Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

Summary

The presented method enables visualization of fluorescently labeled cellular proteins with expansion microscopy leading to a resolution of 70 nm on a conventional microscope.

Abstract

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte foot processes contain actin bundles that bind to cytoskeletal adaptor proteins such as podocin. Those adaptor proteins, such as podocin, link the backbone of the glomerular slit diaphragm, such as nephrin, to the actin cytoskeleton. Studying the localization and function of these and other podocytic proteins is essential for the understanding of the glomerular filter's role in health and disease. The presented protocol enables the user to visualize actin, podocin, and nephrin in cells with super resolution imaging on a conventional microscope. First, cells are stained with a conventional immunofluorescence technique. All proteins within the sample are then covalently anchored to a swellable hydrogel. Through digestion with proteinase K, structural proteins are cleaved allowing isotropical swelling of the gel in the last step. Dialysis of the sample in water results in a 4-4.5-fold expansion of the sample and the sample can be imaged via a conventional fluorescence microscope, rendering a potential resolution of 70 nm.

Introduction

Albuminuria is a surrogate parameter of cardiovascular risk and results from disruption of the glomerular filter¹. The glomerular filter is composed of the fenestrated endothelium, the glomerular basement membrane and the slit diaphragm formed by podocytes. Primary and secondary foot processes of podocytes wrap around the capillary wall of the glomerulum². The delicate structure of foot processes is maintained by cortical actin bundles which also serve as anchors for multiple slit diaphragm proteins and other adaptor proteins². The slit diaphragm's backbone protein is called nephrin and interacts in a homophilic manner with nephrin molecules of opposing podocytes. Via diverse adaptor proteins, nephrin is linked to the actin cytoskeleton^2,3. Mutations in the nephrin-encoding gene NPHS1 lead to nephrotic syndrome of the Finnish type⁴.

One of nephrin's interacting proteins is podocin, a hairpin-like protein of the stomatin family³. Podocin recruits nephrin to lipid rafts and links it to the actin cytoskeleton⁵. Podocin is encoded by the NPHS2 gene. Mutations in NPHS2 lead to steroid-resistant nephrotic syndrome⁶.

To visualize and co-localize actin adaptor proteins, immunofluorescence techniques may be used. Unfortunately, the diffraction barrier of the light limits the resolution of conventional fluorescence microscopes to 200-350 nm⁷. Novel microscopy techniques, e.g., stimulated emission depletion (STED)⁸, photo-activated localization microscopy (PALM)⁹, stochastic optical reconstruction microscopy (STORM or dSTORM) or ground state deletion microscopy followed by individual molecule return (GSDIM)^9,10,11, enable a resolution up to approximately 10 nm. However, these super resolution techniques require highly expensive microscopes, well-trained personnel and are therefore not available in many laboratories.

Expansion microscopy (ExM) is a novel and simple technique that enables super resolution imaging with conventional microscopes and is potentially available to a large research community¹². In protein retention expansion microscopy (proExM), the sample of interest (cells or tissue) is fixed and stained with fluorophores¹³. Proteins within the sample are then covalently anchored by a small molecule (6-((Acryloyl)amino)hexanoic acid, succinimidyl ester, AcX) into a swellable hydrogel¹³. Through enzymatic digestion with proteinase K (ProK), proteins and fluorophores maintain their relative position within the gel after expansion¹³. After swelling of the gel, the sample expands up to 4.5-fold (90-fold volumetric expansion) leading to an effective lateral resolution of approximately 60-70 nm (300 nm/4.5). Modifications of this technique can even allow for a 10-fold expansion (1,000-fold volumetric expansion), rendering a resolution of 20-30 nm on conventional microscopes^14,15,16.

Glomerular structures of mouse and human kidneys have been visualized via ExM¹⁷. Within this paper, we present a detailed proExM protocol to visualize super resolution images of F-actin and the actin-adapter protein podocin within cells using a conventional fluorescence microscope.

