Novel PH-sensitive Red Fluorescent Protein And Its Application In Tracking Exocytosis

The research in transport and exocytosis of vesicles has always been a frontier hotspot in biological research for its important physiological significance.The dysfunction of vesicles can cause many diseases.However,vesicle exocytosis is a sequential multi-step dynamic process regulated by a variety of protein molecules.Moreover,this process involves temporal and spatial coupling and precise regulation of calcium signaling,vesicle fusion and other related events.There are still many unknowns about its complex molecular mechanism.One of the important reason for this blocked research is the lack of corresponding technical tools.How to visualize secretion of vesicles and its coupling events in living cells with high spatiotemporal resolution is a technical difficulty that needs to be broken through.There is important research significance for revealing the molecular mechanism of vesicle secretion and related biological events.At present,the super-resolution tracking of vesicle exocytosis still faces a series of challenges.One of the key technical bottlenecks is the lack of suitable fluorescent protein probes.Super-ecliptic pHLuorin(SEP)is a pH-sensitive green fluorescent protein(GFP),which is very beneficial for non-invasive monitoring of exocytosis events and has been widely used in the imaging of single vesicle fusion.However,due to the acidic environment inside the vesicles,the fluorescence is too low to observe the vesicles before fusion with the cell membrane,so SEP cannot be used to visualize the docking step.Among the available red pH-sensitive fluorescent proteins,none is comparable to SEP for practical applications due to unoptimized pH sensitivity,fluorescence brightness,or severe photochromic behavior.Therefore,there is an urgent need for a pH-sensitive RFP that can simultaneously monitor vesicle docking and fusion,and this RFP is preferably also suitable for high-temporal-resolution imaging.In this dissertation,we designed a bright and photostable pH-sensitive RFP named pHmScarlet.It is very beneficial for monitoring of exocytosis events for its highest pH sensitivity with?26-fold change in fluorescence intensity upon increasing pH from 5.5 to 7.5.To the best of our knowledge,this is the largest fold change ever detected among red FPs in this pH range.The brightness of pHmScarlet in living cells is almost seven times brighter than pHuji.pHmScarlet is more photostable than pHuji,and it does not exhibit apparent photo-switching or photochromic behavior.Using pHmScarlet can simultaneously detect the vesicle docking and fusion steps.In addition,since the emission peak of pHmScarlet is 585 nm,it can be used in combination with SEP to perform dual-color imaging of two individual secretory events.Finally,although the emission wavelength of pHmScarlet is red-shifted compared with SEP,its spatial resolution is high enough to show the ring of vesicle fusion pores using Hessian structured illumination microscope(Hessian-SIM).In this dissertation,we imaged the fusion pore structure labeled by RFP for the first time.In general,the developed pH-sensitive FP(pHmScarlet)is a new RFP with the highest pH sensitivity,and it is very bright and photostable.pHmScarlet is suitable for long-term tracking and super-resolution detection of vesicle secretion events,and it has great potential in numerous applications pertaining to neurons and other secretory cells.pHmScarlet is a powerful tool for neuroscience research in the future.