Name: combining 3d magnetic force actuator and multi-functional fluorescence imaging to study nucleus mechanobiology
Uploaded: 2022-07-05T15:00:00
Description: Watch this Scientific Journal Video about Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology at JoVE.com

This project is funded by UF Gatorade Award Start-up Package (X. T.), the UFHCC Pilot Award (X. T. and Dr. Dietmar Siemann), UF Opportunity Seed Fund (X. T.), and UFHCC University Scholars Program (H. Y. Wang). We sincerely appreciate the intellectual discussions with and the technical support from Dr. Jonathan Licht (UFHCC), Dr. Rolf Renne (UFHCC), Dr. Christopher Vulpe (UFHCC), Dr. Blanka Sharma (BME), Dr. Mark Sheplak (MAE &#38; ECE), Dr. Daniel Ferris (BME), Dr. Malisa Sarntinoranont (MAE), Dr. Ashok Kumar (MAE), Dr. Benjamin Keselowsky (BME), Dr. Brent Gila (RSC), Dr. Philip Feng (ECE), Dr. Gregory A. Hudalla (BME), Dr. Steven Ghivizzani (OSSM), Dr. Yenisel Cruz-Almeida (CDBS), Dr. Roger Fillingim (CD-BS), Dr. Robert Caudle (OMS), Dr. John Neubert (DN-OR), Dr. Justin Hiliard (Neurosurgery), Dr. Tian He (Harvard University), Dr. Youhua Tan (Hong Kong Polytechnic University), Dr. Jessie L-S Au (Institute of Quantitative Systems Pharmacology), Dr. David Hahn (University of Arizona), and Support Team of Nikon (Drs. Jose Serrano-Velez, Larry Kordon, and Jon Ekman). We are deeply grateful for the effective support from all members of Tang&#39;s, Yamaguchi&#39;s, Sharma&#39;s, Au&#39;s, Siemann&#39;s, and Guan&#39;s research laboratories and all staff members of the UF MAE Department.

1. Maintenance of CRISPR/Cas9-engineered B2B cells
<ol>
	<li>Culture B2B cells in a T25 flask with RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.</li>
	<li>Maintain the B2B cells in a humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>Subculture the B2B cells when the confluency reaches 70% to 80%.</li>
	<li>Store the B2B cell line in RPMI-1640 culture medium with 10% (v/v) DMSO in a -80 &#176;C freezer.</li>
	<li>Use the B2B cells with a passage nu...

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)&#160;are&#160;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 ...

