We acknowledge Dr. Harold Dvorak for helpful discussions including the suggestion to try fixation in 37&#160;&#176;C PFA. This work was supported by NIH grant P01 CA92644 and by a contract from the National Foundation for Cancer Research.

1. Cell Culture on Collagen Coated Glass Disk
<ol>
	<li>Place glass disks (12 mm in diameter) in a glass jar (4 oz) and autoclave to sterilize the discs.</li>
	<li>Put 25 ml 70% ethanol in a 50&#160;ml Falcon tube and place it in a cell culture hood. This solution can be reused multiple times until the level of the solution drops to 20 ml.</li>
	<li>Place a sharp forceps with an extra fine point in the 70% ethanol for 5 min before using it to handle the glass disk.</li>
	<li>Carefully remove the forceps out of the tube and close the cap, then gently place the ethanol treated forceps on top of the tube to air dry for 2 min. Do not allow the tip of the forceps to touch anything in the hood.</li>
	<li>Use the sterile forceps to grab one sterile glass disk&#160;out of the glass jar and place it in a well of the 24-well cell culture plate. Repeat until the desired number of wells has been populated with discs.</li>
	<li>Put 500 &#956;l of Bovine collagen solution (50 ng/ml) into each well that contains a glass disk&#160;and place it in a 37 &#176;C, 5% CO2 cell culture incubator for at least 30 min. There is no harm in a longer incubation.</li>
	<li>Harvest HUVEC (Human Umbilical Vein Endothelial Cells) or PC3 (prostate tumor cells) that were already grown in 150&#160;mm cell culture plate through trypsinization and block the trypsin activity using its complete culture medium. Collect cells into a 15&#160;ml Falcon tube.</li>
	<li>Count the cell number and make sure the viability is greater than 90%.</li>
	<li>Pellet the cells by centrifuging at 200 x g for 10 min at 4 &#176;C. Remove the supernatant.</li>
	<li>Use the culture medium to dilute the cells to 105 cells/ml. Place cells on ice.</li>
	<li>Aspirate the collagen in the cell culture hood and gently place 500 &#956;l of cell suspension to each well in which glass disks were precoated with collagen. 5 x 104 cells/well in the 24-well plate will give 60% confluency for HUVEC and 30% confluency for PC3 cells. Note: add more cell suspension for higher cell density.</li>
	<li>Culture the cells in a 37 &#176;C, 5% CO2 incubator for 1 hr, 2 hr, 4 hr, 6 hr, or 24 hr to track nanopodia activities and cell passage. Typically, cells will attach to the glass disk&#160;in the first 30 min, then start to polarize and migrate in the first hour, and perform intercellular interactions and cell division in the following hours.</li>
</ol>
2. Cell Fixation
<ol>
	<li>Each well will need 500 &#956;l 4% PFA (paraformaldehyde) to fix the cells. Either commercially manufactured or freshly made 4% PFA can be used. Calculate the amount of PFA needed based on the number of wells prepared, and place PFA in a 15-ml falcon tube. CAUTION: paraformaldehyde is a suspected carcinogen.</li>
	<li>Put the falcon tube in a Styrofoam stand that was already in place in a 37 &#176;C incubator. Leave the tube in the incubator for 30 min to warm up the PFA to 37 &#176;C.</li>
	<li>Place a heating pad in the culture hood and turn it on.</li>
	<li>Take out the 24-well cell culture plate and the prewarmed PFA out of the incubator and place it on top of the heating pad.</li>
	<li>Use one hand to aspirate the medium from a well, and immediately after use the other hand to gently slide 500 &#956;l PFA into the well through the well edge. For easier aspiration, tilt the culture plate at about 45&#176;, leaning it against the Styrofoam stand so that it is stable at that angle. This will facilitate the aspiration and adding PFA solution to the well without disturbing the cells in culture. This is the most crucial step to preserve the original cellular structure of cell activities.</li>
