scholarly journals A comprehensive analysis of Rab GTPases reveals a role for Rab34 in serum starvation-induced primary ciliogenesis

2020 ◽  
Vol 295 (36) ◽  
pp. 12674-12685 ◽  
Author(s):  
Mai E. Oguchi ◽  
Koki Okuyama ◽  
Yuta Homma ◽  
Mitsunori Fukuda
Keyword(s):  
Membrane Trafficking ◽  
Primary Cilia ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Small Gtpases ◽  
Terminal Region ◽  
Serum Starvation ◽  
Rab Gtpases ◽  
Human Telomerase Reverse Transcriptase ◽  
Specific Sequence

Primary cilia are sensors of chemical and mechanical signals in the extracellular environment. The formation of primary cilia (i.e. ciliogenesis) requires dynamic membrane trafficking events, and several Rab small GTPases, key regulators of membrane trafficking, have recently been reported to participate in ciliogenesis. However, the precise mechanisms of Rab-mediated membrane trafficking during ciliogenesis remain largely unknown. In the present study, we used a collection of siRNAs against 62 human Rabs to perform a comprehensive knockdown screening for Rabs that regulate serum starvation-induced ciliogenesis in human telomerase reverse transcriptase retinal pigment epithelium 1 (hTERT-RPE1) cells and succeeded in identifying Rab34 as an essential Rab. Knockout (KO) of Rab34, but not of Rabs previously reported to regulate ciliogenesis (e.g. Rab8 and Rab10) in hTERT-RPE1 cells, drastically impaired serum starvation-induced ciliogenesis. Rab34 was also required for serum starvation-induced ciliogenesis in NIH/3T3 cells and MCF10A cells but not for ciliogenesis in Madin-Darby canine kidney (MDCK)-II cysts. We then attempted to identify a specific region(s) of Rab34 that is essential for ciliogenesis by performing deletion and mutation analyses of Rab34. Unexpectedly, instead of a specific sequence in the switch II region, which is generally important for recognizing effector proteins (e.g. Rab interacting lysosomal protein [RILP]), a unique long N-terminal region of Rab34 before the conserved GTPase domain was found to be essential. These findings suggest that Rab34 is an atypical Rab that regulates serum starvation-induced ciliogenesis through its unique N-terminal region.

scholarly journals BBS Proteins Affect Ciliogenesis and Are Essential for Hedgehog Signaling, but Not for Formation of iPSC-Derived RPE-65 Expressing RPE-Like Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1345
Author(s):  
Caroline Amalie Brunbjerg Hey ◽  
Lasse Jonsgaard Larsen ◽  
Zeynep Tümer ◽  
Karen Brøndum-Nielsen ◽  
Karen Grønskov ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Dystrophy ◽  
Renal Cysts ◽  
Wild Type ◽  
Bardet Biedl Syndrome ◽  
Induced Pluripotent

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, renal cysts, obesity and polydactyly. BBS genes have been implicated in ciliogenesis, hedgehog signaling and retinal pigment epithelium maturation. BBS1 and BBS5 are members of the BBSome, implicated in cilia transport of proteins, and BBS10 is a member of the chaperonin-complex, mediating BBSome assembly. In this study, involvement of BBS1, BBS5 and BBS10 in ciliogenesis and hedgehog signaling were investigated in BBS-defective patient fibroblasts as well as in RPE-hTERT cells following siRNA-mediated knockdown of the BBS genes. Furthermore, the ability of BBS1-defective induced pluripotent stem-cells (iPSCs) to differentiate into RPE cells was assessed. We report that cells lacking functional BBS5 or BBS10 have a reduced number of primary cilia, whereas cells lacking functional BBS1 display shorter primary cilia compared to wild-type cells. Hedgehog signaling was substantially impaired and Smoothened, a component of hedgehog signaling, was trapped inside the cilia of the BBS-defective cells, even in the absence of Smoothened agonist. Preliminary results demonstrated the ability of BBS1-defective iPSC to differentiate into RPE-65 expressing RPE-like cells. The BBS1−/−-defective RPE-like cells were less pigmented, compared to RPE-like cells differentiated from control iPSCs, indicating an impact of BBS1 on RPE maturation.

