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1.
Front Cell Dev Biol ; 11: 1148773, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37333987

RESUMO

We recently identified the Drosophila ortholog of TTC1 (dTtc1) as an interacting partner of Egalitarian, an RNA adaptor of the Dynein motor. In order to better understand the function of this relatively uncharacterized protein, we depleted dTtc1 in the Drosophila female germline. Depletion of dTtc1 resulted in defective oogenesis and no mature eggs were produced. A closer examination revealed that mRNA cargoes normally transported by Dynein were relatively unaffected. However, mitochondria in dTtc1 depleted egg chambers displayed an extremely swollen phenotype. Ultrastructural analysis revealed a lack of cristae. These phenotypes were not observed upon disruption of Dynein. Thus, this function of dTtc1 is likely to be Dynein independent. Consistent with a role for dTtc1 in mitochondrial biology, a published proteomics screen revealed that dTtc1 interacts with numerous components of electron transport chain (ETC) complexes. Our results indicate that the expression level of several of these ETC components was significantly reduced upon depletion of dTtc1. Importantly, this phenotype was completely rescued upon expression of wild-type GFP-dTtc1 in the depleted background. Lastly, we demonstrate that the mitochondrial phenotype caused by a lack of dTtc1 is not restricted to the germline but is also observed in somatic tissues. Our model suggests that dTtc1, likely in combination with cytoplasmic chaperones, is required for stabilizing ETC components.

2.
Front Cell Dev Biol ; 11: 1045759, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37351276

RESUMO

Sigma 1 Receptor (S1R) is a therapeutic target for a wide spectrum of pathological conditions ranging from neurodegenerative diseases to cancer and COVID-19. S1R is ubiquitously expressed throughout the visceral organs, nervous, immune and cardiovascular systems. It is proposed to function as a ligand-dependent molecular chaperone that modulates multiple intracellular signaling pathways. The purpose of this study was to define the S1R proximatome under native conditions and upon binding to well-characterized ligands. This was accomplished by fusing the biotin ligase, Apex2, to the C terminus of S1R. Cells stably expressing S1R-Apex or a GFP-Apex control were used to map proximal proteins. Biotinylated proteins were labeled under native conditions and in a ligand dependent manner, then purified and identified using quantitative mass spectrometry. Under native conditions, S1R biotinylates over 200 novel proteins, many of which localize within the endomembrane system (endoplasmic reticulum, Golgi, secretory vesicles) and function within the secretory pathway. Under conditions of cellular exposure to either S1R agonist or antagonist, results show enrichment of proteins integral to secretion, extracellular matrix formation, and cholesterol biosynthesis. Notably, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) displays increased binding to S1R under conditions of treatment with Haloperidol, a well-known S1R antagonist; whereas Low density lipoprotein receptor (LDLR) binds more efficiently to S1R upon treatment with (+)-Pentazocine ((+)-PTZ), a classical S1R agonist. Furthermore, we demonstrate that the ligand bound state of S1R correlates with specific changes to the cellular secretome. Our results are consistent with the postulated role of S1R as an intracellular chaperone and further suggest important and novel functionalities related to secretion and cholesterol metabolism.

3.
Invest Ophthalmol Vis Sci ; 63(2): 1, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35103752

RESUMO

Purpose: Sigma 1 receptor (S1R) is expressed in retinal ganglion cells (RGCs) and astrocytes, and its activation is neuroprotective. We evaluated the contribution of S1R within optic nerve head astrocytes (ONHAs) to growth and survival of RGCs in vitro. Methods: Wild-type (WT) RGCs and WT or S1R knockout (S1R KO) ONHAs were cocultured for 2, 4, or 7 days. Total and maximal neurite length, neurite root, and extremity counts were measured. Cell death was measured using a TUNEL assay. Signal transducer and activator of transcription 3 phosphorylation levels were evaluated in ONHA-derived lysates by immunoblotting. Results: The coculture of WT RGCs with WT or S1R KO ONHAs increased the total and maximal neurite length. Neurite root and extremity counts increased at 4 and 7 days when WT RGCs were cocultured with WT or S1R KO ONHAs. At all timepoints, the total and maximal neurite length decreased for WT RGCs in coculture with S1R KO ONHAs compared with WT ONHAs. Root and extremity counts decreased for WT RGCs in coculture with S1R KO ONHAs compared with WT ONHAs at 2 and 7, but not 4 days. RGC apoptosis increased in S1R KO ONHA coculture and S1R KO-conditioned medium, compared with WT ONHA coculture or WT-conditioned medium. S1R KO ONHA-derived lysates showed decreased phosphorylated signal transducer and activator of transcription 3 levels compared with WT ONHA-derived lysates. Conclusions: The absence of S1R within ONHAs has a deleterious effect on RGC neurite growth and RGC survival, reflected in analysis of WT RGC + S1R KO ONHA indirect cocultures. The data suggest that S1R may enhance ganglion cell survival via glia-mediated mechanisms.


