Non-invasive Deactivation and Spatial Control of Intracellular Chromosome with Magnetic Nanoparticle

Abstract

The regulation of cell signaling pathway is important for understanding biological systems. Thus, various techniques to modulate cell signaling by spatial control of cellular components have been developed. Magnetic nanoparticles are emerging as promising candidates in a broad range from cell biology to biomedical application due to their unique characteristics such as remote control and their ability to interact with various cellular and molecular level of interest through their surface decoration. With the advent of advanced nanoscience and nanotechnology in the reliable production and specific tailoring of functional magnetic particles, successful applications were dedicated to a broad range of biomedical applications such as high throughput separation of biomolecules, magnetic resonance image, hyperthermia for cancer therapy, and drug delivery. Recently, fluorescent magnetic nanoparticles inside a living cell were shown to be directly attracted by an external magnetic field. In addition, the signaling transduction pathway was remotely triggered by the functionalized magnetic nanoparticles and the microtubule nucleation and assembly inside Xenopus oocyte was spatiotemporally controlled by magnetic nanoparticles conjugated with key regulatory proteins. Moreover, it is shown that the remote induction of cell death by targeting intracellular lysosome was triggered by functionalized magnetic nanoparticles. In this study, we demonstrate a novel strategy to non-invasively regulate chromosome activity by targeting genetic regulation materials (i.e., oocyte-specific linker histone H1 protein) in live oocyte with the functionalized magnetic nanoparticles and to spatially control the targeted chromosomes by a remote magnetic field. To this end, bacterial magnetic nanoparticles (BMPs, ~50 nm) produced by magnetotactic bacteria (Magnetosome) were used because of their peculiar features, such as high magnetism, high dispersal ability in an aqueous media and good biocompatibility. In addition, BMPs can effectively be conjugated to the diverse biomolecules due to the abundance of amine groups on particles surrounded by lipid membrane. First, we present a novel method to regulate the chromosome activity in mature mouse (Metaphase II) oocytes by targeting the histone H1 protein in chromosome with functionalized magnetic nanoparticles. BMPs were conjugated with histone H1 antibodies (H1-BMPs), specifically targeting the oocyte-specific histone H1 protein. The dose-dependent cytotoxicity test for H1-BMPs was initially conducted before starting experiments. Our data clearly show that the oocytes are able to be tolerated up to the delivery of 1 μg/ml H1-BMPs without any developmental retardation and this concentration is optimal for regulation of a chromosome activities. In the first stage, to verify this approach, we examined whether H1-BMPs have the specific affinity against the chromosomal histone H1 protein in live oocyte. The capability of H1-BMPs for specific targeting was easily detected by confocal microscopy. In addition, we investigated how oocyte developmental process was affected by the magnetic labeling of chromosomes. The parthenogenesis was induced to activate the cell cycle progression. When chromosomes were targeted by H1-BMPs, oocytes were developed up to the 4-cell stage, but began to degrade afterwards and failed to reach the blastocyst stage regardless of the application of magnetic field. Interestingly, in mouse oocyte, it is well known that transcriptional repression in parthenogenesis is proceeded until four cell stage and newly synthesized ribonucleic acid (RNA) begins to be transcriptionally expressed at late four cell stage. Thus, we believed that an abrupt fall in oocyte development after four cell stage is caused by the perturbation of transcriptional activity. Second, we demonstrate the spatial control of targeted chromosomes with H1-BMPs and remaining unbound H1-BMP inside living oocyte via a remote magnetic field. In these experiments, immature oocytes at germinal vesicle breakdown (GVBD) stage were selected for subsequent experiments. For cell cycle arrest which can prevent the spontaneous chromosome movement during experiments, the nocodazole treatment was carried out to prevent. As a first step towards the spatial control of chromosomes, the applied magnetics force was analyzed. To generate strong magnetic fields, the several neodymium magnets in various experimental condition were used. It is calculated that a large force of 780 fN was exerted on single BMP by a strong magnetic field. Based on the specific