Rabphilin silencing causes dilated cardiomyopathy in a Drosophila model of nephrocyte damage

AbstractHeart failure (HF) and the development of chronic kidney disease (CKD) have a direct association. Both can be cause and consequence of the other. Many factors are known, such as diabetes or hypertension, which can lead to the appearance and/or development of these two conditions. However, it is suspected that other factors, namely genetic ones, may explain the differences in the manifestation and progression of HF and CKD among patients. One candidate factor is Rph, a gene expressed in the nervous and excretory system in mammals and Drosophila, encoding a Rab small GTPase family effector protein implicated in vesicular trafficking. We found that Rph is expressed in the Drosophila heart, and the silencing of Rph gene expression in this organ had a strong impact in the organization of fibers and functional cardiac parameters. Specifically, we observed a significant increase in diastolic and systolic diameters of the heart tube, which is a phenotype that resembles dilated cardiomyopathy in humans. Importantly, we also show that silencing of Rabphilin (Rph) expression exclusively in the pericardial nephrocytes, which are part of the flies' excretory system, brings about a non-cell-autonomous effect on the Drosophila cardiac system. In summary, in this work, we demonstrate the importance of Rph in the fly cardiac system and how silencing Rph expression in nephrocytes affects the Drosophila cardiac system.

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Abstract 194: Characterization of Arrhythmogenic Dilated Cardiomyopathy Caused by Novel Filamin C Splice Variant in a Zebrafish Model

Circulation Research ◽

10.1161/res.117.suppl_1.194 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Rene L Begay ◽

Teisha J Rowland ◽

Charles A Tharp ◽

August Martin ◽

Sharon L Graw ◽

...

Keyword(s):

Dilated Cardiomyopathy ◽

Splice Variant ◽

Systolic Dysfunction ◽

Family Members ◽

Muscle Disease ◽

Heart Tube ◽

Zebrafish Model ◽

Cell Stage ◽

Cardiac Phenotype ◽

Filamin C

Although dilated cardiomyopathy (DCM) is a serious and frequent genetic cause of heart failure, only 30-40% of cases can be attributed to a known DCM gene mutation. To identify and confirm additional disease genes involved in DCM, we performed whole exome sequencing in two multigenerational families with DCM, both from the same geographic region of Italy, and found a novel splice variant in the gene encoding filamin-C (FLNC). Previously characterized mutations in FLNC had been primarily linked to skeletal muscle disease, although none of the affected family members displayed skeletal myopathy. To confirm and further characterize the arrhythmogenic DCM phenotype observed in family members, we performed embryonic knockdown experiments using morpholino (MO) treatment in zebrafish (Danio rerio) targeting the FLNC ortholog, filamin Cb (flncb). Following MO injection into 1-2 cell stage zebrafish embryos, 63.4% (78 of 123) of viable flncb MO-injected embryos displayed a cardiac phenotype at 72 hours post fertilization (hpf) (vs. 17.0% [30 of 177] of control MO-injected embryos; p≤0.001). Increases in mortality were observed, with 20.8% (54 of 260) of flncb MO-injected embryos surviving at 7 days post fertilization (vs. 65% [162 of 249] of control embryos; p≤0.001). The flncb MO-injected embryos demonstrated pericardial edema, dysmorphic or dilated cardiac chambers, and abnormal looping of the heart tube suggestive of systolic dysfunction. The flncb MO-injected embryos additionally demonstrated a lower mean stroke volume than controls (0.076 vs. 0.181 nl; p=0.015), a reduced mean cardiac output (10.8 vs. 25 nl/min; p=0.02), and an increase in the fraction of retrograde blood flow over the cardiac cycle (0.42 vs. 0.03; p=0.027). Overall, this flncb MO treatment recapitulated a DCM phenotype similar to the state caused by the human splicing variant, supporting haploinsufficiency as the mechanism leading to DCM in these families. Our findings suggest that approaches to augment endogenous filamin C protein levels may represent a viable treatment strategy that warrants exploration in future studies.

