The authors thank Shu Hashimoto, Aoi Yoshida, and Taeko Ohta at Doshisha University for their generous assistance with the experimental setup. This work was supported by KAKENHI (15H02121, 15K05400, 25103012, 50587441) and by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities. This study was also supported by a Polish grant from the KNOW (Leading National Research Centre) Scientific Consortium &#34;Healthy Animal - Safe Food&#34;, decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015..

1. Preparation of Cells
<ol>
	<li>Maintain NAMRU mouse mammary gland epithelial cells (NMuMG cells) with 5 mL of D-MEM containing 10% (v) fetal bovine serum (FBS) and 1% (v) Penicillin-Streptomycin (P/S) in a 25 cm3 flask. Remove Dulbecco&#39;s modified Eagle&#39;s medium (D-MEM), containing 10% FBS and 1% P/S.</li>
	<li>Add 3-5 mL of 37 &#176;C phosphate-buffered saline (PBS) (-) to the flask. Note that the pH of PBS is 7.1-7.3.</li>
	<li>Remove all the PBS from the flask using an aspirator.</li>
	<li>Add 1.5 mL of 37 &#176;C trypsin (0.25%, w/v) to the flask.</li>
	<li>Incubate the flask for approximately 1-2&#160;minutes at 37 &#176;C in a CO2 incubator.</li>
	<li>Add 3.5 mL of D-MEM containing 10% FBS and 1% P/S to the flask. Pipette to mix.</li>
	<li>Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge it for 3 min at room temperature. Note that the rotation radius and rotation speed of the centrifuge are 16.6 cm and 1500 rpm, respectively. Under this condition, the relative centrifugal force is 417 x g.</li>
	<li>Add 5 mL of fresh D-MEM (10% FBS and 1% P/S) to the flask after aspirating the medium (if required, cryopreserve the cells with cryopreservation solution, following the manufacturer&#39;s directions).</li>
</ol>
2. Preparation of Dextran (DEX)
<ol>
	<li>Prepare 80 mg/mL of DEX solution by mixing 10 mL of D-MEM (10% FBS, 1%P/S) and 0.8 g of DEX. Note that the DEX solution can be filtered with a syringe filter (0.22 &#181;m) after the cells have cultured for a sufficient time.</li>
	<li>Prepare a cell suspension containing 40 mg/mL DEX medium by mixing 200 &#181;L of DEX solution and 200 &#181;L of the cell suspension prepared in 2.1. Note that the number density of cells in the solution is approximately 2.3&#215;105 cells/mL.</li>
</ol>
3. Preparation for Laser and Microscopy
<ol>
	<li>Turn on the laser (continuous wave, 1,064 nm wavelength) (Figure 1a). Note that the use of a laser beam with a wavelength in the red to near-infrared region is most effective; this wavelength region is called the diagnostic and therapeutic window10 since it is minimally absorbed by cells.</li>
	<li>Double-click the software icon.</li>
	<li>Double-click the icons for the &#9312; camera, &#9313; light-emitting diode (LED), &#9314; focus adjust, and &#9315; moving stage. The displays corresponding to &#9312;-&#9315; will show up (Figure 1b).</li>
</ol>
4. Cell Manipulation using the Laser Trapping System
<ol>
	<li>Place 20 &#181;L of the sample prepared in step 2.2 on the bottom cover glass (0.17 mm thickness, size = 30 mm &#215; 40 mm), and cover it with the top cover glass (0.17 mm thickness, size = 18 mm &#215; 18 mm), with separation provided by two spacers (0.17 mm thickness, size = 10 mm &#215; 24 mm), as shown in Figure 2. Note that these glasses do not require a special coating.</li>
	<li>Place the sample cell prepared in step 4.1 on the lower objective Lens (water immersion with ca. 10 &#956;l, magnification = 60X, working distance = 0.28 mm, numerical aperture = 1.2)).</li>
	<li>Attach the upper objective lens (water immersion with ca. 10 &#956;l, magnification = 60X, working distance = 2 mm, numerical aperture = 1.0) at the top of the sample cell.</li>
	<li>Turn on the LED light by clicking icon 2 (Figure 1b).</li>
	<li>Adjust the distance between the sample and the lower objective lens by clicking the icons on panel 3 (Figure 1b) until it is in focus.</li>
	<li>Irradiate the laser beams at Position 1 and Position 2 (Figure 1B) of the sample by clicking icons I, II, and III (Figure 1b).</li>
	<li>Set the intensity of each laser beam to 1,500 mW by entering this value at icon IV (Figure 1b).</li>
	<li>Move the sample stage by clicking icon 4 indicating directions (Figure 1b) until a cell is trapped at Position 1 (Figure 1b).</li>
	<li>Drag the cursor indicating Position 2 (Figure 1b) until another cell is trapped at Position 2.</li>
</ol>
5. Construction of a 3-Dimensional (3D) Cell Structure
<ol>
	<li>Manipulate a single cell so that it is in contact with another cell. Maintain this condition for 300 s; i.e., each cell is exposed to a laser for 300 s.</li>
	<li>Record the X-Y axis shown in Figure 1b.</li>
	<li>Construct an arbitrary 2D cell assembly by trapping and transporting another cell to the cells.</li>
	<li>Construct a 3D cellular assembly by moving the stage up and down.</li>
	<li>Confirm that the assembly remains stable after the laser is switched off.</li>
</ol>