Assuntos
Apoptose , Astrócitos/metabolismo , Neuroproteção/fisiologia , Estresse Oxidativo , Receptores sigma/metabolismo , Doenças Retinianas/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Astrócitos/patologia , Western Blotting , Morte Celular , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Disco Óptico/metabolismo , Disco Óptico/patologia , Doenças Retinianas/patologia , Células Ganglionares da Retina/patologia , Receptor Sigma-1
4.
Dev Biol ; 478: 76-88, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34181915

RESUMO

Egalitarian (Egl) is an RNA adaptor for the Dynein motor and is thought to link numerous, perhaps hundreds, of mRNAs with Dynein. Dynein, in turn, is responsible for the transport and localization of these mRNAs. Studies have shown that efficient mRNA binding by Egl requires the protein to dimerize. We recently demonstrated that Dynein light chain (Dlc) is responsible for facilitating the dimerization of Egl. Mutations in Egl that fail to interact with Dlc do not dimerize, and as such, are defective for mRNA binding. Consequently, this mutant does not efficiently associate with BicaudalD (BicD), the factor responsible for linking the Egl/mRNA complex with Dynein. In this report, we tested whether artificially dimerizing this Dlc-binding mutant using a leucine zipper would restore mRNA binding and rescue mutant phenotypes in vivo. Interestingly, we found that although artificial dimerization of Egl restored BicD binding, it only partially restored mRNA binding. As a result, Egl-dependent phenotypes, such as oocyte specification and mRNA localization, were only partially rescued. We hypothesize that Dlc-mediated dimerization of Egl results in a three-dimensional conformation of the Egl dimer that is best suited for mRNA binding. Although the leucine zipper restores Egl dimerization, it likely does not enable Egl to assemble into the conformation required for maximal mRNA binding activity.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Oócitos/fisiologia , Oogênese , Animais , Drosophila , Proteínas de Drosophila/genética , Feminino , Zíper de Leucina , Proteínas Mutantes/metabolismo , Oócitos/citologia , Ovário/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , RNA Mensageiro/metabolismo
5.
RNA Biol ; 18(12): 2376-2389, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33904382

RESUMO

The Dynein motor is responsible for the localization of numerous mRNAs within Drosophila oocytes and embryos. The RNA binding protein, Egalitarian (Egl), is thought to link these various RNA cargoes with Dynein. Although numerous studies have shown that Egl is able to specifically associate with these RNAs, the nature of these interactions has remained elusive. Egl contains a central RNA binding domain that shares limited homology with an exonuclease, yet Egl binds to RNA without degrading it. Mutations have been identified within Egl that disrupt its association with its protein interaction partners, BicaudalD (BicD) and Dynein light chain (Dlc), but no mutants have been described that are specifically defective for RNA binding. In this report, we identified a series of positively charged residues within Egl that are required for RNA binding. Using corresponding RNA binding mutants, we demonstrate that specific RNA cargoes are more reliant on maximal Egl RNA biding activity for their correct localization in comparison to others. We also demonstrate that specification and maintenance of oocyte fate requires maximal Egl RNA binding activity. Even a subtle reduction in Egl's RNA binding activity completely disrupts this process. Our results show that efficient RNA localization at the earliest stages of oogenesis is required for specification of the oocyte and restriction of meiosis to a single cell.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Oócitos/fisiologia , Oogênese , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Comunicação Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Oócitos/citologia , Ligação Proteica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética
6.
J Cell Sci ; 134(3)2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33408246

