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1.
Commun Biol ; 3(1): 229, 2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393743

RESUMO

Primary adult cardiomyocyte (aCM) represent the mature form of myocytes found in the adult heart. However, culture of aCMs in particular is challenged by poor survival and loss of phenotype, rendering extended in vitro experiments unfeasible. Here, we establish murine aCM culture methods that enhance survival and maintain sarcomeric structure and Ca2+ cycling to enable physiologically relevant contractile force measurements. We also demonstrate genetic and small-molecule manipulations that probe mechanisms underlying myocyte functional performance. Together, these refinements to aCM culture present a toolbox with which to advance our understanding of myocardial physiology.

2.
Acta Crystallogr D Struct Biol ; 75(Pt 12): 1063-1070, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31793900

RESUMO

Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.


Assuntos
Microscopia Crioeletrônica/instrumentação , Substâncias Macromoleculares/química , Proteínas/química , Manejo de Espécimes/instrumentação , Software , Vitrificação
3.
Nat Commun ; 10(1): 3938, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477732

RESUMO

The nematode Caenorhabditis elegans is a bacterivore filter feeder. Through the contraction of the worm's pharynx, a bacterial suspension is sucked into the pharynx's lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels made by surrounding marginal cells. We find that many worm-bioactive small molecules (a.k.a. wactives) accumulate inside of the marginal cells as crystals or globular spheres. Through screens for mutants that resist the lethality associated with one crystallizing wactive we identify a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. We find that expression of sphingomyelin synthase 5 (SMS-5) in the marginal cells is not only sufficient for wactive accumulation but is also important for absorbing exogenous cholesterol, without which C. elegans cannot develop. We conclude that sphingomyelin-rich marginal cells act as a sink to scavenge important nutrients from filtered liquid that might otherwise be shunted back into the environment.


Assuntos
Caenorhabditis elegans/metabolismo , Colesterol/metabolismo , Faringe/metabolismo , Esfingomielinas/metabolismo , Animais , Bactérias/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Cristalização , Interações Hidrofóbicas e Hidrofílicas , Mutação , Faringe/citologia , Esfingomielinas/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
4.
Sci Rep ; 9(1): 7867, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133706

RESUMO

Pathological cardiac hypertrophy is a debilitating condition characterized by deleterious thickening of the myocardium, dysregulated Ca2+ signaling within cardiomyocytes, and contractile dysfunction. Importantly, the nanoscale organization, localization, and patterns of expression of critical Ca2+ handling regulators including dihydropyridine receptor (DHPR), ryanodine receptor 2 (RyR2), phospholamban (PLN), and sarco/endoplasmic reticulum Ca2+-ATPase 2A (SERCA2A) remain poorly understood, especially during pathological hypertrophy disease progression. In the current study, we induced cardiac pathological hypertrophy via transverse aortic constriction (TAC) on 8-week-old CD1 mice, followed by isolation of cardiac ventricular myocytes. dSTORM super-resolution imaging was then used to visualize proteins at nanoscale resolution at two time points and we quantified changes in protein cluster properties using Voronoi tessellation and 2D Fast Fourier Transform analyses. We showed a decrease in the density of DHPR and RyR2 clusters with pressure-overload cardiac hypertrophy and an increase in the density of SERCA2A protein clusters. PLN protein clusters decreased in density in 2-week TAC but returned to sham levels by 4-week TAC. Furthermore, 2D-FFT analysis revealed changes in molecular organization during pathological hypertrophy, with DHPR and RyR2 becoming dispersed while both SERCA2A and PLN sequestered into dense clusters. Our work reveals molecular adaptations that occur in critical SR proteins at a single molecule during pressure overload-induced cardiomyopathy. Nanoscale alterations in protein localization and patterns of expression of crucial SR proteins within the cardiomyocyte provided insights into the pathogenesis of cardiac hypertrophy, and specific evidence that cardiomyocytes undergo significant structural remodeling during the progression of pathological hypertrophy.

