RESUMO
Single particle tracking fluorescence microscopy was used to study two late endosomal proteins, Rab7 and LAMP1, that appear to be highly colocalized in static fluorescence microscopy images. Imaging these proteins simultaneously reveals that Rab7 and LAMP1 undergo periods of separation within the cell. Single particle tracking carried out during these periods of separation shows that Rab7-vesicles have greater velocities, but undergo less efficient transport than LAMP1-vesicles. This research demonstrates the use of single particle tracking as a tool to resolve functional differences in highly colocalized proteins in intact live cells.
Assuntos
Endossomos/ultraestrutura , Proteína 1 de Membrana Associada ao Lisossomo/análise , Microscopia de Fluorescência/métodos , Proteínas rab de Ligação ao GTP/análise , Células HeLa , Humanos , proteínas de unión al GTP Rab7RESUMO
In nanomedicine, determining the spatial distribution of particles and drugs, together and apart, at high resolution within tissues, remains a major challenge because each must have a different label or detectable feature that can be observed with high sensitivity and resolution. We prepared nanoparticles capable of enzyme-directed assembly of particle therapeutics (EDAPT), containing an analogue of the Pt(II)-containing drug oxaliplatin, an 15N-labeled monomer in the hydrophobic block of the backbone of the polymer, the near-infrared dye Cy5.5, and a peptide that is a substrate for tumor metalloproteinases in the hydrophilic block. When these particles reach an environment rich in tumor associated proteases, the hydrophilic peptide substrate is cleaved, causing the particles to accumulate through a morphology transition, locking them in the tumor extracellular matrix. To evaluate the distribution of drug and EDAPT carrier in vivo, the localization of the isotopically labeled polymer backbone was compared to that of Pt by nanoscale secondary ion mass spectrometry (NanoSIMS). The correlation of NanoSIMS with super-resolution fluorescence microscopy revealed the release of the drug from the nanocarrier and colocalization with cellular DNA within tumor tissue. The results confirmed the dependence of particle accumulation and Pt(II) drug delivery on the presence of a Matrix Metalloproteinase (MMP) substrate and demonstrated antitumor activity. We conclude that these techniques are powerful for the elucidation of the localization of cargo and carrier, and enable a high-resolution assessment of their performance following in vivo delivery.
RESUMO
Direct polymerization of an oxaliplatin analogue was used to reproducibly generate amphiphiles in one pot, which consistently and spontaneously self-assemble into well-defined nanoparticles (NPs). Despite inefficient drug leakage in cell-free assays, the NPs were observed to be as cytotoxic as free oxaliplatin in cell culture experiments. We investigated this phenomenon by super-resolution fluorescence structured illumination microscopy (SIM) and nanoscale secondary ion mass spectrometry (NanoSIMS). In combination, these techniques revealed NPs are taken up via endocytic pathways before intracellular release of their cytotoxic cargo. As with other drug-carrying nanomaterials, these systems have potential as cellular delivery vehicles. However, high-resolution methods to track nanocarriers and their cargo at the micro- and nanoscale have been underutilized in general, limiting our understanding of their interactions with cells and tissues. We contend this type of combined optical and isotopic imaging strategy represents a powerful and potentially generalizable methodology for cellular tracking of nanocarriers and their cargo.
Assuntos
Antineoplásicos/química , Complexos de Coordenação/química , Portadores de Fármacos/química , Nanopartículas/química , Imagem Óptica/métodos , Compostos Organoplatínicos/química , Piridinas/química , Células A549 , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Liberação Controlada de Fármacos , Endocitose , Fluorescência , Células HeLa , Humanos , Compostos Organoplatínicos/farmacologia , Tamanho da Partícula , Polímeros/química , Piridinas/farmacologia , Propriedades de SuperfícieRESUMO
The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells "overloaded" while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. Here, we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantum dots (QDs), showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with up-regulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly down-regulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong up-regulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis, and organelle activities. In contrast, strategies unique to carboxylated QDs showed up-regulation of DNA repair and RNA activities and decreased regulation of cell division, coupled in some cases with up-regulation of stress responses and ATP-related functions. Together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified proactive defenses or repairs of the NP insults.