scholarly journals Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Martin Steger ◽  
Federico Diez ◽  
Herschel S Dhekne ◽  
Pawel Lis ◽  
Raja S Nirujogi ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Protein Transport ◽  
Serum Starvation ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Rab Protein ◽  
Affinity Enrichment ◽  
Fold Reduction ◽  
Cilia Formation ◽  
Conserved Residue

We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

scholarly journals 2-IPMA Ameliorates PM2.5-Induced Inflammation by Promoting Primary Ciliogenesis in RPE Cells

Molecules ◽  
2021 ◽  
Vol 26 (17) ◽  
pp. 5409 ◽  
Author(s):  
Ji Yeon Choi ◽  
Ji-Eun Bae ◽  
Joon Bum Kim ◽  
Doo Sin Jo ◽  
Na Yeon Park ◽  
...  
Keyword(s):  
Primary Cilium ◽  
Primary Cilia ◽  
Inflammatory Responses ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Ros Generation ◽  
Rpe Cells ◽  
Tnf Α ◽  
External Stresses ◽  
Stress Kinase

Primary cilia mediate the interactions between cells and external stresses. Thus, dysregulation of primary cilia is implicated in various ciliopathies, e.g., degeneration of the retina caused by dysregulation of the photoreceptor primary cilium. Particulate matter (PM) can cause epithelium injury and endothelial dysfunction by increasing oxidative stress and inflammatory responses. Previously, we showed that PM disrupts the formation of primary cilia in retinal pigment epithelium (RPE) cells. In the present study, we identified 2-isopropylmalic acid (2-IPMA) as a novel inducer of primary ciliogenesis from a metabolite library screening. Both ciliated cells and primary cilium length were increased in 2-IPMA-treated RPE cells. Notably, 2-IPMA strongly promoted primary ciliogenesis and restored PM2.5-induced dysgenesis of primary cilia in RPE cells. Both excessive reactive oxygen species (ROS) generation and activation of a stress kinase, JNK, by PM2.5 were reduced by 2-IPMA. Moreover, 2-IPMA inhibited proinflammatory cytokine production, i.e., IL-6 and TNF-α, induced by PM2.5 in RPE cells. Taken together, our data suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in RPE cells.

scholarly journals Cell Stiffening Contributes to Complement-mediated Injury of Choroidal Endothelial Cells in Early AMD

2021 ◽  
Author(s):  
Andrea P Cabrera ◽  
Jonathan Stoddard ◽  
Irene Santiago Tierno ◽  
Nikolaos Matisioudis ◽  
Mahesh Agarwal ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Membrane Attack Complex ◽  
Small Gtpases ◽  
Age Related Macular Degeneration ◽  
Cell Stiffness ◽  
Age Related ◽  
Dry Amd ◽  
Choroidal Vessels

Age-related macular degeneration (AMD) is the leading cause of blindness in the aging population. Yet, no therapies exist for ~85% of all AMD patients who have the dry form that is marked by degeneration of the retinal pigment epithelium (RPE) and underlying choroidal vasculature. As the choroidal vessels are crucial for RPE development and maintenance, understanding how they degenerate may lead to effective therapies for dry AMD. One likely causative factor for choroidal vascular loss is the cytolytic membrane attack complex (MAC) of the complement pathway that is abundant on choroidal vessels of humans with early dry AMD. To examine this possibility, we studied the effect of complement activation on choroidal endothelial cells (ECs) isolated from a rhesus monkey model of early AMD that, we report, exhibits MAC deposition and choriocapillaris endothelial loss similar to that seen in human early AMD. Treatment of choroidal ECs from AMD eyes with complement-competent normal human serum caused extensive actin cytoskeletal injury that was significantly less pronounced in choroidal ECs from young normal monkey eyes. We further show that ECs from AMD eyes are significantly stiffer than their younger counterparts and exhibit peripheral actin organization that is distinct from the longitudinal stress fibers in young ECs. Finally, these differences in complement susceptibility and mechanostructural properties were found to be regulated by the differential activity of small GTPases Rac and Rho because Rac inhibition in AMD cells led to simultaneous reduction in stiffness and complement susceptibility while Rho inhibition in young cells exacerbated complement injury. Thus, by identifying cell stiffness and cytoskeletal regulators Rac and Rho as important determinants of complement susceptibility, the current findings offer a new mechanistic insight into choroidal vascular loss in early AMD that warrants further investigation for assessment of translational potential.

