RESUMO
Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. Although this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphology similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-associated proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphology in a genomically minimal cell.
Assuntos
Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Genoma Bacteriano , Mycoplasma/genética , Biologia Sintética/métodos , Proteínas de Bactérias/antagonistas & inibidores , Sistemas CRISPR-Cas , Engenharia GenéticaRESUMO
Nuclear factor κB (NF-κB), a key activator of inflammation, primes the NLRP3-inflammasome for activation by inducing pro-IL-1ß and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1ß-dependent inflammation, enhancing macrophage death. Therefore, the "NF-κB-p62-mitophagy" pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Choque Térmico/metabolismo , Inflamassomos/metabolismo , Mitocôndrias/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Choque Térmico/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1 , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Neurônios GABAérgicos/metabolismo , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteoma/metabolismo , Membranas Sinápticas/metabolismo , Animais , Antígenos CD/metabolismo , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Camundongos , Moléculas de Adesão de Célula Nervosa/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Proteômica , Ratos , Receptores de GABA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tálamo/metabolismoRESUMO
Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.
Assuntos
Anticorpos Monoclonais/imunologia , Fibrinogênio/antagonistas & inibidores , Doenças Neurodegenerativas/imunologia , Animais , Epitopos , Humanos , Inflamação/imunologia , Camundongos , RatosRESUMO
Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Metabolismo Energético , Neoplasias Pulmonares , Mitocôndrias , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Ácidos Graxos/metabolismo , Glucose/metabolismo , Gotículas Lipídicas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/ultraestrutura , Microscopia Eletrônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fosforilação Oxidativa , Fenótipo , Tomografia por Emissão de PósitronsRESUMO
Cristae are high-curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae-shaping proteins have been defined, analogous lipid-based mechanisms have yet to be elucidated. Here, we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the inner mitochondrial membrane against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein-mediated curvatures. This model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that cardiolipin is essential in low-oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of cardiolipin is dependent on the surrounding lipid and protein components of the IMM.
Assuntos
Cardiolipinas , Lipidômica , Cardiolipinas/metabolismo , Membranas Mitocondriais/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the multiple and dominant functions of adenovirus oncoproteins has remained elusive. E4-ORF3 forms a nuclear polymer and simultaneously inactivates p53, PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of E4-ORF3. E4-ORF3 forms a dimer with a central ß core, and its structure is unrelated to known polymers or oncogenes. E4-ORF3 dimer units coassemble through reciprocal and nonreciprocal exchanges of their C-terminal tails. This results in linear and branched oligomer chains that further assemble in variable arrangements to form a polymer network that partitions the nuclear volume. E4-ORF3 assembly creates avidity-driven interactions with PML and an emergent MRN binding interface. This reveals an elegant structural solution whereby a small protein forms a multivalent matrix that traps disparate tumor suppressors.
Assuntos
Proteínas E4 de Adenovirus/química , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Infecções por Adenovirus Humanos/virologia , Linhagem Celular , Células Cultivadas , Cristalografia por Raios X , Humanos , Células Vegetais/virologia , Dobramento de Proteína , Nicotiana/virologiaRESUMO
Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.
Assuntos
Cromotripsia , Evolução Molecular , Amplificação de Genes/genética , Neoplasias/genética , Oncogenes/genética , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA Circular/química , DNA Circular/metabolismo , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , Proteína Quinase Ativada por DNA , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Células HeLa , Humanos , Micronúcleos com Defeito Cromossômico , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Seleção Genética , Sequenciamento Completo do GenomaRESUMO
Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins. Further refinements target specific neuronal subcellular locations of interest. We demonstrate the discovery power of these new tools including the detection of detailed microstructural changes of rabies-labeled neurons in aging and Alzheimer's disease mouse models, live imaging of neuronal activities using calcium indicators, and automated measurement of infected neurons. RVs that encode GFP and ferritin as electron microscopy (EM) and fluorescence microscopy reporters are used for dual EM and mesoscale imaging. These new viral variants significantly expand the scale and power of rabies virus-mediated neural labeling and circuit mapping across multiple imaging scales in health and disease.
