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 ...

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><tbody><tr><td>Glass disks</td><td>Fisher Scientific</td><td>12-545-82</td><td>12 mm diameter</td></tr><tr><td>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><td>Glass slide</td><td>Fisher Scientific</td><td>12-544-1</td><td>Fisherfinest Premium Plain Glass Microscope Slides</td></tr><tr><td>50 ml Falcon tube</td><td>BD Falcon</td><td>352070</td><td>50 ml high-clarity polypropylene conical centrifuge tube, 16,000 rcf rating. Sterile.</td></tr><tr><td>15 ml Falcon tube with Styrofoam rack</td><td>Corning</td><td>430790</td><td>Corning 15 ml PP Centrifuge Tubes</td></tr><tr><td>Sharp forceps</td><td>Fisher Scientific</td><td>13-812-42</td><td>Dissecting Extra Fine Pointed Splinter Forceps</td></tr><tr><td>24-well Cell culture plate</td><td>BD Bioscience</td><td>353047</td><td>24-well Cell Culture Plate</td></tr><tr><td>150 mm Cell culture plate</td><td>BD Bioscience</td><td>353025</td><td>150 mm cell culture plate</td></tr><tr><td>19 G 1 &amp;frac12; Syringe needle</td><td>BD Bioscience</td><td>309635</td><td>19 G 1 &amp;frac12;</td></tr><tr><td>Ethanol</td><td>Decon Laboratories, Inc.</td><td>2701</td><td>Decon&#039;s Pure Ethanol 200 Proof</td></tr><tr><td>Collagen solution</td><td>BD Bioscience</td><td>354249</td><td>Collagen I, High Concentration, rat tail, 100 mg</td></tr><tr><td>Trypsin/EDTA</td><td>Cellgro</td><td>25-053-CI</td><td>0.25% Trypsin-EDTA 1x</td></tr><tr><td>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><td>10x PBS</td><td>Affymetrix</td><td>75889 5 LT</td><td>PBS, 10x Solution, pH 7.4</td></tr><tr><td>PFA (paraformaldehyde)</td><td>Affymetrix</td><td>19943 1 LT</td><td>4% in PBS</td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F4135-500ML</td><td>Fetal Bovine Serum</td></tr><tr><td>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 Supplement &amp;amp; Growth Factors</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Triton X-100</td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S2002-25G</td><td>Solubilized in water. 4% stock solution and working concentration is 0.4%.</td></tr><tr><td>Mouse anti-human TM4SF1 antibody&amp;nbsp;</td><td>Millipore</td><td>MAB3127</td><td>Epitope is located in extracellular domain</td></tr><tr><td>Mouse anti-human CD9 antibody&amp;nbsp;</td><td>BD Bioscience</td><td>555370</td><td>Epitope is located in extracellular domain</td></tr><tr><td>Alexa Fluor 488 Donkey anti-mouse 2nd antibody</td><td>Life Technologies</td><td>A-21202</td><td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L)</td></tr><tr><td>Phalloidin</td><td>Chemicon</td><td>90324</td><td>Rhodamine-conjugated Phalloidin</td></tr><tr><td>Anti-fade mounting media&amp;nbsp;</td><td>Life Technologies</td><td>P-36931</td><td>ProLong Gold Antifade Reagent with DAPI</td></tr><tr><td>70% Ethanol&amp;nbsp;</td><td></td><td></td><td>17.5 ml of ethanol (200 proof)&lt;br/&gt; 7.5 ml of double-deionized water</td></tr><tr><td>10x PBS (make 200 ml)</td><td></td><td></td><td>20 ml of 10x PBS&lt;br/&gt; 180 ml of double-deionized water</td></tr><tr><td>20% Triton X-100 (make 20 ml)&amp;nbsp;</td><td></td><td></td><td>4 ml Triton X-100&lt;br/&gt; 16 ml double-deionized water</td></tr><tr><td>4% NaN&lt;sub&gt;3&lt;/sub&gt; (make 25 ml)</td><td></td><td></td><td>1 g of NaN&lt;sub&gt;3&lt;/sub&gt;&lt;br/&gt; 25 ml of double-deionized water</td></tr><tr><td>50 ng/ml Bovine collagen solution in PBS (make 5 ml)</td><td></td><td></td><td>Stock solution of 50 &amp;mu;g/ml (50 ml)&lt;br/&gt; 830 &amp;mu;l of Collagen I (3 mg)&lt;br/&gt; 50 ml PBS</td></tr><tr><td>ICC Blocking Buffer (50 ml)</td><td></td><td></td><td>49 ml PBS&lt;br/&gt; 2% FBS (add 1 ml)&lt;br/&gt; 0.04% NaN&lt;sub&gt;3&lt;/sub&gt; (add 100 &amp;mu;l of 4% NaN&lt;sub&gt;3&lt;/sub&gt;)</td></tr><tr><td>ICC Blocking Buffer/0.01% Triton X-100 (make 50 ml)</td><td></td><td></td><td>50 ml of ICC Blocking Buffer&lt;br/&gt; 25 &amp;mu;l of 20% Triton X-100</td></tr><tr><td>HUVEC&amp;nbsp;</td><td>Lonza</td><td>C2517A</td><td>&lt;a href=&quot;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&quot;&gt;www.lonza.com&lt;/a&gt;</td></tr><tr><td>PC3</td><td>ATCC</td><td>CRL-1435</td><td>&lt;a href=&quot;http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7&quot;&gt;www.atcc.org&lt;/a&gt;</td></tr><tr><td>Cell culture hood</td><td>NuAIRE</td><td>Nu-425-600</td><td>NU-425 (Series 60) Biological Safety Cabinet&amp;nbsp;</td></tr><tr><td>37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; Cell culture incubator&amp;nbsp;</td><td>CellStar</td><td>QWJ300DABA</td><td>Cellstar CO&lt;sub&gt;2&lt;/sub&gt; Water Jacketed Incubator</td></tr><tr><td>Heating pad&amp;nbsp;</td><td>K&amp;amp;H Manufacturing</td><td>1020</td><td>K&amp;amp;H Lectro Kennel Heated Pad with Free Fleece Cover (&lt;a href=&quot;http://www.amazon.com/Lectro-Kennel-Heat-Pad-22-5x28-5-In/dp/B000PSRN20/ref=pd_bxgy_petsupplies_img_y&quot;&gt;www.amazon.com&lt;/a&gt;)</td></tr><tr><td>Centrifuge</td><td>Sorvall</td><td>T6000B</td><td>Sorvall T6000 (B) Benchtop Centrifuge</td></tr><tr><td>Hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629</td><td>Bright-Line Hemocytometer</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:
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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 ...