targeting test, we attempted to rotate cell using a remote magnetic field. The orientation of cell is dictated and arranged by the direction of a magnetic field. Next, we attempted to move the chromosomes located around the center of an oocyte by an external magnetic field. Without the antibodies, the BMPs did not conjugate with the chromosomes and the chromosomes did not move together with the BMPs in the presence of the magnetic field. The green spots from the FITC-conjugated BMPs moved in the direction of the magnetic field, whereas the blue spots from the DAPI-stained chromosomes remained close to the center. On the other hand, with antibody, the blue spots (chromosomes) coincided with the green spots (H1-BMPs) that noticeably moved to the top. For a more detailed analysis, chromosomes movement was quantitatively assessed by using image process. The mass center of the blue spots was calculated and used as a value that represents the entire distance moved. We compare the distances with and without. A substantial difference was observed between the cases with (20.59 ± 2.47 μm) and without (9.18 ± 4.23 μm) antibodies. To gain further evidence for chromosome movement by a remote magnetic field, we observed the time course motion of the chromosomes in live oocyte. To observe lateral movement, we prevented the rotation and drift of the living oocyte by anchoring it to the substrate. To achieve this goal, we developed a vacuum-assisted microfluidic device for trapping the oocyte on a transparent substrate and tested it. The oocytes were Hoechst-stainied before immobilized on the trapping hole. When the negative pressure was applied to the microfluidic device, oocytes were rapidly attracted to and fixed on the holes. A magnetic field was applied from the side of the trapped oocyte. The sequence of captured images shows the lateral motion of the chromosomes toward the applied magnetic field. The velocity of chromosome movement by a remote magnetic force was calculated as ~70 nm/min. Our data shows that the movement of the chromosomes linked to the H1-BMPs can be severely hindered by the meshwork of actin filament bundles. Recently, although human stem cell was generated by somatic cell nuclear transfer (SCNT), the enucleation process is still important factor in SCNT. It is well known that the invasive enucleation (removal of human oocyte genome) leads to the failure of embryonic development after genome exchange due to the loss of meiosis-specific factors associated with the spindle removal during physical enucleation process. Thus, we suggest the non-invasive method for SCNT without enucleation, by targeting chromosome with H1-BMPs. To achieve it, two possibilities have to be pre-verified. First, there are no interaction between chromosomes in somatic cell and unbounded H1-BMPs in oocyte after somatic cell – oocyte fusion. In previous work, we showed the possibility to control the both H1-BMPs and chromosome attached to H1-BMPs by using external magnetic field. Through previous result, the position of unbounded H1-BMPs can be controlled before somatic cell chromosomes are fused with oocyte chromosomes. Second, to deactivate chromosome activity, specific time (10 hrs) is necessary to target chromosome with H1-BMPs. Thus, it is required to investigate whether oocyte move to blastocyst stage after delay in activation for 10 hrs. Thus, it is investigated whether oocyte move to blastocyst stage after delay of activation for 10 hrs. It is shown that oocytes are well developed and reached the blastocyst stage with ~ 20 % despite of delay of activation time. Second there are no interaction between chromosomes in somatic cell and unbounded H1-BMPs in oocyte after somatic cell – oocyte fusion. These results show the possibility to apply our method to SCNT process. In the study described above, we demonstrate the possibility to non-invasively regulate a chromosome activity by the magnetic labeling, leading to arrest of embryonic development in parthenogenesis due to abnormal transcriptional activity. Through result, our non-invasive technique can be a candidate of alternative methods in somatic cell nuclear transfer (SCNT) that suffers from cell damage during enucleation process. To validate the potential application, the long range displacement of chromosomes is verified in oocyte by external magnetic field. We showed the traction of whole chromosomes in an oocyte across the entire cell span using functionalized magnetic nanoparticles (H1-BMPs). Procedures and techniques were well developed. In addition, our technique will provide a useful tool for investigating cellular functions associated with the spatiotemporal distribution of such components.