Fig 1.
Expression of horseradish peroxidase variants in neuroblastoma cells.
(a) WT HRP transfection did not result in detectable HRP reaction product. (b) eGFP was reliably detected. Most HRP variants with enhanced catalytic activity were HRP-positive in bright field images: (c) 13A7_N175S), (d) H1-8H10, (e) H1-6E1, and (f) H2-10G5_M83I.
Table 1.
List of HRP variants and their corresponding mutations.
Fig 2.
Co-transfection of NB41a cells with eGFP and various HRP mutants.
The top row shows the bright-field images after HRP cytochemistry, and the bottom row depicts the corresponding epi-fluorescence image (eGFP).
Fig 3.
Comparison of HRP variants targeted to the endoplasmic reticulum (ER).
(a) Comparison of transfection rates after HRP and eGFP co-transfection in neuroblastoma cells. Only the co-expression of WT HRP and eGFP was significant different (t-test p 0.001). (b) Cumulative histogram of the density of neuroblastoma cell body transfected with various HRP mutants. eHRP produced the highest densities. Both 10G5 and 6E1 were significantly different compared to WT and 13A5LE was significantly different to 10G5 and 6E1. eHRP produced the highest densities. The Kolmogorov-Smirnov test was used to determine significant differences. Comparison of eHRP (c) with eGFP (d). The black arrowheads point to examples where the eHRP density produces a stronger signal than eGFP (white arrowheads in (d)).
Fig 4.
Live imaging correlated with light-electron microscopy.
Cultured hippocampal neurons were transfected with eGFP (a) and ER-targeted eHRP (b). The arrowheads point to the HRP-labeled dendrite. (c) Electron micrograph of the same HRP-labeled dendrite as in (b). The ultrastructure is optimally preserved. The arrows point to the labeled ER. (d) Unlabeled (bottom) and labeled (top) dendrites when imaged in the EM. Note the high signal-to-noise ratio of the HRP-positive ER (arrows) compared to the ER in the unlabeled dendrite (d). Both dendrites are innervated by presynaptic boutons (s). Bars in (c) and (d) = 500 nm.
Fig 5.
High resolution fluorescent image of eHRP labeled neurons in culture.
eHRP was targeted to the endoplasmic reticulum and visualized with Amplex Red.
Fig 6.
Targeting eHRP to the plasma membrane.
(a) Neuroblastoma cells transfected with eHRP targeted to the plasma membrane in the light microscope. In eHRP positive cells many more fine processes can be resolved. (b) Electron micrograph of two neighboring eHRP positive cells. eHRP reaction product is found evenly distributed on the extracellular side of the plasma membrane. Even on the finest filopodia (arrowhead) are labeled. The arrow points to the thin labeled extracellular space between a process and a cell body. Bar = 300 nm.
Fig 7.
Neuronal cells expressing a fusion protein of eHRP and synaptotagmin 1 (eHRPsyt1).
(a) Numerous vesicles (arrows) of various sizes are HRP-positive in a young cultured hippocampal neuron. At some locations eHRPsyt1 can be found at the plasma membrane (arrowhead). Bar = 200 nm. (b) An electron micrograph of an eHRPsyt1-positive presynaptic bouton. Note the numerous synaptic vesicles distributed throughout the synaptic vesicle cluster. Some labeled synaptic vesicles are also docked at the active zone (to the left). Bar = 200 nm.
Fig 8.
Quintuple labeling of neuroblastoma cells in a single sample.
Each unique label and/or combination of labeling corresponds to a unique color. The three single labelings are in red, green and blue. The double labelings are in yellow, turquoise, and magenta. The triple labeling is in white. (a) An unlabeled cell. The horizontal arrowheads point at the endoplasmic reticulum, the vertical arrowheads at small vesicles with an electron-lucent lumen. The arrows demarcate the plasma membrane. (b) A cell expressing ER-targeted eHRP (arrowheads). (c) Expression of eHRPsyt1. Note the numerous small eHRP-positive vesicles in the cytosol (e.g. arrowheads). (d) Single expression of eHRP targeted to the plasma membrane. The arrows point at the labeled surface of the cell. (e) Double expression ER-targeted eHRP and eHRPsyt1. The horizontal arrowheads point to the labeled ER and the vertical arrowheads to vesicle labeling. (f) Double labeling with eHRP targeted to the plasma membranes and eHRPsyt1. The arrows point at the labeled cell surface and the arrowheads to labeled vesicles. (g) Double labeling of ER-targeted eHRP and eHRP targeted to the plasma membrane. Again, the arrows indicate the labeled plasma membrane and the horizontal arrowheads the labeled ER. (h) Triple labeling with all three fusion proteins. Horizontal arrows mark the labeled ER, vertical arrows labeled vesicles, and arrows point at the labeled cell surface. Bars = 500 nm in (a), 200 nm in (b), 1 μm in (c), 200 nm in (d), 500 nm in (e), 200 nm in (f), 200 nm in (g), 1 μm in (h).