scholarly journals Loss of Motor Protein MYO1C Causes Rhodopsin Mislocalization and Results in Impaired Visual Function

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1322
Author(s):  
Ashish K. Solanki ◽  
Manas R. Biswal ◽  
Stephen Walterhouse ◽  
René Martin ◽  
Altaf A. Kondkar ◽  
...  
Keyword(s):  
Visual Function ◽  
Direct Interaction ◽  
Retinal Cell ◽  
Cell Physiology ◽  
Binding Assays ◽  
Ultrastructural Examination ◽  
Direct Role ◽  
Progressive Loss ◽  
Cone Opsins ◽  
Punctate Staining

Unconventional myosins, linked to deafness, are also proposed to play a role in retinal cell physiology. However, their direct role in photoreceptor function remains unclear. We demonstrate that systemic loss of the unconventional myosin MYO1C in mice, specifically causes rhodopsin mislocalization, leading to impaired visual function. Electroretinogram analysis of Myo1c knockout (Myo1c-KO) mice showed a progressive loss of photoreceptor function. Immunohistochemistry and binding assays demonstrated MYO1C localization to photoreceptor inner and outer segments (OS) and identified a direct interaction of rhodopsin with MYO1C. In Myo1c-KO retinas, rhodopsin mislocalized to rod inner segments (IS) and cell bodies, while cone opsins in OS showed punctate staining. In aged mice, the histological and ultrastructural examination of the phenotype of Myo1c-KO retinas showed progressively shorter photoreceptor OS. These results demonstrate that MYO1C is important for rhodopsin localization to the photoreceptor OS, and for normal visual function.

scholarly journals Motor Protein MYO1C is Critical for Photoreceptor Opsin Trafficking and Visual Function

2020 ◽  
Author(s):  
Ashish K. Solanki ◽  
Stephen Walterhouse ◽  
René Martin ◽  
Elisabeth Obert ◽  
Ehtesham Arif ◽  
...  
Keyword(s):  
Visual Function ◽  
Direct Interaction ◽  
Retinal Cell ◽  
Cell Physiology ◽  
Binding Assays ◽  
Ultrastructural Examination ◽  
Direct Role ◽  
Progressive Loss ◽  
Cone Opsins ◽  
Punctate Staining

AbstractUnconventional myosins linked to deafness are also proposed to play a role in retinal cell physiology. However, their direct role in photoreceptor function remains unclear. We demonstrate that systemic loss of the unconventional myosin MYO1C in mice specifically affected opsin trafficking, leading to loss of visual function. Electroretinogram analysis of Myo1c knockout (Myo1c-KO) mice showed a progressive loss of photoreceptor function. Immunohistochemistry and binding assays demonstrated MYO1C localization to photoreceptor inner and outer segments (OS) and identified a direct interaction of rhodopsin with the MYO1C cargo domain. In Myo1c-KO retinas, rhodopsin mislocalized to rod inner segments (IS) and cell bodies, while cone opsins in OS showed punctate staining. In aged mice, the histological and ultrastructural examination of the phenotype of Myo1c-KO retinas showed progressively shorter photoreceptor OS. These results demonstrate that MYO1C is critical for opsin trafficking to the photoreceptor OS and for normal visual function.

scholarly journals Conditional Loss of the Exocyst Component exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction, Photo-Receptor Cell Degeneration, and Decreased Visual Function.

2021 ◽  
Author(s):  
Bärbel Rohrer ◽  
Elisabeth Obert ◽  
Yujing Dang ◽  
Yanhui Su ◽  
Xiaofeng Zuo ◽  
...  
Keyword(s):  
Visual Function ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Photoreceptor Cells ◽  
Retinal Cell ◽  
Cell Degeneration ◽  
Genetic Ablation ◽  
Pigmented Epithelium ◽  
Cone Opsins ◽  
Retinal Thinning

To characterize the mechanisms by which the highly-conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on exoc5 (aka sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5-/- zebrafish showed smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post fertilization, loss of rod and cone opsins were observed in zebrafish Tg:exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function, and loss of visual photoreceptor pigments. This retinal phenotype in Exoc5-/- mice was present at 20-weeks, and the phenotype was more severe at 27-weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. As RPE cells are “downstream” of photoreceptor cells in the visual process, these data suggest exocyst-mediated retrograde communication and dependence between the RPE and photoreceptors.

scholarly journals Conditional Loss of the Exocyst Component Exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction, Photoreceptor Cell Degeneration, and Decreased Visual Function

2021 ◽  
Vol 22 (10) ◽  
pp. 5083
Author(s):  
Bärbel Rohrer ◽  
Manas R. Biswal ◽  
Elisabeth Obert ◽  
Yujing Dang ◽  
Yanhui Su ◽  
...  
Keyword(s):  
Visual Function ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Cell ◽  
Cell Degeneration ◽  
Genetic Ablation ◽  
Pigmented Epithelium ◽  
Cone Opsins ◽  
And Function ◽  
Retinal Thinning

To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5−/− zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5−/− mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.

Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2413-2419 ◽  
Author(s):  
José L. Alonso-Lebrero ◽  
Juan M. Serrador ◽  
Carmen Domı́nguez-Jiménez ◽  
Olga Barreiro ◽  
Alfonso Luque ◽  
...  
Keyword(s):  
Adhesion Molecules ◽  
Adhesion Molecule ◽  
Direct Interaction ◽  
Intercellular Adhesion ◽  
Actin Binding ◽  
Intercellular Adhesion Molecule ◽  
Human Neutrophils ◽  
Binding Assays ◽  
Glutathione S Transferase ◽  
Amino Terminal

Abstract In response to the chemoattractants interleukin 8, C5a,N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM-3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin-radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants.

scholarly journals Reinvestigation of the dysbindin subunit of BLOC-1 (biogenesis of lysosome-related organelles complex-1) as a dystrobrevin-binding protein

2006 ◽  
Vol 395 (3) ◽  
pp. 587-598 ◽  
Author(s):  
Ramin Nazarian ◽  
Marta Starcevic ◽  
Melissa J. Spencer ◽  
Esteban C. Dell'Angelica
Keyword(s):  
Recombinant Proteins ◽  
Binding Protein ◽  
Coiled Coil ◽  
Direct Interaction ◽  
Muscle Pathology ◽  
Binding Assays ◽  
Genetic Studies ◽  
Binding Interface ◽  
Lysosome Related Organelles

Dysbindin was identified as a dystrobrevin-binding protein potentially involved in the pathogenesis of muscular dystrophy. Subsequently, genetic studies have implicated variants of the human dysbindin-encoding gene, DTNBP1, in the pathogeneses of Hermansky–Pudlak syndrome and schizophrenia. The protein is a stable component of a multisubunit complex termed BLOC-1 (biogenesis of lysosome-related organelles complex-1). In the present study, the significance of the dystrobrevin–dysbindin interaction for BLOC-1 function was examined. Yeast two-hybrid analyses, and binding assays using recombinant proteins, demonstrated direct interaction involving coiled-coil-forming regions in both dysbindin and the dystrobrevins. However, recombinant proteins bearing the coiled-coil-forming regions of the dystrobrevins failed to bind endogenous BLOC-1 from HeLa cells or mouse brain or muscle, under conditions in which they bound the Dp71 isoform of dystrophin. Immunoprecipitation of endogenous dysbindin from brain or muscle resulted in robust co-immunoprecipitation of the pallidin subunit of BLOC-1 but no specific co-immunoprecipitation of dystrobrevin isoforms. Within BLOC-1, dysbindin is engaged in interactions with three other subunits, named pallidin, snapin and muted. We herein provide evidence that the same 69-residue region of dysbindin that is sufficient for dystrobrevin binding in vitro also contains the binding sites for pallidin and snapin, and at least part of the muted-binding interface. Functional, histological and immunohistochemical analyses failed to detect any sign of muscle pathology in BLOC-1-deficient, homozygous pallid mice. Taken together, these results suggest that dysbindin assembled into BLOC-1 is not a physiological binding partner of the dystrobrevins, likely due to engagement of its dystrobrevin-binding region in interactions with other subunits.

scholarly journals The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading

2019 ◽  
Vol 4 ◽  
pp. 117
Author(s):  
Maria Shamin ◽  
Tomasz H. Benedyk ◽  
Stephen C. Graham ◽  
Janet E. Deane
Keyword(s):  
Lipid Transfer Protein ◽  
Direct Interaction ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
Binding Assays ◽  
Lipid Transfer Proteins ◽  
Lipid Antigens ◽  
Saposin B ◽  
Protein Components ◽  
Lipid Loading