	<li>Repeat the process until all wells with a glass disk&#160;have received PFA. Note: all steps that need to change pre-existing solution from the well will apply same aspiration procedures.</li>
	<li>Place the plate in 37 &#176;C for 5&#160;min.</li>
	<li>Take the plate back to the culture hood and tilt against Styrofoam as described in step 2.5. Use one hand to gently transfer the PFA from the well into a collection tube; use the other hand to immediately afterward place at least 500 &#956;l room temperature&#160;PBS in the well. After all wells have been washed, return and repeat the PBS washing once more in every well. Empty the collected PFA into a hazardous waste container.</li>
	<li>Prepare ICC blocking buffer by adding 2% Fetal Bovine Serum to PBS. 0.04% sodium azide (dilute from 20% stock solution) is added as a preservative. CAUTION: sodium azide is a toxic compound. The buffer can be stored in 4 &#176;C for a long period of time without bacterial contamination.</li>
	<li>With the culture plate still tilted against a stand, once again cycle through each well aspirating to remove PBS and immediately afterward adding 500 &#956;l of ICC blocking buffer. Cells are now ready for immunofluorescence staining. If necessary, the fixed cells can be stored in a 4 &#176;C refrigerator for a week without significant loss of TM4SF1 protein.</li>
</ol>
3. TM4SF1 Immunofluorescence Staining of Nanopodia
<ol>
	<li>In each well, replace the 500 &#956;l ICC blocking buffer with a new blocking buffer containing 0.01% Triton X-100 and let it sit at room temperature for 1 hr. Do not use higher than 0.03% Triton X-100 as it will remove TM4SF1 from cell membranes. PBS washed cells from step 2.10 can be directly moved to the blocking buffer containing Triton if the staining is ready to be started right away.</li>
	<li>Dilute primary anti-TM4SF1 (or anti-CD9) antibody to 0.5 &#956;g/ml in ICC blocking buffer.</li>
	<li>In each well, remove the ICC/0.01% Triton buffer and replace it with 300 &#956;l of the anti-TM4SF1-antibody solution. Incubate 2 hr at&#160;room temperature or leave overnight&#160;at 4 &#176;C.</li>
	<li>Wash each well with no less than 500 &#956;l PBS. Repeat 3x; each time give at least 5 min of incubation time.</li>
	<li>Prepare a secondary antibody and phalloidin solution in ICC blocking buffer, using a 1,000 times dilution of Alexa 488&#160;labeled donkey anti-mouse secondary antibody (2 &#956;g/ml final concentration) and 1,000x dilution of phalloidin (50 ng/ml final concentration).</li>
	<li>Remove PBS and add 300 &#956;l of the secondary antibody and phalloidin solution to each well and incubate for 2 hr or leave it overnight&#160;at 4 &#176;C.</li>
	<li>Repeat PBS washing steps with at least a 5 min incubation per wash. In the last wash, leave each well in PBS for 1 hr.</li>
	<li>Drop a single drop (~10 &#956;l) of anti-fade mounting media on a glass slide.</li>
	<li>Bend the tip of a 19 G 1&#160;&#189; in syringe needle 90&#176;&#160;by pressing gently on a hard surface. Use one hand to hold bended needle and gently lift up a glass disk&#160;from the well,&#160;and use the other hand to grasp the disc using the sharp forceps.</li>
	<li>Turn the glass disk&#160;face down letting the cell side contact the mounting media on the glass slide. Gently place the glass disk&#160;and let it dry overnight&#160;in a dark place at&#160;room temperature.</li>
	<li>Proceed to image the immunofluorescence stained nanopodia, or store the slides in a slide box at -20 &#176;C. The slides will remain viewable for a few months in -20 &#176;C.</li>
</ol>