Targeting of Rab GTPases to cellular membranes

2005 ◽  
Vol 33 (4) ◽  
pp. 652-656 ◽  
Author(s):  
B.R. Ali ◽  
M.C. Seabra
Keyword(s):  
Membrane Trafficking ◽  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Small Gtpases ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Cellular Membranes ◽  
Structural Determinants ◽  
Cellular Processes

Rab proteins are members of the superfamily of Ras-like small GTPases and are involved in several cellular processes relating to membrane trafficking and organelle mobility throughout the cell. Like other small GTPases, Rab proteins are initially synthesized as soluble proteins and for membrane attachment they require the addition of lipid moiety(ies) to specific residues of their polypeptide chain. Despite their well-documented roles in regulating cellular trafficking, Rab proteins own trafficking is still poorly understood. We still need to elucidate the molecular mechanisms of their recruitment to cellular membranes and the structural determinants for their specific cellular localization. Recent results indicate that Rab cellular targeting might be Rab-dependent, and this paper briefly reviews our current knowledge of this process.

scholarly journals Rab GTPases: Switching to Human Diseases

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 909 ◽  
Author(s):  
Noemi Antonella Guadagno ◽  
Cinzia Progida
Keyword(s):  
Membrane Trafficking ◽  
Intracellular Transport ◽  
Small Gtpases ◽  
Regulatory Proteins ◽  
Human Diseases ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Intracellular Membrane ◽  
And Migration ◽  
And Control

Rab proteins compose the largest family of small GTPases and control the different steps of intracellular membrane traffic. More recently, they have been shown to also regulate cell signaling, division, survival, and migration. The regulation of these processes generally occurs through recruitment of effectors and regulatory proteins, which control the association of Rab proteins to membranes and their activation state. Alterations in Rab proteins and their effectors are associated with multiple human diseases, including neurodegeneration, cancer, and infections. This review provides an overview of how the dysregulation of Rab-mediated functions and membrane trafficking contributes to these disorders. Understanding the altered dynamics of Rabs and intracellular transport defects might thus shed new light on potential therapeutic strategies.

scholarly journals Emerging Roles of Small GTPases in Islet β-Cell Function

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1503
Author(s):  
Rajakrishnan Veluthakal ◽  
Debbie C. Thurmond
Keyword(s):  
Insulin Secretion ◽  
Membrane Trafficking ◽  
Cell Function ◽  
Mitochondrial Dynamics ◽  
Small Gtpases ◽  
Rab Gtpases ◽  
Β Cells ◽  
Β Cell ◽  
Cytoskeletal Dynamics ◽  
Glucose Stimulated Insulin Secretion

Several small guanosine triphosphatases (GTPases) from the Ras protein superfamily regulate glucose-stimulated insulin secretion in the pancreatic islet β-cell. The Rho family GTPases Cdc42 and Rac1 are primarily involved in relaying key signals in several cellular functions, including vesicle trafficking, plasma membrane homeostasis, and cytoskeletal dynamics. They orchestrate specific changes at each spatiotemporal region within the β-cell by coordinating with signal transducers, guanine nucleotide exchange factors (GEFs), GTPase-activating factors (GAPs), and their effectors. The Arf family of small GTPases is involved in vesicular trafficking (exocytosis and endocytosis) and actin cytoskeletal dynamics. Rab-GTPases regulate pre-exocytotic and late endocytic membrane trafficking events in β-cells. Several additional functions for small GTPases include regulating transcription factor activity and mitochondrial dynamics. Importantly, defects in several of these GTPases have been found associated with type 2 diabetes (T2D) etiology. The purpose of this review is to systematically denote the identities and molecular mechanistic steps in the glucose-stimulated insulin secretion pathway that leads to the normal release of insulin. We will also note newly identified defects in these GTPases and their corresponding regulatory factors (e.g., GDP dissociation inhibitors (GDIs), GEFs, and GAPs) in the pancreatic β-cells, which contribute to the dysregulation of metabolism and the development of T2D.

