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Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.

機(jī)譯：腺苷酸激酶及其同工型的細(xì)胞表征：雙光子激發(fā)熒光成像和熒光相關(guān)光譜。

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摘要

Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the mobility of the target protein in live cells. This technology offers advantages in studying protein interactions and function in the cell.
機(jī)譯：腺苷酸激酶（AK）是一種普遍存在的酶，可調(diào)節(jié)細(xì)胞中腺嘌呤核苷酸的穩(wěn)態(tài)。來自鼠細(xì)胞的AK1beta（長型）與AK1（短型）具有相同的蛋白質(zhì)序列，只是在其N端添加了18個(gè)氨基酸殘基。假設(shè)這些殘基充當(dāng)?shù)鞍踪|(zhì)脂質(zhì)修飾和蛋白質(zhì)靶向質(zhì)膜的信號(hào)。為了更好地了解這些AK同工型的細(xì)胞功能，我們使用了幾種現(xiàn)代熒光技術(shù)來表征AK酶的這兩種同工型。我們?nèi)诤狭税|(zhì)腺苷酸激酶（AK1）及其同工型（AK1beta）與增強(qiáng)的綠色熒光蛋白（EGFP），并在HeLa細(xì)胞中表達(dá)了嵌合蛋白。使用雙光子激發(fā)掃描熒光成像，我們能夠直接可視化活細(xì)胞中AK1-EGFP和AK1beta-EGFP的定位。 AK1beta-EGFP主要位于質(zhì)膜上，而AK1-EGFP分布在整個(gè)細(xì)胞中，除了在核膜和某些囊泡中有痕量。我們?cè)诨罴?xì)胞的特定域中進(jìn)行了熒光相關(guān)光譜測(cè)量和光子計(jì)數(shù)直方圖分析。對(duì)于AK1-EGFP，我們?cè)诩?xì)胞質(zhì)中僅觀察到一種擴(kuò)散成分。對(duì)于AK1beta-EGFP，我們?cè)谫|(zhì)膜上觀察到兩個(gè)不同的擴(kuò)散成分。一個(gè)對(duì)應(yīng)于自由擴(kuò)散蛋白，而另一個(gè)代表與膜結(jié)合的AK1beta-EGFP。相對(duì)于EGFP，AK1-EGFP的擴(kuò)散速率降低了1.8倍，比我們預(yù)期的AK1-EGFP自由擴(kuò)散的速率高50％。為了排除低聚物形成的可能性，我們進(jìn)行了光子計(jì)數(shù)直方圖分析，以直接分析AK1-EGFP和EGFP之間的亮度差異。根據(jù)我們的分析，我們得出結(jié)論，細(xì)胞質(zhì)AK1-EGFP是單體的。熒光相關(guān)光譜被證明是定量研究靶蛋白在活細(xì)胞中遷移率的強(qiáng)大技術(shù)。這項(xiàng)技術(shù)在研究細(xì)胞中蛋白質(zhì)相互作用和功能方面具有優(yōu)勢(shì)。

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