5.
Cytoskeleton (Hoboken) ; 76(1): 63-72, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30176126

RESUMO

Septins are a conserved family of GTPases that associate with numerous components of the cytoskeleton and the inner leaflet of the plasma membrane. These proteins are involved in many biological processes, including cell division and membrane trafficking, and serving as a scaffolding component of the cytoskeleton used to recruit other proteins and form diffusion barriers to maintain the composition of membrane domains. In order to carry out their cellular functions, septins undergo interactions via their NC or G interfaces to form heteromeric rod-like structures that can polymerize into filaments and associate laterally into bundles. While electron microscopy studies of affinity-tagged and purified Saccharomyces cerevisiae septin complexes have provided evidence for this periodic organization and in-registry lateral bundling in vitro, the in-vivo arrangement of stress fiber-associated septin bundles in mammalian cells remains poorly characterized. We report here on a direct stochastic optical reconstruction microscopy and photoactivated localization microscopy study of the 2D spatial distribution of septins in mammalian cells. From simulated and experimental results, we show the effects of labeling method, labeling efficiency, and fluorescent emitter photophysics on image reconstruction and interpretation. Our experimental results are consistent with septin organization by polymerization of hetero-octamers and an approximate 30-35 nm periodicity between subsequent units of SEPT2-SEPT2 or SEPT9-SEPT9.


Assuntos
Septinas/metabolismo , Animais , Mamíferos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Nat Microbiol ; 3(12): 1472-1485, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30478389

RESUMO

Plasma membrane integrity is essential for the viability of eukaryotic cells. In response to bacterial pore-forming toxins, disrupted regions of the membrane are rapidly repaired. However, the pathways that mediate plasma membrane repair are unclear. Here we show that autophagy-related (ATG) protein ATG16L1 and its binding partners ATG5 and ATG12 are required for plasma membrane repair through a pathway independent of macroautophagy. ATG16L1 is required for lysosome fusion with the plasma membrane and blebbing responses that promote membrane repair. ATG16L1 deficiency causes accumulation of cholesterol in lysosomes that contributes to defective membrane repair. Cell-to-cell spread by Listeria monocytogenes requires membrane damage by the bacterial toxin listeriolysin O, which is restricted by ATG16L1-dependent membrane repair. Cells harbouring the ATG16L1 T300A allele associated with inflammatory bowel disease were also found to accumulate cholesterol and be defective in repair, linking a common inflammatory disease to plasma membrane integrity. Thus, plasma membrane repair could be an important therapeutic target for the treatment of bacterial infections and inflammatory disorders.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Listeria monocytogenes/efeitos dos fármacos , Animais , Autofagia , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Toxinas Bacterianas/toxicidade , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Colesterol/metabolismo , Modelos Animais de Doenças , Exocitose , Células HeLa , Proteínas de Choque Térmico/toxicidade , Proteínas Hemolisinas/toxicidade , Humanos , Listeria monocytogenes/metabolismo , Lisossomos , Masculino , Camundongos
7.
J Biol Chem ; 293(38): 14723-14739, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30061153

RESUMO

Glycogen synthase kinase 3ß (GSK3ß) phosphorylates and thereby regulates a wide range of protein substrates involved in diverse cellular functions. Some GSK3ß substrates, such as c-Myc and Snail, are nuclear transcription factors, suggesting the possibility that GSK3ß function is controlled through its nuclear localization. Here, using ARPE-19 and MDA-MB-231 human cell lines, we found that inhibition of mTOR complex 1 (mTORC1) leads to partial redistribution of GSK3ß from the cytosol to the nucleus and to a GSK3ß-dependent reduction of the levels of both c-Myc and Snail. mTORC1 is known to be controlled by metabolic cues, such as by AMP-activated protein kinase (AMPK) or amino acid abundance, and we observed here that AMPK activation or amino acid deprivation promotes GSK3ß nuclear localization in an mTORC1-dependent manner. GSK3ß was detected on several distinct endomembrane compartments, including lysosomes. Consistently, disruption of late endosomes/lysosomes through a perturbation of RAS oncogene family member 7 (Rab7) resulted in loss of GSK3ß from lysosomes and in enhanced GSK3ß nuclear localization as well as GSK3ß-dependent reduction of c-Myc levels. These findings indicate that the nuclear localization and function of GSK3ß is suppressed by mTORC1 and suggest a link between metabolic conditions sensed by mTORC1 and GSK3ß-dependent regulation of transcriptional networks controlling cellular biomass production.