<ol>
	<li>Chhabra, E. S., Higgs, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+faces+of+actin:+matching+assembly+factors+with+cellular+structures.">The many faces of actin: matching assembly factors with cellular structures.</a> Nat. Cell Biol. 9, 1110-1121 (2007).</li><li>Shih, S. C., 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=The+L6+protein+TM4SF1+is+critical+for+endothelial+cell+function+and+tumor+angiogenesis.">The L6 protein TM4SF1 is critical for endothelial cell function and tumor angiogenesis.</a> Cancer Res. 69, 3272-3277 (2009).</li><li>Zukauskas, 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=TM4SF1:+a+tetraspanin-like+protein+necessary+for+nanopodia+formation+and+endothelial+cell+migration.">TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration.</a> Angiogenesis. 14, 345-354 (2011).</li><li>Hellstrom, I., Beaumier, P. L., Hellstrom, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effects+of+L6,+an+IgG2a+antibody+that+reacts+with+most+human+carcinomas.">Antitumor effects of L6, an IgG2a antibody that reacts with most human carcinomas.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 7059-7063 (1986).</li><li>Jang, Y. Y., Ye, Z., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+and+stem+cell+research.">Molecular imaging and stem cell research.</a> Mol. Imag. 10, 111-122 (2011).</li><li>Hemler, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraspanin+functions+and+associated+microdomains.">Tetraspanin functions and associated microdomains.</a> Nat. Rev. Mol. Cell Biol. 6, 801-811 (2005).</li><li>Vasile, E., Tomita, Y., Brown, L. F., Kocher, O., Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+thymosin+beta-10+by+early+passage+and+senescent+vascular+endothelium+is+modulated+by+VPF/VEGF:+evidence+for+senescent+endothelial+cells+in+vivo+at+sites+of+atherosclerosis.">Differential expression of thymosin beta-10 by early passage and senescent vascular endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis.</a> FASEB. 15, 458-466 (2001).</li><li>Itoh, T. J., Hotani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+and+the+regulation+by+microtubule-associated+proteins+(MAPs).">Microtubule dynamics and the regulation by microtubule-associated proteins (MAPs).</a> Uchu Seibutsu Kagaku. 18, 116-117 (2004).</li><li>Caplow, M., Shanks, J., Ruhlen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-jump+studies+of+microtubule+dynamic+instability.">Temperature-jump studies of microtubule dynamic instability.</a> J. Biol. Chem. 263, 10344-10352 (1988).</li><li>Pollice, A. 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=Sequential+paraformaldehyde+and+methanol+fixation+for+simultaneous+flow+cytometric+analysis+of+DNA,+cell+surface+proteins,+and+intracellular+proteins.">Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.</a> Cytometry. 13, 432-444 (1992).</li><li>Macarulla, J. 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=Membrane+solubilization+by+the+non-ionic+detergent+triton+X-100.+A+comparative+study+including+model+and+cell+membranes.">Membrane solubilization by the non-ionic detergent triton X-100. A comparative study including model and cell membranes.</a> Revista Espanola de Fisiologia. 45 Suppl, 1-8 (1989).</li><li>Koley, D., Bard, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triton+X-100+concentration+effects+on+membrane+permeability+of+a+single+HeLa+cell+by+scanning+electrochemical+microscopy+(SECM).">Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM).</a> Proc. Natl. Acad. Sci. U.S.A. 107, 16783-16787 (2010).</li><li>Chang, Y. W., 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=CD13+(aminopeptidase+N)+can+associate+with+tumor-associated+antigen+L6+and+enhance+the+motility+of+human+lung+cancer+cells.">CD13 (aminopeptidase N) can associate with tumor-associated antigen L6 and enhance the motility of human lung cancer cells.</a> Int. J. Cancer. 116, 243-252 (2005).</li></ol>