<ol>
	<li>Aloysious, N., Nair, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+survival+and+function+of+islet-like+clusters+differentiated+from+adipose+stem+cells+on+a+three-dimensional+natural+polymeric+scaffold:+an+in+vitro+study.">Enhanced survival and function of islet-like clusters differentiated from adipose stem cells on a three-dimensional natural polymeric scaffold: an in vitro study.</a> Tissue Engineering Part A. 20 (9-10), 1508-1522 (2014).</li><li>Fatehullah, A., Tan, S. H., Barker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+as+an+in+vitro+model+of+human+development+and+disease.">Organoids as an in vitro model of human development and disease.</a> Nature Cell Biology. 18 (3), 246-254 (2016).</li><li>Kirkham, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+assembly+of+complex+cellular+microenvironments+using+holographic+optical+tweezers.">Precision assembly of complex cellular microenvironments using holographic optical tweezers.</a> Scientific Reports. 5, 8577 (2015).</li><li>Liu, Y., Rayatpisheh, S., Chew, S. Y., Chan-Park, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+endothelial+cells+on+3D+cultured+smooth+muscle+cells+in+a+biomimetic+hydrogel.">Impact of endothelial cells on 3D cultured smooth muscle cells in a biomimetic hydrogel.</a> ACS Applied Materials &amp; Interfaces. 4 (3), 1378-1387 (2012).</li><li>Liu, Z. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dextran-based+hydrogel+formed+by+thiol-Michael+addition+reaction+for+3D+cell+encapsulation.">Dextran-based hydrogel formed by thiol-Michael addition reaction for 3D cell encapsulation.</a> Colloids and Surfaces B. 128, 140-148 (2015).</li><li>McKee, C., Perez-Cruet, M., Chavez, F., Chaudhry, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+three-dimensional+culture+system+for+long-term+expansion+of+embryonic+stem+cells.">Simplified three-dimensional culture system for long-term expansion of embryonic stem cells.</a> World Journal of Stem Cells. 7 (7), 1064-1077 (2015).</li><li>Sadovska, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+heterotypic+spheroid+model+for+studying+extracellular+vesicle-mediated+tumor+and+immune+cell+communication.">A novel 3D heterotypic spheroid model for studying extracellular vesicle-mediated tumor and immune cell communication.</a> Biochemical and Biophysical Research Communications. , (2017).</li><li>Srinivasan, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Salivary+Human+Stem/Progenitor+Cells+Undergo+Microenvironment-Driven+Acinar-Like+Differentiation+in+Hyaluronate+Hydrogel+Culture.">Primary Salivary Human Stem/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture.</a> Stem Cells Translational Medicine. 6 (1), 110-120 (2017).</li><li>Yoshida, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+living+cells+to+construct+a+3D+single-cell+assembly+without+an+artificial+scaffold.">Manipulating living cells to construct a 3D single-cell assembly without an artificial scaffold.</a> Polymers. 9, 319 (2017).</li><li>Anderson, R. R., Parrish, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+photothermolysis:+precise+microsurgery+by+selective+absorption+of+pulsed+radiation.">Selective photothermolysis: precise microsurgery by selective absorption of pulsed radiation.</a> Science. 220, 524-527 (1983).</li><li>Hashimoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+Stable+Cell-Cell+Contact+without+a+Solid/Gel+Scaffold:+Non-invasive+Manipulation+by+Laser+under+Depletion+Interaction+with+a+Polymer.">Formation of Stable Cell-Cell Contact without a Solid/Gel Scaffold: Non-invasive Manipulation by Laser under Depletion Interaction with a Polymer.</a> Chemical Physics Letters. 655, 11-16 (2016).</li><li>Rauch, P., J&#228;hnke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Tweezers+for+Quantitative+Force+Measurements+and+Live+Cell+Experiments.">Optical Tweezers for Quantitative Force Measurements and Live Cell Experiments.</a> Microscopy Today. 22, 26-30 (2014).</li><li>Ichikawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tilt+control+in+optical+tweezers.">Tilt control in optical tweezers.</a> Journal of Biomedical Optics. 13, 010503 (2008).</li></ol>

The authors have nothing to disclose.

