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

Summary

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.

Abstract

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 μm 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 μm and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 μm 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 °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.

Introduction

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 μm in diameter), elongated (up to 50-100 μm 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 cells^2,3.

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 migration^2,3. 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/µm 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 communications^2,3. 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 and for the complete lack of knowledge of TM4SF1 enrichment in 100-300 μm 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 °C 4% PFA and fix cells in a 37 °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 cells^2,3. 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.

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Protocol

1. Cell Culture on Collagen Coated Glass Disk

1. Place glass disks (12 mm in diameter) in a glass jar (4 oz) and autoclave to sterilize the discs.
2. Put 25 ml 70% ethanol in a 50 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.
3. Place a sharp forceps with an extra fine point in the 70% ethanol for 5 min before using it to handle the glass disk.
4. 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.
5. Use the sterile forceps to grab one sterile glass disk 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.
6. Put 500 μl of Bovine collagen solution (50 ng/ml) into each well that contains a glass disk and place it in a 37 °C, 5% CO₂ cell culture incubator for at least 30 min. There is no harm in a longer incubation.
7. Harvest HUVEC (Human Umbilical Vein Endothelial Cells) or PC3 (prostate tumor cells) that were already grown in 150 mm cell culture plate through trypsinization and block the trypsin activity using its complete culture medium. Collect cells into a 15 ml Falcon tube.
8. Count the cell number and make sure the viability is greater than 90%.
9. Pellet the cells by centrifuging at 200 x g for 10 min at 4 °C. Remove the supernatant.
10. Use the culture medium to dilute the cells to 10⁵ cells/ml. Place cells on ice.
11. Aspirate the collagen in the cell culture hood and gently place 500 μl 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.
12. Culture the cells in a 37 °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 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.

2. Cell Fixation

1. Each well will need 500 μl 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.
2. Put the falcon tube in a Styrofoam stand that was already in place in a 37 °C incubator. Leave the tube in the incubator for 30 min to warm up the PFA to 37 °C.
3. Place a heating pad in the culture hood and turn it on.
4. 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.
5. Use one hand to aspirate the medium from a well, and immediately after use the other hand to gently slide 500 μl PFA into the well through the well edge. For easier aspiration, tilt the culture plate at about 45°, 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.
6. Repeat the process until all wells with a glass disk have received PFA. Note: all steps that need to change pre-existing solution from the well will apply same aspiration procedures.
7. Place the plate in 37 °C for 5 min.
8. 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 μl room temperature 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.
9. 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 °C for a long period of time without bacterial contamination.
10. With the culture plate still tilted against a stand, once again cycle through each well aspirating to remove PBS and immediately afterward adding 500 μl of ICC blocking buffer. Cells are now ready for immunofluorescence staining. If necessary, the fixed cells can be stored in a 4 °C refrigerator for a week without significant loss of TM4SF1 protein.

3. TM4SF1 Immunofluorescence Staining of Nanopodia

1. In each well, replace the 500 μl 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.
2. Dilute primary anti-TM4SF1 (or anti-CD9) antibody to 0.5 μg/ml in ICC blocking buffer.
3. In each well, remove the ICC/0.01% Triton buffer and replace it with 300 μl of the anti-TM4SF1-antibody solution. Incubate 2 hr at room temperature or leave overnight at 4 °C.
4. Wash each well with no less than 500 μl PBS. Repeat 3x; each time give at least 5 min of incubation time.
5. Prepare a secondary antibody and phalloidin solution in ICC blocking buffer, using a 1,000 times dilution of Alexa 488 labeled donkey anti-mouse secondary antibody (2 μg/ml final concentration) and 1,000x dilution of phalloidin (50 ng/ml final concentration).
6. Remove PBS and add 300 μl of the secondary antibody and phalloidin solution to each well and incubate for 2 hr or leave it overnight at 4 °C.
7. 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.
8. Drop a single drop (~10 μl) of anti-fade mounting media on a glass slide.
9. Bend the tip of a 19 G 1 ½ in syringe needle 90° by pressing gently on a hard surface. Use one hand to hold bended needle and gently lift up a glass disk from the well, and use the other hand to grasp the disc using the sharp forceps.
10. Turn the glass disk face down letting the cell side contact the mounting media on the glass slide. Gently place the glass disk and let it dry overnight in a dark place at room temperature.
11. Proceed to image the immunofluorescence stained nanopodia, or store the slides in a slide box at -20 °C. The slides will remain viewable for a few months in -20 °C.

