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1.
Int J Mol Sci ; 22(15)2021 Jul 28.
Article in English | MEDLINE | ID: mdl-34360830

ABSTRACT

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.


Subject(s)
Alcohol Oxidoreductases/metabolism , Co-Repressor Proteins/metabolism , Eye Proteins/metabolism , Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Photoreceptor Cells, Vertebrate/metabolism , Protein Transport , Animals , Chromatography, Liquid , Cilia , Eye Proteins/genetics , Immunoprecipitation , Mice , Mice, Knockout , Protein Binding , Proteomics , Rats , Synapses , Tandem Mass Spectrometry
2.
Adv Exp Med Biol ; 854: 223-30, 2016.
Article in English | MEDLINE | ID: mdl-26427415

ABSTRACT

Mutations in the TULP1 gene are associated with early-onset retinitis pigmentosa (RP); however, the molecular mechanisms related to the deleterious effects of TULP1 mutations remains unknown. Several studies have shown that misfolded proteins secondary to genetic mutations can accumulate within the endoplasmic reticulum (ER), causing activation of the unfolded protein response (UPR) complex followed by cellular apoptosis. We hypothesize that TULP1 mutations produce misfolded protein products that accumulate in the ER and induce cellular apoptosis via the UPR. To test our hypothesis, we first performed three in-silico analyses of TULP1 missense mutations (I459K, R420P and F491L), which predicted misfolded protein products. Subsequently, the three mutant TULP1-GFP constructs and wild-type (wt) TULP1-GFP were transiently transfected into hTERT-RPE-1 cells. Staining of cells using ER tracker followed by confocal microscopy showed wt-TULP1 localized predominantly to the cytoplasm and plasma membrane. In contrast, all three mutant TULP1 proteins revealed cytoplasmic punctate staining which co-localized with the ER. Furthermore, western blot analysis of cells expressing mutant TULP1 proteins revealed induction of downstream targets of the ER-UPR complex, including BiP/GPR-78, phosphorylated-PERK (Thr980) and CHOP. Our in-vitro analyses suggest that mutant TULP1 proteins are misfolded and accumulate within the ER leading to induction of the UPR stress response complex.


Subject(s)
Endoplasmic Reticulum/metabolism , Eye Proteins/genetics , Mutation, Missense , Unfolded Protein Response/genetics , Apoptosis/genetics , Blotting, Western , Cell Line , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/genetics , Eye Proteins/chemistry , Eye Proteins/metabolism , Heat-Shock Proteins/metabolism , Humans , Microscopy, Confocal , Phosphorylation/genetics , Protein Folding , Transcription Factor CHOP/metabolism , eIF-2 Kinase/metabolism
3.
Adv Exp Med Biol ; 854: 605-11, 2016.
Article in English | MEDLINE | ID: mdl-26427465

ABSTRACT

Photoreceptors (PRs) are highly polarized and compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. The PR-specific protein, Tulp1, is localized to the IS and synapse and is hypothesized to be involved in protein trafficking. To better understand the molecular processes that regulate protein trafficking in PRs, we aimed to identify compartment-specific Tulp1 binding partners. Serial tangential sectioning of Long Evans rat retinas was utilized to isolate the IS and synaptic PR compartments. Tulp1 binding partners in each of these layers were identified using co-immunoprecipitation (co-IP) with Tulp1 antibodies. The co-IP eluates were separated by SDS-PAGE, trypsinized into peptide fragments, and proteins were identified by liquid chromatography tandem mass spectrometry. In the IS, potential Tulp1-binding partners included cytoskeletal scaffold proteins, protein trafficking molecules, as well as members of the phototransduction cascade. In the synaptic region, the majority of interacting proteins identified were cytoskeletal. A separate subset of proteins were identified in both the IS and synapse including chaperones and family members of the GTPase activating proteins. Tulp1 has two distinct PR compartment-specific interactomes. Our results support the hypothesis that Tulp1 is involved in the trafficking of proteins from the IS to the OS and the continuous membrane remodeling and vesicle cycling at the synaptic terminal.