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The Small GTPase Arf6: An Overview of Its Mechanisms of Action and of Its Role in Host–Pathogen Interactions and Innate Immunity

International Journal of Molecular Sciences ◽

10.3390/ijms20092209 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2209 ◽

Cited By ~ 13

Author(s):

Tim Van Acker ◽

Jan Tavernier ◽

Frank Peelman

Keyword(s):

Innate Immunity ◽

Phospholipase D ◽

Actin Polymerization ◽

Mechanisms Of Action ◽

Vesicular Trafficking ◽

Small Gtpase ◽

Host Pathogen Interactions ◽

Nadph Oxidases ◽

Different Types ◽

Host Pathogen

The small GTase Arf6 has several important functions in intracellular vesicular trafficking and regulates the recycling of different types of cargo internalized via clathrin-dependent or -independent endocytosis. It activates the lipid modifying enzymes PIP 5-kinase and phospholipase D, promotes actin polymerization, and affects several functionally distinct processes in the cell. Arf6 is used for the phagocytosis of pathogens and can be directly or indirectly targeted by various pathogens to block phagocytosis or induce the uptake of intracellular pathogens. Arf6 is also used in the signaling of Toll-like receptors and in the activation of NADPH oxidases. In this review, we first give an overview of the different roles and mechanisms of action of Arf6 and then focus on its role in innate immunity and host–pathogen interactions.

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Parent-of-origin effects on nuclear chromatin organization and behavior in a Drosophila model for Williams–Beuren Syndrome

Vavilov Journal of Genetics and Breeding ◽

10.18699/vj21.054 ◽

2021 ◽

Vol 25 (5) ◽

pp. 472-485

Author(s):

A. V. Medvedeva ◽

E. V. Tokmatcheva ◽

A. N. Kaminskaya ◽

S. A. Vasileva ◽

E. A. Nikitina ◽

...

Keyword(s):

X Chromosome ◽

Bioinformatic Analysis ◽

Small Gtpase ◽

Model Systems ◽

Lim Kinase ◽

Dna Repeat ◽

Drosophila Model ◽

Parent Of Origin ◽

Origin Effects ◽

Chromosome Bands

Prognosis of neuropsychiatric disorders in progeny requires consideration of individual (1) parent-of-origin effects (POEs) relying on (2) the nerve cell nuclear 3D chromatin architecture and (3) impact of parent-specific miRNAs. Additionally, the shaping of cognitive phenotypes in parents depends on both learning acquisition and forgetting, or memory erasure. These processes are independent and controlled by different signal cascades: the first is cAMPdependent, the second relies on actin remodeling by small GTPase Rac1 – LIMK1 (LIM-kinase 1). Simple experimental model systems such as Drosophila help probe the causes and consequences leading to human neurocognitive pathologies. Recently, we have developed a Drosophila model for Williams–Beuren Syndrome (WBS): a mutant agnts3 of the agnostic locus (X:11AB) harboring the dlimk1 gene. The agnts3 mutation drastically increases the frequency of ectopic contacts (FEC) in specific regions of intercalary heterochromatin, suppresses learning/memory and affects locomotion. As is shown in this study, the polytene X chromosome bands in reciprocal hybrids between agnts3 and the wild type strain Berlin are heterogeneous in modes of FEC regulation depending either on maternal or paternal gene origin. Bioinformatic analysis reveals that FEC between X:11AB and the other X chromosome bands correlates with the occurrence of short (~30 bp) identical DNA fragments partly homologous to Drosophila 372-bp satellite DNA repeat. Although learning acquisition in a conditioned courtship suppression paradigm is similar in hybrids, the middle-term memory formation shows patroclinic inheritance. Seemingly, this depends on changes in miR-974 expression. Several parameters of locomotion demonstrate heterosis. Our data indicate that the agnts3 locus is capable of trans-regulating gene activity via POEs on the chromatin nuclear organization, thereby affecting behavior.