The present study shows a concrete application of our recent reports9,11 on the use of soluble polymers for the construction of 3D single-cell assemblies. Such assemblies are stably formed in the bulk solution when the number of cells is up to 10, and can be held by a single laser beam. Assemblies precipitate on the glass surface when there are more than 10 cells. Although the experiments are still in a primitive stage, we expect that the novel methodology could be a powerful tool for the construction of next-generation 3D single-cell assemblies, which are indispensable for progress in the fields of cell biology and regenerative medicine.
In a solution containing no polymer, cells repel each other due to the electrostatic repulsion arising from the surface charge, the hydration repulsion force, the glycocalyx repulsion effect, and membrane undulation. Our previous study showed that cell pairs can be stable for a long time when the cells are treated with PEG. More importantly, the successful transport of a cell pair to a region without PEG, after the cells had been held in contact for 5 minutes in PEG, suggests that cellular contact is maintained in a stable manner. This is well explained in terms of the depletion effect11, and essentially the same mechanism applies to the cellular assemblies generated using DEX9. Our current results suggest that other kinds of natural macromolecules could also be used to construct stable 3D cellular assemblies.
For the prompt transport of cells, the concentration of polymer is important. Generally, the viscosity of the solution drastically increases when the polymer is dissolved above the overlap concentration. Under this condition, it is difficult to manipulate cells using optical tweezers. Hence, the experiment should be performed below the overlap concentration. For a DEX solution, the overlap concentration is ca. 50 mg/mL (the kinetic viscosity is 5.5 mm2/s). As shown in Ref. 9, a stable cellular assembly was observed when the concentration of DEX was 10 mg/mL to 40 mg/mL. This result suggests that the depletion effect is sufficiently large to maintain stable cell-cell contact even when the DEX concentration is lower than the overlap concentration. It has been shown that the addition of DEX does not affect cell viability up to 40 mg/mL 9.
The establishment of a method for the construction of 3D cellular assemblies is important in the field of regenerative medicine, since mimicking an in vivo cellular microenvironment by structuring single cells may facilitate stem cell-derived tissue formation. So far, we have used the present protocol to construct cellular assemblies using Neuro2A cells9 in addition to NMuMG cells. We hope to establish an experimental methodology for constructing 3D cellular assemblies of a larger number of cells of various morphologies. The optical tweezers system developed by Ichikawa et al.13 would be applicable for this purpose since the orientation of the cells can be controlled. Further trials along these lines should be promising.

While human tissues are composed of several assemblies of cells and can help to maintain homeostasis of the body, single cells by themselves also play important roles via cell-to-cell&#12288;interaction. Therefore, it is important to elucidate how single cells can be stimulated by external signals and how they transfer such signals to other adherent cells. For this purpose, several methods have been established for the construction of single-cell-based 3-dimensional (3D) assemblies1,2,3,4,5,6,7,8. However, the materials that are used to construct cellular assemblies can still be improved. For example, synthetic gels and polymers including polyethylene glycol (PEG) possess certain chemical physicochemical properties and may affect target cells (e.g., toxicity).
We recently reported a novel system that could generate a single-cell-based 3D assembly of cells using dextran (DEX) by establishing stable cell-cell contact9. We considered that this technology could be useful in several research fields, including regenerative medicine and even cancer biology. In this report, we describe how we manipulate single cells and construct 3-dimensional (3D) cellular assemblies in the presence of various hydrophilic biomacromolecules including DEX without an artificial scaffold.