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 stiffness1,2,3,4, interstitial fluid pressure and shear stress5,6,7, surface topology/geometry8,9,10,11,12, and tension/compression stress13,14,15,16. Biophysical signals are converted into biochemical signals and trigger potential downstream changes of gene expression and cell behaviors-a process known as mechanotransduction17,18,19,20,21,22,23,24,25,26,27. To study mechanotransduction processes, a myriad of techniques have been developed to apply mechanical force on the cells, such as atomic force microscopy28, cell stretching device29, bio-MEMS (micro-electromechanical systems) force sensor15,30,31, shear rheology32, and Stereo Vision System33. A recent review summarizes the approaches to apply extracellular mechanical cues and interfere with mechanosensing34. To date, most of these methods apply force on the cell plasma membrane, and cells directly receive these extracellular biophysical signals via membrane receptors such as integrin, cadherin, ion channels, and G-Protein-coupled receptors. Subsequently, they transmit the signal to the intracellular cytoskeleton and nucleus. For example, using yes-associated protein (YAP) translocation as an indicator of mechano-sensing, cells are shown to sense the mechanical signals of substrate stiffness and extracellular tension from the cell membrane and transmit them through the cytoskeleton into the nucleus to induce YAP cytoplasm-to-nucleus translocation28,35.
Recent evidence suggests that the cell nucleus itself is an independent mechano-sensor8,36,37. This is proven by experiments performed on the isolated nucleus harvested from cells, where it was revealed that nuclei adaptively change their stiffness in response to the mechanical force directly applied on them36. During many physiological conditions, nuclei in both tumor and healthy cells sense extracellular biophysical signals and change their mechanical properties and assemblies38,39,40. For example, upon extravasation, the nuclear stiffness of tumor cells decreases and maintains softness for over 24 h38. During migration through confined interstitial space, the nuclei of tumor cells frequently lose and recover their structural integrity39. However, the way in which the nucleus senses the biophysical signal is unknown, although several nuclear-envelope proteins and families of proteins have been found to be involved, such as Lamin A/C and linker of nucleoskeleton and cytoskeleton (LINC) complex38,41. Hence, new non-invasive methods that can directly apply force to the nucleus will decouple the effect of force transmission from the cell-plasma membrane and cytoskeleton, and will help elucidate the previously inaccessible molecular mechanisms of nuclear mechano-sensing.
Research that employed optical tweezers to manipulate organelles42 and microbeads injected into cells43 showed the technological capability of directly applying force on the nucleus. However, the optical-tweezer technique has several limitations: (1) low throughput-optical tweezers often only manipulate one cell or microbead at a time; and (2) potential photodamage and temperature artifact-deformation of nuclear requires tens of pN36, and the corresponding necessary laser power is about 10 mW per pN44,45. Such laser intensity is sufficient to trigger photodamage in the cells and perturb cell functions during the experiment46.
Magnetic force applied through microbeads within living cells shows the potential to directly apply force on the nucleus and overcomes the limitations of optical tweezers. Once microbeads are delivered into the cytoplasm, a magnetic field can exert a magnetic force on multiple microbeads simultaneously in a high-throughput manner. The magnetic field does not influence cell functions47,&#160;but generates force from pN to nN, which is enough to induce nuclear deformation36,48,49.&#160;To date, manipulation of magnetic microbeads has been applied on cell plasma membrane48, inside the cytoplasm50, on F-actin51, inside the nucleus47, and on the isolated nucleus36. However, magnetic manipulation of microbeads has never been used to apply direct mechanical force on the nuclear envelope to study mechanotransduction in the nucleus.
In this paper, a simple technique is developed to non-invasively deliver magnetic microbeads into the cytoplasm and use these microbeads to apply mechanical force on the nucleus (Figure 1). Here, CRISPR/Cas9-engineered human normal B2B cell lines that endogenously express mNeonGreen21-10/11-tagged YAP are used to validate the method. YAP is a mechano-sensitive protein, and the translocation of YAP is regulated by nuclear mechano-sensing14,28. The CRISPR/Cas9-regulated knock-in approach was chosen to tag endogenous YAP with a fluorescent protein (FP) mNeonGreen21-10/11. Although CRISPR editing is known to have incomplete efficiency and off-target effect, the protocols in previous publications integrated fluorescence sorting to select for correct open reading frame insertion52,53,54. With this additional layer of selection, no off-target tagging event was observed in 20+ cell lines previously generated52,53,54,55. This is a split fluorescent protein construct, but in principle, any expressible fluorescent tag could be usable. This labeling approach is superior to transgene or antibody methods. First, unlike the transgene expression, the tagged protein maintains single-copy gene dosage and expresses in the physiological context of the native gene regulatory network, limiting deviations in protein concentration, localization, and interaction. The tagging method used in this study achieves over an order-of-magnitude higher throughput and efficiency than full FP tagging. It also avoids challenges associated with immunofluorescence due to fixation artifacts and the limited availability of high-quality, high-specificity antibodies. Second, the approach used in this paper does minimum perturbation to the cell physiology and enables the real-time revelation of all endogenous YAPs authentically. In contrast, other common transgene methods often lead to overexpression of YAP. The resulting artificial distribution can potentially cause cytotoxicity and affect mechano-sensing of cells56,57,58.
This study presents a protocol to directly apply force on the nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging. In summary, the protocols presented here demonstrate how to (1) deliver magnetic microbeads into the cell while outside the nucleus, (2) manipulate the microbeads to apply magnetic force on the nucleus, (3) perform confocal fluorescent imaging of the cells during manipulation, and (4) quantitatively analyze the YAP nuclear/cytoplasm (N/C) ratio throughout the force application process. The results suggest that (1) through endocytosis, magnetic microbeads can be non-invasively delivered into the cytoplasm of B2B cells within 7 h (Figure 2 and Figure 3); and (2) quantified magnetic force directly applied on the nucleus (Figure 4, Figure 5, and Figure 6) alone can trigger diverse changes of YAP N/C ratio in CRISPR/Cas9-engineered B2B cells (Figure 7 and Figure 8).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05 % Trypsin</td>
			<td>Corning</td>
			<td>25-051-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 flask</td>
			<td>Corning</td>
			<td>156340</td>
			<td></td>
		</tr>
		<tr>
			<td>7-&#181;m mean diameter carbonyl iron microbeads</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>A1R confocal system</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonyl Iron Powder CM</td>
			<td>BASF</td>
			<td>30042253</td>
			<td>Magnetic microbead</td>
		</tr>
		<tr>
			<td>Culture medium (RPMI-1640)</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop Computer</td>
			<td>Dell</td>
			<td></td>
			<td>with Windows 10 operating system</td>
		</tr>
		<tr>
			<td>Environmental chamber TIZB</td>
			<td>Tokai Hit</td>
			<td>TIZB</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji ImageJ</td>
			<td>National Institutes of Health and the Laboratory for Optical and Computational Instrumentation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-bottom petri dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnet</td>
			<td>K&#38;J Magnetics, Inc.</td>
			<td>D99-N52</td>
			<td></td>
		</tr>
		<tr>
			<td>Monochrome&#160;Camera</td>
			<td>FLIR</td>
			<td>BFS-U3-70S7M-C</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements software platform</td>
			<td>Nikon</td>
			<td></td>
			<td>software platform</td>
		</tr>
		<tr>
			<td>Nucleus mask ImageJ macro</td>
			<td></td>
			<td></td>
			<td>https://github.com/KOLIUG/Nuclear mask</td>
		</tr>
		<tr>
			<td>NucSpot Live 650</td>
			<td>Biotium</td>
			<td>#40082</td>
			<td>Nuclear stain</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti2-E inverted microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>XYZ mover (CAD files)</td>
			<td></td>
			<td></td>
			<td>https://github.com/KOLIUG/XYZ-mover</td>
		</tr>
	</tbody>
</table>

combining 3d magnetic force actuator and multi-functional fluorescence imaging to study nucleus mechanobiology

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.

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 &#181;m ...

Watch this Scientific Journal Video about Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology at JoVE.com

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

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.

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The ...

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