Intracellular nanoparticle dynamics affected by cytoskeletal integrity (original) (raw)
Related papers
2016
The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. Cytoskeletal organization and dynamics are known to modulate cytoplasmic transport processes, but how local transport dynamics depends on the highly heterogeneous intracellular organization of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for transport dynamics in different sub-cellular environments. Rapid imaging of non-functionalized QDs revealed two populations with a 100-fold difference in diffusion constant. Depolymerization of actin increased the fast diffusing fraction, suggesting that slow QDs are trapped inside the actin network. When nanobody-functionalized QDs were targeted to different kinesin motor proteins and moved over microtubules, they did not experience strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 bound QDs displayed subtle di...
Non-specific interactions govern cytosolic diffusion of nanosized objects in mammalian cells
Nature Materials, 2018
The diffusivity of macromolecules in the cytoplasm of eukaryotic cells varies over orders of magnitude and dictates the kinetics of cellular processes. However, a general description associating the Brownian or anomalous nature of intracellular diffusion to the architectural and biochemical properties of the cytoplasm has not been achieved. Here, we measure the mobility of individual fluorescent nanoparticles in living mammalian cells to obtain a comprehensive analysis of cytoplasmic diffusion. We identify a correlation between tracer size, its biochemical nature, and its mobility. Inert particles with size equal or below 50 nm behave as Brownian particles diffusing in a medium of low viscosity with negligible effects of molecular crowding. Increasing the strength of non-specific interactions of the nanoparticles within the cytoplasm gradually reduces their mobility and leads to sub-diffusive behaviour. These experimental observations and the transition from Brownian to sub-diffusive motion can be recapitulated in a minimal phenomenological model.Methods Cell culture. HeLa and NIH 3T3 cells were cultured in 1.0 g • l-1 glucose, phenol-red free DMEM (11054020, Gibco) supplemented with 10% (v/v) FBS (10270106, Gibco), 1× GlutaMAX™ (35050061, Gibco), and 100 U • ml-1 of penicillin/streptomycin (15140122, Gibco). Primary normal, adult Human Dermal Fibroblast (HDFa, PCS-201-012™, ATCC ®) were cultured in 4.5 g • l-1 glucose, phenol-red free DMEM (31053028, Gibco) supplemented with 10% (v/v) FCS (CVFSVF00-01, Eurobio), 1 mM Sodium Pyruvate (11360070, Gibco), 100 U • ml-1 penicillin/streptomycin (15140122, Gibco), and 1× GlutaMAX™ (35050061, Gibco). Retinal Pigmented Epithelial (RPE-1) cells were cultivated using DME/F-12 medium (D8437, Sigma-Aldrich) supplemented with 10% (v/v) FBS (10270106, Gibco). Cells were passed every 3 days when reaching 80-90% confluency. Human Mesenchymal Stem Cells (hMSC, MSC-001F, StemCell) were cultivated with MesenCult™ proliferation kit (05411, StemCell) in flask coated with type I collagen (C8919, Sigma-Aldrich). Cells were passed every 10 days when reaching 70% confluency. All cells were maintained at 37°C in humidified, CO 2-controlled (5%) incubators. HeLa cells were purchased from ATCC. NIH 3T3 and hMSC cells were provided by Maïté Coppey-Moisan (Institut Jacques Monod) and originally obtained from ATCC and StemCell, respectively. RPE-1 cells were provided by Bruno Goud (Institut Curie) and originally obtained from ATCC. HDFa cells were provided by Sébastien Letard and Patrice Dubreuil (Centre de Recherche en Cancérologie de Marseille) and originally obtained from ATCC.