RESUMO

Replication-dependent histone mRNAs are the only cellular mRNAs that are not polyadenylated, ending in a stemloop instead of a polyA tail, and are normally regulated coordinately with DNA replication. Stemloop-binding protein (SLBP) binds the 3' end of histone mRNA, and is required for processing and translation. During Drosophila oogenesis, large amounts of histone mRNAs and proteins are deposited in the developing oocyte. The maternally deposited histone mRNA is synthesized in stage 10B oocytes after the nurse cells complete endoreduplication. We report that in wild-type stage 10B oocytes, the histone locus bodies (HLBs), formed on the histone genes, produce histone mRNAs in the absence of phosphorylation of Mxc, which is normally required for histone gene expression in S-phase cells. Two mutants of SLBP, one with reduced expression and another with a 10-amino-acid deletion, fail to deposit sufficient histone mRNA in the oocyte, and do not transcribe the histone genes in stage 10B. Mutations in a putative SLBP nuclear localization sequence overlapping the deletion phenocopy the deletion. We conclude that a high concentration of SLBP in the nucleus of stage 10B oocytes is essential for histone gene transcription.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas de Drosophila , Histonas , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histonas/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Proteínas Supressoras de Tumor , Fatores de Poliadenilação e Clivagem de mRNA
7.
Development ; 148(22)2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-35020877

RESUMO

Numerous motors of the Kinesin family contribute to plus-end-directed microtubule transport. However, almost all transport towards the minus-end of microtubules involves Dynein. Understanding the mechanism by which Dynein transports this vast diversity of cargo is the focus of intense research. In selected cases, adaptors that link a particular cargo with Dynein have been identified. However, the sheer diversity of cargo suggests that additional adaptors must exist. We used the Drosophila egg chamber as a model to address this issue. Within egg chambers, Egalitarian is required for linking mRNA with Dynein. However, in the absence of Egalitarian, Dynein transport into the oocyte is severely compromised. This suggests that additional cargoes might be linked to Dynein in an Egalitarian-dependent manner. We therefore used proximity biotin ligation to define the interactome of Egalitarian. This approach yielded several novel interacting partners, including P body components and proteins that associate with Dynein in mammalian cells. We also devised and validated a nanobody-based proximity biotinylation strategy that can be used to define the interactome of any GFP-tagged protein.


Assuntos
Proteínas de Drosophila/genética , Dineínas/genética , Cinesinas/genética , Oócitos/crescimento & desenvolvimento , Animais , Biotina/química , Polaridade Celular/genética , Drosophila melanogaster/genética , Dineínas/química , Regulação da Expressão Gênica/genética , Cinesinas/química , Microtúbulos/genética , Oócitos/metabolismo , Corpos de Processamento/genética , Mapas de Interação de Proteínas/genética , Transporte Proteico , RNA Mensageiro/genética
8.
Dis Model Mech ; 13(5)2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32152089

RESUMO

A critical barrier in the treatment of endosomal and lysosomal diseases is the lack of understanding of the in vivo functions of the putative causative genes. We addressed this by investigating a key pair of endocytic adaptor proteins, PH domain-containing endocytic trafficking adaptor 1 and 2 (PHETA1/2; also known as FAM109A/B, Ses1/2, IPIP27A/B), which interact with the protein product of OCRL, the causative gene for Lowe syndrome. Here, we conducted the first study of PHETA1/2 in vivo, utilizing the zebrafish system. We found that impairment of both zebrafish orthologs, pheta1 and pheta2, disrupted endocytosis and ciliogenesis in renal tissues. In addition, pheta1/2 mutant animals exhibited reduced jaw size and delayed chondrocyte differentiation, indicating a role in craniofacial development. Deficiency of pheta1/2 resulted in dysregulation of cathepsin K, which led to an increased abundance of type II collagen in craniofacial cartilages, a marker of immature cartilage extracellular matrix. Cathepsin K inhibition rescued the craniofacial phenotypes in the pheta1/2 double mutants. The abnormal renal and craniofacial phenotypes in the pheta1/2 mutant animals were consistent with the clinical presentation of a patient with a de novo arginine (R) to cysteine (C) variant (R6C) of PHETA1. Expressing the patient-specific variant in zebrafish exacerbated craniofacial deficits, suggesting that the R6C allele acts in a dominant-negative manner. Together, these results provide insights into the in vivo roles of PHETA1/2 and suggest that the R6C variant is contributory to the pathogenesis of disease in the patient.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Endocitose , Face/embriologia , Rim/embriologia , Crânio/embriologia , Proteínas de Peixe-Zebra/deficiência , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Sistemas CRISPR-Cas/genética , Catepsina K/metabolismo , Diferenciação Celular , Condrócitos/patologia , Cílios/patologia , Colágeno Tipo II/metabolismo , Genes Dominantes , Células HeLa , Humanos , Morfogênese , Atividade Motora , Mutação/genética , Pronefro/patologia , Doenças não Diagnosticadas/diagnóstico por imagem , Doenças não Diagnosticadas/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo
9.
Development ; 146(15)2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31391195