RESUMO
The rod photoreceptor synapse is the first synapse of dim-light vision and one of the most complex in the mammalian CNS. The components of its unique structure, a presynaptic ribbon and a single synaptic invagination enclosing several postsynaptic processes, have been identified, but disagreements about their organization remain. Here, we have used EM tomography to generate high-resolution images of 3-D volumes of the rod synapse from the female domestic cat. We have resolved the synaptic ribbon as a single structure, with a single arciform density, indicating the presence of one long site of transmitter release. The organization of the postsynaptic processes, which has been difficult to resolve with past methods, appears as a tetrad arrangement of two horizontal cell and two rod bipolar cell processes. Retinal detachment severely disrupts this organization. After 7 d, EM tomography reveals withdrawal of rod bipolar dendrites from most spherules; fragmentation of synaptic ribbons, which lose their tight association with the presynaptic membrane; and loss of the highly branched telodendria of the horizontal cell axon terminals. After detachment, the hilus, the opening through which postsynaptic processes enter the invagination, enlarges, exposing the normally sequestered environment within the invagination to the extracellular space of the outer plexiform layer. Our use of EM tomography provides the most accurate description to date of the complex rod synapse and details changes it undergoes during outer segment degeneration. These changes would be expected to disrupt the flow of information in the rod pathway.SIGNIFICANCE STATEMENT Ribbon-type synapses transmit the first electrical signals of vision and hearing. Despite their crucial role in sensory physiology, the three-dimensional ultrastructure of these synapses, especially the complex organization of the rod photoreceptor synapse, is not well understood. We used EM tomography to obtain 3-D imaging at nanoscale resolution to help resolve the organization of rod synapses in normal and detached retinas. This approach has enabled us to show that in the normal retina a single ribbon and arciform density oppose a tetrad of postsynaptic processes. In addition, it enabled us to provide a 3-D perspective of the ultrastructural changes that occur in response to retinal detachment.
Assuntos
Descolamento Retiniano , Feminino , Animais , Gatos , Microscopia Eletrônica , Sinapses/metabolismo , Retina/ultraestrutura , Células Bipolares da Retina , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , MamíferosRESUMO
NFAT5 is the only known mammalian tonicity-responsive transcription factor with an essential role in cellular adaptation to hypertonic stress. It is also implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity, but the underlying mechanisms remain elusive. Here, we demonstrate that NFAT5 enters the nucleus via the nuclear pore complex. We found that NFAT5 utilizes a unique nuclear localization signal (NFAT5-NLS) for nuclear import. siRNA screening revealed that only karyopherin ß1 (KPNB1), but not karyopherin α, is responsible for the nuclear import of NFAT5 via direct interaction with the NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is driven by exportin-T (XPOT), where the process requires RuvB-like AAA-type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified an unconventional tonicity-dependent nucleocytoplasmic trafficking pathway for NFAT5 that represents a critical step in orchestrating rapid cellular adaptation to change in extracellular tonicity. These findings offer an opportunity for the development of novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.
Assuntos
Núcleo Celular , Carioferinas , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , DNA Helicases , Humanos , Carioferinas/metabolismo , Mamíferos/metabolismo , Sinais de Localização Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Astrocytes with their specialised morphology are essential for brain homeostasis as metabolic mediators between blood vessels and neurons. In neurodegenerative diseases such as Alzheimer's disease (AD), astrocytes adopt reactive profiles with molecular and morphological changes that could lead to the impairment of their metabolic support and impact disease progression. However, the underlying mechanisms of how the metabolic function of human astrocytes is impaired by their morphological changes in AD are still elusive. To address this challenge, we developed and applied a metabolic multiscale modelling approach integrating the dynamics of metabolic energy pathways and physiological astrocyte morphologies acquired in human AD and age-matched control brain samples. The results demonstrate that the complex cell shape and intracellular organisation of energetic pathways determine the metabolic profile and support capacity of astrocytes in health and AD conditions. Thus, our mechanistic approach indicates the importance of spatial orchestration in metabolism and allows for the identification of protective mechanisms against disease-associated metabolic impairments.
Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Metabolismo EnergéticoRESUMO
Many cancers evade immune rejection by suppressing major histocompatibility class I (MHC-I) antigen processing and presentation (AgPP). Such cancers do not respond to immune checkpoint inhibitor therapies (ICIT) such as PD-1/PD-L1 [PD-(L)1] blockade. Certain chemotherapeutic drugs augment tumor control by PD-(L)1 inhibitors through potentiation of T-cell priming but whether and how chemotherapy enhances MHC-I-dependent cancer cell recognition by cytotoxic T cells (CTLs) is not entirely clear. We now show that the lysine acetyl transferases p300/CREB binding protein (CBP) control MHC-I AgPPM expression and neoantigen amounts in human cancers. Moreover, we found that two distinct DNA damaging drugs, the platinoid oxaliplatin and the topoisomerase inhibitor mitoxantrone, strongly up-regulate MHC-I AgPP in a manner dependent on activation of nuclear factor kappa B (NF-κB), p300/CBP, and other transcription factors, but independently of autocrine IFNγ signaling. Accordingly, NF-κB and p300 ablations prevent chemotherapy-induced MHC-I AgPP and abrogate rejection of low MHC-I-expressing tumors by reinvigorated CD8+ CTLs. Drugs like oxaliplatin and mitoxantrone may be used to overcome resistance to PD-(L)1 inhibitors in tumors that had "epigenetically down-regulated," but had not permanently lost MHC-I AgPP activity.
Assuntos
Apresentação de Antígeno/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , NF-kappa B/metabolismo , Neoplasias/tratamento farmacológico , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linfócitos T CD8-Positivos , Proliferação de Células , Quimioterapia Combinada , Humanos , Imunoterapia/métodos , Camundongos , NF-kappa B/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Oxaliplatina/farmacologia , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Fatores de Transcrição de p300-CBP/genéticaRESUMO
Deleterious Ca2+ accumulation is central to hypoxic cell death in the brain of most mammals. Conversely, hypoxia-mediated increases in cytosolic Ca2+ are retarded in hypoxia-tolerant naked mole-rat brain. We hypothesized that naked mole-rat brain mitochondria have an enhanced capacity to buffer exogenous Ca2+ and examined Ca2+ handling in naked mole-rat cortical tissue. We report that naked mole-rat brain mitochondria buffer >2-fold more exogenous Ca2+ than mouse brain mitochondria, and that the half-maximal inhibitory concentration (IC50 ) at which Ca2+ inhibits aerobic oxidative phosphorylation is >2-fold higher in naked mole-rat brain. The primary driving force of Ca2+ uptake is the mitochondrial membrane potential (Δψm ), and the IC50 at which Ca2+ decreases Δψm is â¼4-fold higher in naked mole-rat than mouse brain. The ability of naked mole-rat brain mitochondria to safely retain large volumes of Ca2+ may be due to ultrastructural differences that support the uptake and physical storage of Ca2+ in mitochondria. Specifically, and relative to mouse brain, naked mole-rat brain mitochondria are larger and have higher crista density and increased physical interactions between adjacent mitochondrial membranes, all of which are associated with improved energetic homeostasis and Ca2+ management. We propose that excessive Ca2+ influx into naked mole-rat brain is buffered by physical storage in large mitochondria, which would reduce deleterious Ca2+ overload and may thus contribute to the hypoxia and ischaemia-tolerance of naked mole-rat brain. KEY POINTS: Unregulated Ca2+ influx is a hallmark of hypoxic brain death; however, hypoxia-mediated Ca2+ influx into naked mole-rat brain is markedly reduced relative to mice. This is important because naked mole-rat brain is robustly tolerant against in vitro hypoxia, and because Ca2+ is a key driver of hypoxic cell death in brain. We show that in hypoxic naked mole-rat brain, oxidative capacity and mitochondrial membrane integrity are better preserved following exogenous Ca2+ stress. This is due to mitochondrial buffering of exogenous Ca2+ and is driven by a mitochondrial membrane potential-dependant mechanism. The unique ultrastructure of naked mole-rat brain mitochondria, as a large physical storage space, may support increased Ca2+ buffering and thus hypoxia-tolerance.
