The Section on Organelle Biology investigates the global principles underlying secretory membrane trafficking, sorting and compartmentalization within eukaryotic cells. Live cell imaging of green fluorescent protein (GFP) fusion proteins in combination with photobleaching and photoactivation techniques are being used to investigate the subcellular localization, mobility, transport routes and binding interactions of a variety of proteins with important roles in the organization and regulation of membrane traffic and compartmentalization. Quantitative measurements of these protein characteristics are used in kinetic modeling and simulation experiments in order to test mechanistic hypotheses related to protein and organelle dynamics. Among the topics currently under study include: growth and maintenance of endoplasmic reticulum (ER) and Golgi morphology in mammalian cells and in developing Drosophila embryos; the mechanism(s) of secretory protein transport into and out of the Golgi apparatus; membrane binding/dissociation kinetics of trafficking machinery and its regulation; the generation and maintenance of cell polarity; and, organelle breakdown and reassembly during mitosis. We have also recently developed a photoactivatable GFP, whose mechanism of photoactivation is currently being investigated. Development of a Photoactivatable GFP Photoactivation is the rapid conversion of photoactivatable molecules to a fluorescent state by intense irradiation and is a useful method for marking and monitoring selected molecules within cells. Previous efforts to develop a photoactivatable protein capable of high optical contrast when photoactivated under physiological conditions have had limited success. By mutagenizing wild type GFP (wtGFP), we have developed a variant of GFP that allows selective marking of proteins through photoactivation. The wtGFP normally exists as a mixed population of neutral phenols and anionic phenolates producing its major 397 nm and minor 475 nm absorbance peaks, respectively. Upon intense illumination of the protein with ultraviolet or ~400 nm light, the chromophore population undergoes photoconversion and shifts predominantly to the anionic form, giving rise to an increase in minor peak absorbance. This produces a ~3-fold increase in fluorescence upon excitation at 488 nm. The photoactivatable variant of wild type GFP that we developed (called PA-GFP for ?photo? and ?activatable?) contains a histidine substitution at the T203 position of GFP that results in its having a negligible minor absorbance peak. Photoconversion with ~400 nm irradiation produces a large increase in absorbance at the minor peak and thus a more noticeable optical contrast under 488 nm excitation. Upon photoactivation of living cells, PA-GFP exhibited an optical enhancement of nearly two orders of magnitude, making it suitable to mark specific protein or cell populations. The speed with which an optical signal was obtained and the absence of signal from newly synthesized proteins, furthermore, indicated PA-GFP photoactivation was a preferable labeling method to photobleaching for studying the temporal and spatial dynamics of proteins. We used the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange. Our results suggest that the photoactivatable variant of GFP, PA-GFP, has the potential for addressing many fundamental questions in cell and developmental biology. Dissection of COPI dynamics in vivo Cytosolic coat proteins that bind reversibly to membranes have a central role in membrane transport within the secretory pathway. One well studied example is COPI or coatomer, a heptameric protein complex that is recruited to membranes by the GTP-binding protein Arf1. Assembly into an electron-dense coat then helps in budding off membrane to be transport between the ER and Golgi. To study COPI dynamics in vivo, variants of GFP were fused to the carboxy terminus of the eCOPI subunit of coatomer and expressed in ldlF cells, which contain a mutated eCOP that at 40oC is degraded, causing coatomer inactivity and growth inhibition. The cells grew indefinitely at 40oC, indicating that eCOP-GFP could substitute functionally for endogenous eCOPI in coatomer complexes in these cells. The identity and behavior of coatomer-containing membranes, assessed by eCOP-YFP labeling, were studied in dual-color time-lapse experiments in ldlF cells co-expressing cyan fluorescent protein (CFP)-tagged secretory cargo markers. eCOP-YFP was present on juxtanuclear Golgi membranes and on pre-Golgi transport intermediates containing secretory cargo, and it was depleted from retrograde (Golgi-to-ER) transport intermediates. The pre-Golgi intermediates remained brightly labeled with eCOP-YFP as they tracked into the Golgi. To gain insight into the role of COPI on anterograde (ER-to-Golgi) transport intermediates we used photobleaching techniques to investigate the characteristics of COPI binding and release from membranes. Upon photobleaching either Golgi or pre-Golgi structures expressing eCOP-GFP in ldlF cells, fluorescence recovered onto these structures exponentially with a half-time of 35 s. This indicated that coatomer binding and dissociation from membranes occurs continuously. To test whether this activity is coupled to vesicle budding, we measured the kinetics of coat exchange on and off membranes at low temperatures, in which vesicle transport is nonexistent. We found that at all temperatures down to 4oC, eCOP-GFP underwent binding and release from Golgi membranes, with no abrupt change in the kinetics on reaching temperatures at which vesicle budding is slowed or inhibited. The data thus indicated that the cycling of coatomer on and off membranes can be uncoupled from vesicle formation, and that feedback from productive vesicle budding is not necessary for COPI dissociation. Based on these findings, we proposed that membrane binding and release of COPI serves to initiate and stabilize lateral sorting and segregation of cargo into membrane domains that progressively differentiate into pleiomorphic membrane transport intermediates and/or vesicles for membrane transport in the ER/Golgi system. Arf1 regulation of COPI dynamics COPI is known to be recruited to membranes by the GTP binding protein Arf1 and GTP hydrolysis of GTP-bound Arf1 is believed to be necessary for COPI release from membranes. To gain further insight into Arf1 regulation of COPI dynamics, we asked if Arf1 GTP hydrolysis is sufficient for COPI to dissociate from membranes; that is, do Arf1 and COPI dissociate together from membranes. To address this question, we utilized a functional Arf1-CFP chimera that allowed us to visualize the behavior of Arf1 and coatomer in the same cell. FRAP experiments revealed the half-time for Golgi membrane dissociation of Arf1-CFP (13s) was significantly faster than for eCOP-YFP (30 s) suggesting Arf1 and coatomer dissociate independently from Golgi membranes. This was supported by results from experiments using BFA, which inhibits Arf1 and COPI recruitment to membranes, allowing the dissociation of these proteins to be observed in the absence of rebinding. On treatment with BFA, Arf1-CFP dissociated significantly faster (T1/2 13 s) than eCOP-YFP (t1/2 30s). These findings indicated, therefore, that Arf1 and coatomer dissociation from Golgi membranes are regulated differently. A mathematical formulation of COPI and Arf1 membrane binding and dissociation kinetics was able to fit simultaneously both Arf1 and COPI photobleaching data and the Arf1 and COPI release kinetics following BFA treatment, providing new quantitative estimates of the lifetime of these molecules on membranes and their bi
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