RESUMO

A conserved mechanism of polarity establishment is the localization of mRNA to specific cellular regions. Although it is clear that many mRNAs are transported along microtubules, much less is known about the mechanism by which these mRNAs are linked to microtubule motors. The RNA binding protein Egalitarian (Egl) is necessary for localization of several mRNAs in Drosophila oocytes and embryos. Egl also interacts with Dynein light chain (Dlc) and Bicaudal-D (BicD). The role of Dlc and BicD in mRNA localization has remained elusive. Both proteins are required for oocyte specification, as is Egl. Null alleles in these genes result in an oogenesis block. In this report, we used an shRNA-depletion strategy to overcome the oogenesis block. Our findings reveal that the primary function of Dlc is to promote Egl dimerization. Loss of dimerization compromises the ability of Egl to bind RNA. Consequently, Egl is not bound to cargo, and is not able to efficiently associate with BicD and the Dynein motor. Our results therefore identify the key molecular steps required for assembling a localization-competent mRNP.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Dineínas/metabolismo , Oócitos/citologia , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Dineínas/genética , Microtúbulos/metabolismo , Oogênese/genética , Oogênese/fisiologia , Ligação Proteica/fisiologia , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
10.
Curr Biol ; 27(19): 3010-3016.e3, 2017 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-28966088

RESUMO

Retrotransposons are a pervasive class of mobile elements present in the genomes of virtually all forms of life [1, 2]. In metazoans, these are preferentially active in the germline, which, in turn, mounts defenses that restrain their activity [3, 4]. Here we report that certain classes of retrotransposons ensure transgenerational inheritance by invading presumptive germ cells before they are formed. Using sensitized Drosophila and zebrafish models, we found that diverse classes of retrotransposons migrate to the germ plasm, a specialized region of the oocyte that prefigures germ cells and specifies the germline of descendants in the fertilized egg. In Drosophila, we found evidence for a "stowaway" model, whereby Tahre retroelements traffic to the germ plasm by mimicking oskar RNAs and engaging the Staufen-dependent active transport machinery. Consistent with this, germ plasm determinants attracted retroelement RNAs even when these components were ectopically positioned in bipolar oocytes. Likewise, vertebrate retrotransposons similarly migrated to the germ plasm in zebrafish oocytes. Together, these results suggest that germ plasm targeting represents a fitness strategy adopted by some retrotransposons to ensure transgenerational propagation.


Assuntos
Drosophila melanogaster/genética , Oócitos/metabolismo , Retroelementos/genética , Peixe-Zebra/genética , Animais , Hereditariedade/genética , Oócitos/crescimento & desenvolvimento , RNA Mensageiro/metabolismo
11.
PLoS One ; 12(9): e0184421, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28898265