Dynamics and mechanisms of quantum dot nanoparticle cellular uptake
Journal of Nanobiotechnology, 2010
Background: The rapid growth of the nanotechnology industry and the wide application of various nanomaterials have raised concerns over their impact on the environment and human health. Yet little is known about the mechanism of cellular uptake and cytotoxicity of nanoparticles. An array of nanomaterials has recently been introduced into cancer research promising for remarkable improvements in diagnosis and treatment of the disease. Among them, quantum dots (QDs) distinguish themselves in offering many intrinsic photophysical properties that are desirable for targeted imaging and drug delivery.
In Vivo Nano-imaging of Membrane Dynamics in Metastatic Tumor Cells Using Quantum Dots
Journal of Biological Chemistry, 2010
Changes in membrane morphology and membrane protein dynamics based on its fluidity are critical for cancer metastasis. However, this subject has remained unclear because the spatial precision of previous in vivo imaging has been limited to the micrometer level and single-molecule imaging is impossible. Here, we have imaged the membrane dynamics of tumor cells in mice with a spatial precision of 7-9 nm under a confocal microscope. A metastasis-promoting factor on the cell membrane, protease-activated receptor 1 (PAR1), was labeled with quantum dots conjugated with an anti-PAR1 antibody. Movements of cancer cells and PAR1 during metastasis were clearly observed in vivo. Images used to assess PAR1 dynamics were taken of representative cells for four stages of metastasis; i.e., cancer cells far from blood vessels in tumor, near the vessel, in the bloodstream, and adherent to inner vascular surface in the normal tissues near tumor were photographed. The diffusion speed of PAR1 in static cells far from tumor blood vessels was slower than in moving cells near the vessels and in the bloodstream. The diffusion speed of cells adhering to the inner vascular surface in the normal tissues was also very slow. Cells formed membrane protrusion during migration. The PAR1 diffusion speed on these pseudopodia was faster than in other membrane regions in the same cell. Thus, the dynamics of PAR1 movement showed that membrane fluidity increases during intravasation, reaches a peak in the vessel, decreases during extravasation and is also higher at locally formed pseudopodia. During metastasis, cancer cells detach from the parent tumor, invade surrounding connective tissue and blood vessels, are transported in the bloodstream, and invade other organs after extravasation (1, 2). Membrane dynamics are significantly altered in metastatic cancer cells (3).
Journal of Biomedical Nanotechnology, 2014
The practical use of quantum dots (QD) as diagnostic, visualizing and therapeutic nano-agents depends on the understanding of fundamental mechanisms of their entrance and trafficking within cells. Here we show that CdSe/ZnS carboxylic-coated QD (COOH-QD) enter fibroblast cells via lipid raft/caveolin-mediated endocytosis, pass early sorting endosomes and accumulate in the multivesicular bodies, but not in the lysosomes. Later phase of their endocytosis leads to the generation of lipid raft/caveolin-dependent endocytosis inhibition that prevents intracellular uptake of new COOH-QD, but not the QD coupled with platelet-derived growth factor BB (PDGF-QD). PDGF-QD enter fibroblasts by the clathrin-mediated endocytosis and undergo similar intracellular trafficking as COOH-QD, yet they accumulate in lysosomes in contrast to COOH-QD. The PDGF-QD activate PDGF receptor-and are mitogenic, however, COOH-QD suppress cell migration and chemotaxis. Data show that surface coating of QD with the biologically active proteins redirects their intracellular traffic routes and changes their biological activity.
In vitro sliding of actin filaments labelled with single quantum dots
Biochemical and Biophysical Research Communications, 2004
We recently refined the in vitro motility assay for studies of actomyosin function to achieve rectified myosin induced sliding of actin filaments. This paves the way, both for detailed functional studies of actomyosin and for nanotechnological applications. In the latter applications it would be desirable to use actin filaments for transportation of cargoes (e.g., enzymes) between different predetermined locations on a chip. We here describe how single quantum dot labelling of isolated actin filaments simultaneously provides handles for cargo attachment and bright and photostable fluorescence labels facilitating cargo detection and filament tracking. Labelling was achieved with preserved actomyosin function using streptavidin-coated CdSe quantum dots (Qdots). These nanocrystals have several unique physical properties and the present work describes their first use for functional studies of isolated proteins outside the cell. The results, in addition to the nanotechnology developments, open for new types of in vitro assays of isolated biomolecules. .se (A. M a ansson), lars.monte-lius@ftf.lth.se (L. Montelius).
Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection
International Journal of Nanomedicine, 2013
Background: The purpose of this study was to elucidate the mechanism of natural uptake of nonfunctionalized quantum dots in comparison with microinjected quantum dots by focusing on their time-dependent accumulation and intracellular localization in different cell lines. Methods: The accumulation dynamics of nontargeted CdSe/ZnS carboxyl-coated quantum dots (emission peak 625 nm) was analyzed in NIH3T3, MCF-7, and HepG2 cells by applying the methods of confocal and steady-state fluorescence spectroscopy. Intracellular colocalization of the quantum dots was investigated by staining with Lysotracker ®. Results: The uptake of quantum dots into cells was dramatically reduced at a low temperature (4°C), indicating that the process is energy-dependent. The uptake kinetics and imaging of intracellular localization of quantum dots revealed three accumulation stages of carboxyl-coated quantum dots at 37°C, ie, a plateau stage, growth stage, and a saturation stage, which comprised four morphological phases: adherence to the cell membrane; formation of granulated clusters spread throughout the cytoplasm; localization of granulated clusters in the perinuclear region; and formation of multivesicular body-like structures and their redistribution in the cytoplasm. Diverse quantum dots containing intracellular vesicles in the range of approximately 0.5-8 µm in diameter were observed in the cytoplasm, but none were found in the nucleus. Vesicles containing quantum dots formed multivesicular body-like structures in NIH3T3 cells after 24 hours of incubation, which were Lysotracker-negative in serum-free medium and Lysotracker-positive in complete medium. The microinjected quantum dots remained uniformly distributed in the cytosol for at least 24 hours. Conclusion: Natural uptake of quantum dots in cells occurs through three accumulation stages via a mechanism requiring energy. The sharp contrast of the intracellular distribution after microinjection of quantum dots in comparison with incubation as well as the limited transfer of quantum dots from vesicles into the cytosol and vice versa support the endocytotic origin of the natural uptake of quantum dots. Quantum dots with proteins adsorbed from the culture medium had a different fate in the final stage of accumulation from that of the protein-free quantum dots, implying different internalization pathways.
ACS Nano, 2013
Nanoporous polymer particles (NPPs) prepared by mesoporous silica templating show promise as a new class of versatile drug/gene delivery vehicles owning to their high payload capacity, functionality, and responsiveness. Understanding the cellular dynamics of such particles, including uptake, intracellular trafficking and distribution, is an important requirement for their development as therapeutic carriers. Herein, we examine the spatiotemporal map of the cellular processing of submicron-sized disulfide-bonded poly(methacrylic acid) (PMA SH) NPPs in HeLa cells using both flow cytometry and fluorescence microscopy. The data show that the PMA SH NPPs are transported from the early endosomes to the lysosomes within a few minutes. Upon cell division, the lysosome-enclosed PMA SH NPPs are distributed asymmetrically between two daughter cells. Statistical analysis of cells during cytokinesis suggests that partitioning of particles is biased with an average segregation deviation of 60%. Further, two-dimensional difference gel electrophoresis (2D-DIGE) analysis reveals that 127 out of 3059 identified spots are differentially regulated upon exposure to the PMA SH NPPs. Pathway analysis of the proteomics data suggests that ubiquitylation, a reversible modification of cellular proteins with ubiquitin, plays a central role in overall cellular responses to the particles. These results provide important insights into the cellular dynamics and heterogeneity of NPPs, as well as the mechanisms that regulate the motility of these particles within cells, all which have important implications for drug susceptibility characteristics in cancer cells using particle-based carriers.