RESUMO
Proteins are involved in all tasks of life, and their characterization is essential to understand the underlying mechanisms of biological processes. We present a method called "differential visual proteomics" geared to study proteome-wide structural changes of proteins and protein-complexes between a disturbed and an undisturbed cell or between two cell populations. To implement this method, the cells are lysed and the lysate is prepared in a lossless manner for single-particle electron microscopy (EM). The samples are subsequently imaged in the EM. Individual particles are computationally extracted from the images and pooled together, while keeping track of which particle originated from which specimen. The extracted particles are then aligned and classified. A final quantitative analysis of the particle classes found identifies the particle structures that differ between positive and negative control samples. The algorithm and a graphical user interface developed to perform the analysis and to visualize the results were tested with simulated and experimental data. The results are presented, and the potential and limitations of the current implementation are discussed. We envisage the method as a tool for the untargeted profiling of the structural changes in the proteome of single-cells as a response to a disturbing force.
Assuntos
Proteoma/genética , Proteômica/métodos , Análise de Célula Única/métodos , Relação Estrutura-Atividade , AlgoritmosRESUMO
The interactions of proteins, membranes, nucleic acid, and metabolites shape a cell's phenotype. These interactions are stochastic, and each cell develops differently, making it difficult to synchronize cell populations. Consequently, studying biological processes at the single- or few-cell level is often necessary to avoid signal dilution below the detection limit or averaging over many cells. We have developed a method to study metabolites and proteins from a small number of or even a single adherent eukaryotic cell. Initially, cells are lysed by short electroporation and aspirated with a microcapillary under a fluorescent microscope. The lysate is placed on a carrier slide for further analysis using liquid-chromatography mass spectrometry (LC-MS) and/or reverse-phase protein (RPPA) approach. This method allows for a correlative measurement of (i) cellular structures and metabolites and (ii) cellular structures and proteins on the single-cell level. The correlative measurement of cellular structure by light-microscopy, metabolites by LC-MS, and targeted protein detection by RPPA was possible on the few-cell level. We discuss the method, potential applications, limitations, and future improvements.
RESUMO
Pseudomonas aeruginosa, a leading cause of severe hospital-acquired pneumonia, causes infections with up to 50% mortality rates in mechanically ventilated patients. Despite some knowledge of virulence factors involved, it remains unclear how P. aeruginosa disseminates on mucosal surfaces and invades the tissue barrier. Using infection of human respiratory epithelium organoids, here we observed that P. aeruginosa colonization of apical surfaces is promoted by cyclic di-GMP-dependent asymmetric division. Infection with mutant strains revealed that Type 6 Secretion System activities promote preferential invasion of goblet cells. Type 3 Secretion System activity by intracellular bacteria induced goblet cell death and expulsion, leading to epithelial rupture which increased bacterial translocation and dissemination to the basolateral epithelium. These findings show that under physiological conditions, P. aeruginosa uses coordinated activity of a specific combination of virulence factors and behaviours to invade goblet cells and breach the epithelial barrier from within, revealing mechanistic insight into lung infection dynamics.
Assuntos
Células Caliciformes , Infecções por Pseudomonas , Pseudomonas aeruginosa , Mucosa Respiratória , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/fisiologia , Células Caliciformes/microbiologia , Células Caliciformes/metabolismo , Humanos , Mucosa Respiratória/microbiologia , Mucosa Respiratória/citologia , Infecções por Pseudomonas/microbiologia , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo III/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Organoides/microbiologia , Translocação BacterianaRESUMO
The dynamic rearrangement of the actin cytoskeleton plays a key role in several cellular processes such as cell motility, endocytosis, RNA processing and chromatin organization. However, the supramolecular actin structures involved in the different processes remain largely unknown. One of the less studied forms of actin is the lower dimer (LD). This unconventional arrangement of two actin molecules in an antiparallel orientation can be detected by chemical crosslinking at the onset of polymerization in vitro. Moreover, evidence for a transient incorporation of LD into growing filaments and its ability to inhibit nucleation of F-actin filament assembly implicate that the LD pathway contributes to supramolecular actin patterning. However, a clear link from this actin species to a specific cellular function has not yet been established. We have developed an antibody that selectively binds to LD configurations in supramolecular actin structures assembled in vitro. This antibody allowed us to unveil the LD in different mammalian cells. In particular, we show an association of the antiparallel actin arrangement with the endocytic compartment at the cellular and ultrastructural level. Taken together, our results strongly support a functional role of LD in the patterning of supramolecular actin assemblies in mammalian cells.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/química , Actinas/ultraestrutura , Mamíferos/metabolismo , Citoesqueleto de Actina/química , Animais , Linhagem Celular , Movimento Celular , Endocitose , Imunofluorescência/métodos , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão e Varredura , Microscopia Imunoeletrônica/métodos , Modelos Moleculares , Células PC12 , Polímeros/química , Estrutura Terciária de Proteína , Coelhos , RatosRESUMO
Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.
