살아있는 세포 내에서 형광물질을 이용한 막단백질 단일추적 분석 연구

Abstract

Translational diffusion of protein and lipid molecules in cellular membrane plays a fundamental role in live cell, participating in every biochemical process and cellular function taking place in the membrane. The diffusion of the molecules on the membrane can be studied theoretically as an action of Brownian motion, by regarding the membrane as an infinite plane sheet of viscous fluid and the protein or lipid molecule as a cylinder diffusing about in the sheet. In the crowded and complex environment of cellular membrane the trajectories exhibit various diffusive behaviors, such as a free diffusion of Brownian motion, anomalous motion, confined motion, or directed motion. As these motions reflect the status of molecules on the membrane, characterizing and distinguishing these trajectories of molecules on cellular membrane is critical to understand the biochemical process and cellular function. To visualize the motion of molecule diffusion in the membrane, fluorescent molecules were used in a wide range of biophysical studies. The development of the fluorescence microscopy in the past few years enabled the direct observation of single biomolecule motions in a living cell. While traditional biochemistry methods to observe diffusivity only measures the average behaviors of an ensemble of biomolecules, single-molecule techniques provided information of individual molecules where the dynamic transitions between status of different conformations and properties could be gathered. Although the analysis of individual trajectory reveals the biochemical process of the molecule observed, the number of trajectories that could be gathered from live cell by single molecule technique was limited due to photo-bleaching property of fluorescent molecules. To overcome the problem of photo-bleaching, photoactivatable property of fluorescent molecule was used in the single-particle tracking (SPT), which was known as super-resolution technique. Super-resolution techniques using photoactivatable fluorescent particle were first developed to image individual molecules distributed closer than classical diffraction limit. The technique was later applied to SPT to achieve high density tracking of single particle in live cell by using photoactivatable property to control density of particles. High density tracking gives a large population of trajectory which each trajectory provides information on dynamics of single molecule. The information altogether shows heterogeneity of motions based on their complex environment in living cell and could be used to reveal the cellular function of the target molecule in various conditions. Here, I used high density SPT method to gather diffusive information of membrane protein analyze the cellular function of live cell using diffusive property of individual trajectory. Three different techniques were used to achieve high density tracking; stochastic optical reconstruction microscopy (STORM) and photo-activated localization microscopy (PALM), the existing super-resoultion technique used for high density tracking, points accumulation for imaging in nanoscale topography (PAINT), a super-resolution technique that has not been used for high density tracking, and photoconversion of far-red fluorophore, a new method for high density tracking. First, diffusion-based molecular colocalization analysis using SPT with STORM and PALM was utilized to collect information of highly dense membrane protein diffusing on the plasma membrane. By adding an extra dimension of diffusivity, a criterion for dividing true positive and false positive was obtained. High density SPT enabled a population of trajectories where the portion of trajectories that were immobile was sufficient to calculate robust subset percentage of CCPs. Although SPT using STORM uses additive and SPT PALM acquires short trajectories, the diffusion property obtained by these methods are enough when the threshold is simple (immobilized) and imaging time is short (few minutes). In this work, the pre-allocation process of CCPs for receptors in the resting state was observed by utilizing the diffusion-based molecular colocalization analysis. Next, another super-resolution technique known as PAINT was applied to achieve high density SPT. While SPT by photoactivatable dye is suitable for gathering dynamics data of a single target, it is a different story when the target number is increased. Using photoactivatable localization microscopy for simultaneous multi-color SPT is limited by several problems; photo-conversion structure limits excitation and emission channel for more color light, imaging buffer condition needs to be synchronized for all fluorophores, and light-induced density control is coincided. To overcome these issues, PAINT was applied SPT in this work to observe multiple target at a same time. Binding and detaching of DNA oligonucleotides was used to control density of tracked particles and DNA strands binding specifically to its complementary strand regulated each target density by simply adjusting each concentration of imaging DNA strands in the media. Using this technique, I aimed to gain individual dynamics information of the different target protein simultaneously to reveal the interactions between the tracked particles. However, the nonspecific binding of DNA to live cell membrane was inevitable, and the false positive signal trajectories by the membrane bound DNA oligo affected the analysis of target protein diffusion status. Last, the photoconversion of fluorophore was applied to SPT to achieve high density of trajectories throughout time without using additives. Recent observations that blueshifted derivatives of Cyanine dyes are formed via photoconversion have raised concerns as to the potential artifacts in multicolor imaging. Here, I show the mechanism for the photoconversion of Cyanine5 (Cy5) to Cyanine3 (Cy3) that occurs upon photoexcitation during fluorescent imaging, as well as its possibility to be used in SPT method. The formal C2H2 excision from Cy5 occurs mainly through an intermolecular pathway involving a combination of bond cleavage and reconstitution, while unambiguously confirming the identity of the fluorescent photoproduct of Cy5 to be Cy3 using various spectroscopic tools. The deletion of a two methine unit from the polymethine chain, which results in the formation of blueshifted products, commonly occurs in other cyanine dyes, such as Alexa Fluor 647 (AF647). The formation of a blueshifted congener dye can obscure the multicolor fluorescence imaging, leading to misinterpretation of the data. I, however, demonstrate that the potentially deleterious photoconversion can be exploited to develop a new photoactivation method for high-density SPT in a living cell without using ultraviolet (UV) illumination and cell-toxic additives.