RESUMO

The sigma 1 receptor (S1R) is a unique transmembrane protein that has been shown to regulate neuronal differentiation and cellular survival. It is expressed within several cell types throughout the nervous system and visceral organs, including neurons and glia within the eye. S1R ligands are therapeutic targets for diseases ranging from neurodegenerative conditions to neoplastic disorders. However, effects of S1R activation and inhibition within glia cells are not well characterized. Within the eye, the astrocytes at the optic nerve head are crucial to the health and survival of the neurons that send visual information to the brain. In this study, we used the S1R-specific agonist, (+)-pentazocine, to evaluate S1R activation within optic nerve head-derived astrocytes (ONHAs). Treatment of ONHAs with (+)-pentazocine attenuated the level and duration of stress-induced ERK phosphorylation following oxidative stress exposure and promoted survival of ONHAs. These effects were specific to S1R activation because they were not observed in ONHAs that were depleted of S1R using siRNA-mediated knockdown. Collectively, our results suggest that S1R activation suppresses ERK1/2 phosphorylation and protects ONHAs from oxidative stress-induced death.


Assuntos
Astrócitos/metabolismo , Nervo Óptico/metabolismo , Receptores sigma/metabolismo , Analgésicos Opioides/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Células HeLa , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nervo Óptico/citologia , Estresse Oxidativo , Pentazocina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores sigma/agonistas , Receptor Sigma-1
12.
Results Probl Cell Differ ; 63: 149-168, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28779317

RESUMO

Messenger RNA (mRNA) localization is a powerful and prevalent mechanism of post-transcriptional gene regulation, enabling the cell to produce protein at the exact location at which it is needed. The phenomenon of mRNA localization has been observed in many types of cells in organisms ranging from yeast to man. Thus, the process appears to be widespread and highly conserved. Several model systems have been used to understand the mechanism by which mRNAs are localized. One such model, and the focus of this chapter, is the egg chamber of the female Drosophila melanogaster. The polarity of the developing Drosophila oocyte and resulting embryo relies on the specific localization of three critical mRNAs: gurken, bicoid, and oskar. If these mRNAs are not localized during oogenesis, the resulting progeny will not survive. The study of these mRNAs has served as a model for understanding the general mechanisms by which mRNAs are sorted. In this chapter, we will discuss how the localization of these mRNAs enables polarity establishment. We will also discuss the role of motor proteins in the localization pathway. Finally, we will consider potential mechanisms by which mRNAs can be anchored at their site of localization. It is likely that the lessons learned using the Drosophila oocyte model system will be applicable to mRNAs that are localized in other organisms as well.


Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Oócitos/metabolismo , RNA Mensageiro/metabolismo , Animais , Polaridade Celular , Proteínas de Drosophila/genética , Feminino , Proteínas de Homeodomínio/genética , Oogênese/genética , Transporte de RNA , RNA Mensageiro/análise , Transativadores/genética , Fator de Crescimento Transformador alfa/genética
13.
J Cell Sci ; 129(22): 4252-4264, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27802167

RESUMO

Recent studies have revealed that diverse cell types use mRNA localization as a means to establish polarity. Despite the prevalence of this phenomenon, much less is known regarding the mechanism by which mRNAs are localized. The Drosophila melanogaster oocyte provides a useful model for examining the process of mRNA localization. oskar (osk) mRNA is localized at the posterior of the oocyte, thus restricting the expression of Oskar protein to this site. The localization of osk mRNA is microtubule dependent and requires the plus-end-directed motor Kinesin-1. Unlike most Kinesin-1 cargoes, localization of osk mRNA requires the Kinesin heavy chain (Khc) motor subunit, but not the Kinesin light chain (Klc) adaptor. In this report, we demonstrate that a newly discovered isoform of Tropomyosin 1, referred to as Tm1C, directly interacts with Khc and functions in concert with this microtubule motor to localize osk mRNA. Apart from osk mRNA localization, several additional Khc-dependent processes in the oocyte are unaffected upon loss of Tm1C. Our results therefore suggest that the Tm1C-Khc interaction is specific for the osk localization pathway.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Cinesinas/metabolismo , Músculos/metabolismo , Transporte de RNA , Tropomiosina/metabolismo , Animais , Feminino , Células Germinativas , Proteínas de Fluorescência Verde/metabolismo , Mutação/genética , Ligação Proteica , Isoformas de Proteínas/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Genetics ; 203(1): 415-32, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27017624