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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

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Biochemistry

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To understand the regulation of cell motility, we must be able to measure it easily and consistently under different conditions. Here we describe a method for measuring and quantifying single-cell and bulk motility of HaCaT keratinocytes using a nuclear stain. This method includes a MATLAB script for analyzing TrackMate output files to calculate displacements, motility rates, and trajectory angles in single cells and in bulk for an imaging site. This motility analysis script allows for quick, straightforward, and scalable analysis of cell motility rates from TrackMate data and could be broadly used to identify and study the regulation of motility in epithelial cells. We also provide a MATLAB script for reorganizing microscopy videos collected on a microscope and converting them to TIF stacks, which can be analyzed using the ImageJ TrackMate plugin in bulk. Using this methodology to explore the roles of adherens junctions and actin cytoskeletal dynamics in regulating cell motility in HaCaT keratinocytes, we demonstrate evidence that Arp2/3 activity is required for the elevated motility seen after &#945;-catenin depletion in HaCaT keratinocytes.

This protocol for high-throughput measurement of cell motility in HaCaT keratinocytes describes methods for collecting and processing images of cell nuclei and performing particle tracking using the ImageJ plugin TrackMate.

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...

Types of Membrane Protrusions

The protrusion of the cell surface is an initial step for several cellular processes, including cell migration, phagocytosis, and neurite outgrowth. These membrane protrusions are a result of cytoskeletal rearrangement. The most&#160; widely observed cell protrusions include lamellipodia, pseudopodia, filopodia, microvilli, invadopodia, and podosomes. These protrusions can be of two types &#8212; static or dynamic.
The microvilli, an example of stable protrusions, are finger-like projections with a generally stable actin architecture. These brush-like extensions are found on the apical surface of epithelial cells lining internal organs, such as the intestine. They greatly increase the surface area for enhanced nutrient absorption.
Contrastingly, dynamic protrusions are primarily involved in cell migration. The majority of these structures use actin polymerization as the driving force for membrane deformation. Additionally, actin binds myosin motor proteins to form contractile bundles. The myosin motors pull on the actin filaments, thus rearranging them during migration. Based on their appearance, actin-dependent protrusions are classified as lamellipodia, pseudopodia, filopodia, and invadopodia.
Many migrating cells like keratinocytes or fibroblasts exhibit lamellipodia, which are thin, sheet-like projections at the leading edge of these cells. They are involved in the rapid migration of cells to sites of injury for repair and regeneration. Lamellipodia&#160; can extend bundled actin filaments to form finger-like projections called filopodia. For example,&#160; neurons form these outgrowths to sense environmental signals and rewire the nervous system after trauma.
Other membrane protrusions include the podosomes&#160; and invadopodia. While podosomes are transient peg-like structures developed by migrating cells like macrophages, invadopodia are projections produced by metastatic cells on the extracellular matrix. Additionally, invadopodia secrete proteolytic enzymes, degrading the extracellular matrix to break tissue architecture and metastasize into adjacent tissues. Lastly, blebs are spherical membrane projections formed due to internal hydrostatic pressure. They do not depend on the actin cytoskeleton for their enlargement. Fibroblasts and immune cells exhibit bleb formation for migration and other cellular processes such as apoptosis.