Assuntos
Núcleo Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Compartimento Celular , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Endossomos/metabolismo , Glicogênio Sintase Quinase 3 beta/química , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Serina/metabolismo
8.
Mol Pharmacol ; 94(2): 917-925, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29903751

RESUMO

Deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cystic fibrosis (CF)-causing mutation. Recently, ORKAMBI, a combination therapy that includes a corrector of the processing defect of F508del-CFTR (lumacaftor or VX-809) and a potentiator of channel activity (ivacaftor or VX-770), was approved for CF patients homozygous for this mutation. However, clinical studies revealed that the effect of ORKAMBI on lung function is modest and it was proposed that this modest effect relates to a negative impact of VX-770 on the stability of F508del-CFTR. In the current studies, we showed that this negative effect of VX-770 at 10 µM correlated with its inhibitory effect on VX-809-mediated correction of the interface between the second membrane spanning domain and the first nucleotide binding domain bearing F508del. Interestingly, we found that VX-770 exerted a similar negative effect on the stability of other membrane localized solute carriers (SLC26A3, SLC26A9, and SLC6A14), suggesting that this negative effect is not specific for F508del-CFTR. We determined that the relative destabilizing effect of a panel of VX-770 derivatives on F508del-CFTR correlated with their predicted lipophilicity. Polarized total internal reflection fluorescence microscopy on a supported lipid bilayer model shows that VX-770, and not its less lipophilic derivative, increased the fluidity of and reorganized the membrane. In summary, our findings show that there is a potential for nonspecific effects of VX-770 on the lipid bilayer and suggest that this effect may account for its destabilizing effect on VX-809- rescued F508del-CFTR.


Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Fibrose Cística/genética , Quinolonas/farmacologia , Transportadores de Sulfato/química , Aminofenóis/química , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Linhagem Celular , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Liofilização , Células HEK293 , Humanos , Microscopia de Fluorescência , Mutação , Estabilidade Proteica/efeitos dos fármacos , Quinolonas/química
9.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(4): 447-457, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29343430

RESUMO

The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology. Most notably, cardiolipin directly binds to mitochondrial proteins and helps assemble and stabilize mitochondrial multi-protein complexes. Despite their importance for mitochondrial health, how the proteins involved in cardiolipin biosynthesis are organized and embedded in mitochondrial membranes has not been investigated in detail. Here we show that human PGS1 and CLS1 are constituents of large protein complexes. We show that PGS1 forms oligomers and associates with CLS1 and PTPMT1. Using super-resolution microscopy, we observed well-organized nanoscale structures formed by PGS1. Together with the observation that cardiolipin and CLS1 are not required for PGS1 to assemble in the complex we predict the presence of a PGS1-centered cardiolipin-synthesizing scaffold within the mitochondrial inner membrane. Using an unbiased proteomic approach we found that PGS1 and CLS1 interact with multiple cardiolipin-binding mitochondrial membrane proteins, including prohibitins, stomatin-like protein 2 and the MICOS components MIC60 and MIC19. We further mapped the protein-protein interaction sites between PGS1 and itself, CLS1, MIC60 and PHB. Overall, this study provides evidence for the presence of a cardiolipin synthesis structure that transiently interacts with cardiolipin-dependent protein complexes.


