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 ...

In this method, the force is directly applied to the nucleus. This decouples the force transmission effect from the cell plasma membrane and cytoskeleton, revealing the molecular mechanisms of nuclear mechanosensing. The force is applied within the living cells in a non-invasive way.Compared to optical tweezers, the magnetic field and magnetic force do not affect the cell functions and have higher throughput. Begin culturing the cells with magnetic microbeads by weighing 0.2 grams of carbonyl iron microbeads having seven micrometer of mean diameter. Suspend the microbeads in one milliliter RPMI 1640 culture medium using a pipette.Then take the Petri dish with B2B cells to the biosafety cabinet and quickly add 200 microliters of the medium containing microbeads into the Petri dish. Put the Petri dish in an incubator until microbeads are internalized by the cells. Open the inverted microscope and using confocal fluorescence imaging, determine the optimal time for the internalization of the cell lines every six hours by visualizing the microbead, nuclear, and cell boundary.The internalized microbeads will be present within the cell boundary. For small force application and live-cell imaging, open the software application Elements. To define the configuration magnetic find, click the one over two button to set the scanning speed to one frame per two seconds.Set pinhole size to 1.2 Airy unit. Check only the FITC channel and set PMT HV as 70, offset as zero, laser intensity as 10. To define the configuration magnetic YAP nucleus, set the scanning speed to one frame per four seconds by clicking the one over four button.Then click the 1.2 Airy unit button to set the pinhole size to 1.2 Airy unit and check the FITC channel. Set PMT HV as 70, offset as zero, laser intensity as 10. For imaging the nucleus boundary and nuclear stain intensity, check the cyanine 5 channel.Do not click the 1.2 Airy unit button and set PMT HV to 70, offset to zero and laser intensity to 10. The pinhole size will be optimized for three-dimensional YES-associated protein imaging. Next, turn on the DIA through Elements.Open spin view, use a brightfield and adjust the focus of the object to get a clear in-focus image of cells. Use a 10X objective to find appropriate multiple single cells in three conditions, such as a cell with a single microbead inside, with multiple microbeads inside, and without any microbead inside. Then switch to 40X objective and name this position with the appropriate position number.Open Elements, click on magnetic find, remove interlock, then scan tabs, then select the top and bottom buttons to adjust the Z position of the focal plane for setting the lower and upper limit for the Z stack of the selected cells. Stop scanning by clicking scan again. Switch to magnetic YAP nucleus configuration and set the filename as before_small_force.nd2.Click on the run button with the recorded Z stack. Switch to the right light path and turn on DIA. Open spin view and click on the recording button.Move the magnet to 46 millimeters above the Petri dish bottom by spinning the knob of the magnet moving device. Save and check the video to confirm the microbeads displacement induced by magnetic force. Repeat the scanning procedure by setting the filename as after_small_force.nd2.Next, switch to the right light path, turn on DIA and open spin view before clicking on the recording button. Spin the magnet moving device knob up to 120 millimeters above the Petri dish bottom and save a brightfield image sequence or video. Repeat the steps for scanning by setting the filename to before_large_force.nd2.The microbeads cannot emit fluorescence under laser excitation in the FITC or cyanine 5 channel. Thus, the internalized microbeads were identified by the dark hollow located in the confocal imaging of fluorescence of YES-associated protein and nucleus. Nuclear circularity showed no significant difference between control cells and cells with microbead internalization.The YES-associated protein cell nucleus-to-cytoplasm ratio of control cells co-cultured with microbead but without internalization and cells with microbead internalization also showed no significant difference. The nucleus deformation and de-polymerization of actin were caused by the compression force applied by the microbeads in cytoskeleton-containing cells. The quantitative imaging analysis calibration curve provided by the quantitative AFM force displacement relationship.Based on this relationship, the beads applied force was estimated. The nucleus deformation and YES-associated protein translocation were observed on application or the release of the magnetic force. The intensity changes in YES-associated protein in the green channel and no change in the nucleus staining in the red channel confirmed that the magnetic force-induced nuclear deformation triggered by the YES-associated protein translocation.The quantification of the net change of the YES-associated protein nucleus-to-cytoplasm ratio within two groups confirmed that the magnetic force applied to the microbeads within the cytoplasm induced the YES-associated protein translocation and changed the YES-associated protein nucleus-to-cytoplasm ratio. Before applying force and imaging, ensure that the cells have internalized the magnetic microbeads and the beads are within the cell boundary.

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Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...