IMAGING AND TRACKING OF SINGLE CELL FLUORESCENT PROBES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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成为平面的共轭连接体连接时，则可能通过键发生从供体到受体的快速能量转移。通键能量转移在力学上不同于FRET的F？rster基，并且没有已知要求给体片段的发射与受体部分的吸收重叠。因此，适当设计的通键能量转移盒可以通过一个或多个施主部分在方便的波长(例如488 nm：来自Ar激光的激发)吸收光子，并通过共轭连接物将能量快速传递到在更长波长下发射的受体片段。在该方案中，对施主吸收波长和受主发射波长之间的差异没有限制。因此，有可能设计出在短波长上强吸收，在几个波长(由受体的化学性质控制)上以非常相似的强度发出明亮的染料，这些波长相隔许多波数，即具有极好的分辨率。在共轭体系中，将一个以上的给体与受体偶联有助于吸收更多的光，从而增加发射的强度。总之，通过键能量转移盒有可能提高由工作在单一波长的激光光源激发的几个探针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，但需要主动运输。虽然一些蛋白质也可以通过显微注射、脂质体、病毒载体和电穿孔转移到细胞内，但这些方法费时费力，而且效率往往很低。最近开发的一种方法涉及一种名为“Chariot”的多肽(活性基序，加利福尼亚州卡尔斯巴德)，克服了这些问题。Chariot与蛋白质形成多肽的非共价复合体，并促进它们向细胞内的转运。Chariot肽无细胞毒性，不依赖转运蛋白或特定受体穿过质膜，从而避免了溶酶体的降解途径。Chariot多肽具有很高的转运效率(65-95%)，并且已经被证明可以快速地共运输大的荧光蛋白。一旦内化，荧光蛋白-战车多肽复合体迅速解离，从而允许荧光标记的蛋白质继续其细胞内的目标，而战车多肽被迅速降解。 利用PEP1多肽(和其他载体系统)将蛋白质/通过键盒结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系统输入细胞的蛋白质是否在胞浆中是游离的；它们可能被包裹在细胞内的小泡中。我们研究的目标之一是通过在该中心进行的单分子研究来阐明这一点。

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.