Fig 1.
Activation of the Unfolded Protein Response (UPR) complex during misfolded protein accumulation within the ER.
Accumulation of misfolded proteins in the ER lumen causes the chaperone protein BiP to dissociate from the three stress transducers PERK, IRE1 and ATF6. Loss of BiP binding, allows for either oligomerization or phosphorylation of the ER transducers, resulting in activation of their corresponding downstream signaling pathways. Prolonged signaling by the ER transducers and an inability of the cell to eliminate misfolded proteins via the endoplasmic reticulum associated degradation (ERAD) pathway leads to an upregulation of the pro-apoptotic transcription factor CHOP.
Table 1.
In-silico analysis of TULP1 mutations on protein stability and folding.
Fig 2.
Co-localization of mutant TULP1 protein with the ER-resident protein Calnexin.
Immunofluorescent localization of GFP-fused TULP1 (green) and Calnexin (red) in WT and mutant TULP1 expressing cells. Nuclei were stained using DAPI (blue) (A) WT-TULP1 expressing cells displayed predominant plasma membrane and some cytoplasmic staining patterns. (B-E) In contrast all four mutant TULP1 expressing cell lines (D94Y, R420P, I459K and F491L) displayed punctate co-localization patterns with the ER-resident marker Calnexin (merge: yellow). Scale bar = 10 μM. Approximately 100 cells per transfection were counted. Experiments were repeated twice.
Fig 3.
Retention of mutant TULP1 in the ER of fractionated cells.
(A) Overview of the subcellular fractionation procedure used for the isolation of ER microsomes. (B) Western blot analysis of ER microsomes isolated from GFP-fused WT or mutant TULP1 expressing hTERT-RPE-1 cells. Antibodies against Calnexin and Calreticulin were used to determine the ER fractions; whereas antibodies against Golgin97 and COX IV were used to determine the presence of Golgi or mitochondria, respectively. Expression of mutant TULP1 proteins (D94Y, R420P, I459K and F491L) was retained within the ER. Total cell lysate from untransfected HEK293T or hTERT-RPE-1 cells were used as controls. Actin was used a protein loading control.
Fig 4.
Induction of the ER-UPR complex in cells expressing mutant TULP1.
(A) Western blot analysis using antibodies against BiP, phosphorylated PERK (pPERK), XBP1 and XBP1s, showed induction of UPR stress proteins in cells expressing mutant forms of TULP1. Actin was used a protein loading control. Western blot experiments were repeated twice. (B) Quantification of ER-stress protein markers was performed using densitometry in mutant TULP1 expressing cells compared to WT-TULP1 expressing cells. The relative band intensity of each protein was normalized to Actin. Data is presented as arbitrary units from two separate western blot densitometry analyses. Statistical significance was calculated using the two-tailed Student’s t-test. * = p< 0.001. Since the untransfected or WT-TULP1 expressing cells revealed no activation of pPERK protein, the pPERK densitometry value was arbitrary set to 1 (arbitrary units/ pixel intensity) for statistical calculations.
Fig 5.
Mutant TULP1 expressing cells undergo apoptosis.
(A) Confocal images of untransfected, WT and mutant TULP1 expressing cells stained with TUNEL (red). (B) Quantification of TUNEL-positive nuclei showed statistically significant differences between untransfected, WT and mutant TULP1 expressing cells (p<0.0001). (C) Western blot analysis using antibodies against the pro-apoptotic transcription factor CHOP protein indicate significant induction of CHOP in mutant TULP1 expressing cell lines as compared to WT-TULP1. Approximately 200 cells from five randomly selected fields of cells from each transfection were evaluated.
Fig 6.
Co-localization and induction of UPR markers in mutant TULP1 expressing photoreceptors.
P1 WT mice retinas were co-transfected with GFP-fused (green) plasmids and a mCherry tagged ER reporter (mCh-Sec61b: red). Retinas were harvested and sectioned at P30 (A) Confocal images of retinas show co-localization (merge: yellow) of GFP-fused mutant D94Y-TULP1 and F491L-TULP1 with mCh-Sec61b, the ER resident protein, in the inner segments. Scale bar = 10μM. (B-C) Activation of the ER-UPR stress markers in transgenic mice expressing mutant TULP1 protein. qRT-PCR indicates a 1.3–5 fold increase of multiple ER-UPR stress markers in mutant D94Y-TULP1 (B) or mutant F491L-TULP1 (C) retinas vs WT-TULP1 injected retinas. qRT-PCR analysis was normalized to GAPDH (as a mRNA quantity control) and GFP (as a transfection efficiency control). The ΔΔCt method was employed to calculate fold changes. Samples were assayed in triplicates for each ER-UPR marker and qRT-PCR experiments were repeated twice. Statistical significance was assessed by using the two-tailed Student’s t-test. * = p<0.001; n.s. not statistically significant.