Background: Lipid antigens are presented on the surface of cells by the CD1 family of glycoproteins, which have structural and functional similarity to MHC class I molecules. The hydrophobic lipid antigens are embedded in membranes and inaccessible to the lumenal lipid-binding domain of CD1 molecules. Therefore, CD1 molecules require lipid transfer proteins for lipid loading and editing. CD1d is loaded with lipids in late endocytic compartments, and lipid transfer proteins of the saposin family have been shown to play a crucial role in this process. However, the mechanism by which saposins facilitate lipid binding to CD1 molecules is not known and is thought to involve transient interactions between protein components to ensure CD1-lipid complexes can be efficiently trafficked to the plasma membrane for antigen presentation. Of the four saposin proteins, the importance of Saposin B (SapB) for loading of CD1d is the most well-characterised. However, a direct interaction between CD1d and SapB has yet to be described. Methods: In order to determine how SapB might load lipids onto CD1d, we used purified, recombinant CD1d and SapB and carried out a series of highly sensitive binding assays to monitor direct interactions. We performed equilibrium binding analysis, chemical cross-linking and co-crystallisation experiments, under a range of different conditions. Results: We could not demonstrate a direct interaction between SapB and CD1d using any of these binding assays. Conclusions: This work establishes comprehensively that the role of SapB in lipid loading does not involve direct binding to CD1d. We discuss the implication of this for our understanding of lipid loading of CD1d and propose several factors that may influence this process.

Metastable Ions as a Guide to Reaction Mechanisms. Loss of ’OH from Substituted Nitrobenzene Molecular Ions

1974 ◽  
Vol 29 (3) ◽  
pp. 507-512
Author(s):  
T. Keough ◽  
J. H. Beynon ◽  
R. G. Cooks ◽  
C. Chang ◽  
R. H. Shapiro
Keyword(s):  
Nitro Group ◽  
Hydrogen Transfer ◽  
Direct Interaction ◽  
Kinetic Energy Release ◽  
Molecular Ions ◽  
Time Range ◽  
Direct Role ◽  
Ion Fragmentation ◽  
Distinct Process ◽  
Metastable Ions

Loss of ‘OH from the molecular ions of nitrobenzenes having various substituents in the m- and p-positions does not involve substituent randomization and reaction through the ortho-isomer; the kinetic energy release accompanying metastable ion fragmentation shows this latter to be an entirely distinct process. The origin of the hydrogen lost as 'OH in the m- and p-compounds is the ring (mainly the position ortho to the nitro group in methyl p-nitrobenzoate). However, the hydro­gens of the substituent do play a direct role in the reaction as shown by an isotope effect upon the abundances of metastable peaks. Hydrogen atom abstraction by the ionized nitro group apparently occurs independently of participation by the substituent on the nitrobenzene but a second hydrogen transfer from this substituent to the ring precedes fragmentation. Direct interaction between groups o ' the aromatic ring that are not ortho to each other therefore apparently does not occur. Inversion of the rate constants for two reactions, NO' loss and CH2O loss from the methyl p-nitrobenzoate molecular ion in the metastable ion time range, has been observed following isotopic substitution.

scholarly journals Evaluation of different iron sources and their influence in biofilm formation by the dental pathogen Actinobacillus actinomycetemcomitans

2007 ◽  
Vol 56 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Eric R. Rhodes ◽  
Christopher J. Shoemaker ◽  
Sharon M. Menke ◽  
Richard E. Edelmann ◽  
Luis A. Actis
Keyword(s):  
Biofilm Formation ◽  
Storage Proteins ◽  
Cell Envelope ◽  
Direct Interaction ◽  
Iron Chelator ◽  
Actinobacillus Actinomycetemcomitans ◽  
Binding Assays ◽  
Dot Blot ◽  
Oral Infections ◽  
Cell Responses

Actinobacillus actinomycetemcomitans, a pathogen associated with oral and extra-oral infections, requires iron to grow under limiting conditions. Although incapable of producing siderophores, this pathogen could acquire iron by direct interaction with compounds such as haemin, haemoglobin, lactoferrin and transferrin. In this work the ability of different A. actinomycetemcomitans strains to bind and use different iron sources was tested. None of the strains tested used haemoglobin, lactoferrin or transferrin as sole sources of iron. However, all of them used FeCl3 and haemin as iron sources under chelated conditions. Dot-blot binding assays showed that all strains bind lactoferrin, haemoglobin and haemin, but not transferrin. Insertion inactivation of hmsF, which encodes a predicted cell-envelope protein related to haemin-storage proteins produced by other pathogens, reduced haemin and Congo red binding drastically without affecting haemin utilization as an iron source under chelated conditions. Biofilm assays showed that all strains tested attached to and formed biofilms on plastic under iron-rich and iron-chelated conditions. However, scanning electron microscopy showed that smooth strains formed simpler biofilms than rough isolates. Furthermore, the incubation of rough cells in the presence of FeCl3 or haemin resulted in the formation of more aggregates and microcolonies compared with the fewer cell aggregates formed when cells were grown in the presence of the synthetic iron chelator dipyridyl. These cell responses to changes in extracellular iron concentrations may reflect those that this pathogen expresses under the conditions it encounters in the human oral cavity.