scholarly journals Retinosomes

2004 ◽  
Vol 166 (4) ◽  
pp. 447-453 ◽  
Author(s):  
Yoshikazu Imanishi ◽  
Volker Gerke ◽  
Krzysztof Palczewski
Keyword(s):  
Membrane Trafficking ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Structural Proteins ◽  
Lipid Bodies ◽  
Metabolic Intermediate ◽  
Hydrophobic Substances ◽  
Adipose Differentiation ◽  
Specific Tissue ◽  
Tissue Origin

Lipid bodies form autonomous intracellular structures in many model cells and in some cells of specific tissue origin. They contain hydrophobic substances, a set of structural proteins such as perilipin or adipose differentiation-related protein, enzymes implicated in lipid metabolism, and proteins that participate in signaling and membrane trafficking. Retinosomes, particles reminiscent of lipid bodies, have been identified in retinal pigment epithelium as distinct structures compartmentalizing a metabolic intermediate involved in regeneration of the visual chromophore. These observations suggest that lipid bodies, including retinosomes, carry out specific functions that go beyond those of mere lipid storage organelles.

scholarly journals Chronically shortened rod outer segments accompany photoreceptor cell death in Choroideremia

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242284
Author(s):  
Ingrid P. Meschede ◽  
Thomas Burgoyne ◽  
Tanya Tolmachova ◽  
Miguel C. Seabra ◽  
Clare E. Futter
Keyword(s):  
Cell Death ◽  
Membrane Trafficking ◽  
Outer Segment ◽  
Photoreceptor Cell ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Membrane Traffic ◽  
Rod Outer Segments ◽  
Rab Protein ◽  
Outer Segments

X-linked choroideremia (CHM) is a disease characterized by gradual retinal degeneration caused by loss of the Rab Escort Protein, REP1. Despite partial compensation by REP2 the disease is characterized by prenylation defects in multiple members of the Rab protein family that are master regulators of membrane traffic. Remarkably, the eye is the only organ affected in CHM patients, possibly because of the huge membrane traffic burden of the post mitotic photoreceptors, which synthesise outer segments, and the adjacent retinal pigment epithelium that degrades the spent portions each day. In this study, we aimed to identify defects in membrane traffic that might lead to photoreceptor cell death in CHM. In a heterozygous null female mouse model of CHM (Chmnull/WT), degeneration of the photoreceptor layer was clearly evident from increased numbers of TUNEL positive cells compared to age matched controls, small numbers of cells exhibiting signs of mitochondrial stress and greatly increased microglial infiltration. However, most rod photoreceptors exhibited remarkably normal morphology with well-formed outer segments and no discernible accumulation of transport vesicles in the inner segment. The major evidence of membrane trafficking defects was a shortening of rod outer segments that was evident at 2 months of age but remained constant over the period during which the cells die. A decrease in rhodopsin density found in the outer segment may underlie the outer segment shortening but does not lead to rhodopsin accumulation in the inner segment. Our data argue against defects in rhodopsin transport or outer segment renewal as triggers of cell death in CHM.

scholarly journals Membrane trafficking in the retinal pigment epithelium at a glance

2020 ◽  
Vol 133 (16) ◽  
pp. jcs238279
Author(s):  
Tina Storm ◽  
Thomas Burgoyne ◽  
Clare E. Futter
Keyword(s):  
Retinal Pigment Epithelium ◽  
Membrane Trafficking ◽  
Pigment Epithelium ◽  
Scattered Light ◽  
Retinal Pigment ◽  
Retinal Disease ◽  
Neural Retina ◽  
Photoreceptor Outer Segment ◽  
Waste Products ◽  
Photoreceptor Outer Segments

ABSTRACTThe retinal pigment epithelium (RPE) is a highly specialised pigmented monolayer sandwiched between the choroid and the photoreceptors in the retina. Key functions of the RPE include transport of nutrients to the neural retina, removal of waste products and water from the retina to the blood, recycling of retinal chromophores, absorption of scattered light and phagocytosis of the tips of the photoreceptor outer segments. These functions place a considerable membrane trafficking burden on the RPE. In this Cell Science at a Glance article and the accompanying poster, we focus on RPE-specific adaptations of trafficking pathways. We outline mechanisms underlying the polarised expression of membrane proteins, melanosome biogenesis and movement, and endocytic trafficking, as well as photoreceptor outer segment phagocytosis and degradation. We also briefly discuss theories of how dysfunction in trafficking pathways contributes to retinal disease.

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