Protocol

1. Splitting and seeding of cells

1. Warm up sterile Dulbecco's Modified Eagle's Medium (DMEM) including 10% fetal calf serum (FCS), sterile Phosphate buffered saline (PBS) and sterile trypsin to 37 °C. Activate the clean bench.
2. Prepare a 6-well plate by adding one sterile glass cover slip (10 mm) to each well using sterile forceps.
3. Put a 10 cm cell culture dish with Cos7 cells under the clean bench. Under the clean bench, aspirate the cells' medium using a vacuum device.
4. Hold the angulated cell culture dish in one hand and add 10 mL of sterile PBS to the side of the cell culture dish to avoid rinsing off cells. Put the cell culture dish down so that PBS rinses the complete cell culture dish.
5. Remove the PBS and add 1 mL of trypsin to the middle of the cell culture dish and incubate for 5 min at 37 °C.
6. Stop the trypsinization reaction by adding 10 mL of DMEM including 10% FCS and pipette the cell solution up and down with a pipettor to manually separate the cells.
7. Analyze the cell number per milliliter using a counting chamber.
8. Seed 68,000 cells in 2 mL medium per well onto the glass cover slips in the 6-well plate. Distribute the cells within the 6-well plate by cautiously shaking the plate horizontally and vertically.
   NOTE: Cells should lie scattered.
9. Incubate the cells overnight in a 37 °C incubator with 5% CO₂.

2. Transfection of cells

1. Prepare two sterile 1.5 mL tubes (one for diluted DNA (A) and one for diluted reagent for cationic lipid transfection (B)). Pipette 0.75 µg of nephrin and 0.75 µg of podocin cDNA expression plasmid per well into one 1.5 mL tube and dilute it into 100 µL of reduced serum medium per well. Add 3 µL of cationic lipid transfection reagent per well to the second 1.5 mL tube and dilute with 100 µL of reduced serum medium. Incubate both reactions for 5 min at room temperature.
2. Combine both reactions (A and B) to a DNA-lipid complex and incubate for 20 min at room temperature. Cautiously add 200 µL of the DNA-lipid complex to each well.
3. Incubate the transfected cells at 37 °C with 5% CO₂ for 48 h without changing the medium.

3. Immunolabeling of cellular structures

1. Prepare a fixing solution (4% (w/v) paraformaldehyde in PBS, 1 mL/well), the permeabilization solution (0.5% (w/v) Triton X-100 in PBS, 1 mL/well), and a blocking solution (5% (v/v) bovine serum albumin in PBS, 1 mL/well).
2. Remove the medium with a vacuum device. Add 2 mL of PBS to each well to remove extra medium. Aspirate the PBS completely.
   NOTE: To avoid washing away cells with PBS, make sure to pipette PBS not onto the glass cover slip directly.
3. Fix cells with 4% (w/v) paraformaldehyde (PFA) dissolved in PBS for 10 min at room temperature.
   NOTE: Alternatively, fix cells in 3% (v/v) glyoxal-ethanol¹⁸.
4. Discard PFA and wash cells twice in PBS (2 mL each) by avoiding direct pipetting onto the glass cover slips. Permeabilize the fixed cells with Triton X-100 0.5% (w/v) in PBS for 10 min at room temperature.
5. Remove the permeabilization solution and wash twice with PBS as indicated in step 3.2.
6. Block the cells by adding 1 mL of 5% (v/v) BSA in PBS for 1 h at room temperature. Incubate the cells with 200 µL of the primary antibody (anti-podocin antibody 1:200 in 1% (v/v) BSA in PBS) overnight at 4 °C.
   NOTE: Alternatively, incubate the primary antibody for 1 h at room temperature.
7. Remove the primary antibody and wash three times with PBS as in step 3.2. Add the secondary antibody (goat anti-rabbit Alexa 488 1:1000 in 1% (v/v) BSA in PBS) for 1 h at room temperature. Keep the cells in the dark using a box.
8. Discard the secondary antibody and wash with PBS three times.
9. Remove PBS and incubate the cells with 200 µL of anti-nephrin antibody 1:100 in 1% (v/v) BSA in PBS for 1 h at room temperature. Wash with PBS three times. Keep the cells in the dark using a box.
10. Remove PBS and incubate with the secondary antibody donkey anti-guinea pig 633, 1:200, for 1 h at room temperature. Wash with PBS three times. Keep the cells in the dark using a box.