RESUMO

The Drosophila egg chamber provides a useful model for examining mechanisms by which cell fates are specified and maintained in the context of a complex tissue. The egg chamber is also an excellent model for understanding the mechanism by which cytoskeletal filaments are organized and the critical interplay between cytoskeletal organization, polarity establishment, and cell fate specification. Previous work has shown that Egalitarian (Egl) is required for specification and maintenance of oocyte fate. Mutants in egl either completely fail to specify an oocyte, or if specified, the oocyte eventually reverts back to nurse cell fate. Due to this very early role for Egl in egg chamber maturation, it is unclear whether later stages of egg chamber development also require Egl function. In this report, we have depleted Egl at specific stages of egg chamber development. We demonstrate that in early-stage egg chambers, Egl has an additional role in organization of oocyte microtubules. In the absence of Egl function, oocyte microtubules completely fail to reorganize. As such, the localization of microtubule motors and their cargo is disrupted. In addition, Egl also appears to function in regulating the translation of critical polarity determining messenger RNAs (mRNAs). Finally, we demonstrate that in midstage egg chambers, Egl does not appear to be required for microtubule organization, but rather for the correct spatial localization of oskar, bicoid, and gurken mRNAs.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/genética , Oogênese/genética , Animais , Drosophila/citologia , Drosophila/fisiologia , Proteínas de Drosophila/genética , Feminino , Microtúbulos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
Invest Ophthalmol Vis Sci ; 57(2): 453-61, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26868747

RESUMO

PURPOSE: To evaluate, in vivo, the effects of the sigma-1 receptor (σR1) agonist, (+)-pentazocine, on N-methyl-D-aspartate (NMDA)-mediated retinal excitotoxicity. METHODS: Intravitreal NMDA injections were performed in C57BL/6J mice (wild type [WT]) and σR1-/- (σR1 knockout [KO]) mice. Fellow eyes were injected with phosphate-buffered saline (PBS). An experimental cohort of WT and σR1 KO mice was administered (+)-pentazocine by intraperitoneal injection, and untreated animals served as controls. Retinas derived from mice were flat-mounted and labeled for retinal ganglion cells (RGCs). The number of RGCs was compared between NMDA and PBS-injected eyes for all groups. Apoptosis was assessed using TUNEL assay. Levels of extracellular-signal-regulated kinases (ERK1/2) were analyzed by Western blot. RESULTS: N-methyl-D-aspartate induced a significant increase in TUNEL-positive nuclei and a dose-dependent loss of RGCs. Mice deficient in σR1 showed greater RGC loss (≈80%) than WT animals (≈50%). (+)-Pentazocine treatment promoted neuronal survival, and this effect was prevented by deletion of σR1. (+)-Pentazocine treatment resulted in enhanced activation of ERK at the 6-hour time point following NMDA injection. The (+)-pentazocine-induced ERK activation was diminished in σR1 KO mice. CONCLUSIONS: Targeting σR1 activation prevented RGC death while enhancing activation of the mitogen-activated protein kinase (MAPK), ERK1/2. Sigma-1 receptor is a promising therapeutic target for retinal neurodegenerative diseases.


Assuntos
Apoptose/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/toxicidade , N-Metilaspartato/toxicidade , Antagonistas de Entorpecentes/farmacologia , Pentazocina/farmacologia , Receptores sigma/metabolismo , Degeneração Retiniana/prevenção & controle , Células Ganglionares da Retina/patologia , Animais , Western Blotting , Contagem de Células , Relação Dose-Resposta a Droga , Marcação In Situ das Extremidades Cortadas , Injeções Intraperitoneais , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Receptor Sigma-1
16.
J Cell Sci ; 129(1): 166-77, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26567222