No conflicts of interest declared.

Nanopodia are thin cellular membrane channels that firmly attach to matrix through TMED and can extend more than 100 &#956;m from a polarized mobile cell to sense the environment and mediate intercellular interactions2,3. Nanopodia adhere to matrix so firmly that residues are left behind as the cell moves away (Figures 1 and 2). Nanopodia thus allow us to study how cells sense their environment and determine the path of cell movement, and how gene expression or drug treatment changes movement. However, due to the fragility of these thin (100-300 &#956;m width) membrane structures, caution needs to be taken in order to preserve them.
In order to faithfully record cellular activities without disturbing nanopodia, and to study how experimental conditions affect nanopodia activities and the path of cell movement, one should first verify that cells grow effectively in complete medium before pursuing the experiment. Normal healthy primary endothelial cells, such as HUVEC, in fresh EGM2-MV complete culture medium will normally divide approximately every 18 hr during their log phase of growth within six passages of their culture. One should avoid the use of HUVEC that are greater than passage-6 as higher passage numbers will lead to increased numbers of senescent or binuclear cells in culture7 and will affect cells&#39; ability to polarize and produce nanopodia2. PC3 prostate tumor cells are more stable to cultures (unpublished observation), but one should still avoid the use of cells more than ten passages from their removal from the frozen state.
Healthy adherent cells will typically attach to collagen-coated discs within the first 30 min after they are seeded, followed soon afterward by cell polarization. Thus one should expect to see most endothelial cells in a polarized state at the end of the first hour of incubation. If a majority of endothelial cells do not attach to the glass surface, then check whether (a) collagen has passed its expiration date, (b) trypsinization during cell harvest was too stringent, or (c) cells are unhealthy. PC3 cells polarize slower than endothelial cells, but should demonstrate a polarized morphology within 2 hr after seeding. If contamination occurs in culture, then examine whether glass disks or forceps were properly sterilized. Alternatively, one can consider using chamber slides; the protocol described here suggests the use of glass disks in 24-well culture plates in order to save cost and to more easily handle a large number of samples.
Conventional immunostaining methods, use of PBS to wash cells before adding room temperature fixative, easily disturb fine cellular structures like nanopodia and will destroy them during the staining process. This is largely due to the fact that physical stresses like temperature fluctuations during cell fixation or rigorous PBS washing during the staining process are the main causes of fragmentation or removal of nanopodial structures. With three simple methodological changes - (i) do not disturb cell polarization state by washing with PBS before fixation, (ii) fix cells directly in 37 &#176;C prewarmed PFA and incubate cells at 37 &#176;C during fixation, and (ii) gently wash cells in room temperature PBS - nanopodia are largely preserved. Figure 1 shows how PFA and PBS temperatures affect preservation of nanopodia in cultured endothelial cells; Figure 2A shows that TM4SF1 expression is heterogeneous in PC3 cells and that cells with the strongest TM4SF1 staining display the longest nanopodia. Temperature is known to affect F-actin activities and tubulin integrity8,9. Thus fixing cells at the same temperature in which cells were grown (this is 37 &#176;C in the protocol described here) is likely to better preserve the polarized cellular state (Figures 1A and 1B) and intercellular junctions (Figure 1C), permitting more faithful recording