scholarly journals Neurofascin induces neurites by heterophilic interactions with axonal NrCAM while NrCAM requires F11 on the axonal surface to extend neurites.

1996 ◽  
Vol 135 (4) ◽  
pp. 1059-1069 ◽  
Author(s):  
H Volkmer ◽  
R Leuschner ◽  
U Zacharias ◽  
F G Rathjen
Keyword(s):  
Direct Interaction ◽  
Binding Activity ◽  
Soluble Form ◽  
Binding Assays ◽  
Binding Studies ◽  
Experimental Conditions ◽  
Neurite Extension ◽  
Ig Superfamily ◽  
Inert Surface ◽  
Axonal Extension

Neurofascin and NrCAM are two axon-associated transmembrane glycoproteins belonging to the L1 subgroup of the Ig superfamily. In this study, we have analyzed the interaction of both proteins using neurite outgrowth and binding assays. A neurofascin-Fc chimera was found to stimulate the outgrowth of tectal cells when immobilized on an inert surface but not as a soluble form using polylysine as substrate. Antibody blocking experiments demonstrate that neurite extension on immobilized neurofascin is mediated by NrCAM on the axonal surface. Under the reverse experimental conditions where NrCAM induces neurite extension, F11, and not neurofascin, serves as axonal receptor. Binding studies using transfected COS7 cells and immunoprecipitations reveal a direct interaction between neurofascin and NrCAM. This binding activity was mapped to the Ig domains within neurofascin. The neurofascin-NrCAM binding can be modulated by alternative splicing of specific stretches within neurofascin. These studies indicate that heterophilic interactions between Ig-like proteins implicated in axonal extension underlie a regulation by the neuron.

STIM1 negatively regulates Ca2+ release from the sarcoplasmic reticulum in skeletal myotubes

2013 ◽  
Vol 453 (2) ◽  
pp. 187-200 ◽  
Author(s):  
Keon Jin Lee ◽  
Jin Seok Woo ◽  
Ji-Hye Hwang ◽  
Changdo Hyun ◽  
Chung-Hyun Cho ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Direct Interaction ◽  
Dominant Negative ◽  
Wild Type ◽  
Triple Mutant ◽  
Direct Role ◽  
Independent Manner ◽  
C Terminus ◽  
The Times

STIM1 (stromal interaction molecule 1) mediates SOCE (store-operated Ca2+ entry) in skeletal muscle. However, the direct role(s) of STIM1 in skeletal muscle, such as Ca2+ release from the SR (sarcoplasmic reticulum) for muscle contraction, have not been identified. The times required for the maximal expression of endogenous STIM1 or Orai1, or for the appearance of puncta during the differentiation of mouse primary skeletal myoblasts to myotubes, were all different, and the formation of puncta was detected with no stimulus during differentiation, suggesting that, in skeletal muscle, the formation of puncta is a part of the differentiation. Wild-type STIM1 and two STIM1 mutants (Triple mutant, missing Ca2+-sensing residues but possessing the intact C-terminus; and E136X, missing the C-terminus) were overexpressed in the myotubes. The wild-type STIM1 increased SOCE, whereas neither mutant had an effect on SOCE. It was interesting that increases in the formation of puncta were observed in the Triple mutant as well as in wild-type STIM1, suggesting that SOCE-irrelevant puncta could exist in skeletal muscle. On the other hand, overexpression of wild-type or Triple mutant, but not E136X, attenuated Ca2+ releases from the SR in response to KCl [evoking ECC (excitation–contraction coupling) via activating DHPR (dihydropyridine receptor)] in a dominant-negative manner. The attenuation was removed by STIM1 knockdown, and STIM1 was co-immunoprecipitated with DHRP in a Ca2+-independent manner. These results suggest that STIM1 negatively regulates Ca2+ release from the SR through the direct interaction of the STIM1 C-terminus with DHPR, and that STIM1 is involved in both ECC and SOCE in skeletal muscle.

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