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Results

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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Discussion

Nanopodia are thin cellular membrane channels that firmly attach to matrix through TMED and can extend more than 100 μm from a polarized mobile cell to sense the environment and mediate intercellular interactions^2,3. 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 ...

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Materials

Name	Company	Catalog Number	Comments
Glass disks	Fisher Scientific	12-545-82	12 mm diameter
Glass jar	Fisher Scientific	02-912-310	Certified Clean Clear Glass Straight-Sided Jars, 4 oz
Glass slide	Fisher Scientific	12-544-1	Fisherfinest Premium Plain Glass Microscope Slides
50 ml Falcon tube	BD Falcon	352070	50 ml high-clarity polypropylene conical centrifuge tube, 16,000 rcf rating. Sterile.
15 ml Falcon tube with Styrofoam rack	Corning	430790	Corning 15 ml PP Centrifuge Tubes
Sharp forceps	Fisher Scientific	13-812-42	Dissecting Extra Fine Pointed Splinter Forceps
24-well Cell culture plate	BD Bioscience	353047	24-well Cell Culture Plate
150 mm Cell culture plate	BD Bioscience	353025	150 mm cell culture plate
19 G 1 ½ Syringe needle	BD Bioscience	309635	19 G 1 ½
Ethanol	Decon Laboratories, Inc.	2701	Decon's Pure Ethanol 200 Proof
Collagen solution	BD Bioscience	354249	Collagen I, High Concentration, rat tail, 100 mg
Trypsin/EDTA	Cellgro	25-053-CI	0.25% Trypsin-EDTA 1x
DMEM	Life Technologies	11965-092	DMEM high glucose (1x), liquid, with L-glutamine, without sodium pyruvate
10x PBS	Affymetrix	75889 5 LT	PBS, 10x Solution, pH 7.4
PFA (paraformaldehyde)	Affymetrix	19943 1 LT	4% in PBS
FBS	Sigma-Aldrich	F4135-500ML	Fetal Bovine Serum
EGM2-MV	Lonza	CC-3162	EGM-2 BulletKit, EBM-2 Basal Medium 500 ml and EGM-2 SingleQuot Kit Supplement & Growth Factors
Triton X-100	Sigma-Aldrich	T8787	Triton X-100
NaN3	Sigma-Aldrich	S2002-25G	Solubilized in water. 4% stock solution and working concentration is 0.4%.
Mouse anti-human TM4SF1 antibody	Millipore	MAB3127	Epitope is located in extracellular domain
Mouse anti-human CD9 antibody	BD Bioscience	555370	Epitope is located in extracellular domain
Alexa Fluor 488 Donkey anti-mouse 2nd antibody	Life Technologies	A-21202	Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L)
Phalloidin	Chemicon	90324	Rhodamine-conjugated Phalloidin
Anti-fade mounting media	Life Technologies	P-36931	ProLong Gold Antifade Reagent with DAPI
70% Ethanol			17.5 ml of ethanol (200 proof) 7.5 ml of double-deionized water
10x PBS (make 200 ml)			20 ml of 10x PBS 180 ml of double-deionized water
20% Triton X-100 (make 20 ml)			4 ml Triton X-100 16 ml double-deionized water
4% NaN3 (make 25 ml)			1 g of NaN3 25 ml of double-deionized water
50 ng/ml Bovine collagen solution in PBS (make 5 ml)			Stock solution of 50 μg/ml (50 ml) 830 μl of Collagen I (3 mg) 50 ml PBS
ICC Blocking Buffer (50 ml)			49 ml PBS 2% FBS (add 1 ml) 0.04% NaN3 (add 100 μl of 4% NaN3)
ICC Blocking Buffer/0.01% Triton X-100 (make 50 ml)			50 ml of ICC Blocking Buffer 25 μl of 20% Triton X-100
HUVEC	Lonza	C2517A	www.lonza.com
PC3	ATCC	CRL-1435	www.atcc.org
Cell culture hood	NuAIRE	Nu-425-600	NU-425 (Series 60) Biological Safety Cabinet
37 °C, 5% CO2 Cell culture incubator	CellStar	QWJ300DABA	Cellstar CO2 Water Jacketed Incubator
Heating pad	K&H Manufacturing	1020	K&H Lectro Kennel Heated Pad with Free Fleece Cover (www.amazon.com)
Centrifuge	Sorvall	T6000B	Sorvall T6000 (B) Benchtop Centrifuge
Hemocytometer	Sigma-Aldrich	Z359629	Bright-Line Hemocytometer