Ruijing Tang

• Water transport through the water channels, aquaporins (AQPs), is involved in epithelial fluid secretion and absorption, cell migration, brain edema, adipocyte metabolism, and other physiological or pathological functions. Modulation of AQP function has therapeutic potential in edema, cancer, obesity, brain injury, glaucoma, etc. The function of AQPs is in response to the osmotic gradient that is formed by the concentration differences of ions or small molecules. In terms of brain edema, it is a pathophysiological condition, resulting fromWater transport through the water channels, aquaporins (AQPs), is involved in epithelial fluid secretion and absorption, cell migration, brain edema, adipocyte metabolism, and other physiological or pathological functions. Modulation of AQP function has therapeutic potential in edema, cancer, obesity, brain injury, glaucoma, etc. The function of AQPs is in response to the osmotic gradient that is formed by the concentration differences of ions or small molecules. In terms of brain edema, it is a pathophysiological condition, resulting from dysfunction of the plasma membrane that causes a disorder of intracellular ion homeostasis and thus increases intracellular fluid content. Optogenetics can be used to regulate ion transport easily by light with temporal and spatial precision. Therefore, if we control the cell ion influx, boosting the water transport through AQPs, this will help to investigate the pathological mechanisms in e.g. brain edema. To this end, I investigated the possibility for optogenetic manipulating water transport in Xenopus oocytes. The main ions in Xenopus oocyte cytoplasm are ~10 mM Na+, ~50 mM Cl- and ~100 mM K+, similar to the mammalian cell physiological condition. Three light-gated channels, ChR2-XXM 2.0 (light-gated cation channel), GtACR1 (light-gated anion channel) and SthK-bPAC (light-gated potassium channel), were used in my study to regulate ion transport by light and thus manipulate the osmotic gradient and water transport. To increase water flow, I also used coexpression of AQP1. When expressing ChR2-XXM 2.0 and GtACR1 together, mainly Na+ influx was triggered by ChR2-XXM2.0 under blue light illumination, which then made the membrane potential more positive and facilitated Cl- influx by GtACR1. Due to this inward movement of Na+ and Cl-, the osmotic gradient was formed to trigger water influx through AQP1. Large amounts of water uptake can speedily increase the oocyte volume until membrane rupture. Next, when co-expressing GtACR1 and SthK-bPAC, water efflux will be triggered with blue light because of the light-gated KCl efflux and then oocyte shrinking could be observed. I also developed an optogenetic protein purification method based on a light-induced protein interactive system. Currently, the most common protein purification method is based on affinity chromatography, which requires different chromatography columns and harsh conditions, such as acidic pH 4.5 - 6 and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. The change in conditions could influence the activity of target proteins. So, an easy and flexible protein purification method based on the photo-induced protein interactive system iLID was designed, which regulates protein binding with light in mild conditions and does not require a change of solution composition. For expression in E. coli, the blue light-sensitive part of iLID, the LOV2 domain, was fused with a membrane anchor and expressed in the plasma membrane, and the other binding partner, SspB, was fused with the protein of interest (POI), expressed in the cytosol. The plasma membrane fraction and the soluble cytosolic fraction of E. coli can be easily separated by centrifugation. The SspB-POI can be then captured to the membrane fraction by light stimulation and released to clean buffer in the dark after washing. This method does not require any specific column and functions in mild conditions, which are very flexible at scale and will facilitate extensive protein engineering and purification of proteins, sensitive to changed buffer conditions.…
• Der Wassertransport durch die Wasserkanäle, Aquaporine (AQPs), ist unter anderem an der Sekretion und Absorption der Epithelflüssigkeit, der Zellmigration, dem Hirnödem und dem Adipozytenstoffwechsel beteiligt. Die Modulation der AQP-Funktion hat therapeutisches Potenzial bei Ödemen, Krebs, Fettleibigkeit, Hirnverletzungen, Glaukom usw. Die Funktion von AQPs reagiert auf den osmotischen Gradienten, der durch Konzentrationsunterschiede von Ionen oder kleinen Molekülen gebildet wird. In Bezug aufDer Wassertransport durch die Wasserkanäle, Aquaporine (AQPs), ist unter anderem an der Sekretion und Absorption der Epithelflüssigkeit, der Zellmigration, dem Hirnödem und dem Adipozytenstoffwechsel beteiligt. Die Modulation der AQP-Funktion hat therapeutisches Potenzial bei Ödemen, Krebs, Fettleibigkeit, Hirnverletzungen, Glaukom usw. Die Funktion von AQPs reagiert auf den osmotischen Gradienten, der durch Konzentrationsunterschiede von Ionen oder kleinen Molekülen gebildet wird. In Bezug auf das Hirnödemhandelt es sich um einen pathophysiologischen Zustand, der aus einer Funktionsstörung der Plasmamembran resultiert und eine Störung der intrazellulären Ionenhomöostase verursacht und somit den intrazellulären Wassergehalt erhöht. Die Optogenetik kann verwendet werden, um den Ionentransport durch Licht mit zeitlicher und räumlicher Präzision leicht zu regulieren. Wenn wir also den Ioneneinstrom in die Zelle erhöhen, wird dies den Wasserimport durch AQPs fördern. ...…