The protrusion of the cell surface is an initial step for several cellular processes, including cell migration, phagocytosis, and neurite outgrowth. These ...

Education

JoVE Core

Cell Biology

Unit 1

Cell Polarization and Migration

KEY TERMS AND CONCEPTS

Mechanism of Lamellipodia Formation

Cells migrating in response to external stimuli form lamellipodia, which are thin membrane protrusions supported by a mesh of linked, branched, or unbranched actin filaments. These actin filaments interact with myosin motor proteins, creating the dynamic actomyosin complex within the cytoskeleton. Contractility, or the ability to generate contractile stress, is inherent to the actomyosin complex. It helps cells detect the stiffness of the surrounding ECM and exert contractile force for migration.
At the leading edge, the binding of an activated Rac GTPase to the WAVE complex initiates lamellipodia formation. Subsequently, the WAVE complex changes conformation to prime actin nucleators like the Arp2/3 complex for polymerization and branching of filaments. Additionally, members of the formin family localize at the sites of lamellipodia formation, elongating the unbranched filaments. The force generated by continuous actin polymerization pushes the membrane outwards and forms the membrane protrusion. The actin mesh at the leading edge contributes to the formation of focal adhesion points on the ECM, preventing the retraction of lamellipodia.
After lamellipodia formation, the dissociation of filaments begins at the rear end of the dynamic actin meshwork. The actin monomers steadily detach from the minus ends and attach to the plus ends of filaments in the actin network, resulting in treadmilling. This cyclic assembly and disassembly causes&#160; the forward movement of the entire actin filament network even though the position of individual filaments remains stationary to the substratum.
Behind the dynamic lamellipodial meshwork lies a stable network of bundled linear actin filaments called the lamella. This actin network supports the lamellipodium by developing mature and strong adhesion sites, which resist cell compression during migration. Additionally, stable actin filaments of the lamella associate with myosin proteins and use their inherent contractibility to propel the cell forward.

Cells migrating in response to external stimuli form lamellipodia, which are thin membrane protrusions supported by a mesh of linked, branched, or ...

Mechanism of Filopodia Formation

Filopodia are thin, actin-rich cellular protrusions that play an important role in many fundamental cellular functions. They vary in their occurrence, length, and positioning in different cell types, suggesting their diverse roles.
Their main function is to guide migrating cells during normal tissue morphogenesis or cancer metastasis by recognizing and making initial contacts with the extracellular matrix. However, they can also act as stationary cell anchors or help to establish communication between two cells. For example, the formation of filopodial bridges between two adjacent endothelial or epithelial cells is important for the subsequent establishment of adherens junctions. In addition, filopodia can also help the cells to reach and internalize distant targets, such as pathogens.
Filopodia Formation and Disassembly
Actin nucleation, elongation, and bundling are critical for filopodia formation and function. The filopodia core comprises 12-20 actin filaments spaced 12 nm apart. Bundling of these filaments by fascin is crucial to give filopodia the rigidity to maintain its elongated shape.
The disassembly of actin filaments during the retraction phase involves periodic helical and rotational motion of the actin shaft and is regulated by several factors. For instance, capping proteins promote filopodial retraction by shielding the polymerizing ends of the filaments from further elongation. RhoA kinase activity also regulates actin polymerization within filopodia.

Filopodia are thin, actin-rich cellular protrusions that play an important role in many fundamental cellular functions. They vary in their occurrence, ...

