IMAGING AND TRACKING OF SINGLE CELL FLUORESCENT PROBES

单细胞荧光探针的成像和跟踪

• 批准号: 7955433
• 负责人: KEVIN BURGESS
• 金额: $ 0.96万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955433
• 关键词: Active Biological Transport; Cell membrane; Cells; Chariot peptide; Chemicals; Complex; Computer Retrieval of Information on Scientific Projects Database; Coupling; Cytosol; Degradation Pathway; Diffusion; Dyes; Electroporation; Encapsulated; Energy Transfer; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Funding; Grant; Image; Institution; Laboratories; Lasers; Life; Light; Liposomes; Methods; Microinjections; Nature; Optics; Peptides; Photons; Process; Protein Import; Proteins; Research; Research Personnel; Resolution; Resources; Scheme; Source; System; Time; United States National Institutes of Health; Vesicle; Viral Vector; Work; absorption; base; cytotoxic; design; receptor; single molecule

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The central hypothesis of this work is that when a donor and acceptor systems are connected via a conjugated linker that does not allow them to become planar then rapid energy transfer from the donor to the acceptor may occur through bonds. Through-bond energy transfer is mechanistically different to the F¿rster basis for FRET, and there is no known requirement for overlap of the emission of the donor fragment with the absorption of the acceptor part. Thus, appropriately designed through-bond energy transfer cassettes could absorb photons via a donor part, or parts, at a convenient wavelength (eg 488 nm: excitation from an Ar-laser), transfer the energy rapidly through the conjugated linker to the acceptor fragment that emits at a far longer wavelength. There is no constraint on the difference between the donor absorption and the acceptor emission wavelengths in this scheme. It therefore is possible to design dyes that absorb strongly at a short wavelength and emit brightly with very similar intensities at several wavelengths (governed by the chemical nature of the acceptor) that are many wavenumbers apart, ie with excellent resolution. Coupling more than one donor in a conjugated system with an acceptor facilitates absorption of more light thereby increasing the intensity of the emission. In summary, through bond energy transfer cassettes have the potential to increase both the resolution and fluorescence intensities obtained from several probes excited by a laser source operating at a single wavelength. Proteins generally cannot enter cells by passive diffusion, but require active transport. While some proteins can also be transported into cells by microinjection, entrapped in liposomes, viral vectors, and electroporation, such methods are laborious, time consuming, and often have low efficiencies. A recently developed method involving a peptide called "Chariot" (Active Motif, Carlsbad, CA) overcomes these problems. Chariot non-covalently complexes with proteins to peptides and facilitates their transport into cells. The Chariot peptide is non-cytotoxic, and crosses plasma membranes independent of transporters or specific receptors, thus avoiding the lysosomal degradative pathway. The Chariot peptide has high transport efficiency (65-95%) and has already been shown to rapidly co-transport large fluorescent proteins. Once internalized, the fluorescent protein-Chariot peptide complex rapidly dissociates, thereby allowing the fluorescent-tagged protein to proceed to its intracellular target while the Chariot peptide is rapidly degraded. Use of the Pep1 peptide (and other carrier systems) to transfer protein/through-bond cassette conjugates into living cells opens new vistas of research. It is not yet evident that proteins imported into cells using the Chariot system are free in the cytosol; they could be encapsulated in intracellular vesicles. One of the objectives of our research is to elucidate this with single molecule studies performed at the center.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 这项工作的中心假设是，当捐助者和受体系统通过共轭接头连接时，该连接器不允许它们成为平面，然后通过债券从捐助者到受体的快速能量转移。贯穿键的能量转移在机械上与FRET的f基础不同，并且尚无已知要求将捐赠者片段与受体部分重叠的要求。这样，适当设计的整个键传递盒可以通过方便的波长（例如488 nm：从AR-激动器引起的兴奋）通过供体部件或零件吸收照片，从而通过连接的接头迅速将能量传递到散发出较长波长的受体片段。在该方案中，供体缓解和受体发射波长之间的差异没有限制。可以设计在短波长下强烈吸收的染料，并在几个波长（由受体的化学性质约束）下明亮地发射，它们分开了许多波数，即具有出色的分辨率。与受体的共轭系统中，将多个捐赠者耦合，综合促进了更多的光，从而摘要，通过键转移盒有可能增加从单个波长的激光源激发的几个问题获得的分辨率和荧光强度。 蛋白质通常不能通过被动扩散进入细胞，而需要主动运输。虽然某些蛋白质也可以通过显微注射转运到细胞中，以脂质体，病毒载体和电穿孔输入，但这种方法是实验室，耗时，并且通常具有较低的效率。最近开发的方法涉及一种称为“战车”（Active Motif，Carlsbad，CA）的肽，以克服这些问题。战车与蛋白质无比复合物以肽并促进其运输到细胞中。战车肽是非环毒性的，并且跨越质膜独立于转运蛋白或特定受体，从而避免了溶酶体降解途径。战车肽具有较高的运输效率（65-95％），并且已经显示出迅速共同转发大型荧光蛋白。一旦内化，荧光蛋白 - 特征肽复合物迅速解离，从而使荧光标记的蛋白在战车肽迅速降解的同时继续前进到其细胞内靶标。 使用Pep1 Pepperide（和其他载体系统）将蛋白质/透过键的盒式盒子转移到活细胞中，从而打开了新的研究远景。尚无证据表明，使用战车系统在细胞质中免费进口到细胞中的蛋白质是免费的。它们可以封装在细胞内蔬菜中。我们研究的目标之一是通过在中心进行的单分子研究来阐明这一点。