Assuntos
Nanopartículas , Pontos Quânticos , RNA/química , Linhagem Celular , Expressão Gênica , HumanosRESUMO
Cerium oxide nanoparticles (CNPs) have been shown to induce diverse biological effects, ranging from toxic to beneficial. The beneficial effects have been attributed to the potential antioxidant activity of CNPs via certain redox reactions, depending on their oxidation state or Ce(3+)/Ce(4+) ratio. However, this ratio is strongly dependent on the environment and age of the nanoparticles and it is unclear whether and how the complex intracellular environment impacts this ratio and the possible redox reactions of CNPs. To identify any changes in the oxidation state of CNPs in the intracellular environment and better understand their intracellular reactions, we directly quantified the oxidation states of CNPs outside and inside intact hydrated cells and organelles using correlated scanning transmission x-ray and super resolution fluorescence microscopies. By analyzing hundreds of small CNP aggregates, we detected a shift to a higher Ce(3+)/Ce(4+) ratio in CNPs inside versus outside the cells, indicating a net reduction of CNPs in the intracellular environment. We further found a similar ratio in the cytoplasm and in the lysosomes, indicating that the net reduction occurs earlier in the internalization pathway. Together with oxidative stress and toxicity measurements, our observations identify a net reduction of CNPs in the intracellular environment, which is consistent with their involvement in potentially beneficial oxidation reactions, but also point to interactions that can negatively impact the health of the cells.
Assuntos
Cério/química , Células Epiteliais/química , Nanopartículas Metálicas/química , Organelas/química , Teste de Materiais , OxirreduçãoRESUMO
Interrogating polarized growth is technologically challenging due to extensive cellular branching and uncontrollable environmental conditions in conventional assays. Here we present a robust and high-performance microfluidic system that enables observations of polarized growth with enhanced temporal and spatial control over prolonged periods. The system has built-in tunability and versatility to accommodate a variety of scientific applications requiring precisely controlled environments. Using the model filamentous fungus, Neurospora crassa, our microfluidic system enabled direct visualization and analysis of cellular heterogeneity in a clonal fungal cell population, nuclear distribution and dynamics at the subhyphal level, and quantitative dynamics of gene expression with single hyphal compartment resolution in response to carbon source starvation and exchange. Although the microfluidic device is demonstrated on filamentous fungi, the technology is immediately extensible to a wide array of other biosystems that exhibit similar polarized cell growth, with applications ranging from bioenergy production to human health.
Assuntos
Análise em Microsséries , Técnicas Analíticas Microfluídicas/métodos , Neurospora crassa/crescimento & desenvolvimento , Carbono/metabolismo , Regulação Fúngica da Expressão Gênica , Hifas/fisiologia , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de Fluorescência , Microscopia de Vídeo , Morfogênese , Neurospora crassa/metabolismoRESUMO
Airborne nanoparticles (NPs) that enter the respiratory tract are likely to reach the alveolar region. Accumulating observations support a role for zinc oxide (ZnO) NP dissolution in toxicity, but the majority of in-vitro studies were conducted in cells exposed to NPs in growth media, where large doses of dissolved ions are shed into the exposure solution. To determine the precise intracellular accumulation dynamics and fate of zinc ions (Zn(2+)) shed by airborne NPs in the cellular environment, we exposed alveolar epithelial cells to aerosolized NPs at the air-liquid interface (ALI). Using a fluorescent indicator for Zn(2+), together with organelle-specific fluorescent proteins, we quantified Zn(2+) in single cells and organelles over time. We found that at the ALI, intracellular Zn(2+) values peaked 3 h post exposure and decayed to normal values by 12 h, while in submerged cultures, intracellular Zn(2+) values continued to increase over time. The lowest toxic NP dose at the ALI generated peak intracellular Zn(2+) values that were nearly three-folds lower than the peak values generated by the lowest toxic dose of NPs in submerged cultures, and eight-folds lower than the peak values generated by the lowest toxic dose of ZnSO4 or Zn(2+). At the ALI, the majority of intracellular Zn(2+) was found in endosomes and lysosomes as early as 1 h post exposure. In contrast, the majority of intracellular Zn(2+) following exposures to ZnSO4 was found in other larger vesicles, with less than 10% in endosomes and lysosomes. Together, our observations indicate that low but critical levels of intracellular Zn(2+) have to be reached, concentrated specifically in endosomes and lysosomes, for toxicity to occur, and point to the focal dissolution of the NPs in the cellular environment and the accumulation of the ions specifically in endosomes and lysosomes as the processes underlying the potent toxicity of airborne ZnO NPs.
