A model showing steps involved in E50K-OPTN-induced death of retinal cells.

<p>In cells expressing wild type optineurin, it plays a role in autophagy and also in TFR trafficking and recycling. This maintains homeostasis that is necessary for survival of normal cells. In retinal cells expressing E50K-OPTN, autophagosomes are formed but autophagy flux (autolysosome formation) is inhibited that leads to accumulation of large autophagosomes. TFR recycling is also impaired in E50K expressing cells. In both these processes TBC1D17 plays a crucial role. Impaired autophagy as well as impaired TFR recycling leads to loss of cellular homeostasis that results in cell death. This cell death can be largely prevented by blocking TBC1D17 function and partly by overexpressing TFR.</p

E50K-OPTN-induced inhibition of autophagic flux is rescued by TBC1D17 knockdown.

<p>The cells plated on coverslips were cotransfected with mCherry-GFP-LC3B and indicated plasmids. After 28 hours of transfection, cells were treated with EBSS for 2 hours. (A) Images showing representative fields with autophagosomes (yellow) and autolysosomes (red). Scale bar: 10 µm. Bar diagram showing number of autophagosomes (yellow) (B) and autolysosomes (red) (C) per cell in EBSS treated cells (mean±SD). n = 40 cells from 2 independent experiments. **p<0.01, ***p<0.001.</p

E50K-OPTN-Induced Retinal Cell Death Involves the Rab GTPase-Activating Protein, TBC1D17 Mediated Block in Autophagy

<div><p>The protein optineurin coded by <i>OPTN</i> gene is involved in several functions including regulation of endocytic trafficking, autophagy and signal transduction. Certain missense mutations in the gene <i>OPTN</i> cause normal tension glaucoma. A glaucoma-causing mutant of optineurin, E50K, induces death selectively in retinal cells. This mutant induces defective endocytic recycling of transferrin receptor by causing inactivation of Rab8 mediated by the GTPase-activating protein, TBC1D17. Here, we have explored the mechanism of E50K-induced cell death. E50K-OPTN-induced cell death was inhibited by co-expression of a catalytically inactive mutant of TBC1D17 and also by shRNA mediated knockdown of TBC1D17. Endogenous TBC1D17 colocalized with E50K-OPTN in vesicular structures. Co-expression of transferrin receptor partially protected against E50K-induced cell death. Overexpression of the E50K-OPTN but not WT-OPTN inhibited autophagy flux. Treatment of cells with rapamycin, an inducer of autophagy, reduced E50K-OPTN-induced cell death. An LC3-binding-defective mutant of E50K-OPTN showed reduced cell death, further suggesting the involvement of autophagy. TBC1D17 localized to autophagosomes and inhibited autophagy flux dependent on its catalytic activity. Knockdown of TBC1D17 rescued cells from E50K-mediated inhibition of autophagy flux. Overall, our results suggest that E50K mutant induced death of retinal cells involves impaired autophagy as well as impaired transferrin receptor function. TBC1D17, a GTPase-activating protein for Rab GTPases, plays a crucial role in E50K-induced impaired autophagy and cell death.</p></div

Involvement of autophagy in E50K-induced cell death.

<p>(A) Cells plated in 24-well plate were infected with control or WT OPTN or E50K adenoviruses and allowed to express for 24 hours. Protein lysates were subjected to western blotting with p62, LC3-II and HA antibodies. Actin was used as a loading control. (B) Cells plated on coverslips were transfected with GFP-E50K plasmids. After 12 hours of transfection, cells were treated either with DMSO or with 1 µM rapamycin (Rapa) for 20 hours. At the end of 32 hours of overexpression, cells were fixed and mounted to score for cell death. Bar diagram depicts percentage of cells showing E50K-induced cell death in the presence and absence of rapamycin. Data represents mean ± SD of 5 experiments. ***p<0.001 (Student’s t-test). (C) Cells plated in 35-mm dishes were transfected with GFP-E50K and after 12 hours of transfection, cells were treated with either DMSO or rapamycin (Rapa) (1 µM) for 20 hours. At the end of 32 hours of overexpression, lysates were made and subjected to western blot with cleaved caspase-3 and GFP antibodies. Actin was used as a loading control.</p

E50K-OPTN-induced cell death is dependent on UBD and LC3-interacting region.

<p>(A) Schematic of optineurin protein showing domains LIR (LC3 interacting region) and UBD (ubiquitin binding domain), ZF (zinc finger) and CC (coiled coil). (B) Cells plated on coverslips were transfected with 300ng of GFP-E50K or GFP-E50K-D474N or GFP-E50K-F178A. After 32 hours of transfection, cells were fixed and cell death was determined in the respective expressing cells. Bar diagram showing quantitation of cell death in the presence of each of the mutants after subtracting background cell death in non-expressing cells. Data represent mean ± SD of 3 experiments. **p<0.01 (Student’s t-test) (C) Cells were transfected with 3 µg of GFP control plasmid or GFP-WT or GFP-E50K or GFP-E50K-F178A plasmids and overexpressed for 32 hours. Western blot was done with cleaved caspase-3 and GFP antibodies. GAPDH was used as a loading control. UT, untransfected (D) The cells were transfected with 3 µg of control plasmid or HA-WT or HA-E50K or HA-E50K-D474N plasmids and overexpressed for 32 hours. Western blot was done with cleaved caspase-3 and HA antibodies.</p

Effect of TFR expression on E50K-OPTN-induced cell death.