RESUMO

Nervous wreck (Nwk) is a conserved F-BAR protein that attenuates synaptic growth and promotes synaptic function in Drosophila. In an effort to understand how Nwk carries out its dual roles, we isolated interacting proteins using mass spectrometry. We report a conserved interaction between Nwk proteins and BAR-SH3 sorting nexins, a family of membrane-binding proteins implicated in diverse intracellular trafficking processes. In mammalian cells, BAR-SH3 sorting nexins induce plasma membrane tubules that localize NWK2, consistent with a possible functional interaction during the early stages of endocytic trafficking. To study the role of BAR-SH3 sorting nexins in vivo, we took advantage of the lack of genetic redundancy in Drosophila and employed CRISPR-based genome engineering to generate null and endogenously tagged alleles of SH3PX1. SH3PX1 localizes to neuromuscular junctions where it regulates synaptic ultrastructure, but not synapse number. Consistently, neurotransmitter release was significantly diminished in SH3PX1 mutants. Double-mutant and tissue-specific-rescue experiments indicate that SH3PX1 promotes neurotransmitter release presynaptically, at least in part through functional interactions with Nwk, and might act to distinguish the roles of Nwk in regulating synaptic growth and function.


Assuntos
Sequência Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nexinas de Classificação/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Córtex Cerebral/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Mutação/genética , Neurogênese , Junção Neuromuscular/metabolismo , Neurônios/metabolismo , Neurotransmissores/metabolismo , Ligação Proteica , Transporte Proteico , Sinapses/ultraestrutura
17.
Biol Open ; 4(11): 1448-61, 2015 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-26459243

RESUMO

The mammalian Sorting Nexin 9 (Snx9) family consists of three paralogs: Snx9, Snx18 and Snx33. Most of the published literature to date has centered on the role of Snx9 in clathrin-mediated endocytosis (CME). Snx9 contains an Sh3 domain at its N-terminus and has been shown to interact with Dynamin and actin nucleation factors via this domain. In addition to the Sh3 domain, Snx9 also contains a C-terminal BAR domain. BAR domains are known to sense and/or induce membrane curvature. In addition to endocytosis, recent studies have implicated the Snx9 family in diverse processes such as autophagy, macropinocytosis, phagocytosis and mitosis. The Snx9 family is encoded by a single gene in Drosophila called sh3px1. In this report, we present our initial characterization of sh3px1. We found that depletion of Sh3px1 from Drosophila Schneider 2 (S2) cells resulted in defective lamellipodia formation. A similar phenotype has been reported upon depletion of Scar, the actin nucleation factor implicated in forming lamellipodia. In addition, we demonstrate that over-expression of Sh3px1 in S2 cells results in the formation of tubules as well as long protrusions. Formation of these structures required the C-terminal BAR domain as well as the adjacent Phox homology (PX) domain of Sh3px1. Furthermore, efficient protrusion formation by Sh3px1 required the actin nucleation factor Wasp. Tubules and protrusions were also generated upon over-expressing the mammalian orthologs Snx18 and Snx33 in S2 cells. By contrast, over-expressing Snx9 mostly induced long tubules.

18.
Genetics ; 201(2): 631-49, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26265702

RESUMO

Dynactin is a multi-subunit complex that functions as a regulator of the Dynein motor. A central component of this complex is Dynamitin/p50 (Dmn). Dmn is required for endosome motility in mammalian cell lines. However, the extent to which Dmn participates in the sorting of cargo via the endosomal system is unknown. In this study, we examined the endocytic role of Dmn using the Drosophila melanogaster oocyte as a model. Yolk proteins are internalized into the oocyte via clathrin-mediated endocytosis, trafficked through the endocytic pathway, and stored in condensed yolk granules. Oocytes that were depleted of Dmn contained fewer yolk granules than controls. In addition, these oocytes accumulated numerous endocytic intermediate structures. Particularly prominent were enlarged endosomes that were relatively devoid of Yolk proteins. Ultrastructural and genetic analyses indicate that the endocytic intermediates are produced downstream of Rab5. Similar phenotypes were observed upon depleting Dynein heavy chain (Dhc) or Lis1. Dhc is the motor subunit of the Dynein complex and Lis1 is a regulator of Dynein activity. We therefore propose that Dmn performs its function in endocytosis via the Dynein motor. Consistent with a role for Dynein in endocytosis, the motor colocalized with the endocytic machinery at the oocyte cortex in an endocytosis-dependent manner. Our results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor along with its regulators, Dynactin and Lis1, functions to ensure efficient endocytic uptake and maturation.