Assuntos
Células Epiteliais/metabolismo , Exposição por Inalação/análise , Espaço Intracelular/metabolismo , Nanopartículas Metálicas/administração & dosagem , Alvéolos Pulmonares/metabolismo , Óxido de Zinco/farmacocinética , Zinco/farmacocinética , Animais , Técnicas de Cultura de Células , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/química , Células Epiteliais/efeitos dos fármacos , Espaço Intracelular/química , Espaço Intracelular/efeitos dos fármacos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Zinco/análise , Zinco/química , Zinco/toxicidade , Óxido de Zinco/administração & dosagem , Óxido de Zinco/química , Óxido de Zinco/toxicidadeRESUMO
Quantum dots (QDs) and other nanoparticles require delivery and targeting for most intracellular applications. Despite many advances, intracellular delivery and targeting remains inefficient with many QDs remaining bound to the plasma membrane rather than internalized into the cell. The fluorescence resulting from these extracellular QDs results in a background signal that competes with intracellular QDs of interest. We present two methods for the reduction and discrimination of signal resulting from plasma membrane-bound QDs. The first method, a photophysical approach, uses an extracellular quencher to greatly reduce the fluorescence signal from extracellular QDs. This method is compatible with fast, widefield, fluorescence imaging in live cells. Results are presented for two extracellular quenchers, QSY-21 and trypan blue, used in combination with 655 nm emitting QDs. The use of an extracellular quencher can be extended to a wide variety of fluorophores. The second method uses transmission electron microscopy (TEM) to image thin (60-70 nm) slices of resin-embedded cells. The use of sectioned cells and high-resolution TEM makes it possible to discriminate between plasma membrane-bound and intracellular QDs. To overcome the difficulties associated with using TEM to image individual QDs in cells, we have utilized a silver enhancement method that significantly improves the contrast of QDs in TEM images.
Assuntos
Espaço Intracelular/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Pontos Quânticos , Transporte Biológico , Linhagem Celular , Prata/química , Coloração e Rotulagem , Fatores de TempoRESUMO
The endo-lysosomal pathway is essential for intracellular transport and the degradation of extracellular cargo. The relationship between three populations of endo-lysosomal vesicles--Rab7-positive, LAMP1-positive, and both Rab7- and LAMP1-postive--was probed with fluorescence microscopy and single particle tracking. Of specific interest was determining if these vesicles were intermediate or terminal vesicles in the transport of extracellular cargo. We find that the major organelle in the endo-lysosomal pathway, both in terms of population and cargo transport, is positive for Rab7 and LAMP1. Dextran, a fluid phase cargo, shifts from localization within all three populations of vesicles at 30 minutes and 1 hour to primarily LAMP1- and Rab7/LAMP1-vesicles at longer times. This demonstrates that LAMP1- and Rab7/LAMP1-vesicles are terminal vesicles in the endo-lysosomal pathway. We tested two possible mechanisms for this distribution of cargo, delivery to mannose 6-phosphate receptor (M6PR)-negative vesicles and the fusion dynamics of individual vesicles. We find no correlation with M6PR but do find that Rab7-vesicles undergo significantly fewer fusion events than LAMP1- or Rab7/LAMP1-vesicles suggesting that the distribution of fluid phase cargo is driven by vesicle dynamics.