Assuntos
Cardiolipinas/biossíntese , Cardiolipinas/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multienzimáticos/metabolismo , Detergentes/farmacologia , Células HEK293 , Humanos , Imunoprecipitação , Microscopia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos
10.
APL Bioeng ; 2(2): 026105, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31069302

RESUMO

Parkinson's disease neurodegenerative brain tissue exhibits two biophysically distinct α-synuclein fiber isoforms-single stranded fibers that appear to be steric-zippers and double-stranded fibers with an undetermined structure. Herein, we describe a ß-helical homology model of α-synuclein that exhibits stability in probabilistic and Monte Carlo simulations as a candidate for stable prional dimer conformers in equilibrium with double-stranded fibers and cytotoxic pore assemblies. Molecular models of ß-helical pore assemblies are consistent with α-synucleinA53T transfected rat immunofluorescence epitope maps. Atomic force microscopy reveals that α-synuclein peptides aggregate into anisotropic fibrils lacking the density or circumference of a steric-zipper. Moreover, fibrillation was blocked by mutations designed to hinder ß-helical but not steric-zipper conformations. ß-helical species provide a structural basis for previously described biophysical properties that are incompatible with a steric-zipper, provide pathogenic mechanisms for familial human α-synuclein mutations, and offer a direct cytotoxic target for therapeutic development.

11.
Mol Biol Cell ; 28(19): 2479-2491, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28768827

RESUMO

Proteasomes are essential for protein degradation in proliferating cells. Little is known about proteasome functions in quiescent cells. In nondividing yeast, a eukaryotic model of quiescence, proteasomes are depleted from the nucleus and accumulate in motile cytosolic granules termed proteasome storage granules (PSGs). PSGs enhance resistance to genotoxic stress and confer fitness during aging. Upon exit from quiescence PSGs dissolve, and proteasomes are rapidly delivered into the nucleus. To identify key players in PSG organization, we performed high-throughput imaging of green fluorescent protein (GFP)-labeled proteasomes in the yeast null-mutant collection. Mutants with reduced levels of ubiquitin are impaired in PSG formation. Colocalization studies of PSGs with proteins of the yeast GFP collection, mass spectrometry, and direct stochastic optical reconstitution microscopy of cross-linked PSGs revealed that PSGs are densely packed with proteasomes and contain ubiquitin but no polyubiquitin chains. Our results provide insight into proteasome dynamics between proliferating and quiescent yeast in response to cellular requirements for ubiquitin-dependent degradation.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Citosol/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Biochim Biophys Acta Proteins Proteom ; 1865(11 Pt B): 1687-1695, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28844737

RESUMO

Identifying the key structural and dynamical determinants that drive the association of biomolecules, whether in solution, or perhaps more importantly in a membrane environment, has critical implications for our understanding of cellular dynamics, processes, and signaling. With recent advances in high-resolution imaging techniques, from the development of new molecular labels to technical advances in imaging methodologies and platforms, researchers are now reaping the benefits of being able to directly characterize and quantify local dynamics, structures, and conformations in live cells and tissues. These capabilities are providing unique insights into association stoichiometries, interactions, and structures on sub-micron length scales. We previously examined the role of lipid headgroup chemistry and phase state in guiding the formation of pseudoisocyanine (PIC) dye J-aggregates on supported planar bilayers [Langmuir, 25, 10719]. We describe here how these same J-aggregates can report on the in situ formation of organellar membrane domains in live cells. Live cell hyperspectral confocal microscopy using GFP-conjugated GTPase markers of early (Rab5) and late (Rab7) endosomes revealed that the PIC J-aggregates were confined to domains on either the limiting membrane or intralumenal vesicles (ILV) of late endosomes, known to be enriched in the anionic lipid bis(monoacylglycero)phosphate (BMP). Correlated confocal fluorescence - atomic force microscopy performed on endosomal membrane-mimetic supported planar lipid bilayers confirmed BMP-specific templating of the PIC J-aggregates. These data provide strong evidence for the formation of BMP-rich lipid domains during multivesicular body formation and portend the application of structured dye aggregates as markers of cellular membrane domain structure, size, and formation.


Assuntos
Endossomos/metabolismo , Glicerofosfatos/metabolismo , Membranas Intracelulares/metabolismo , Microdomínios da Membrana/metabolismo , Monoglicerídeos/metabolismo , Animais , Células CHO , Cricetulus , Endossomos/ultraestrutura , Células Hep G2 , Humanos , Membranas Intracelulares/ultraestrutura , Microdomínios da Membrana/ultraestrutura , Camundongos , Microscopia de Força Atômica , Microscopia Confocal , Células NIH 3T3 , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
13.
J Biol Chem ; 292(34): 14292-14307, 2017 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-28698382

RESUMO

Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability.