of cellular activities at the time when cells are removed for experimental studies. Unlike on the retracting side, nanopodia projections at the cell&#39;s leading front are short lived, in part because the cells are constantly moving and overrun leading edge nanopodia as they move forward2,3. Thus the best time to observe nanopodia at the leading front is not long after cells are plated, ideally at around 30-60 min after seeding, a time when cells have attached to matrix and are only beginning to initiate movement. Live cell imaging is an alternative approach which enables a precise record of nanopodia activities at the leading front.
PFA is not the only reagent used in fixation; organic solvents like methanol, ethanol, and acetone are also frequently used in cell fixation for immunostaining10. The best fixative is often decided by the epitope location of a specific antibody used in the staining (unpublished data). For the mouse anti-human TM4SF1 antibody that recognizes extracellular domains of the TM4SF1 (Millipore; used in this study), PFA is the only fixative which reveals TMED in immune-staining, indicating that other organic solvents destroy the epitope. In contrast, rabbit anti-human TM4SF1 polyclonal antibody from Acris (data not shown), whose epitope is located in eighteen amino acids near the N-terminal region, works also with ethanol fixed cells. However, the Acris antibody does not yield good TM4SF1 staining, probably because the N-terminal epitope is preoccupied by TM4SF1 interactions with cytosolic molecule(s) which partially cover the epitope.
Triton X-100 is a nonionic surfactant and is often used in immune-staining with the aim of permeabilizing cell membrane and reducing surface tension of aqueous solutions11,12. However, this capability also brings the risk of solubilizing membrane proteins. TM4SF1 is completely removed from the cell surface by Triton X-100 concentrations greater than 0.05% 2,3. For this reason, only a 1-hr treatment with 0.01% Triton X-100 before addition of the primary antibody is used in the method described here. This treatment permeabilizes the membrane enough to enable peri-nuclear TM4SF1 to be well assessed by the antibodies, while preserving most TM4SF1 on the cell membrane. For double staining of TM4SF1 with proteins located in the nucleus, use 0.03% Triton X-100 in the preincubation stage if 0.01% Triton does not work. This will facilitate Triton X-100 puncturing of the nuclear membrane.
TM4SF1 is highly specific to endothelial cells and tumor cells; most other cell types either do not express TM4SF1 or express it at a very low level2,3. However, classic tetraspanins - a family that encompasses thirty-three members including CD9, CD81, and CD151 - are ubiquitously expressed6, and can also be found in membrane domains in nanopodia2,3 (Figure 3A). Thus, staining classic tetraspanins through the same immune-staining method used for TM4SF1 can enable the studies of nanopodia in cell types that do not express TM4SF1. PC3 tumor cells that express TM4SF1 highly tend to move continuously in a single direction, whereas PC3 cells that weakly express TM4SF1 do not have a clearly defined direction of movement (Figure 2A and unpublished data). TM4SF1 expression level in tumor cells is known to be correlated with their metastatic potential13, and investigations are currently underway linking tumor cells&#39; ability to express TM4SF1 and produce nanopodia to their metastatic potential.
In summary, mastery of effective methods for staining nanopodia enables investigators to track the path of cell movement through nanopodia residues, study how cells sense their environment for movement, how cell movement is modified by alteration of the expression levels of genes (either overexpression or knockdown of genes of interest). Nanopodia represent a new tool for investigation of cell polarization and cell movement.