Subject(s)
Carrier Proteins/metabolism , Eye Proteins/metabolism , Photoreceptor Cells, Vertebrate/metabolism , Retinal Photoreceptor Cell Inner Segment/metabolism , Animals , Antibodies/immunology , Antibodies/metabolism , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Eye Proteins/genetics , Eye Proteins/immunology , Immunoprecipitation , Mice, Inbred C57BL , Mice, Knockout , Protein Binding , Protein Transport , Rats, Long-Evans , Retinal Photoreceptor Cell Outer Segment/metabolism , Synapses/metabolism , Tandem Mass Spectrometry
4.
Adv Exp Med Biol ; 801: 511-8, 2014.
Article in English | MEDLINE | ID: mdl-24664738

ABSTRACT

Tubby-like protein-1 (Tulp1) is a photoreceptor-specific protein involved in the transport of specific proteins from the inner segment (IS) to the outer segment (OS) in photoreceptor cells. Mutations in the human TULP1 gene cause an early onset form of retinitis pigmentosa. Our previous work has shown an association between Tulp1 and the microtubule-associated protein, MAP1B. An allele of Mtap1a, which encodes the MAP1A protein, significantly delays photoreceptor degeneration in Tulp1 mutant mice. MAP1 proteins are important in stabilizing microtubules in neuronal cells, but their role in photoreceptors remains obscure. To investigate the relationship between Tulp1 and MAP1 proteins, we performed western blots, immunoprecipitations (IP), immunohistochemistry and proximity ligand assays (PLA) in wild-type and tulp1-/- mouse retinas. Our IP experiments provide evidence that Tulp1 and MAP1B interact while PLA experiments localize their interaction to the outer nuclear layer and IS of photoreceptors. Although MAP1A and MAP1B protein levels are not affected in the tulp1-/- retina, they are no longer localized to the OS of photoreceptors. This may be the cause for disorganized OSs in tulp1-/- mice, and indicate that their transport to the OS is Tulp1-dependent.


Subject(s)
Eye Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Retina/metabolism , Retinal Degeneration/metabolism , Retinal Photoreceptor Cell Outer Segment/metabolism , Animals , Biological Transport/physiology , Eye Proteins/genetics , Humans , Mice , Mice, Knockout , Photoreceptor Cells, Vertebrate/metabolism , Retinal Degeneration/genetics
5.
Obesity (Silver Spring) ; 22(5): 1246-55, 2014 May.
Article in English | MEDLINE | ID: mdl-24376179

ABSTRACT

OBJECTIVE: To determine the cellular architecture of the inflammatory infiltrate in adipose tissue from obese mice, and identify the source of inflammatory cytokines in adipose tissue at a single cell level. METHODS: Adipose tissue from diet-induced obese mice was digested by collagenase treatment and fractionated by density centrifugation to obtain an adipocyte floating layer and a pellet of stromal vascular cells. The cellular architecture of the adipocyte-macrophage interaction in both intact white adipose tissue (WAT) and the separated density gradient floating layer fraction was analyzed by confocal immunohistochemistry. Cytokine expression was detected by semi-quantitative real time PCR and immunohistochemical analysis. RESULTS: Three dimensional image analysis of WAT and the separated "adipocyte" floating layer revealed lipid-engorged macrophages, macrophages in contact with lipid droplets and sheath-like assemblies of macrophages surrounding adipocytes. The macrophages immunostained for TNFα and to a lesser extent for the immunoregulatory cytokine IL-10. TNFα staining was associated only with macrophages indicating that macrophages and not adipocytes are the source of TNFα expression in the adipocyte floating layer. CONCLUSION: Macrophages form assemblies that tightly adhere to and cover adipocytes and lipid droplets. TNFα found in low density adipocyte preparations is due to contamination with macrophages.