Assuntos
Endocitose/genética , Endossomos/genética , Proteínas Associadas aos Microtúbulos/genética , Oócitos/metabolismo , Animais , Citoesqueleto/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Complexo Dinactina , Dineínas/biossíntese , Dineínas/genética , Endossomos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Oócitos/crescimento & desenvolvimento , Transporte Proteico/genética
19.
Invest Ophthalmol Vis Sci ; 55(6): 3375-84, 2014 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-24812552

RESUMO

PURPOSE: To evaluate the effects of the σ 1 receptor (σR1) agonist, (+)-pentazocine, on lipopolysaccharide (LPS)-induced inflammatory changes in retinal microglia cells. METHODS: Retinal microglia cells were isolated from Sprague-Dawley rat pups. Cells were treated with LPS with or without (+)-pentazocine and with or without the σR1 antagonist BD1063. Morphologic changes were assayed. Cell viability was assessed by using MTT assay. Supernatant levels of tumor necrosis factor α (TNF-α), interleukin 10, (IL-10), monocyte chemoattractant protein-1 (MCP-1), and nitric oxide (NO) were determined. Reactive oxygen species (ROS) formation was assayed, and levels of mitogen-activated protein kinases (MAPKs) were analyzed by using Western blot. RESULTS: The σR1 protein was expressed in retinal microglia. Incubation with LPS and/or (+)-pentazocine did not alter cell viability or σR1 protein levels. Incubation with LPS for 24 hours induced a marked change in microglial morphology and a significant increase in secreted levels of TNF-α, IL-10, MCP-1, and NO. Pretreatment with (+)-pentazocine inhibited the LPS-induced morphologic changes. Release of TNF-α, IL-10, MCP-1, and NO was reduced with (+)-pentazocine. Intracellular ROS formation was suppressed with (+)-pentazocine. Phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was reduced in the presence of (+)-pentazocine. The σR1 antagonist BD1063 blocked the (+)-pentazocine-mediated inhibition of LPS-induced morphologic changes. In addition, BD1063 treatment blocked (+)-pentazocine-mediated suppression of LPS-induced TNF-α, IL-10, MCP-1, NO, and intracellular ROS release. CONCLUSIONS: Treatment with (+)-pentazocine suppressed inflammatory responses of retinal microglia and inhibited LPS-induced activation of ERK/JNK MAPK. In neurodegenerative disease, (+)-pentazocine may exert neuroprotective effects through manipulation of microglia.


Assuntos
Microglia/efeitos dos fármacos , Pentazocina/farmacologia , Receptores sigma/biossíntese , Células Ganglionares da Retina/patologia , Retinite/tratamento farmacológico , Animais , Western Blotting , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Microglia/metabolismo , Microglia/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Piperazinas/farmacologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores sigma/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Retinite/metabolismo , Retinite/patologia , Receptor Sigma-1
20.
PLoS One ; 8(11): e80605, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244700

RESUMO

In order for eukaryotic cells to function properly, they must establish polarity. The Drosophila oocyte uses mRNA localization to establish polarity and hence provides a genetically tractable model in which to study this process. The spatial restriction of oskar mRNA and its subsequent protein product is necessary for embryonic patterning. The localization of oskar mRNA requires microtubules and microtubule-based motor proteins. Null mutants in Kinesin heavy chain (Khc), the motor subunit of the plus end-directed Kinesin-1, result in oskar mRNA delocalization. Although the majority of oskar particles are non-motile in khc nulls, a small fraction of particles display active motility. Thus, a motor other than Kinesin-1 could conceivably also participate in oskar mRNA localization. Here we show that Dynein heavy chain (Dhc), the motor subunit of the minus end-directed Dynein complex, extensively co-localizes with Khc and oskar mRNA. In addition, immunoprecipitation of the Dynein complex specifically co-precipitated oskar mRNA and Khc. Lastly, germline-specific depletion of Dhc resulted in oskar mRNA and Khc delocalization. Our results therefore suggest that efficient posterior localization of oskar mRNA requires the concerted activities of both Dynein and Kinesin-1.


Assuntos
Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Oócitos/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Drosophila , Proteínas de Drosophila/genética , Dineínas/genética , Feminino , Ribonucleoproteínas/genética
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