4. Expansion microscopy

1. Preparation
   1. To form the spacers for the gelation chamber, cut glass cover slips #1.0 and #1.5 into 5 mm stripes (four each per glass slide) using a diamond knife. Position the #1.5 mm cover slip stripes so that they form a square of 2.5 cm length on a glass slide and place them into a staining chamber (Figure 2A1-C1). Pipette a droplet of ddH₂O into the corners of the square to adhere the glass cover slip stripes to each other and to the glass slide (Figure 2A2-C2).
      NOTE: Avoid complete drying of ddH₂O as the adhesion force will get lost. If needed, apply more droplets of ddH₂O. Wait for approximately 20 min so that #1.5 mm cover slips are stably attached before starting with step 4.1.2.
   2. Place four #1.0 glass cover slip stripes per cover slip onto the #1.5 mm cover slips. Adhere the stripes by pipetting a droplet on each #1.5 mm cover slip stripe (Figure 2A3-C3).
      NOTE: The gel's thickness should be at least 0.15 mm to facilitate handling in the progress of the protocol. However, to avoid excessive gel on top of the cells, keep the spacers height close to the cell substrate. By using #1.5 and #1.0 cover slip stripes as spacers, the spacers height will be approximately 0.3 mm. The cells adhere to the cover glass (height 0.12 mm). Therefore, the gel will be thick enough to handle but excessive gel on top of the cells is avoided using #1.0 and #1.5 cover glass stripes as spacers.
   3. For the gelation chamber lid, wrap a cover glass (#1.5) with paraffin film. Avoid any folds or dirt on the paraffin film (Figure 2A5-C5).
2. Anchoring and polymerization (gelation)
   1. Prepare the anchoring buffer (see Table 1). For anchoring treatment, replace PBS with 250 µL of anchoring buffer per well directly onto the glass cover slip and incubate for 3 h at room temperature. Keep substrate in the dark using a box.
      ​NOTE: Alternatively, incubate overnight at room temperature. Prepare fresh anchoring buffer for every experiment and wait for 10-15 min until it is dissolved properly. 6-((Acryloyl)amino)hexanoic acid, succinimidyl ester (AcX) should be replaced every 4-5 months to ensure adequate anchoring.
   2. Remove the anchoring buffer and wash once with 1.5 mL PBS per well.
   3. To stain actin fibres, thaw an ExM compatible phalloidin-solution and incubate the phalloidin (5 µL of phalloidin diluted in 195 µL of 1% (v/v) BSA in PBS/well) for 45 min at room temperature. Keep samples in the dark using a box.
   4. In the meantime, dissolve sodium acrylate in ddH₂O using a stirring device. On ice, prepare the monomer solution (see Table 1).
      NOTE: Dissolved sodium acrylate should be a clear and colorless solution. If the solution is yellow, replace with new sodium acrylate.
   5. Prepare the gelling solution on ice (see Table 1). Pipette ammoniumperoxidsulfate (APS) into the gelling solution right before the gelling solution is applied to the gelation chamber.
   6. Remove phalloidin from the cells and wash with 1.5 mL of PBS twice at room temperature. Leave 1.5 mL PBS within the well to facilitate removal of the sample on the glass cover slip.
   7. Place the cells on the cover glass into the gelling chamber using forceps and a cannula to lift the cover glass slip from the 6-well plate.
      NOTE: The cells should be on the top of the glass cover slip. The glass cover slip should not touch the spacers.
   8. Add APS to the gelling solution and vortex shortly. Pipette 200 µL of gelling solution on the sample (Figure 2A4-C4). Cautiously close the gelation chamber by avoiding air bubbles within the gel (Figure 2A5-C5).
   9. Incubate the gelling chamber for at least 1 h at 37 °C to polymerize the gel in the wet staining chamber.
3. Homogenization (digestion)
   1. Take the staining chamber out of the incubator. To open the gelation chamber lid, introduce a razor blade between the lid and the spacer. Remove the lid cautiously. Remove the spacers with the razor blade and eliminate all extra gel by cutting it with the razor blade.
   2. Put the slide with the gel and cover glass into a dish filled with PBS. By shaking gently, remove the detached cover glass from the gel. To remove the gel from the dish easily, put the slide below the gel to attach the gel to the slide.
   3. With the gel on the slide, divide the gel in small pieces (quarter of the gel is divided into two to three pieces) using the razor blade. Gently push one piece of gel into a well of a 6-well plate with glass bottom and enfold it by a paintbrush. Keep the gel moisturized with a little amount of PBS using the paintbrush to avoid dehydration of the gel.
      NOTE: The cells face downwards.
   4. Using an inverted microscope, take overview images with low numerical aperture to determine the expansion factor after expansion.
   5. Prepare the digestion buffer (see Table 1). Dilute Proteinase K to 4 U/mL in digestion buffer to receive the digestion solution.
      NOTE: Digestion buffer without Proteinase K can be stored at 4 °C for 1-2 weeks.
   6. Add 500 µL of the digestion solution to each well and immerse the gel within the solution. Let it digest overnight at room temperature and close the lid keeping the samples in the dark.
      NOTE: Alternatively, let it digest at 37 °C for 1 h.
4. Expansion
   1. Remove the digestion solution with a pipette and discard it. Add 1 mL of ddH₂O. Incubate the immersed gel for 10 min at room temperature.
   2. Remove the water and add 1 mL of fresh ddH₂O. Wait for 10 min and continue exchanging water every 10 min until a plateau of expansion is reached.
      NOTE: Sample expansion up to 4.5-fold is achievable. The gel becomes optically clear.
5. Imaging
   1. Remove the water from the gel and directly start microscopy. Using an inverted microscope, primarily use an air objective (low magnification) to find imaged cells in the pre-expansion state (step 4.3.4).
   2. Switch to a 40x (oil/water) and 63x objective for better resolution. Excite with the wavelength of interest and take the image via the camera.
6. Validation
   1. Take an overview image of the sample. Find and match the same structures within the sample that were imaged in step 4.3.4. Use the channel with the best signal-to-noise ratio for validation (Figure 3 A-B).
      NOTE: Adjust the imaging parameters to achieve similar brightness as in the image acquired in the non-expanded state (step 4.3.4).
   2. Overlay pre- and post-expansion images by rotating and shifting them with ImageJ. Use the distance measuring tool in ImageJ to measure the distances between clearly identifiable structures. Measure at least 10 different structures.
      NOTE: Alternatively, use a Python script to measure distances as described in¹⁴.
   3. Calculate the expansion factor by dividing post-/pre-expansion measurements.
   4. To determine distortions, take images with a higher numerical aperture (Figure 4A-B). Overlay these images and analyze them with ImageJ or as described in¹⁵.
      NOTE: Determination of distortions should be performed routinely but not necessarily on every sample.

