Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology

Podsumowanie

This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging.

Streszczenie

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The cellular mechano-sensation of extracellular mechanical stimuli is believed to be through the membrane receptors, the associated protein complex, and the cytoskeleton. Recent advances in mechanobiology demonstrate that the cell nucleus in cytoplasm itself can independently sense mechanical stimuli simultaneously. However, a mechanistic understanding of how the cell nucleus senses, transduces, and responds to mechanical stimuli is lacking, mainly because of the technical challenges in accessing and quantifying the nucleus mechanics by conventional tools. This paper describes the design, fabrication, and implementation of a new magnetic force actuator that applies precise and non-invasive 3D mechanical stimuli to directly deform the cell nucleus. Using CRISPR/Cas9-engineered cells, this study demonstrates that this tool, combined with high-resolution confocal fluorescent imaging, enables the revelation of the real-time dynamics of a mechano-sensitive yes-associated protein (YAP) in single cells as a function of nucleus deformation. This simple method has the potential to bridge the current technology gap in the mechanobiology community and provide answers to the knowledge gap that exists in the relation between nucleus mechanotransduction and cell function.

Wprowadzenie

This study aims to develop and apply a new technique to elucidate nucleus mechanobiology by combining the magnetic actuators that apply mechanical force directly on the cell nucleus and the confocal fluorescence microscopy that simultaneously images the structural and functional subcellular changes. Cells sense extracellular biophysical signals including tissue stiffness^1,2,3,4, interstitial fluid pressure and shear stress^5,6,7, surface topology/geo....

Protokół

1. Maintenance of CRISPR/Cas9-engineered B2B cells

1. Culture B2B cells in a T25 flask with RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.
2. Maintain the B2B cells in a humidified incubator at 37 °C with 5% CO₂.
3. Subculture the B2B cells when the confluency reaches 70% to 80%.
4. Store the B2B cell line in RPMI-1640 culture medium with 10% (v/v) DMSO in a -80 °C freezer.
5. Use the B2B cells with a passage number less than 10 in the experiments.

2. Cell culture

1. Seed the cells onto a glass-bott....

Wyniki

Design of a magnet-moving device and application of magnetic force
To apply force on the nucleus through the magnetic microbeads, a magnet-moving device was designed and built to control the spatial position of the magnet. The magnet-moving device contains a central frame, three knobs, and rails to move the attached magnet in x, y, and z directions independently at the spatial resolution of 1.59 mm per cycle (Figure 1A). Once the magnet is moved close to the 7 µm .......

Dyskusje

Internalization of magnetic microbeads (section 2.2) is critical because extracellular microbeads cannot apply force directly to the nucleus. Force application and imaging (section 5.3) are critical steps in this experiment, and the force needed to deform the nucleus and induce meaningful biological consequences might be sample-dependent. The force magnitude in this experiment (0.8 nN and 1.4 nN) can be further increased to trigger nuclear mechano-sensing in less sensitive cells.

To .......

Materiały

Name	Company	Catalog Number	Comments
0.05 % Trypsin	Corning	25-051-CI
25 cm2 flask	Corning	156340
7-µm mean diameter carbonyl iron microbeads	N/A	N/A
A1R confocal system	Nikon
Carbonyl Iron Powder CM	BASF	30042253	Magnetic microbead
Culture medium (RPMI-1640)	Gibco	11875093
Desktop Computer	Dell		with Windows 10 operating system
Environmental chamber TIZB	Tokai Hit	TIZB
Fetal bovine serum (FBS)	Gibco	26140
Fiji ImageJ	National Institutes of Health and the Laboratory for Optical and Computational Instrumentation
Glass-bottom petri dish	MatTek	P35G-1.5-14-C
Magnet	K&J Magnetics, Inc.	D99-N52
Monochrome Camera	FLIR	BFS-U3-70S7M-C
NIS-Elements software platform	Nikon		software platform
Nucleus mask ImageJ macro			https://github.com/KOLIUG/Nuclear mask
NucSpot Live 650	Biotium	#40082	Nuclear stain
Penicillin-streptomycin	Gibco	15140122
Phosphate buffered saline (PBS)	Gibco	10010023
Ti2-E inverted microscope	Nikon
XYZ mover (CAD files)			https://github.com/KOLIUG/XYZ-mover