During polarization for movement, animal cells extend a variety of dynamic, membrane protrusions from their surfaces, including filopodia, lamellipodia, retraction fibers, and ruffles1. Recently added to this list were nanopodia, a newly recognized type of thin (100-300 &#956;m in diameter), elongated (up to 50-100 &#956;m long) membrane projection that provide membrane channels for the extension of F-actin structures such as filopodia and retraction fiber, and that stain positively with TM4SF1 (Transmembrane-4-L-six-family-1) in cultured endothelial and tumor cells2,3.
TM4SF1 is a protein with tetraspanin-like topology that was originally known as a tumor cell antigen4 before the discovery that the molecule is an endothelial cell biomarker that plays an essential role in endothelial cell proliferation and migration2,3. Immunofluorescence staining revealed that TM4SF1 is localized to perinuclear vesicles and to the plasma membrane, and is enriched in TM4SF1 enriched microdomains (TMED). TMED anchor nanopodia to matrix and present in a regularly spaced banded pattern of 1-3 TMED/&#181;m length of nanopodia. Nanopodia typically extend from a cell's leading front and trailing rear during cell polarization for movement. Due to the firm adherent nature of TMED, nanopodia are unable to retract back into the cell as it moves away; abandoned nanopodia residues thus trace out the path of cellular movement. Nanopodia provide membrane channels for F-actin extension and retraction, and are sites of intercellular interactions and communications2,3. These characteristics mean that nanopodia provide a unique opportunity to study the mechanisms underlying F-actin assembly during cellular polarization, cellular sensing of the environment, determination of the path and direction of cell movement, and intercellular interactions and communications.
Due to the highly hydrophobic nature of TMED and the thin and fragile membranous nature of nanopodia, special care needs to be taken in order to preserve TMED and nanopodia. The destruction of nanopodia and removal of TMED by common laboratory methods is a possible reason why only sixty-three publications have appeared on TM4SF1 since its first discovery in 19861&#160;and for the complete lack of knowledge of TM4SF1 enrichment in 100-300 &#956;m microdomains on the cell surface until the report of TM4SF1 in endothelial cells in 20092.
Conventional immunostaining methods commonly use organic solvents like ethanol, methanol, or acetone to fix cells and use 0.1% or higher Triton X-100 concentration to permeabilize cells5. Studies described here implemented three major changes to the conventional method to reveal TMED and nanopodia: (i) use 37 &#176;C 4% PFA and fix cells in a 37 &#176;C incubator, (ii) apply gentle room temperature PBS washing, and (iii) use less than 0.03% Triton X-100 only briefly to permeabilize cells before addition of primary antibody, as Triton X-100 higher than 0.03% will extract TM4SF1.
All tetraspanins form microdomains on the cell membrane6 and some colocalize with TMED in endothelial and tumor cells2,3. As tetraspanins like CD9, CD81, and CD151 are ubiquitously expressed, the staining protocol described here can be extended to many different cell types that lack TM4SF1 for the studies of nanopodia function.

