Nanophotonic Approach to Imaging Exocytosis

胞吐作用成像的纳米光子方法

基本信息

批准号：
7885125
负责人：
ZYGMUNT GRYCZYNSKI
金额：
$ 18.92万
依托单位：
UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2012-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7885125
关键词：
Address Biochemistry Biological Biological Process Biology Biophysics Breast Buffers Calcium Calcium Signaling Caliber Cancer Detection Cancerous Cell Proliferation Cell membrane Cell physiology Cell surface Cells Cellular biology Chemicals Clinical Medicine Colloids Communication Complex Deposition Development Diffusion Discipline Dyes Endocytosis Environment Epithelial Cells Event Exocytosis Extracellular Space Fluorescence Fluorescence Microscopy Fluorescence Spectroscopy Generations Goals Gold Grant Growth Hormones Image Imaging Techniques Immunochemistry In Vitro Ionic Strengths Kinetics Label Life Lung Malignant Neoplasms Malignant neoplasm of lung Membrane Membrane Proteins Methodology Methods Molecular Molecular Profiling Monitor Muscle Fibers Nanostructures Nanotechnology Neoplasm Metastasis Neurotransmitters Nutrient P2X-receptor Pathology Pathway interactions Physiological Play Positioning Attribute Process Proteins Pump Purinergic P1 Receptors Quinacrine Recording of previous events Recycling Regulation Research Personnel Resolution Role Secretory Vesicles Signal Transduction Signaling Molecule Silicon Dioxide Silver Solutions Spectrum Analysis Stimulus Structure Surface System Systems Biology Technology Testing Transmembrane Transport Vesicle Waste Products Work autocrine base cancer cell cell growth cell motility directional cell experience fluorophore imaging modality improved molecular imaging neurotransmitter uptake novel novel strategies photonics plasmonics public health relevance receptor function response tool transmission process tumor uptake

项目摘要

DESCRIPTION (provided by applicant): Fluorescence based imaging has evolved into a discipline in its own right with numerous applications in biology, clinical medicine and cancer detection. Many fundamental processes of a living cell take place at the cell surface plasma membrane, including secretion of hormones and neurotransmitters, uptake of nutrients, generation and transmission of chemical and electrical signals. Vesicular exocytosis is an important mechanism by which signaling molecules, such as hormones and neurotransmitters cross the surface membrane. The process of exocytosis-endocytosis is also involved in the recycling of membrane proteins between the plasma membrane and intracellular compartments. The ability to selectively visualize such processes and the sub-membrane structures in living cells would be tremendously helpful in improving our understanding of the fundamental principles of cell physiology and pathology. However, today no imaging method, with some exception for TIRF, is capable of directly visualizing in live cells the exocytotic events that involve transport and secretion of vesicular cargo across the plasma membrane. The size of the cell membrane is only ~10 nm and the diameter of many secretory vesicles is often well below 50 nm, thus all imaging modalities lack sufficient resolution. The long-term goal of this R21 application is to develop and validate a novel approach for monitoring membrane processes by utilizing enormous fluorescence signal enhancement resulting from near field interactions of fluorophores with surface deposited metallic nanostructures. This application builds on our recent discovery that self assembled colloidal structures (SACS) produce up to 1000- fold fluorescence signal enhancement for closely positioned fluorophores. Our immediate goal is to apply this nanophotonic phenomenon in combination with TIRF microscopy for monitoring exocytotic process with unprecedented sensitivity. This will allow close following of vesicle recruitment to the plasma membrane and kinetics of cargo release even for the smallest vesicles that typically constitute the largest portion of the secretory vesicular pool. The biological problem we want to address is the exocytotic ATP release from non-excitable, cancerous lung epithelial cells. TIRF microscopy will visualize vesicles loaded with fluorescently-tagged ATP by evanescence field excitation in distances up to 200 nm. Following the exocytotic release into the extracellular space the labelled cargo will be positioned within the enhancement field of the metallic nanostructure, producing a huge, easy to detect signal that disappears due to free diffusion of the fluorophore into the extracellular space. PUBLIC HEALTH RELEVANCE: ATP release is suggested to take place at the leading edge of a migrating cell, contributing to a coordination of many processes that are involved in a directional cell movement. Proposed nanophotonic approach will allow studying these complex processes with unprescedented sensitivity and help to better understand regulation of tumour metastasis (tumour spread) and invasiveness.

描述（由申请人提供）：基于荧光的成像已经发展成为一门独立的学科，在生物学、临床医学和癌症检测中具有广泛的应用。活细胞的许多基本过程都在细胞表面质膜上发生，包括激素和神经递质的分泌、营养物质的摄取、化学和电信号的产生和传输。囊泡胞吐作用是信号分子（例如激素和神经递质）穿过表面膜的重要机制。胞吐作用-内吞作用的过程也涉及质膜和细胞内区室之间膜蛋白的再循环。选择性地可视化活细胞中的这些过程和亚膜结构的能力将极大地有助于提高我们对细胞生理学和病理学基本原理的理解。然而，如今除了 TIRF 之外，没有任何成像方法能够直接观察活细胞中涉及囊泡货物跨质膜运输和分泌的胞吐事件。细胞膜的尺寸仅为约 10 nm，许多分泌囊泡的直径通常远低于 50 nm，因此所有成像方式都缺乏足够的分辨率。 R21 应用的长期目标是开发和验证一种监测膜过程的新方法，该方法利用荧光团与表面沉积的金属纳米结构的近场相互作用产生的巨大荧光信号增强。该应用建立在我们最近的发现之上，即自组装胶体结构 (SACS) 可为紧密定位的荧光团产生高达 1000 倍的荧光信号增强。我们的近期目标是将这种纳米光子现象与 TIRF 显微镜相结合，以前所未有的灵敏度监测胞吐过程。这将允许密切跟踪囊泡募集到质膜和货物释放的动力学，即使对于通常构成分泌囊泡库最大部分的最小囊泡也是如此。我们想要解决的生物学问题是非兴奋性癌性肺上皮细胞的胞吐 ATP 释放。 TIRF 显微镜将通过消逝场激发在最远 200 nm 的距离上可视化装载有荧光标记 ATP 的囊泡。在胞吐释放到细胞外空间后，标记的货物将被定位在金属纳米结构的增强场内，产生巨大的、易于检测的信号，该信号由于荧光团自由扩散到细胞外空间而消失。公共健康相关性：ATP 释放被认为发生在迁移细胞的前缘，有助于协调定向细胞运动中涉及的许多过程。所提出的纳米光子方法将允许以前所未有的灵敏度研究这些复杂的过程，并有助于更好地了解肿瘤转移（肿瘤扩散）和侵袭性的调节。