Subject(s)
Adipocytes/ultrastructure , Adipose Tissue, White/cytology , Macrophages/ultrastructure , Adipocytes/cytology , Animals , Cell Separation , Inflammation , Interleukin-10/metabolism , Macrophages/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Obese , Microscopy, Confocal , Obesity , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
6.
Invest Ophthalmol Vis Sci ; 54(12): 7223-33, 2013 Nov 01.
Article in English | MEDLINE | ID: mdl-24106123

ABSTRACT

PURPOSE: To determine the molecular basis and the pathologic consequences of a chemically induced mutation in the translational vision research models 89 (tvrm89) mouse model with ERG defects. METHODS: Mice from a G3 N-ethyl-N-nitrosourea mutagenesis program were screened for behavioral abnormalities and defects in retinal function by ERGs. The chromosomal position for the recessive tvrm89 mutation was determined in a genome-wide linkage analysis. The critical region was refined, and candidate genes were screened by direct sequencing. The tvrm89 phenotype was characterized by circling behavior, in vivo ocular imaging, detailed ERG-based studies of the retina and RPE, and histological analysis of these structures. RESULTS: The tvrm89 mutation was localized to a region on chromosome 9 containing Myo6. Sequencing identified a T→C point mutation in the codon for amino acid 480 in Myo6 that converts a leucine to a proline. This mutation does not confer a loss of protein expression levels; however, mice homozygous for the Myo6(tvrm89) mutation display an abnormal iris shape and attenuation of both strobe-flash ERGs and direct-current ERGs by 4 age weeks, neither of which is associated with photoreceptor loss. CONCLUSIONS: The tvrm89 phenotype mimics that reported for Myosin6-null mice, suggesting that the mutation confers a loss of myosin 6 protein function. The observation that homozygous Myo6(tvrm89) mice display reduced ERG a-wave and b-wave components, as well as components of the ERG attributed to RPE function, indicates that myosin 6 is necessary for the generation of proper responses of the outer retina to light.


Subject(s)
Iris/physiology , Myosin Heavy Chains/physiology , Retinal Diseases , Animals , DNA Mutational Analysis , Electroretinography , Genetic Linkage , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mutation, Missense , Myosin Heavy Chains/genetics , Ophthalmoscopy , Phenotype , Retinal Diseases/genetics , Retinal Diseases/physiopathology
7.
Vis Neurosci ; 30(4): 129-39, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23746204

ABSTRACT

Dynamin proteins are involved in vesicle generation, providing mechanical force to excise newly formed vesicles from membranes of cellular compartments. In the brain, dynamin-1, dynamin-2, and dynamin-3 have been well studied; however, their function in the retina remains elusive. A retina-specific splice variant of dynamin-1 interacts with the photoreceptor-specific protein Tubby-like protein 1 (Tulp1), which when mutated causes an early onset form of autosomal recessive retinitis pigmentosa. Here, we investigated the role of the dynamins in the retina, using immunohistochemistry to localize dynamin-1, dynamin-2, and dynamin-3 and immunoprecipitation followed by mass spectrometry to explore dynamin-1 interacting proteins in mouse retina. Dynamin-2 is primarily confined to the inner segment compartment of photoreceptors, suggesting a role in outer segment protein transport. Dynamin-3 is present in the terminals of photoreceptors and dendrites of second-order neurons but is most pronounced in the inner plexiform layer where second-order neurons relay signals from photoreceptors. Dynamin-1 appears to be the dominant isoform in the retina and is present throughout the retina and in multiple compartments of the photoreceptor cell. This suggests that it may function in multiple cellular pathways. Surprisingly, dynamin-1 expression and localization did not appear to be disrupted in tulp1−/− mice. Immunoprecipitation experiments reveal that dynamin-1 associates primarily with proteins involved in cytoskeletal-based membrane dynamics. This finding is confirmed by western blot analysis. Results further implicate dynamin-1 in vesicular protein transport processes relevant to synaptic and post-Golgi pathways and indicate a possible role in photoreceptor stability.


Subject(s)
Dynamin I/physiology , Retina/physiology , Animals , Antibodies/chemistry , Blotting, Western , Cytoskeleton/metabolism , Dynamin I/genetics , Dynamin I/metabolism , Dynamin II/genetics , Dynamin II/metabolism , Dynamin II/physiology , Dynamin III/genetics , Dynamin III/metabolism , Dynamin III/physiology , Eye Proteins/genetics , Immunohistochemistry , Immunoprecipitation , Mice , Mice, Inbred C57BL , Mice, Knockout , Photoreceptor Cells, Vertebrate/physiology
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