<table border="0" cellpadding="0" cellspacing="0" width="1597">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass discs&#160;</td>
			<td style="width:160px;">Fisher Scientific</td>
			<td style="width:133px;">12-545-82&#160;</td>
			<td style="width:983px;">12-mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass jar</td>
			<td>Fisher Scientific</td>
			<td>02-912-310</td>
			<td>Certified Clean Clear Glass Straight-Sided Jars, 4 oz.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass slide</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">12-544-1</td>
			<td>Fisherfinest Premium Plain Glass Microscope Slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50-ml falcon tube&#160;</td>
			<td style="width:160px;">BD Falcon</td>
			<td style="width:133px;">352070</td>
			<td>50-ml high-clarity polypropylene conical centrifuge tube, 16,000 rcf rating. Sterile.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">15-ml falcon tube with Styrofoam rack</td>
			<td style="width:160px;">Corning</td>
			<td style="width:133px;">430790</td>
			<td>Corning 15-ml PP Centrifuge Tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sharp forceps&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">13-812-42</td>
			<td>Dissecting Extra-Fine-Pointed Splinter Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">24-well cell culture plate</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">353047</td>
			<td>24-well Cell Culture Plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">150-mm cell culture plate&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">353025</td>
			<td>150-mm cell culture plate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">19G 1 &#189; syringe needle&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">309635</td>
			<td>19G 1 &#189;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Reagent</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2701</td>
			<td>Decon&#39;s Pure Ethanol 200 Proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">collagen solution&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">354249</td>
			<td>Collagen I, High Concentration, rat tail, 100 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">trypsin/EDTA</td>
			<td style="width:160px;">Cellgro</td>
			<td style="width:133px;">25-053-CI</td>
			<td><a name="RANGE!D15">0.25% Trypsin-EDTA 1X</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DMEM</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">75889 5 LT</td>
			<td style="width:983px;">PBS, 10X Solution, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PFA (paraformaldehyde)</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">19943 1 LT</td>
			<td>4% in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500ML</td>
			<td><a name="RANGE!D19">Fetal Bovine Serum</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">EGM2-MV</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>EGM-2 BulletKit, EBM-2 Basal Medium 500 ml and EGM-2 SingleQuot Kit Suppl. &#38; Growth Factors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">T8787</td>
			<td>Triton X-100</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaN3</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">S2002-25G</td>
			<td>solubilized in water. 4% stock solution and working concentration is 0.4%.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human TM4SF1 antibody&#160;</td>
			<td style="width:160px;">Millipore</td>
			<td style="width:133px;">MAB3127</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human CD9 antibody&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">555370</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Alexa Fluor 488 Donkey anti-mouse 2nd antibody</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">A-21202</td>
			<td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Phalloidin</td>
			<td style="width:160px;">Chemicon</td>
			<td style="width:133px;">90324</td>
			<td>Rhodamine-conjugated Phalloidin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">anti-fade mounting media&#160;</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">P-36931</td>
			<td>ProLong Gold Antifade Reagent with DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Buffers and solutions</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">70% ethanol&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td>17.5 ml of ethanol (200 proof) 
			7.5 ml of double deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS (make 200 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20 ml of 10X PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">180 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20% Triton X-100 (make 20 ml)&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">4 ml</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">16 ml double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">4% NaN3 (make 25 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">1 g of NaN3</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">50 ng/ml bovine collagen solution in PBS (make 5 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Make stock soln. of 50 &#956;g/ml (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">830 &#956;l of Collage I (3 mg)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml PBS&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ICC Blocking Buffer (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">49 ml PBS</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2% FBS (add 1 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">0.04% NaN3 (add 100 &#956;l of 4% NaN3)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">ICC Blocking Buffer/0.01% Triton X-100 (make 50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml of ICC Blocking Buffer&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 &#956;l of 20% Triton X-100</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Cell</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">HUVEC&#160;</td>
			<td style="width:160px;">Lonza&#160;</td>
			<td>C2517A</td>
			<td><a href="http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx">http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PC3</td>
			<td style="width:160px;">ATCC</td>
			<td style="width:133px;">CRL-1435</td>
			<td><a href="http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7">http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Equipment</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">cell culture hood</td>
			<td style="width:160px;">NuAIRE</td>
			<td style="width:133px;">Nu-425-600</td>
			<td>NU-425 (Series 60) BIOLOGICAL SAFETY CABINET&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">37 &#176;C, 5% CO2 cell culture incubator&#160;</td>
			<td style="width:160px;">CellStar</td>
			<td style="width:133px;">QWJ300DABA&#160;</td>
			<td>Cellstar CO2 Water Jacketed Incubator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">heating pad&#160;</td>
			<td>K&#38;H Manufacturing</td>
			<td style="width:133px;">1020</td>
			<td style="width:983px;">K&#38;H Lectro Kennel Heated Pad with Free Fleece Cover (http://www.amazon.com/Lectro-Kennel-Heat-Pad-22-5x28-5-In/dp/B000PSRN20/ref=pd_bxgy_petsupplies_img_y)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">centrifuge</td>
			<td style="width:160px;">Sorvall&#160;</td>
			<td style="width:133px;">T6000B&#160;</td>
			<td>Sorvall T6000 (B) Benchtop Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">Z359629</td>
			<td>Bright-Line Hemacytometer</td>
		</tr>
	</tbody>
</table>