Assuntos
Cavéolas/metabolismo , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Modelos Biológicos , Fosfatidilserinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Cavéolas/química , Cavéolas/ultraestrutura , Caveolina 1/antagonistas & inibidores , Caveolina 1/química , Caveolina 1/genética , Linhagem Celular , Membrana Celular/química , Membrana Celular/ultraestrutura , Rastreamento de Células , Cricetulus , Humanos , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mesocricetus , Microscopia Eletrônica de Transmissão , Microscopia de Vídeo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/química , Transporte Proteico , Interferência de RNA , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Imagem com Lapso de Tempo
14.
J Cell Sci ; 130(15): 2579-2590, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28600323

RESUMO

Retromer is a multimeric protein complex that mediates endosome-to-trans-Golgi network (TGN) and endosome-to-plasma membrane trafficking of integral membrane proteins. Dysfunction of this complex has been linked to Alzheimer's disease and Parkinson's disease. The recruitment of retromer to endosomes is regulated by Rab7 (also known as RAB7A) to coordinate endosome-to-TGN trafficking of cargo receptor complexes. Rab7 is also required for the degradation of internalized integral membrane proteins, such as the epidermal growth factor receptor (EGFR). We found that Rab7 is palmitoylated and that this modification is not required for membrane anchoring. Palmitoylated Rab7 colocalizes efficiently with and has a higher propensity to interact with retromer than nonpalmitoylatable Rab7. Rescue of Rab7 knockout cells by expressing wild-type Rab7 restores efficient endosome-to-TGN trafficking, while rescue with nonpalmitoylatable Rab7 does not. Interestingly, Rab7 palmitoylation does not appear to be required for the degradation of EGFR or for its interaction with its effector, Rab-interacting lysosomal protein (RILP). Overall, our results indicate that Rab7 palmitoylation is required for the spatiotemporal recruitment of retromer and efficient endosome-to-TGN trafficking of the lysosomal sorting receptors.


Assuntos
Endossomos/metabolismo , Lipoilação , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Endossomos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , Rede trans-Golgi/genética
15.
Development ; 144(7): 1350-1361, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28213553

RESUMO

Embryos extend their anterior-posterior (AP) axis in a conserved process known as axis elongation. Drosophila axis elongation occurs in an epithelial monolayer, the germband, and is driven by cell intercalation, cell shape changes, and oriented cell divisions at the posterior germband. Anterior germband cells also divide during axis elongation. We developed image analysis and pattern-recognition methods to track dividing cells from confocal microscopy movies in a generally applicable approach. Mesectoderm cells, forming the ventral midline, divided parallel to the AP axis, while lateral cells displayed a uniform distribution of division orientations. Mesectoderm cells did not intercalate and sustained increased AP strain before cell division. After division, mesectoderm cell density increased along the AP axis, thus relieving strain. We used laser ablation to isolate mesectoderm cells from the influence of other tissues. Uncoupling the mesectoderm from intercalating cells did not affect cell division orientation. Conversely, separating the mesectoderm from the anterior and posterior poles of the embryo resulted in uniformly oriented divisions. Our data suggest that mesectoderm cells align their division angle to reduce strain caused by mechanical forces along the AP axis of the embryo.


Assuntos
Padronização Corporal , Divisão Celular , Rastreamento de Células/métodos , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Animais , Automação , Fenômenos Biomecânicos , Contagem de Células , Forma Celular , Ectoderma/citologia , Mesoderma/citologia
16.
ACS Nano ; 11(2): 1131-1135, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28112892

RESUMO

With new approaches in imaging-from new tools or reagents to processing algorithms-come unique opportunities and challenges to our understanding of biological processes, structures, and dynamics. Although innovations in super-resolution imaging are affording novel perspectives into how molecules structurally associate and localize in response to, or in order to initiate, specific signaling events in the cell, questions arise as to how to interpret these observations in the context of biological function. Just as each neighborhood in a city has its own unique vibe, culture, and indeed density, recent work has shown that membrane receptor behavior and action is governed by their localization and association state. There is tremendous potential in developing strategies for tracking how the populations of these molecular neighborhoods change dynamically.


