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Particle tracking of intracellular trafficking of octaarginine-modified liposomes: a comparative study with adenovirus.

ABSTRACT: It is previously reported that octaarginine (R8)-modified liposome (R8-Lip) was taken up via macropinocytosis, and subsequently delivered to the nuclear periphery. In the present study, we investigated the mechanism for the cytoplasmic transport of R8-Lips, comparing with that for adenovirus. Treatment with microtubule-disruption reagent (nocodazole) inhibited the transfection activity of plasmid DNA (pDNA)-encapsulating R8-Lip more extensively than that of adenovirus. The directional transport of R8-Lips along green fluorescent protein (GFP)-tagged microtubules was observed; however, the velocity was slower than those for adenovirus or endosomes that were devoid of R8-Lips. These directional motions were abrogated in R8-Lips by nocodazole treatment, whereas adenovirus continued to undergo random motion. This finding suggests that the nuclear access of R8-Lip predominantly involves microtubule-dependent transport, whereas an apparent diffusive motion is also operative in nuclear access of adenovirus. Furthermore, quantum dot-labeled pDNA underwent directional motion concomitantly with rhodamine-labeled lipid envelopes, indicating that the R8-Lips were subject to microtubule-dependent transport in the intact form. Dual particle tracking of carriers and endosomes revealed that R8-Lip was directionally transported, associated with endosomes, whereas this occurs after endosomal escape in adenovirus. Collectively, the findings reported herein indicate that vesicular transport is a key factor in the cytoplasmic transport of R8-Lips.

SUBMITTER: Akita H

PROVIDER: S-EPMC2890115 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Particle tracking of intracellular trafficking of octaarginine-modified liposomes: a comparative study with adenovirus.

Akita Hidetaka H Enoto Kaoru K Masuda Tomoya T Mizuguchi Hiroyuki H Tani Tomomi T Harashima Hideyoshi H

Molecular therapy : the journal of the American Society of Gene Therapy 20100309 5

It is previously reported that octaarginine (R8)-modified liposome (R8-Lip) was taken up via macropinocytosis, and subsequently delivered to the nuclear periphery. In the present study, we investigated the mechanism for the cytoplasmic transport of R8-Lips, comparing with that for adenovirus. Treatment with microtubule-disruption reagent (nocodazole) inhibited the transfection activity of plasmid DNA (pDNA)-encapsulating R8-Lip more extensively than that of adenovirus. The directional transport ...[more]

PMID: 20216528

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Project description:Label-free imaging techniques such as differential interference contrast (DIC) allow the observation of cells and large subcellular structures in their native, unperturbed states with minimal exposure to light. The development of robust computational image-analysis routines is vital to quantitative label-free imaging. The reliability of quantitative analysis of time-series microscopy data based on single-particle tracking relies on accurately detecting objects as distinct from the background, i.e., segmentation. Typical approaches to segmenting DIC images either involve converting images to those resembling phase contrast, mimicking the optics of DIC object formation, or using the morphological properties of objects. Here, we describe MATLAB based, single-particle tracking tool with a GUI for mobility analysis of objects from in vitro and in vivo DIC time-series microscopy. The tool integrates contrast enhancement with multiple modified Gaussian filters, automated threshold detection for segmentation and minimal distance-based two-dimensional single-particle tracking. We compare the relative performance of multiple filters and demonstrate the utility of the tool for DIC object tracking (DICOT). We quantify subcellular dynamics of a time series of Caenorhabditis elegans embryos in the one-celled stage by detecting birefringent yolk granules in the cytoplasm with high precision. The resulting two-dimensional map of oscillatory dynamics of granules quantifies the cytoplasmic flows driven by anaphasic spindle oscillations. The frequency of oscillations across the anterior-posterior (A-P) and transverse axes of the embryo correspond well with the reported frequency of spindle oscillations. We validate the quantitative accuracy of our method by tracking the in vitro diffusive mobility of micron-sized beads in glycerol solutions. Estimates of the diffusion coefficients of the granules are used to measure the viscosity of a dilution series of glycerol. Thus, our computational method is likely to be useful for both intracellular mobility and in vitro microrheology.

Project description:Following receptor binding and internalization, intracellular trafficking of adenovirus (Ad) among subgroups B and C is different, with significant amounts of Ad serotype 7 (Ad7) (subgroup B) virions found in cytoplasm during the initial hours of infection while Ad5 (subgroup C) virions rapidly translocate to the nucleus. To evaluate the role of the fiber in these differences, we examined intracellular trafficking of Ad5, Ad7, and Ad5f7 (a chimeric vector composed of the Ad5 capsid with the fiber replaced by the Ad7 fiber) by conjugating Ad capsids directly with Cy3 fluorescent dye, permitting the trafficking of the capsids to be examined by fluorescence microscopy. The human lung carcinoma cell line A549 was infected with Cy3-conjugated viruses for 10 min followed by a 1-h incubation. Ad5 virions rapidly translocated to the nucleus (within 1 h of infection), while Ad7 virions were widely distributed in the cytoplasm at the same time point. Interestingly, chimeric Ad5f7 virions behaved similarly to Ad7 but not Ad5. In this regard, the percentages of nuclear localization of Ad5, Ad7, and Ad5f7 at 1 h following infection were 72% +/- 4%, 32% +/- 6%, and 38% +/- 2%, respectively. Consistent with these observations, fluorescence in situ hybridization demonstrated that most of the Ad5 DNA was detected at the nucleus after 1 h, but at the same time point, DNA of Ad7 and Ad5f7 was distributed in both the nucleus and cytoplasm. Quantification of the kinetics of Ad genomic DNA delivery to the nucleus using a fluorogenic probe-based PCR assay (TaqMan PCR) demonstrated that the percentages of nuclear association of Ad5 DNA and Ad5f7 DNA at 1 h postinfection were 80% +/- 13% and 43% +/- 1%, respectively. Although it has been generally accepted that Ad fiber protein mediates attachment of virions to cells and that fibers dissociate during endocytic uptake, these data suggest that in addition to mediating binding to the cell surface, fiber likely modulates intracellular trafficking as well.

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Particle tracking of intracellular trafficking of octaarginine-modified liposomes: a comparative study with adenovirus.

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Particle tracking of intracellular trafficking of octaarginine-modified liposomes: a comparative study with adenovirus.

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