Cancer Cell Migration through Invadopodia

Invadosome is a broad category of cell surface structures with proteolytic activity that&#160; degrades the extracellular matrix (ECM). Invadosomes are present in normal cell types, including macrophages, endothelial cells, and neurons, as well as tumor cells. Although the macrophage podosomes and tumor cell invadopodia are classified as invadosomes, they have different structures, molecular pathways, and functions. Podosomes are short structures that last for a few minutes. However, invadopodia can last for hours and are many microns in length.
In tumors, invadopodia are essential for cell intravasation and extravasation through blood vessels. They are similar to lamellipodia and filopodia, with a filamentous actin core shaped by actin nucleators and regulatory factors. The lifecycle of an invadopodium is initiated by complex processes involving various signaling cascades that remodel the cytoskeleton and cell membrane. The actin cytoskeleton is reorganized and assembled into new filaments and branches, which requires the activity of actin nucleators such as formins or the ARP2/3 complex.
The Arp2/3 complex requires nucleation-promoting factors (NPF) for optimal activity as it is slow to spontaneously initiate new actin branches. Cortactin, an NPF and scaffold protein, recruits ARP2/3 to filaments, allowing branching of the actin network. Additionally, cortactin can stabilize the newly generated branches. The formin proteins induce the elongation of unbranched actin filaments, while filament bundling is coordinated by fascin. Together these events promote actin filament polymerization and maturation of the invadopodium.&#160;
After the invadopodium stabilizes, kinesins use the neighboring microtubules as tracks to transport vesicles with proteases from the Golgi network. These vesicles release enzymes such as&#160; matrix metalloproteases, cathepsins, and serine proteases from the membrane of the invadopodia to degrade the surrounding ECM. Lastly, the actin core is disassembled to retract the invadopodium. Several proteins are implicated in the retraction pathway, but it is unclear how these proteins interact during the final stage of the invadopodium life cycle.&#160;

Invadosome is a broad category of cell surface structures with proteolytic activity that&#160; degrades the extracellular matrix (ECM). Invadosomes are ...

Cell Migration

Cell migration, the process by which cells move from one location to another, is essential for the proper development and viability of organisms throughout their life. When cells are not able to migrate properly to their ordained locations, various disorders may occur. For example, disruption in cell migration causes chronic inflammatory diseases such as arthritis.

<h4>General Mechanism</h4>

Generally, cellular migration begins when a cell, such as a fibroblast, responds to an external-polarizing-chemical signal. As a result, one end extends itself as a protrusion called the leading edge, which attaches itself to substrates via secreted adhesive compounds, in its microenvironment. The trailing edge&mdash;the area that serves as the back of the cell&mdash;also adheres to substrates to anchor the cell. After adhesion, the cell is propelled towards its destination by a sequence of contractions that are generated by cytoskeletal motility structures. Then, the adhesive attachment at the trailing edge gets released. These steps are repeated cyclically until the fibroblast reaches its destination.

<h4>Polarization</h4>

There is a diversity in the different types of signaling molecules that initiate cell migration. They illicit two types of responses: chemokinetic and chemotactic. Chemokinesis refers to movement that occurs when signaling molecules either symmetrically or asymmetrically stimulate cell migration without dictating the directionality of the resultant movement. Chemotaxis refers to a movement where a gradient of soluble (chemotactic) or substrate-bound (haptotactic) signaling molecules dictates the directionality of cellular movement.

Membrane receptors such as G-protein coupled receptors (GPCR) and receptor tyrosine kinase receptors (RTK) detect external signaling molecules and cause an accumulation of phosphatidylinositol (3,4,5) triphosphate (PIP3) at the leading edge. The accumulation of PIP3 then leads to the activation of Rho-family Ras-like small proteins called Rac, Cdc42, and Rho. Rac and/or Cdc42 cause cytoskeletal changes such as actin polymerization at the leading edge while Rho causes actin-myosin contractions at the trailing edge. As a result of actin polymerization, protrusions are generated at the leading edge.

<h4>Types of Protrusions</h4>

Actin serves as a physical scaffold for protrusions. Consequently, the shape of protrusion structures varies depending on how actin is assembled. Two commonly studied types of protrusions are lamellipodia and filopodia. Lamellipodia are broad, sheet-like protrusions that contain a branched network of thin, short actin filaments. When lamellipodia lift away from the substrate and move backward, a notably distinct ruffling movement occurs. Lamellipodia protrusions can be found in cells like fibroblasts, immune cells, and neurons. Filopodia are thin-finger-like protrusions that emanate from cell membranes. They are often observed in cells, such as neurons, working in tandem with lamellipodia during migration.