Lipid Dip-Pen Nanolithography for Model Bio-Membrane Systems

本提案的总体目标是融合自顶向下和自底向上的策略来组装与免疫转导和细胞粘附相关的纳米级仿生脂膜。具体地说，我们的目标是开发用于表面脂质高通量图案化的工具，并了解脂质沉积和组织的基本原理。随后，我们将利用这些能力来探索几何定义和化学定制的脂质结构如何影响细胞过程。越来越多的证据表明，生物膜的各种特性强烈依赖于二维和三维的异质性（如蛋白质聚类、脂筏和质膜曲率）。此外，许多细胞信号传导过程在亚细胞长度尺度上响应信号传导剂。因此，为了研究空间分辨的、超分子的生物结构-功能关系，在纳米到微米尺度上询问这些信号事件变得越来越重要。我们之前已经开发了Dip-Pen纳米光刻（DPN），这是一种直接写入的化学沉积技术，能够产生具有纳米尺寸特征的大规模图案，并且在NSF-ICC资助的前三年，开发了沉积脂质的工具包。这种脂质DPN （L-DPN）目前允许两种或多种不同的膜组分在表面上集成，具有亚细胞纳米级的空间分辨率和每分钟平方厘米的吞吐量。此外，我们已经证明L-DPN能够研究细胞与模拟生物结构的图案表面的相互作用。本研究旨在利用L-DPN产生的这些仿生脂质模式来研究与分子生物学和生物化学相关的两种模型细胞信号系统：IgE的免疫肥大细胞激活；以及钙粘蛋白介导的上皮细胞粘附。为了充分实现这些研究，将根据沉积参数和成分分布制定更多的多组分脂质油墨并对其进行表征。目标将是双重的：第一，回答有关细胞表面相互作用的具体基本问题；第二，开发一种新工具，使研究人员能够研究这些具有高空间控制的纳米级相互作用。

The overall objective of this proposal is to merge top-down and bottom-up strategies to assemble nanoscale biomimetic lipid membranes relevant to immune transduction and cell adhesion. Specifically, we aim to develop tools for high throughput patterning of lipids on surfaces and to understand the fundamentals of lipid deposition and organization. Subsequently, we will use these capabilities to probe how geometrically-defined and chemically-tailored lipid structures affect cellular processes.There is growing evidence that the various properties of biological membranes strongly depend on both two- and three-dimensional heterogeneities (e.g. protein clustering, lipid rafts, and plasma membrane curvature). Furthermore, many cellular signaling processes respond to signaling agents at sub-cellular length scales. For studying spatially-resolved, supramolecular biological structure-function relationships, it is therefore increasingly important to interrogate these signaling events at the nanometer to micron length scales. We have previously developed Dip-Pen Nanolithography (DPN), a direct-write chemical deposition technique, capable of generating large-scale patterns with nanometer size features, and in the first three years of NSF-ICC funding, a toolkit for depositing lipids was developed. This lipid DPN (L-DPN) currently allows for the integration of two or more different membrane components on a surface with sub-cellular nanoscale spatial resolution and square centimeter per minute throughput. In addition, we have shown that L-DPN enables the investigation of cellular interactions with patterned surfaces that mimic biological structures.This proposal aims to exploit these biomimetic lipid patterns generated by L-DPN to examine two model cell signaling systems that are relevant to molecular biology and biochemistry: immunological mast cell activation by IgE; and cadherin-mediated epithelial cell adhesion. To fully enable these studies, additional multi-component lipid inks will be formulated and characterized according to deposition parameters and component distribution. The goals will be twofold: first, to answer specific fundamental questions related to cell-surface interactions; and second, to develop a new tool that will allow researchers to study these nanoscale interactions with high spatial control.