Spatiotemporal Control of Actomyosin Contractility by MRCKβ Signaling Drives Phagocytosis

Phagocytosis requires myosin-generated contractile force to regulate actin dynamics. However, little is known about the molecular mechanisms that guide this complex morphodynamic process. Here we show that particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3 in the retinal pigment epithelium (RPE), triggering activation of MRCKβ and its co-effector N-WASP that cooperate to deform the membrane into cups. Continued MRCKβ activity then drives recruitment of a mechanosensing bridge enabling transmission of the cytoskeletal force required for cup closure and particle internalization. MRCKβ is also required for Fc receptor-mediated phagocytosis by macrophages. In vivo, MRCKβ is essential for RPE phagocytosis of photoreceptor debris and, hence, retinal integrity. MerTK-independent activation of MRCKβ signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. Thus, conserved MRCKβ signaling controls spatiotemporal regulation of actomyosin contractility to guide actomyosin dynamics-driven phagocytosis and represents the principle phagocytic effector pathway downstream of MerTK.

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Ocular Toxoplasmosis: Mechanisms of Retinal Infection and Experimental Models

Parasitologia ◽

10.3390/parasitologia1020007 ◽

2021 ◽

Vol 1 (2) ◽

pp. 50-60

Author(s):

Veronica Rodriguez Fernandez ◽

Giovanni Casini ◽

Fabrizio Bruschi

Keyword(s):

Ex Vivo ◽

Cell Damage ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Treatment Strategies ◽

Experimental Models ◽

Ocular Toxoplasmosis ◽

Cell Infection

Ocular toxoplasmosis (OT) is caused by the parasite Toxoplasma gondii and affects many individuals throughout the world. Infection may occur through congenital or acquired routes. The parasites enter the blood circulation and reach both the retina and the retinal pigment epithelium, where they may cause cell damage and cell death. Different routes of access are used by T. gondii to reach the retina through the retinal endothelium: by transmission inside leukocytes, as free parasites through a paracellular route, or after endothelial cell infection. A main feature of OT is the induction of an important inflammatory state, and the course of infection has been shown to be influenced by the host immunogenetics. On the other hand, there is evidence that the T. gondii phenotype also has an impact on the distribution of the pathology in different areas. Although considerable knowledge has been acquired on OT, a deeper knowledge of its mechanisms is necessary to provide new, more targeted treatment strategies. In particular, in addition to in vitro and in vivo experimental models, organotypic, ex vivo retinal explants may be useful in this direction.

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OCT imaging of rod mitochondrial respiration in vivo

Experimental Biology and Medicine ◽

10.1177/15353702211013799 ◽

2021 ◽

pp. 153537022110137

Author(s):

Bruce A Berkowitz ◽

Haohua Qian

Keyword(s):

Signaling Pathway ◽

Mitochondrial Respiration ◽

Pigment Epithelium ◽

Retinal Pigment ◽

The Body ◽

Subretinal Space ◽

Outer Retina ◽

Water Efflux ◽

Resolution Imaging

There remains a need for high spatial resolution imaging indices of mitochondrial respiration in the outer retina that probe normal physiology and measure pathogenic and reversible conditions underlying loss of vision. Mitochondria are involved in a critical, but somewhat underappreciated, support system that maintains the health of the outer retina involving stimulus-evoked changes in subretinal space hydration. The subretinal space hydration light–dark response is important because it controls the distribution of vision-critical interphotoreceptor matrix components, including anti-oxidants, pro-survival factors, ions, and metabolites. The underlying signaling pathway controlling subretinal space water management has been worked out over the past 30 years and involves cGMP/mitochondria respiration/pH/RPE water efflux. This signaling pathway has also been shown to be modified by disease-generating conditions, such as hypoxia or oxidative stress. Here, we review recent advances in MRI and commercially available OCT technologies that can measure stimulus-evoked changes in subretinal space water content based on changes in the external limiting membrane-retinal pigment epithelium region. Each step within the above signaling pathway can also be interrogated with FDA-approved pharmaceuticals. A highlight of these studies is the demonstration of first-in-kind in vivo imaging of mitochondria respiration of any cell in the body. Future examinations of subretinal space hydration are expected to be useful for diagnosing threats to sight in aging and disease, and improving the success rate when translating treatments from bench-to-bedside.

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CIB2 regulates mTORC1 signaling and is essential for autophagy and visual function

Nature Communications ◽

10.1038/s41467-021-24056-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Saumil Sethna ◽

Patrick A. Scott ◽

Arnaud P. J. Giese ◽

Todd Duncan ◽

Xiaoying Jian ◽

...

Keyword(s):

Visual Function ◽

Molecular Mechanisms ◽

Neurodegenerative Disorder ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Negative Regulator ◽

Age Related Macular Degeneration ◽

Age Related ◽

Mtorc1 Signaling ◽

Marked Accumulation

AbstractAge-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aβ, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling—a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to ‘nucleotide empty’ or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.

