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 intera....

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. ....

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