Assuntos
Algoritmos , Imagem Óptica , Biologia , Células HeLa , Humanos , Estrutura Molecular
17.
J Cell Biol ; 216(2): 367-377, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28108526

RESUMO

Lipid exchange between the endoplasmic reticulum (ER) and peroxisomes is necessary for the synthesis and catabolism of lipids, the trafficking of cholesterol, and peroxisome biogenesis in mammalian cells. However, how lipids are exchanged between these two organelles is not understood. In this study, we report that the ER-resident VAMP-associated proteins A and B (VAPA and VAPB) interact with the peroxisomal membrane protein acyl-CoA binding domain containing 5 (ACBD5) and that this interaction is required to tether the two organelles together, thereby facilitating the lipid exchange between them. Depletion of either ACBD5 or VAP expression results in increased peroxisome mobility, suggesting that VAP-ACBD5 complex acts as the primary ER-peroxisome tether. We also demonstrate that tethering of peroxisomes to the ER is necessary for peroxisome growth, the synthesis of plasmalogen phospholipids, and the maintenance of cellular cholesterol levels. Collectively, our data highlight the importance of VAP-ACBD5-mediated contact between the ER and peroxisomes for organelle maintenance and lipid homeostasis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Homeostase , Humanos , Proteínas de Membrana/genética , Microscopia Confocal , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas de Transporte Vesicular/genética
18.
Biophys J ; 111(6): 1141-1142, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27653472
19.
Structure ; 24(7): 1095-109, 2016 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-27265850

RESUMO

Amyloids are fibrillar protein superstructures that are commonly associated with diseases in humans and with physiological functions in various organisms. The precise mechanisms of amyloid formation remain to be elucidated. Surprisingly, we discovered that a bacterial Escherichia coli chaperone-like ATPase, regulatory ATPase variant A (RavA), and specifically the LARA domain in RavA, forms amyloids under acidic conditions at elevated temperatures. RavA is involved in modulating the proper assembly of membrane respiratory complexes. LARA contains an N-terminal loop region followed by a ß-sandwich-like folded core. Several approaches, including nuclear magnetic resonance spectroscopy and molecular dynamics simulations, were used to determine the mechanism by which LARA switches to an amyloid state. These studies revealed that the folded core of LARA is amyloidogenic and is protected by its N-terminal loop. At low pH and high temperatures, the interaction of the N-terminal loop with the folded core is disrupted, leading to amyloid formation.


Assuntos
Adenosina Trifosfatases/química , Amiloide/química , Proteínas de Escherichia coli/química , Motivos de Aminoácidos , Agregados Proteicos , Domínios Proteicos
20.
Nat Commun ; 7: 11714, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27226243

RESUMO

Cell migration is fundamental for both physiological and pathological processes. Migrating cells usually display high dynamics in morphology, which is orchestrated by an integrative array of signalling pathways. Here we identify a novel pathway, we term lateral signalling, comprised of the planar cell polarity (PCP) protein Pk1 and the RhoGAPs, Arhgap21/23. We show that the Pk1-Arhgap21/23 complex inhibits RhoA, is localized on the non-protrusive lateral membrane cortex and its disruption leads to the disorganization of the actomyosin network and altered focal adhesion dynamics. Pk1-mediated lateral signalling confines protrusive activity and is regulated by Smurf2, an E3 ubiquitin ligase in the PCP pathway. Furthermore, we demonstrate that dynamic interplay between lateral and protrusive signalling generates cyclical fluctuations in cell shape that we quantify here as shape volatility, which strongly correlates with migration speed. These studies uncover a previously unrecognized lateral signalling pathway that coordinates shape volatility during productive cell migration.


Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Forma Celular/fisiologia , Transdução de Sinais/fisiologia , Actomiosina/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Interferência de RNA , Imagem com Lapso de Tempo/métodos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
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