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The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

Journal of Cell Science ◽

10.1242/jcs.91.2.303 ◽

1988 ◽

Vol 91 (2) ◽

pp. 303-312

Author(s):

N.M. McKechnie ◽

M. Boulton ◽

H.L. Robey ◽

F.J. Savage ◽

I. Grierson

Keyword(s):

Retinal Pigment Epithelium ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Cytokeratin 18 ◽

Rpe Cells ◽

Human Retinal Pigment Epithelium ◽

Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

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Lipid Signaling in Retinal Pigment Epithelium Cells Exposed to Inflammatory and Oxidative Stress Conditions. Molecular Mechanisms Underlying Degenerative Retinal Diseases

Retinal Degenerative Diseases - Advances in Experimental Medicine and Biology ◽

10.1007/978-3-030-27378-1_47 ◽

2019 ◽

pp. 289-293

Author(s):

Vicente Bermúdez ◽

Paula E. Tenconi ◽

Norma M. Giusto ◽

Melina V. Mateos

Keyword(s):

Oxidative Stress ◽

Retinal Pigment Epithelium ◽

Molecular Mechanisms ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Stress Conditions ◽

Retinal Diseases ◽

Retinal Pigment Epithelium Cells ◽

Epithelium Cells ◽

And Oxidative Stress

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JAM-C maintains VEGR2 expression to promote retinal pigment epithelium cell survival under oxidative stress

Thrombosis and Haemostasis ◽

10.1160/th16-11-0885 ◽

2017 ◽

Vol 117 (04) ◽

pp. 750-757

Author(s):

Xin Jia ◽

Chen Zhao ◽

Qishan Chen ◽

Yuxiang Du ◽

Lijuan Huang ◽

...

Keyword(s):

Oxidative Stress ◽

Retinal Pigment Epithelium ◽

Cell Survival ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelium Cell ◽

Photoreceptor Cells ◽

Rpe Cells

SummaryJunctional adhesion molecule-C (JAM-C) has been shown to play critical roles during development and in immune responses. However, its role in adult eyes under oxidative stress remains poorly understood. Here, we report that JAM-C is abundantly expressed in adult mouse retinae and choroids in vivo and in cultured retinal pigment epithelium (RPE) and photoreceptor cells in vitro. Importantly, both JAM-C expression and its membrane localisation are downregulated by H2O2-induced oxidative stress. Under H2O2-induced oxidative stress, JAM-C is critically required for the survival of human RPE cells. Indeed, loss of JAM-C by siRNA knockdown decreased RPE cell survival. Mechanistically, we show that JAM-C is required to maintain VEGFR2 expression in RPE cells, and VEGFR2 plays an important role in keeping the RPE cells viable since overexpression of VEGFR2 partially restored impaired RPE survival caused by JAM-C knockdown and increased RPE survival. We further show that JAM-C regulates VEGFR2 expression and, in turn, modulates p38 phosphorylation. Together, our data demonstrate that JAM-C plays an important role in maintaining VEGR2 expression to promote RPE cell survival under oxidative stress. Given the vital importance of RPE in the eye, approaches that can modulate JAM-C expression may have therapeutic values in treating diseases with impaired RPE survival.

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Induction of Heat Shock Protein 70 in Mouse RPE as an In Vivo Model of Transpupillary Thermal Stimulation

International Journal of Molecular Sciences ◽

10.3390/ijms21062063 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2063

Author(s):

Mooud Amirkavei ◽

Marja Pitkänen ◽

Ossi Kaikkonen ◽

Kai Kaarniranta ◽

Helder André ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Response ◽

Pigment Epithelium ◽

Retinal Pigment ◽

In Vivo Model ◽

Tunel Staining ◽

Long Duration ◽

Shock Response

The induction of heat shock response in the macula has been proposed as a useful therapeutic strategy for retinal neurodegenerative diseases by promoting proteostasis and enhancing protective chaperone mechanisms. We applied transpupillary 1064 nm long-duration laser heating to the mouse (C57Bl/6J) fundus to examine the heat shock response in vivo. The intensity and spatial distribution of heat shock protein (HSP) 70 expression along with the concomitant probability for damage were measured 24 h after laser irradiation in the mouse retinal pigment epithelium (RPE) as a function of laser power. Our results show that the range of heating powers for producing heat shock response while avoiding damage in the mouse RPE is narrow. At powers of 64 and 70 mW, HSP70 immunostaining indicates 90 and 100% probability for clearly elevated HSP expression while the corresponding probability for damage is 20 and 33%, respectively. Tunel staining identified the apoptotic regions, and the estimated 50% damaging threshold probability for the heating (ED50) was ~72 mW. The staining with Bestrophin1 (BEST1) demonstrated RPE cell atrophy with the most intense powers. Consequently, fundus heating with a long-duration laser provides an approachable method to develop heat shock-based therapies for the RPE of retinal disease model mice.

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