RESUMO
Extrinsic inhibitors at sites of blood-brain barrier disruption and neurovascular damage contribute to remyelination failure in neurological diseases. However, therapies to overcome the extrinsic inhibition of remyelination are not widely available and the dynamics of glial progenitor niche remodelling at sites of neurovascular dysfunction are largely unknown. By integrating in vivo two-photon imaging co-registered with electron microscopy and transcriptomics in chronic neuroinflammatory lesions, we found that oligodendrocyte precursor cells clustered perivascularly at sites of limited remyelination with deposition of fibrinogen, a blood coagulation factor abundantly deposited in multiple sclerosis lesions. By developing a screen (OPC-X-screen) to identify compounds that promote remyelination in the presence of extrinsic inhibitors, we showed that known promyelinating drugs did not rescue the extrinsic inhibition of remyelination by fibrinogen. In contrast, bone morphogenetic protein type I receptor blockade rescued the inhibitory fibrinogen effects and restored a promyelinating progenitor niche by promoting myelinating oligodendrocytes, while suppressing astrocyte cell fate, with potent therapeutic effects in chronic models of multiple sclerosis. Thus, abortive oligodendrocyte precursor cell differentiation by fibrinogen is refractory to known promyelinating compounds, suggesting that blockade of the bone morphogenetic protein signalling pathway may enhance remyelinating efficacy by overcoming extrinsic inhibition in neuroinflammatory lesions with vascular damage.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Oligodendroglia/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Animais , Barreira Hematoencefálica/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Células Precursoras de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , Medula Espinal/metabolismoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein, and LRRK2 mutants are recognized risk factors for Parkinson's disease (PD). Although the precise mechanisms that control LRRK2 regulation and function are unclear, the importance of the kinase domain is strongly implicated, since 2 of the 5 most common familial LRRK2 mutations (G2019S and I2020T) are localized to the conserved DFGψ motif in the kinase core, and kinase inhibitors are under development. Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives LRRK2 activation. LRRK2 is quite unusual in that the highly conserved Phe in the DFGψ motif, which is 1 of the 4 R-spine residues, is replaced with tyrosine (DY2018GI). A Y2018F mutation creates a hyperactive phenotype similar to the familial mutation G2019S. The hydroxyl moiety of Y2018 thus serves as a "brake" that stabilizes an inactive conformation; simply removing it destroys a key hydrogen-bonding node. Y2018F, like the pathogenic mutant I2020T, spontaneously forms LRRK2-decorated microtubules in cells, while the wild type and G2019S require kinase inhibitors to form filaments. We also explored 3 different mechanisms that create kinase-dead pseudokinases, including D2017A, which further emphasizes the highly synergistic role of key hydrophobic and hydrophilic/charged residues in the assembly of active LRRK2. We thus hypothesize that LRRK2 harbors a classical protein kinase switch mechanism that drives the dynamic activation of full-length LRRK2.
Assuntos
Domínio Catalítico , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Simulação de Dinâmica Molecular , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutação de Sentido IncorretoRESUMO
The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide a vast surface for efficient photon capture. The formation of each disc is initiated by a ciliary membrane evagination driven by an unknown molecular mechanism reportedly requiring actin polymerization. Since a distinct F-actin network resides precisely at the site of disc morphogenesis, we employed a unique proteomic approach to identify components of this network potentially driving disc morphogenesis. The only identified actin nucleator was the Arp2/3 complex, which induces the polymerization of branched actin networks. To investigate the potential involvement of Arp2/3 in the formation of new discs, we generated a conditional knockout mouse lacking its essential ArpC3 subunit in rod photoreceptors. This knockout resulted in the complete loss of the F-actin network specifically at the site of disc morphogenesis, with the time course of ArpC3 depletion correlating with the time course of F-actin loss. Without the actin network at this site, the initiation of new disc formation is completely halted, forcing all newly synthesized membrane material to be delivered to the several nascent discs whose morphogenesis had already been in progress. As a result, these discs undergo uncontrolled expansion instead of normal enclosure, which leads to formation of unusual, large membrane whorls. These data suggest a model of photoreceptor disc morphogenesis in which Arp2/3 initiates disc formation in a "lamellipodium-like" mechanism.
RESUMO
Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.
Assuntos
Proteínas de Membrana/deficiência , Morfogênese/genética , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Distrofias de Cones e Bastonetes/genética , Distrofias de Cones e Bastonetes/patologia , Distrofias de Cones e Bastonetes/veterinária , Modelos Animais de Doenças , Cães , Espaço Extracelular/metabolismo , Proteínas do Olho/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Retinose Pigmentar/genética , Retinose Pigmentar/patologiaRESUMO
As biomedical imaging datasets expand, deep neural networks are considered vital for image processing, yet community access is still limited by setting up complex computational environments and availability of high-performance computing resources. We address these bottlenecks with CDeep3M, a ready-to-use image segmentation solution employing a cloud-based deep convolutional neural network. We benchmark CDeep3M on large and complex two-dimensional and three-dimensional imaging datasets from light, X-ray, and electron microscopy.