Results

The concept and timing of this proExM protocol is depicted in Figure 1. On day 5, transfected cells are fixed and stained with fluorescent antibodies targeting the protein of interest (Figure 1A,B). On day 6, treatment with AcX leads to formation of amine groups on all proteins (including fluorophores) (Figure 1A,B)¹². Upon polymerization of t...

Discussion

The presented method enables the investigator to visualize cellular proteins, e.g., podocin, nephrin, and cytoskeletal components, e.g., F-actin. Within this protocol, transfected cos7 cells are used as a model to study interaction of slit diaphragm proteins with F-actin. Unfortunately, immortalized podocyte cell lines do not express sufficient endogenous amounts of slit diaphragm proteins¹⁹.

With this method, cellular proteins can be visualized with nanoscale resolutio...

Materials

Name	Company	Catalog Number	Comments
Acrylamide >99%	Sigma-Aldrich	A3553-100G
6-((Acryloyl)amino)hexanoic acid, succinimidyl ester, Acryloyl-X, SE	invitrogen	A-20770	store up to 4 months
APS	Sigma-Aldrich	A3678-25G
Deckgläser (cover glasses)	Engelbrecht	K12432	24x32mm #1.0
Diamont cutter	VWR	201-0392	for cutting the cover slips
Guanidine HCl	Sigma-Aldrich	G3272-100G	8M Stock can be kept at RT
Marten hair paintbrush	Leon Hardy	3 (770)
"Menzel" Deckgläser (cover glasses)	Thermo Fischer	15654786	24x24mm #1.5
N,N`-Methylenbisacrylamide	Sigma-Aldrich	M7256-25G
Objektträger UniMark	Marienfeld	703010
Proteinase K	New England Biolabs	P8107S
Sodium Acrylate	Sigma-Aldrich	408220	check purity
Sodium Bicarbonate	Sigma-Aldrich	S5761
Staining chamber			produced at the university's workshop
TEMED	ROTH	2367.1
6-Well glass bottom plates	Cellvis	P06-1.5H-N
Antibodies
Actin-ExM 546	chrometra	non-available	1:40
Anti Podocin produced in rabbit	Sigma	P-0372-200UL	1:200
Donkey anti guinea-pig CF633	Sigma	SAB4600129-50UL	1:200
Goat anti rabbit 488	Life Technologies	A11034	1:1000
Guinea pig anti nephrin	Origene	BP5030	1:100
Software
FIJI
Visiview
microscope
AXIO Observer Z1	Zeiss	non-available