IMAGING AND TRACKING OF SINGLE CELL FLUORESCENT PROBES

单细胞荧光探针的成像和跟踪

• 批准号: 8362566
• 负责人: KEVIN BURGESS
• 金额: $ 1.3万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362566
• 关键词: Active Biological Transport; Cell membrane; Cells; Chariot peptide; Chemicals; Complex; Coupling; Cytosol; Degradation Pathway; Diffusion; Dyes; Electroporation; Encapsulated; Energy Transfer; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Funding; Grant; Image; Laboratories; Lasers; Life; Light; Liposomes; Methods; Microinjections; National Center for Research Resources; Nature; Optics; Peptides; Photons; Principal Investigator; Process; Protein Import; Proteins; Research; Research Infrastructure; Resolution; Resources; Scheme; Source; System; Time; United States National Institutes of Health; Vesicle; Viral Vector; Work; absorption; base; cost; design; receptor; single molecule

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 这项工作的中心假设是，当供体和受体系统通过不允许它们变成平面的共轭连接体连接时，可能会通过键发生从供体到受体的快速能量转移。与 F 不同??供体片段的发射与受体部分的吸收重叠没有已知的要求，因此，适当设计的通过键合能量转移盒可以通过一个或多个供体部分吸收光子。方便的波长（例如 488 nm：来自 Ar 激光的激发），通过共轭连接体将能量快速转移到以更长波长发射的受体片段。对供体之间的差异没有限制。因此，可以设计出在短波长下吸收并在相隔许多波数的几个波长（由受体的化学性质决定）下以非常相似的强度发射的染料。具有出色的分辨率，在共轭系统中将多个供体与受体结合有利于吸收更多的光，从而增加发射强度。 总之，通过键合能量转移盒有可能增加两者。由在单一波长下工作的激光源激发的多个探针获得的分辨率和荧光强度。 蛋白质一般不能通过被动扩散进入细胞，而需要主动转运，虽然有些蛋白质也可以通过显微注射、脂质体包埋、病毒载体和电穿孔转运到细胞内，但此类方法费力、耗时，且效率往往较低。最近开发的一种涉及称为“Chariot”（Active Motif，卡尔斯巴德，加利福尼亚州）的肽的方法克服了这些问题，Chariot 与蛋白质非共价复合，并促进了肽的形成。 Chariot 肽不具有细胞毒性，并且不依赖于转运蛋白或特定受体而穿过质膜，因此避免了溶酶体降解途径。Chariot 肽具有高转运效率 (65-95%)，并且已被证明可以实现这一点。一旦内化，荧光蛋白-Chariot 肽复合物就会快速共转运，从而使荧光标记的蛋白能够继续到达其细胞内靶标。 Chariot 肽迅速降解。 使用 Pep1 肽（和其他载体系统）将蛋白质/直通键盒缀合物转移到活细胞中开辟了新的研究前景。目前尚不清楚使用 Chariot 系统导入细胞的蛋白质是否在细胞质中游离。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。