Images
U2OS cells expressing a Red Fluorescent Protein (RFP) fused to Nuclear-Localization sequence (NLS) have RFP-NLS always localized in cell nuclei. Local heating of cells to 50ºC for a few seconds via millimeter wave radiation (area is encircled) lead to immediate and irreversible leakage of RFP-NLS from nucleus to the cell cytoplasm (Fig. 6, left).
Borderline region ("death-life" borderline) imaged with a 20x objective (Fig. 7) Cells in the upper part of the image were heated to 50ºC for 3 seconds. The nuclear pores of the heated cells lost their selectivity as the RFP-NLS protein leaked out of the nuclei. However, the nuclear envelopes of the heated cells were not compromised as DNA labeled with a DNA stain Hoechst still resides inside the nuclei. The heated cells became round and started to detach from the culture dish bottom. The "death-life" borderline is very narrow: it is practically in the cell size range (Fig. 7, low panel).
Application of millimeter wave radiation to two closely adjacent regions (Fig. 8). The cells in regions 702, 704 are completely eliminated via millimeter wave radiation irradiation. These two regions 702, 704 are in a very close proximity to each other (~250 µm). However, the cells between the regions 702, 704 survived the treatment and continue growing.Left image is acquired using bright field contrast, right image depicts GFP fluorescence as the cells are expressing GFP-tubulin.
In Movie 1 cells expressing GFP-tubulin were exposed to millimeter wave radiation. Normally GFP-tubulin never enter nuclear interior and reside in the cytoplasm. The absence of NLS sequence and high molecular weight of GFP-tubulin of ~ 127 kDa do not allow this protein to pass through the nuclear pore complex by passive diffusion. Heating to 50ºC via millimeter wave radiation destroys nuclear pore barrier properties and lets GFP-tubulin penetrate inside the nucleus (Movies 1; Fig. 9).
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Movies 1
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The graphs in Figure 9 show the fluorescence intensity of the regions of interest (dotted red and blue circles inside the nuclei of two cells) as a function of time. The red on the graph shows the average intensity in red circle and blue curve on the graph shows the average intensity in blue circle. The sharp drop in the GFP fluorescence after application of 94 GHz MMW (arrow at 3 seconds) is attributed to a drop in the GFP quantum yield when the temperature rises. The beginning of GFP-tubulin entry into the nucleus indicates damage in the barrier function of the nuclear envelops.
To remove or "erase" unwanted cells around a region of interest containing desired cells the millimeter wave field should be applied around that region of interest. Fig. 2-3 shows an example where the desired cells (2 cell with green fluorescence) need to be isolated from surrounding wild type cells.
For convenience the cells in Fig. 2-3 were grown in plasticware with a grid (Fig. 3) - Ibidi µ-Dish 35 mm, high Grid-500 Glass Bottom with 500 mm grid repeat distance. The image in Fig. 4 shows the location of the desired cells (square U18) at 4x magnification and the aftermath of the millimeter wave application (W-band) around the square U18 (where 1 cells of interest). The heated cells outside the square U18 became round and started to detach from the well bottom. Wash out with fresh cell medium removes the detached cells (Fig. 5). The millimeter wave application can be repeated to remove more unwanted cells from the square U18.
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Fig. 8.
Fig.5 shows cell patch containing 2 desired cells surrounding by a cell-free area: upper left: 2 hours after heating (4x); upper right: 18 hours later (4x). Bottom images are taken at 10x magnification at the same time interval as upper ones: desired cells are divided at least one time within isolated area. CellEraser can be used further for complete "erasing" of all unwanted cells leaving only the cells of interest to grow in the well. Thus, CellEraser allows isolating practically one desired cell in millions of surrounding unwanted cells.
Fig. 9
Movie 1 Cells with leaky nuclei can't proliferate/divide but they are still alive enough long time (some >24 h). They become rounded, detach from the substrate and start their "last dance" before death via apoptosis. Most likely an apoptosis happens via cGAS–STING signal activation. DNA sensor cGAS is normally located only in cell cytoplasm and never enter cell nucleus. When NPC is permanently opened cGAS enters the nucleus and can be activated by interaction with endogenous DNA. The cGAS-STING pathway activates the transcription factor TBK1-IRF3 and NF-κB pathways ultimately leading to cell apoptosis. After cell detached from substrate (1-1.5h) simple medium change will wash them out and form cell-free area in which surrounding cells start migration (see Movie 2). If cells are not washed they will never attach to a substrate and will be displaced by cells migrating inside of the area.
Movie 2. Wound-healing assay performed via CellEraser™. 16 hours time-lapse of cell migration inside of cell-free circle (red) produced by CellEraser™.
The wound-healing assay is simple, inexpensive, and one of the earliest developed methods to study directional cell migration in vitro. This method mimics cell migration during wound healing in vivo. The basic steps involve creating a "wound" (a hole) in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the wound, and comparing the images to quantify the migration rate of the cells. It is particularly suitable for studies of the effects of cell-matrix and cell-cell interactions on cell migration. Usually the wound is created by mechanical scratch of cell monolayer, via application of biocompatible hydrogel to bottom that creates a circular area across which cells may migrate following gel removal or via placement of special silicone inserts to the cell culture surface before cell seeding (when the insert is removed it creates well defined empty regions with no cells). CellEraser™ could be used to make precise circular holes in cell monolayer without creating any mechanical or chemical disturbance of the surrounding cells. The high temperature (50°C) will induce cell detachment from culture well bottom so the cells that surround the empty circle will immediately start to migrate inside of cell-free area (see Movie 2).
