A similar increase was seen in GPHN.FingR-GFP staining between induced versus uninduced, consistent with a coordinated upregulation of FingR expression. To quantify the relative fidelity with which GPHN.FingR-GFP-labeled uninduced versus induced cells, we calculated the
ratio of total Gephyrin staining versus GPHN.FingR-GFP staining at individual puncta. We found that the ratio of total Gephyrin staining versus GPHN.FingR-GFP staining was 1.40 ± 0.03 (n = 200 puncta, 10 cells) for uninduced versus 1.47 ± 0.06 (n = 200 puncta, 10 cells) for induced cells. The two ratios are not significantly selleck chemicals llc different (p = 0.15, Wilcoxon), indicating that GPHN.FingR-GFP labeled Gephyrin with similar fidelity in the uninduced versus induced cells, a result that is consistent with the transcriptional regulation system responding to the increase in target with an appropriately graded increase in FingR production. To test whether
the transcriptional regulation system could work for two FingRs simultaneously, we coexpressed PSD95.FingR-GFP and GPHN.FingR-mKate2 for 7 days. Both had independently regulated transcriptional control systems. PSD95.FingR-GFP was fused to the CCR5L zinc finger (Mani Ribociclib et al., 2005), and GPHN.FingR-mKate2was fused to the IL2RG2L zinc finger (Gabriel et al., 2011). PSD95.FingR-GFP and GPHN.FingR-mKate2 each expressed in a distinctly punctate manner with very little background or overlap between the two (Figure 3H), indicating Thymidine kinase that each transcriptional feedback system worked efficiently and independently. To this point our experiments have concentrated on using FingRs to visualize the localization of endogenous proteins at single points in time. However, because FingRs can be visualized in living cells,
it should be possible to use them to observe trafficking of their endogenous target proteins. To visualize trafficking of Gephyrin we used lentivirus to express transciptionally controlled GPHN.FingR-GFP in neurons in culture for 7 days. Time-lapse imaging of these cultures revealed numerous vesicles moving in both directions in the cell body, axons, and dendrites (Figures 3I and 3J). Interestingly, axonal vesicles appeared more elongated and moved at higher velocity than dendritic vesicles, hitting speeds of ∼7 μm.s−1 (Movie S1). Thus, GPHN.FingR-GFP can be used to visualize trafficking of endogenous Gephyrin in addition to its localization. To test whether FingRs label their endogenous targets specifically in cortical neurons in culture, we expressed either transcriptionally controlled PSD95.FingR-GFP or GPHN.FingR-GFP along with siRNA against either PSD-95 or Gephyrin. Cells in which either endogenous PSD-95 or endogenous Gephyrin was knocked down expressed extremely low levels of the corresponding FingR (Figures 4A–4D, 4I–4L, and S3). In contrast, cells expressing either PSD95.FingR or GPHN.