<table border="0" cellpadding="0" cellspacing="0" style="width:933px;" width="933">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Microscope IX71</td>
			<td style="width:124px;">Olympus</td>
			<td style="width:136px;">IX71</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Dextran(200,000; molecular biology-grade)</td>
			<td style="width:124px;">Wako</td>
			<td style="width:136px;">CAS.NO&#160; 9004-54-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Laser Trapping System (NanoTracker 2)</td>
			<td style="width:124px;">JPK Instruments</td>
			<td style="width:136px;">S/N T-05-0200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Upper Objective Lens</td>
			<td style="width:124px;">Olympus</td>
			<td style="width:136px;">LUMPLFLN60XW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Lower Objective Lens</td>
			<td style="width:124px;">Olympus</td>
			<td style="width:136px;">UPLSAPO60XW</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Top Cover Glass</td>
			<td style="width:124px;">MATUNAMI</td>
			<td style="width:136px;">C022401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Intermediate Cover Glass (Spacer)</td>
			<td style="width:124px;">MATUNAMI</td>
			<td style="width:136px;">-</td>
			<td>custom-made (size = 10mm&#215;10mm, thickness = 0.17mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Bottom Cover Glass</td>
			<td style="width:124px;">MATUNAMI</td>
			<td style="width:136px;">C030401</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Camera</td>
			<td style="width:124px;">The Imaging Source</td>
			<td style="width:136px;">DFK 31AF03</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Software</td>
			<td style="width:124px;">JPK Instruments</td>
			<td style="width:136px;">NanoTracker2 PFM software</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">NMuMG cells&#160;</td>
			<td style="width:124px;">RIKEN BRC</td>
			<td style="width:136px;">RCB2868</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">PBS</td>
			<td style="width:124px;">Wako</td>
			<td style="width:136px;">166-23555</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Cell banker</td>
			<td style="width:124px;">Nippon Zenyaku Kogyo</td>
			<td style="width:136px;">ZR621</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">D-MEM</td>
			<td style="width:124px;">Wako Pure Chem. Ind., Japan</td>
			<td style="width:136px;">044-29765</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:279px;">FBS</td>
			<td style="width:124px;">Cell Culture Biosci., Nichirei Biosci. Inc., Japan</td>
			<td style="width:136px;">172012-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Trypsin</td>
			<td style="width:124px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">25200056</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Penicillin-Streptomycin</td>
			<td style="width:124px;">Wako Pure Chem. Ind., Japan</td>
			<td style="width:136px;">161-23181</td>
			<td></td>
		</tr>
	</tbody>
</table>

manipulating living cells to construct stable 3d cellular assembly without artificial scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

Figure 1 shows the microscope and software used in this study. Figure 2 is a schematic representation of the procedure for placing the sample solution containing cells. Figure 3 demonstrates the formation of a pyramid structure using double-beam optical tweezers. If the experiment is successful, these cellular assemblies remain stable even after the laser is switched off.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57815/57815fig1.jpg" /> 
Figure 1: (a) The control system for the Laser Trapping System (NanoTracker2 (11)). The system is activated by turning on the laser switch following steps &#9312;-&#9314;. (b) The software for controlling the Laser Trapping System. The camera, LED light, focus adjust, and moving stage are activated by clicking icons &#9312;, &#9313;, &#9314;, and &#9315;, respectively. The microscopic image is displayed in panel 1. The on/off control for the LED is in panel 2. The focus is controlled in panel 3. The laser beams are irradiated at Positions 1 and 2 by clicking icons I to IV. The details of this Laser Trapping Systemare provided in Ref. (12). <a href="https://www.jove.com/files/ftp_upload/57815/57815fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57815/57815fig2.jpg" /> 
Figure 2: Representative schematic for placing the slide glass. 20 &#181;L of the sample (cell suspension containing dextran) is placed on the slide and used for laser manipulation. <a href="https://www.jove.com/files/ftp_upload/57815/57815fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57815/57815fig3.jpg" /> 
Figure 3: a) Assemblies of epithelial cells (NMuMG) of an intended shape in a medium with DEX (40 mg/mL): a pyramid is shown as an example of a 3D cluster. b) A schematic figure of the pyramid-shaped 3D cellular assembly is also shown. <a href="https://www.jove.com/files/ftp_upload/57815/57815fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold at JoVE.com

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

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6.1K Views.  Doshisha University. We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Video: Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

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Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

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The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. 
Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo F&#246;rster resonance energy transfer measurements, followed by data evaluation.

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

Self-Assembly of Microtubule Tactoids

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is especially the case during mitosis or meiosis, where the microtubules form the spindle during cell division. The spindle is the machinery used to segregate genetic material during cell division. Toward creating self-organized spindles in vitro, we recently developed a technique to reconstitute microtubules into spindle-like assemblies with a minimal set of microtubule-associated proteins and crowding agents. Specifically, MAP65 was used, which is an antiparallel microtubule crosslinker from plants, a homolog of Ase1 from yeast and PRC1 from mammalian organisms. This crosslinker self-organizes microtubules into long, thin, spindle-like microtubule self-organized assemblies. These assemblies are also similar to liquid crystal tactoids, and microtubules could be used as mesoscale mesogens. Here, protocols are presented for creating these microtubule tactoids, as well as for characterizing the shape of the assemblies using fluorescence microscopy and the mobility of the constituents using fluorescence recovery after photobleaching.

This article presents a protocol for the formation of microtubule assemblies in the shape of tactoids using MAP65, a plant-based microtubule crosslinker, and PEG as a crowding agent.

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...