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Functions of Rhotekin, an Effector of Rho GTPase, and its Binding Partners in Mammals

10.20944/preprints201805.0395.v1 ◽

2018 ◽

Author(s):

Hidenori Ito ◽

Rika Morishita ◽

Koh-ichi Nagata

Keyword(s):

Cancer Cells ◽

Rho Gtpase ◽

Small Gtpase ◽

Effector Protein ◽

Binding Motif ◽

Ph Domain ◽

Pleckstrin Homology ◽

Binding Partners ◽

Neuronal Tissues ◽

Per Se

Rhotekin is an effector protein for small GTPase Rho. This protein consists of a Rho binding domain (RBD), a pleckstrin homology (PH) domain, proline-rich regions and a C-terminal PDZ-binding motif. We and other groups have identified various binding partners for Rhotekin and proposed their possible physiological roles. However, functions of Rhotekin per se are largely unknown and information about its physiological roles are fragmentary. In this review, we summarize known features of Rhotekin in neuronal tissues and cancer cells. We also describe characteristics of binding partners for Rhotekin and predicted roles of their interaction.

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Hexa-Longin domain scaffolds for inter-Rab signalling

Bioinformatics ◽

10.1093/bioinformatics/btz739 ◽

2019 ◽

Author(s):

Luis Sanchez-Pulido ◽

Chris P Ponting

Keyword(s):

Primary Cilia ◽

Guanine Nucleotide Exchange Factors ◽

Vesicular Trafficking ◽

Small Gtpase ◽

Supplementary Information ◽

Rab Gtpases ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Hermansky Pudlak Syndrome

Abstract Summary CPLANE is a protein complex required for assembly and maintenance of primary cilia. It contains several proteins, such as INTU, FUZ, WDPCP, JBTS17 and RSG1 (REM2- and RAB-like small GTPase 1), whose genes are mutated in ciliopathies. Using two contrasting evolutionary analyses, coevolution-based contact prediction and sequence conservation, we first identified the INTU/FUZ heterodimer as a novel member of homologous HerMon (Hermansky-Pudlak syndrome and MON1-CCZ1) complexes. Subsequently, we identified homologous Longin domains that are triplicated in each of these six proteins (MON1A, CCZ1, HPS1, HPS4, INTU and FUZ). HerMon complexes are known to be Rab effectors and Rab GEFs (Guanine nucleotide Exchange Factors) that regulate vesicular trafficking. Consequently, INTU/FUZ, their homologous complex, is likely to act as a GEF during activation of Rab GTPases involved in ciliogenesis. Supplementary information Supplementary data are available at Bioinformatics online.

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Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes

Biochemical Journal ◽

10.1042/bj20060950 ◽

2007 ◽

Vol 402 (2) ◽

pp. 229-239 ◽

Cited By ~ 56

Author(s):

Daniela B. Munafó ◽

Jennifer L. Johnson ◽

Beverly A. Ellis ◽

Sophie Rutschmann ◽

Bruce Beutler ◽

...

Keyword(s):

Immunofluorescence Microscopy ◽

Small Gtpase ◽

Effector Protein ◽

Cell Fractionation ◽

Density Fraction ◽

Fractionation Analysis ◽

Minor Population ◽

A Minor ◽

Micro Organisms ◽

Deficient Mice

Neutrophils kill micro-organisms using microbicidal products that they release into the phagosome or into the extracellular space. The secretory machinery utilized by neutrophils is poorly characterized. We show that the small GTPase Rab27a is an essential component of the secretory machinery of azurophilic granules in granulocytes. Rab27a-deficient mice have impaired secretion of MPO (myeloperoxidase) into the plasma in response to lipopolysaccharide. Cell fractionation analysis revealed that Rab27a and the Rab27a effector protein JFC1/Slp1 (synaptotagmin-like protein 1) are distributed principally in the low-density fraction containing a minor population of MPO-containing granules. By immunofluorescence microscopy, we detected Rab27a and JFC1/Slp1 in a minor subpopulation of MPO-containing granules. Interference with the JFC1/Slp1–Rab27a secretory machinery impaired secretion of MPO in permeabilized neutrophils. The expression of Rab27a was dramatically increased when promyelocytic HL-60 cells were differentiated into granulocytes but not when they were differentiated into monocytes. Down-regulation of Rab27a in HL-60 cells by RNA interference did not affect JFC1/Slp1 expression but significantly decreased the secretion of MPO. Neither Rab27a nor JFC1/Slp1 was integrated into the phagolysosome membrane during phagocytosis. Neutrophils from Rab27a-deficient mice efficiently phagocytose zymosan opsonized particles and deliver MPO to the phagosome. We conclude that Rab27a and JFC1/Slp1 permit MPO release into the surrounding milieu and constitute key components of the secretory machinery of azurophilic granules in granulocytes. Our results suggest that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.

