的速度与细胞边缘附近的牵引力呈负相关，在细胞边缘形成FAs，F-肌动蛋白运动迅速。相比之下，在F-肌动蛋白速度较低的较大FA中，F-肌动蛋白速度与牵引应力之间存在直接关系。我们发现，双相开关是由8-10 nm/S的F-肌动蛋白阈值速度决定的，与FA蛋白密度、年龄、应力大小、组装/拆解状态或Rho家族GTPase信号和肌球蛋白II活性多效性扰动引起的亚细胞位置的变化无关。因此，F-肌动蛋白速度是细胞迁移过程中FAs牵引力的基本调节器。 这项工作是与玛格丽特·加德尔(芝加哥大学)、Gaudenz Danuser和Lin Ji(斯克里普斯)、Ulrich Schwartz和Benedikt Sabass(海德堡大学)合作完成的，并被接受出版。 项目4：肌动蛋白应激纤维维持和修复的一种由自新介导的机制。 克莱尔·沃特曼

Vinculin stabilizes nascent adhesions and establishes lamellipodium-lamella border in migrating cells Ingo Thievessen, R. Ross The actin cytoskeleton at the leading edge of migrating cells consists of two actin networks, the lamellipodium (LP), characterized by fast polymerization-driven retrograde actin flow and the lamellum (LM) with slow myosin II (myoII) mediated actin flow. The engagement of LP actin to the ECM via nascent integrin-mediated focal adhesions (FA) establishes the flow velocity gradient between LP and LM. Nascent adhesions then elongate and mature via myoII LM actin flow. How integrins are connected to the retrograde actin flow is not known. Using primary murine embryonic fibroblasts (MEF) deficient for the vinculin gene (Vcl), we sought to test the hypothesis that vinculin mediates the coupling of actin retrograde flow to the ECM in FA . Single Vcl-/- MEF migrated faster than control (Vclfl/fl) MEF and displayed impaired anisotropic spreading. To determine if LP/LM organization were effected by vinculin deletion, we analyzed distributions of phospho-myosin light chain (pMLC), cortactin, and paxillin. This revealed a shift in pMLC distribution towards the cell edge, reduced LP paxillin intensity, and broadening of the contactin band at the leading edge. This suggests loss of delineation between the LP and LM. To test this, we performed spinning disc confocal (SDC) microscopy of MEF containing fluorescent paxillin and actin. This revealed reduction in the rate of formation of short-lived, diffraction limited FA in the LP of Vcl-/- MEF, indicating an impaired stabilization of nascent LP FA. Kymograph analyses of high resolution DIC and quantitative fluorescent speckle SDC microscopy of actin indicate the lack of two distinct velocity zones of retrograde f-actin flow near the leading edge and an increased retrograde flow velocity in the LM region of Vcl-/- MEF. We suggest that vinculin stabilizes nascent FA by coupling to lamellipodial actin flow, thus establishing the flow velocity gradient between LP and LM and promoting the maturation of nascent adhesions. This implicates vinculin as an essential component in linking the dynamic actin cytoskeleton to the ECM during cell migration. This work was done in collaboration with Bob Ross, and has been presented at Cell Biology Society meeting and Gordon Conference. Project 2: Nanoscale architecture of integrin-based cell adhesions Pakorn Kanchanawong Focal adhesions (FAs) mediate cell interactions with their extracellular matrices (ECMs) and consist of integrin ECM receptors linked to the actin cytoskeleton via plasma-membrane-associated protein plaques. Despite their fundamental importance in multicellular organisms, the three-dimensional organization of proteins within FAs is unknown. Here we determine FA molecular architecture by using 3D superresolution microscopy (interferometric Photo-Activated Localization Microscopy) to map nanoscale protein organization. We find that the FAs consist of partially overlapping proteinspecific vertical layers of 15-50 nm thickness, with integrins and actin separated by a 30- 50 nm FA core which is spanned by talin tethers. This reveals a structural basis for FA function whereby a multilaminar core architecture mediates the interdependent cell processes of adhesion, signaling, force transduction, and actin cytoskeletal regulation. This work was done in collaboration with Mike Davidson, Gleb Stengle, Harald Hess and has been revised and resubmitted at Nature. Project 3: Actin dynamics correlate with force transmission through focal adhesions Clare Waterman How focal adhesions (FAs) convert retrograde actin filament (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined Traction Force and Fluorescent Speckle Microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8-10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family GTPase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration. This work was performed in collaboration with Margaret Gardel (University of Chicago), Gaudenz Danuser and Lin Ji(Scripps), Ulrich Schwartz and Benedikt Sabass (Heidelberg University) and was accepted for publication. Project 4:A zyxin-mediated mechanism for actin stress fiber maintenance and repair. Clare Waterman