RESUMO
Neuronal communication relies on the release of neurotransmitters from various populations of synaptic vesicles. Despite displaying vastly different release probabilities and mobilities, the reserve and recycling pool of vesicles co-exist within a single cluster suggesting that small synaptic biomolecular condensates could regulate their nanoscale distribution. Here, we performed a large-scale activity-dependent phosphoproteome analysis of hippocampal neurons in vitro and identified Tau as a highly phosphorylated and disordered candidate protein. Single-molecule super-resolution microscopy revealed that Tau undergoes liquid-liquid phase separation to generate presynaptic nanoclusters whose density and number are regulated by activity. This activity-dependent diffusion process allows Tau to translocate into the presynapse where it forms biomolecular condensates, to selectively control the mobility of recycling vesicles. Tau, therefore, forms presynaptic nano-biomolecular condensates that regulate the nanoscale organization of synaptic vesicles in an activity-dependent manner.
Assuntos
Condensados Biomoleculares , Vesículas Sinápticas , Vesículas Sinápticas/metabolismo , Terminações Pré-Sinápticas/metabolismo , Sinapses/fisiologia , Neurônios/metabolismoRESUMO
Nanofabricated and nanopatterned surfaces have revealed the sensitivity of cell adhesion to nanoscale variations in the spacing of adhesive ligands such as the tripeptide arginine-glycine-aspartic acid (RGD). To date, surface characterisation and cell adhesion are often examined in two separate experiments so that the localisation of ligands and adhesion proteins cannot be combined in the same image. Here we developed self-assembled monolayer chemistry for indium tin oxide (ITO) surfaces for single molecule localisation microscopy (SMLM). Cell adhesion and spreading were sensitive to average RGD spacing. At low average RGD spacing, a threshold exists of 0.8 RGD peptides per µm2 that tether cells to the substratum but this does not enable formation of focal adhesions. These findings suggest that cells can sense and engage single adhesive ligands but ligand clustering is required for cell spreading. Thus, our data reveal subtle differences in adhesion biology that may be obscured in ensemble measurements.
Assuntos
Adesivos/metabolismo , Moléculas de Adesão Celular/metabolismo , Imagem Individual de Molécula/métodos , Animais , Adesão Celular , Cor , Vidro/química , Ligantes , Camundongos , Microscopia , Células NIH 3T3 , Oligopeptídeos/química , Paxilina/metabolismo , Propriedades de Superfície , Compostos de Estanho/químicaRESUMO
Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippocampal presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation; (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes; (iii) the dissection, culturing and transfection of hippocampal neurons in microfluidic devices; and (iv) guidance on how to perform the pulse-chase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take â¼1 week.
Assuntos
Técnicas de Cultura de Células/métodos , Endocitose , Dispositivos Lab-On-A-Chip , Neurônios/citologia , Imagem Óptica/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Sobrevivência Celular , Células Cultivadas , Desenho de Equipamento , Hipocampo/citologia , Imagem Óptica/instrumentação , Perfusão/instrumentação , Perfusão/métodos , Impressão Tridimensional , Ratos , Ratos Sprague-Dawley , Transfecção/instrumentação , Transfecção/métodosRESUMO
Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to detect nanodomains of the cell adhesion-stimulating tripeptide arginine-glycine-aspartic acid (RGD). These domains were formed by the phase separation of block copolymers with controllable spacing on the scale of tens of nanometers. We first determined the topology of the block copolymer with atomic force microscopy (AFM) and then imaged the localization of individual RGD peptides with direct stochastic optical reconstruction microscopy (dSTORM). To compare the data, we analyzed the dSTORM data with DBSCAN (density-based spatial clustering application with noise). The ligand distribution and polymer topology are not necessary identical since peptides may attach to the polymer outside the nanodomains and/or coupling and detection of peptides within the nanodomains is incomplete. We therefore performed simulations to explore the extent to which nanodomains could be mapped with dSTORM. We found that successful detection of nanodomains by dSTORM was influenced by the inter-domain spacing and the localization precision of individual fluorophores, and less by non-specific absorption of ligands to the substratum. For example, under our imaging conditions, DBSCAN identification of nanodomains spaced further than 50 nm apart was largely independent of background localisations, while nanodomains spaced closer than 50 nm required a localization precision of ~11 nm to correctly estimate the modal nearest neighbor distance (NDD) between nanodomains. We therefore conclude that SMLM is a promising technique to directly map the distribution and nanoscale organization of ligands and would benefit from an improved localization precision.
