Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs

Four different tandem EGFPs were constructed to elucidate the nuclear microenvironment by quantifying its diffusional properties in both aqueous solution and the nuclei of living cells. Diffusion of tandem EGFP was dependent on the length of the protein as a rod-like molecule or molecular ruler in solution. On the other hand, we found two kinds of mobility, fast diffusional mobility and much slower diffusional mobility depending on cellular compartments in living cells. Diffusion in the cytoplasm and the nucleoplasm was mainly measured as fast diffusional mobility. In contrast, diffusion in the nucleolus was complex and mainly much slower diffusional mobility, although both the fast and the slow diffusional mobilities were dependent on the protein length. Interestingly, we found that diffusion in the nucleolus was clearly changed by energy depletion, even though the diffusion in the cytoplasm and the nucleoplasm was not changed. Our results suggest that the nucleolar microenvironment is sensitive to energy depletion and very different from the nucleoplasm.     

Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells

We have previously demonstrated that apical Na⁺ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971–6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na⁺ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882–C888); however, it is unknown if the Na⁺ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for modulation of Na⁺ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for regulation of Na⁺ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na⁺ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na⁺ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na⁺ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 ± 5.42%) when compared to control (unstimulated) (f = 14.2 ± 0.77%) and vasopressin-treated (20 min) (f = 12.7 ± 2.4%) monolayers. The twofold increase in mobile fraction of Na⁺ channels corresponds to the average increase in Na⁺ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na⁺ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na⁺ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na⁺ channels.Supported by NIH grants DK37206 (DJB), NS26733 and NS28072 (KJA), DK46705 (PRS) and AHA New York Affiliate grant 91007G (LCS).     

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET

Members of the basic helix-loop-helix （bHLH） gene family play important roles in vertebrate neurogenesis. In this study, confocal microscopy-based fluorescence resonance energy transfer （FRET） is used to monitor bHLH protein-protein interactions under various physiological conditions. Tissue-specific bHLH activators, NeuroD 1, Mash 1, Neurogenin 1 （Ngn 1）, Neurogenin2 （Ngn2）, and ubiquitous expressed E47 protein are tagged with enhanced yellow fluorescence protein （EYFP） and enhanced cyan fluorescence protein （ECFP）, respectively. The subcellular localization and mobility ofbHLH fusion proteins are examined in HEK293 cells. By transient transfection and in ovo electroporation, four pairs of tissue-specific bHLH activators and E47 protein are over-expressed in HEK293 cells and developing chick embryo neural tube. With the acceptor photobleaching method, FRET could be detected between these bHLH protein pairs in the nuclei of transfected cells and developing neural tubes. Mashl/E47 and Ngn2/E47 FRET pairs show higher FRET efficiencies in the medial and the lateral half of chick embryo neural tube, respectively. It suggests that these bHLH protein pairs formed functional DNA-protein complexes with regulatory elements of their downstream target genes in the specific regions. This work will help one understand the behaviours of bHLH factors in vivo.     

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins^1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)^3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination^6-10. FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application^8,10. We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This ''FRAP-FLIP'' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines¹⁰, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.