Force-dependent mechanism of interaction between fibronectin and αvβ6

doi:10.3969/j.issn.2095-4344.1949

Abstract

Abstract:

BACKGROUND: Integrin α_vβ₆ is highly expressed on cancer cells and promotes abnormal proliferation and malignant metastasis of cancer cells. Integrin α_vβ₆ mediates the adhesion and migration of tumor cells through interacting with its ligand, fibronectin. This process is not only regulated by the interaction between integrin α_vβ₆ and fibronectin, but also adjusted by shear stress under blood flow. Therefore, it is significant to study the force-chemical mechanism of integrin α_vβ₆-fibronectin interaction. It will further our understanding of the process of malignant metastasis, and benefit the design of novel α_vβ₆-targeted drugs.

OBJECTIVE: To reveal the force-regulated mechanism of cell adhesion, interaction between integrin α_vβ₆ and fibronectin was quantified under fluid shear stress.

METHODS: The adhesive behaviors of microspheres coated with RGD fibronectin fragment on the integrin α_vβ₆-coated substrate under different shear forces were observed and recorded. To study the mechanism of integrin α_vβ₆ interacting with fibronectin, the lifetime and dissociation rate of integrin αvβ6-fibronectin bond were extracted from the instantaneous velocity-time and cumulative distance-time graphs.

RESULTS AND CONCLUSION: Flow-enhanced phenomenon of integrin α_vβ₆ interacting with fibronectin fragments was observed. Bond lifetime of integrin α_vβ₆-fibronectin increased first and then decreased as fluid shear force increased, while the bond dissociation rate decreased first and then increased. As the fluid shear force increased, the bond dissociation rate decreased first and then increased. The force-dependent lifetime and dissociation rate of integrin α_vβ₆-fibronectin bond showed that the flow-enhanced adhesion phenomenon was regulated by catch-bond mechanism.

CLC Number:

R318.08','1');return false;" target="_blank">
R318.08

R459.9

R-331

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Yang Bishan, Li Na, Li Quhuan. Force-dependent mechanism of interaction between fibronectin and α_vβ₆[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(34): 5462-5467.

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URL: https://www.cjter.com/EN/10.3969/j.issn.2095-4344.1949

https://www.cjter.com/EN/Y2019/V23/I34/5462

Figures/Tables 3

2.1 整合素αvβ6与纤连蛋白片段相互作用的特异性为了检测裹有纤连蛋白片段的微球在底板上的黏附是否为特异性黏附作用，控制剪切力为3 μN/cm2，使包被纤连蛋白片段的微球流经3种不同的流动腔底板，分别为：PBS、牛血清蛋白和牛血清蛋白+整合素αvβ6，其中牛血清蛋白浓度为2%，整合素αvβ6的质量浓度为10 mg/L。用显微镜观察并使用高速摄像机以100帧的速度记录微球在底板上的黏附行为，离线用IPP进行图形回放分析，计算黏附微球数与流经相同视窗的总微球数的比率，即黏附比率。由图2可见，在只经过PBS处理的底板上，较多微球发生黏附，其黏附比率为(6.0±1.7)%。在经过2%牛血清蛋白处理的底板上，发生黏附的微球比率明显下降，下降至(1.2±0.4)%，说明2%牛血清蛋白阻断了微球在底板上的非特异性黏附。当包被纤连蛋白片段的微球在经过2%牛血清蛋白处理的整合素αvβ6铺展的基质上时，黏附事件数相较于牛血清蛋白对照组明显增加，其黏附比率为(6.7±0.8)%，表明在使用2%牛血清蛋白阻断非特异性黏附的条件下，微球在铺有整合素αvβ6的底板上黏附是由整合素αvβ6与纤连蛋白片段的特异性相互作用。 "

2.2 整合素αvβ6与纤连蛋白片段的键生存时间呈力依赖的双态性由以上实验可知，微球在底板上的黏附行为是由整合素αvβ6与纤连蛋白片段所介导的。在特异相互作用下，将根据瞬时速度图和累积位移图通过t2-t1测定整合素αvβ6-纤连蛋白片段分子对的键生存时间。当底板上整合素αvβ6的密度足够低的情况下，微球在底板上的停留时间是由微球上的纤连蛋白片段与底板上的整合素αvβ6成单键所致，因此，计算微球在底板上的停留时间即能提取整合素αvβ6-纤连蛋白片段的键生存时间。为了探究整合素αvβ6-纤连蛋白片段的键生存时间与剪切力的关系，控制剪切力大小为1，2，3，5，7 μN/cm2，将裹有纤连蛋白片段的微球灌注于铺有10 mg/L 整合素αvβ6的基质底板中，使微球在流动腔腔体中流动，观察和记录微球在底板上的黏附行为，见图3A。从瞬时速度-时间图和累积距离-时间图提取生存时间，见图3B，C。由图可见随着流体剪切力的增加，整合素αvβ6与纤连蛋白片段相互作用的键生存时间先增加后减少，见图3D，其峰值为剪切力3 μN/cm2。 2.3 整合素αvβ6与纤连蛋白片段的键解离速率分析研究假设微球上纤连蛋白片段与底板上整合素αvβ6成键为单键，因此微球与底板解离服从一级解离动力学模型，由此模型可知，ln(C)=-kofft，其中，C为复合物的个数，koff为键解离速率，t为键生存时间。在图4中，根据一级解离动力学模型，以键生存时间作为横坐标，对应大于等于该生存时间的微球数的自然对数为纵坐标，作出散点图并进行线性拟合，直线的负斜率即为键解离速率。由图4A，B可见，随着剪切力从1 μN /cm2提高至3 μN/cm2，线性拟合的负斜率下降；随着剪切力从3 μN/cm2提高至7 μN /cm2，负斜率上升。通过拟合直线的斜率，提取出每个剪切力下整合素αvβ6-纤连蛋白片段的键解离速率koff，发现随着剪切力的增加，整合素αvβ6-纤连蛋白片段的键解离速率koff呈先下降后增加的趋势，见图4C，与键生存时间趋势相反，但具有相同的剪切力最低点3 μN/cm2。进一步采用逆反应速率常数的方法计算键的生存时间，结果表明通过2种不同的计算方法得到的键生存时间基本一致，见图4D，即从瞬时速率曲线中t2-t1方法及键解离速率的倒数方法。该结果进一步证明该单键黏附过程服从一级解离动力学模型。"

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Force-dependent mechanism of interaction between fibronectin and α_vβ₆

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