Assuntos
Adesão Celular , Microscopia de Força Atômica/métodos , Oligopeptídeos/química , Imagem Individual de Molécula/métodos , Análise por ConglomeradosRESUMO
Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been hampered by the inability to directly measure signaling activity or protein association at the level of individual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of individual proteins.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imagem Individual de Molécula/métodos , Fibroblastos , Adesões Focais , Ativação Linfocitária , Proteínas de Membrana , Microscopia de Fluorescência/métodos , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Software , Análise EspacialRESUMO
Telomerase is a ribonucleoprotein enzyme, which has a significant role in synthesizing DNA telomeric in eukaryotes. Telomere maintenance can cause to immortalization and malignant transformation of human cells and thereby telomerase activity must be scrutinized as an important factor in most tumor cells. The proliferation of cancer cells or apoptosis induction can be suppressed by telomerase inhibition using different therapeutic agents without any side effects upon normal cells. Natural substances, with anti-tumor effects, such as those derived from plants can be suitable candidates due to their capabilities in preventing some side effects and resistance of tumors with respect to most chemotherapeutic drugs. In this regards, many studies have shown that natural phytochemicals have inhibitory effects on telomerase activity through affecting its subunits and components. Therefore, the aim of this paper is to review the recent studies on these kinds of phytochemicals in terms of property and mechanism. Moreover, strategies for improving the therapeutic efficacy of plant-derived substances such as combination therapy and nanoformulation based approaches are included.
Assuntos
Neoplasias/tratamento farmacológico , Telomerase/metabolismo , Anticarcinógenos/química , Anticarcinógenos/metabolismo , Anticarcinógenos/uso terapêutico , Catequina/análogos & derivados , Catequina/química , Catequina/metabolismo , Catequina/uso terapêutico , Curcumina/química , Curcumina/metabolismo , Curcumina/uso terapêutico , Composição de Medicamentos , Humanos , Compostos Fitoquímicos/química , Compostos Fitoquímicos/metabolismo , Compostos Fitoquímicos/uso terapêutico , Polifenóis/química , Polifenóis/metabolismo , Polifenóis/uso terapêutico , Telomerase/antagonistas & inibidoresRESUMO
BACKGROUND: Despite numerous useful anticancer properties of curcumin, its utility is limited due to its hydrophobic structure. In this study, we investigated the comparative antiproliferative effect of PAMAM encapsulating curcumin with naked curcumin on the T47D breast cancer cell line. MATERIALS AND METHODS: Cytotoxic effects of PAMAM dendrimers encapsulating curcumin and curcumin alone were investigated by MTT assay. After treating cells with different concentrations of both curcumin alone and curcumin encapsulated for 24h, telomerase activity was determined by TRAP assay. RESULTS: While PAMAM dendrimers encapsulating curcumin had no cytotoxicity on cancer cells, they decreased the IC50 for proliferation and also increased the inhibitory effect on telomerase activity. CONCLUSIONS: Considering the non-toxicity in addition to effectiveness for enhancing curcumin anticancer properties, dendrimers could be considered good therapeutic vehicles for this hydrophobic agent.
Assuntos
Antineoplásicos/farmacologia , Materiais Biocompatíveis/química , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Curcumina/farmacologia , Dendrímeros/química , Telomerase/antagonistas & inibidores , Neoplasias da Mama/patologia , Feminino , Humanos , Células Tumorais CultivadasRESUMO
Leptin plays the role of mitogenic factor in the breast carcinogenesis. Therefore, it could be considered as a target for breast cancer therapy. Leptin gene expression could be modulated by activation of estrogen receptors. Silibinin is an herbal compound with anti-cancer activity on prostate and colorectal cancers. Based on the fact that targeting of leptin can be considered as a novel strategy for breast cancer therapy, the aim of this study was the investigation of potentiality of silibinin for inhibition of leptin gene expression and secretion, and its link with expression of estrogen receptors. Cytotoxic effect of silibinin on T47D breast cancer cells was investigated by MTT assay test after 24, 48 and 72 h treatments with different concentrations of silibinin. The levels of leptin, estrogen receptor α and estrogen receptor ß genes expression was measured by reverse-transcription real-time PCR. The amount of secreted leptin in the culture medium was determined by ELISA. Data were statistically analyzed by one-way ANOVA test. Silibinin inhibits growth of T47D cells in a time and dose dependent manner. There was significant difference between control and treated cells in the levels of leptin, estrogen receptor ß expression levels and the quantity of secreted leptin was decreased in the treated cells in comparison to control cells. In conclusion, silibinin inhibits the expression and the secretion of leptin and in the future it might probably be a drug candidate for breast cancer therapy through leptin targeting.