<p>(A) The cells plated on coverslips were transfected with 100ng of GFP-E50K alone or 200ng of TFR alone or cotransfected with 100ng GFP-E50K and 200ng TFR. After 32 hours of overexpression, cells were stained with TFR antibody and cell death was analysed. Data shown here represent cell death in expressing cells after subtracting background cell death from non-expressing cells from 6 experiments (mean±SD). ***p<0.001 (student’s t-test) (B) Cells plated in 35-mm dishes were transfected with 1 µg of GFP-E50K alone or 2 µg of TFR alone or cotransfected with 1 µg GFP-E50K and 2 µg TFR. After 32 hours of overexpression, cell lysates were made and subjected to western blotting with cleaved caspase-3, GFP and TFR antibodies. GAPDH was used as a loading control. UT, untransfected.</p

video_3.mp4

<p>Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), also known as AKAP450, is a cytosolic scaffolding protein involved in the targeted positioning of multiple signaling molecules, which are critical for cellular functioning. Here, we show that CG-NAP is predominantly expressed in human primary T-lymphocytes, localizes in close proximity (<0.2 μm) with centrosomal and Golgi structures and serves as a docking platform for Protein Kinase A (PKA). GapmeR-mediated knockdown of CG-NAP inhibits LFA-1-induced T-cell migration and impairs T-cell chemotaxis toward the chemokine SDF-1α. Depletion of CG-NAP dislocates PKARIIα, disrupts centrosomal and non-centrosomal microtubule nucleation, causes Golgi fragmentation, and impedes α-tubulin tyrosination and acetylation, which are important for microtubule dynamics and stability in migrating T-cells. Furthermore, we show that CG-NAP coordinates PKA-mediated phosphorylation of pericentrin and dynein in T-cells. Overall, our findings provide critical insights into the roles of CG-NAP in regulating cytoskeletal architecture and T-cell migration.</p

video_4.avi

<p>Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), also known as AKAP450, is a cytosolic scaffolding protein involved in the targeted positioning of multiple signaling molecules, which are critical for cellular functioning. Here, we show that CG-NAP is predominantly expressed in human primary T-lymphocytes, localizes in close proximity (<0.2 μm) with centrosomal and Golgi structures and serves as a docking platform for Protein Kinase A (PKA). GapmeR-mediated knockdown of CG-NAP inhibits LFA-1-induced T-cell migration and impairs T-cell chemotaxis toward the chemokine SDF-1α. Depletion of CG-NAP dislocates PKARIIα, disrupts centrosomal and non-centrosomal microtubule nucleation, causes Golgi fragmentation, and impedes α-tubulin tyrosination and acetylation, which are important for microtubule dynamics and stability in migrating T-cells. Furthermore, we show that CG-NAP coordinates PKA-mediated phosphorylation of pericentrin and dynein in T-cells. Overall, our findings provide critical insights into the roles of CG-NAP in regulating cytoskeletal architecture and T-cell migration.</p

Presentation_1.PDF

<p>Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), also known as AKAP450, is a cytosolic scaffolding protein involved in the targeted positioning of multiple signaling molecules, which are critical for cellular functioning. Here, we show that CG-NAP is predominantly expressed in human primary T-lymphocytes, localizes in close proximity (<0.2 μm) with centrosomal and Golgi structures and serves as a docking platform for Protein Kinase A (PKA). GapmeR-mediated knockdown of CG-NAP inhibits LFA-1-induced T-cell migration and impairs T-cell chemotaxis toward the chemokine SDF-1α. Depletion of CG-NAP dislocates PKARIIα, disrupts centrosomal and non-centrosomal microtubule nucleation, causes Golgi fragmentation, and impedes α-tubulin tyrosination and acetylation, which are important for microtubule dynamics and stability in migrating T-cells. Furthermore, we show that CG-NAP coordinates PKA-mediated phosphorylation of pericentrin and dynein in T-cells. Overall, our findings provide critical insights into the roles of CG-NAP in regulating cytoskeletal architecture and T-cell migration.</p

video_1.mp4

<p>Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), also known as AKAP450, is a cytosolic scaffolding protein involved in the targeted positioning of multiple signaling molecules, which are critical for cellular functioning. Here, we show that CG-NAP is predominantly expressed in human primary T-lymphocytes, localizes in close proximity (<0.2 μm) with centrosomal and Golgi structures and serves as a docking platform for Protein Kinase A (PKA). GapmeR-mediated knockdown of CG-NAP inhibits LFA-1-induced T-cell migration and impairs T-cell chemotaxis toward the chemokine SDF-1α. Depletion of CG-NAP dislocates PKARIIα, disrupts centrosomal and non-centrosomal microtubule nucleation, causes Golgi fragmentation, and impedes α-tubulin tyrosination and acetylation, which are important for microtubule dynamics and stability in migrating T-cells. Furthermore, we show that CG-NAP coordinates PKA-mediated phosphorylation of pericentrin and dynein in T-cells. Overall, our findings provide critical insights into the roles of CG-NAP in regulating cytoskeletal architecture and T-cell migration.</p