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Protein kinase G signaling disrupts Rac1-dependent focal adhesion assembly in liver specific pericytes

AJP Cell Physiology ◽

10.1152/ajpcell.00038.2011 ◽

2011 ◽

Vol 301 (1) ◽

pp. C66-C74 ◽

Cited By ~ 16

Author(s):

Chittaranjan Routray ◽

Chunsheng Liu ◽

Usman Yaqoob ◽

Daniel D. Billadeau ◽

Kenneth D. Bloch ◽

...

Keyword(s):

Focal Adhesion ◽

Stellate Cells ◽

Small Gtpase ◽

Protein Kinase G ◽

Effector Protein ◽

Phosphorylation Sites ◽

Paracrine Signaling ◽

Inhibitory Effects ◽

And Migration ◽

Fa Markers

Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMP-dependent kinase (PKG) via NO/cGMP paracrine signaling. Although PKG is implicated in integrin-mediated cell adhesion to extracellular matrix, whether or how PKG signaling regulates the assembly of focal adhesion complexes (FA) and migration of HSC is not known. With the help of complementary molecular and cell biological approaches, we demonstrate here that activation of PKG signaling in HSC inhibits vascular tubulogenesis, migration/chemotaxis, and assembly of mature FA plaques, as assessed by vascular tubulogenesis assays and immunofluorescence localization of FA markers such as vinculin and vasodilator-stimulated phosphoprotein (VASP). To determine whether PKG inhibits FA assembly by phosphorylation of VASP at Ser-157, Ser-239, and Thr-278, we mutated these putative phosphorylation sites to alanine (VASP3A, phosphoresistant mutant) or aspartic acid (VASP3D, phosphomimetic), respectively. Data generated from these two mutants suggest that the effect of PKG on FA is independent of these three phosphorylation sites. In contrast, activation of PKG inhibits the activity of small GTPase Rac1 and its association with the effector protein IQGAP1. Moreover, PKG activation inhibits the formation of a trimeric protein complex containing Rac1, IQGAP1, and VASP. Finally, we found that expression of a constitutively active Rac1 mutant abolishes the inhibitory effects of PKG on FA formation. In summary, our data suggest that activation of PKG signaling in pericytes inhibits FA formation by inhibiting Rac1.

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Ras recruits mitotic exit regulator Lte1 to the bud cortex in budding yeast

The Journal of Cell Biology ◽

10.1083/jcb.200301128 ◽

2003 ◽

Vol 161 (5) ◽

pp. 889-897 ◽

Cited By ~ 46

Author(s):

Satoshi Yoshida ◽

Ryuji Ichihashi ◽

Akio Toh-e

Keyword(s):

Small Gtpase ◽

Mitotic Exit ◽

Effector Protein ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Downstream Effector ◽

Nucleotide Exchange Factor ◽

Family Protein

ACdc25 family protein Lte1 (low temperature essential) is essential for mitotic exit at a lowered temperature and has been presumed to be a guanine nucleotide exchange factor (GEF) for a small GTPase Tem1, which is a key regulator of mitotic exit. We found that Lte1 physically associates with Ras2-GTP both in vivo and in vitro and that the Cdc25 homology domain (CHD) of Lte1 is essential for the interaction with Ras2. Furthermore, we found that the proper localization of Lte1 to the bud cortex is dependent on active Ras and that the overexpression of a derivative of Lte1 without the CHD suppresses defects in mitotic exit of a Δlte1 mutant and a Δras1 Δras2 mutant. These results suggest that Lte1 is a downstream effector protein of Ras in mitotic exit and that the Ras GEF domain of Lte1 is not essential for mitotic exit but required for its localization.

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