nanopodia - thin, fragile membrane projections with roles in cell movement and intercellular interactions

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 &#956;m diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per &#956;m and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 &#956;m from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 &#176;C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.

For Step 1:
If cells (such as HUVEC and PC3 used in this study) are able to grow normally, cells will attach to the collagen-coated disc within 30 min after they are seeded, polarize and become mobile soon afterward, and extend nanopodia ahead of their path of movement. Figures 1A and 1B respectively shows a polarized and proliferating HUVEC. Figure 2A shows polarized PC3 cells in a mobile state.
For Step 2:
With prewarming of 4% PFA to 37 &#176;C, fixing cells at 37 &#176;C for 20 min, and gentle washing with room temperature&#160;PBS, cellular states should be well preserved. Generally, isolated individual HUVEC (Figure 1A,&#160;1) or PC3 (Figure 2A) cells will show a polarized morphology, unless cells are in the state of cell division (Figure 1B,&#160;1). Intercellular interactions leading to cell junction formation (Figure 1C,&#160;1) are also commonly observed. Nanopodia should appear on the cell periphery, and F-actin will often be present in portions of the nanopodia proximal to the cell. Cold temperatures cause F-actin to retract back into cells leaving behind nanopodia membrane channels. Suboptimal temperature, as shown in examples using 4 &#176;C PFA fixation and cold PBS washing, will disturb and destroy nanopodia structure in polarized cells (Figure&#160;1A,&#160;2), proliferating cells (Figure&#160;1B,&#160;2), and artificially produce gaps at intercellular junctions (Figure&#160;1C,&#160;2).
For Step 3:
Many membrane-bound proteins including TM4SF1 are very sensitive to Triton X-100 and will be removed from the cell surface if concentrations higher than 0.03% are used2,3. In order to preserve most of cell surface TM4SF1, PFA fixed cells should only be treated with 0.01% Triton X-100 containing blocking buffer for an hour before changing to anti-TM4SF1 primary antibody that has been diluted in a regular blocking buffer that does not contain Triton X-100. As nanopodia are thin and long membrane projections and attach to matrix through TMED, TMED intensity will likely need to be enhanced through brightness and contrast functions in image processing software like Photoshop in order to reveal and track the path of cell movement through nanopodia residues (Figure 1,&#160;2 insets).
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/51320/51320fig1highres.jpg" src="/files/ftp_upload/51320/51320fig1.jpg" /> 
Figure 1.&#160;Nanopodia projections during endothelial cell movement, cell division, and junction formation. HUVEC were freshly cultured for 6 hr after plating at 60% confluence on collagen-coated glass disk. Cells were fixed using (1) the modified method where cells were fixed in 37 &#176;C 4% PFA directly without prewashing in PBS, and placed in a 37 &#176;C incubator during the 20 min fixation time, or (2) a conventional staining method where the cells were washed with room temperature PBS twice and fixed in 4% PFA for 20 min at room temperature. Nanopodia, thin and long membrane extensions that are marked by an intermittent, banded pattern of TMED (white arrows) and that often enclose phalloidin-stained F-actin (pink arrows) in portions proximal to the cell, were immunofluorescence stained using anti-TM4SF1 Ab. Three representative HUVEC images were captured to show the appearance of nanopodia during (A) polarization, (B) cell division, and (C) intercellular junction formation. Inset boxes show higher magnifications of nanopodia. During polarized (A) or retracted cellular states (B, C), the nanopodia projections were preserved by directly fixing cells in 37 &#176;C PFA, whereas the structures were largely removed by washing and fixing the cells in room temperature PBS and PFA. (C) 37 &#176;C PFA fixation preserves intercellular junctions (blue arrows), whereas junctions are disturbed by colder temperatures that cause cells to retract, creating a gap between cells bridged by nanopodia of which most contain F-actin (pink arrows) and some do not (white arrows) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51320/51320fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/51320/51320fig2highres.jpg" src="/files/ftp_upload/51320/51320fig2.jpg" /> 
Figure 2.&#160;Nanopodia demonstrate the direction of PC3 tumor cell movement. (A) PC3 tumor cells were freshly cultured for 6 hr after plating at 60% confluence on collagen-coated glass disk. Cells were fixed directly in 37 &#176;C 4% PFA and placed in a 37 &#176;C incubator for 20 min to preserve nanopodia. Nanopodia (white arrows) and F-actin (pink arrows) were immunofluorescence stained using anti-TM4SF1 Ab and phalloidin, respectively. The inset box shows nanopodia at higher magnification. This representative image shows that TM4SF1 expression in PC3 cells is heterogeneous; one PC3 cell with strong TM4SF1 staining (yellow arrow) produced long nanopodia and is surrounded by two weakly TM4SF1-stained PC3 cells (red arrows). Nanopodia residues at the trailing edge (ib) indicate the direction of PC3 cell movement during the 6 hr cell culture period. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51320/51320fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51320/51320fig3highres.jpg" src="/files/ftp_upload/51320/51320fig3.jpg" /> 
Figure 3.&#160;Visualization of nanopodia through tetraspanin CD9. (A)HUVEC were freshly cultured for 6 hr after plating at 60% confluency on a collagen-coated glass disk. Cells were fixed in 37 &#176;C 4% PFA and nanopodia (white arrows) were revealed through anti-CD9 Ab. F-actin (pink arrows) was stained using phalloidin. This representative image shows that CD9 also localizes to nanopodia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51320/51320fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions at JoVE.com

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Nanopodia are thin but fragile membrane channels that extend up to 100 &#956;m from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 &#176;C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

nanopodia-thin-fragile-membrane-projections-with-roles-cell-movement

Nanyang Technological University

Research

JoVE Journal

Biology

10.2K Views.  Harvard Medical School. Nanopodia are thin but fragile membrane channels that extend up to 100 μm from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 °C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

Video: Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...