Assuntos
Membranas Intracelulares/ultraestrutura , Corpos de Lewy/ultraestrutura , Doença por Corpos de Lewy/patologia , Lipídeos de Membrana/análise , Organelas/ultraestrutura , Doença de Parkinson/patologia , alfa-Sinucleína/análise , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Hipocampo/química , Hipocampo/ultraestrutura , Humanos , Imageamento Tridimensional , Corpos de Lewy/química , Doença por Corpos de Lewy/metabolismo , Mesencéfalo/química , Mesencéfalo/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica/métodos , Microscopia de Fluorescência , Doença de Parkinson/metabolismo , Substância Negra/química , Substância Negra/ultraestrutura , Sequenciamento do ExomaRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD), and currently one of the most promising therapeutic targets for drug design in Parkinson's disease. In contrast, LRRK1, the closest homologue to LRRK2, does not play any role in PD. Here, we use cryo-electron microscopy (cryo-EM) and single particle analysis to gain structural insight into the full-length dimeric structures of LRRK2 and LRRK1. Differential scanning fluorimetry-based screening of purification buffers showed that elution of the purified LRRK2 protein in a high pH buffer is beneficial in obtaining high quality cryo-EM images. Next, analysis of the 3D maps generated from the cryo-EM data show 16 and 25 Å resolution structures of full length LRRK2 and LRRK1, respectively, revealing the overall shape of the dimers with two-fold symmetric orientations of the protomers that is closely similar between the two proteins. These results suggest that dimerization mechanisms of both LRRKs are closely related and hence that specificities in functions of each LRRK are likely derived from LRRK2 and LRRK1's other biochemical functions. To our knowledge, this study is the first to provide 3D structural insights in LRRK2 and LRRK1 dimers in parallel.
Assuntos
Microscopia Crioeletrônica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Soluções Tampão , Detergentes/farmacologia , Humanos , Imageamento Tridimensional , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , SoluçõesRESUMO
Neurodevelopmental diseases such as the Rett syndrome (RTT) have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used in vitro cultures of neurons derived from murine embryonic stem cells missing the methyl-CpG-binding protein 2 (MECP2) gene (MeCP2-/y) and from wild type cells of the corresponding background. Cultures were assessed using whole-cell patch-clamp electrophysiology and immunofluorescence. We studied the functional maturation of developing neurons and the activity of the synaptic connections they formed. Neurons exhibited minor differences in the developmental patterns for their intrinsic parameters, such as resting membrane potential and excitability; with the MeCP2-/y cells showing a slightly accelerated development, with shorter action potential half-widths at early stages. There was no difference in the early phase of synapse development, but as the cultures matured, significant deficits became apparent, particularly for inhibitory synaptic activity. MeCP2-/y embryonic stem cell-derived neuronal cultures show clear developmental deficits that match phenotypes observed in slice preparations and thus provide a compelling tool to further investigate the mechanisms behind RTT pathophysiology.
RESUMO
Murine stem cell-derived neurons have been used to study a wide variety of neuropsychiatric diseases with a hereditary component, ranging from autism to Alzheimer's. While a significant amount of data on their molecular biology has been generated, there is little data on the physiology of these cultures. Different mouse strains show clear differences in behavioral and other neurobiologically relevant readouts. We have studied the physiology of early differentiation and network formation in neuronal cultures derived from three different mouse embryonic stem cell lines. We have found largely overlapping patterns with some significant differences in the timing of the functional milestones. Neurons from R1 showed the fastest development of intrinsic excitability, while E14Tg2a and J1 were slower. This was also reflected in an earlier appearance of synaptic activity in R1 cultures, while E14Tg2a and J1 were delayed by up to 2 days. In conclusion, stem cells from all backgrounds could be successfully differentiated into functioning neural networks with similar developmental patterns. Differences in the timing of specific milestones, suggest that control cell lines and time-points should be carefully chosen when investigating genetic alterations that lead to subtle deficits in neuronal function.