Name: imaging of podocytic proteins nephrin, actin, and podocin with expansion microscopy
Uploaded: 2021-04-23T14:30:00
Description: Watch this Scientific Journal Video about Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy at JoVE.com

The authors would like to thank Blanka Duvnjak and Nikola Kuhr for their excellent technical assistance.

1. Splitting and seeding of cells
<ol>
	<li>Warm up sterile Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) including 10% fetal calf serum (FCS), sterile Phosphate buffered saline (PBS) and sterile trypsin to 37 &#176;C. Activate the clean bench.</li>
	<li>Prepare a 6-well plate by adding one sterile glass cover slip (10 mm) to each well using sterile forceps.</li>
	<li>Put a 10 cm cell culture dish with Cos7 cells under the clean bench. Under the clean bench, aspirate the cells&#39; medium using a vacuum device.</li...

<ol>
	<li>Matsushita, K., 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=Association+of+estimated+glomerular+filtration+rate+and+albuminuria+with+all-cause+and+cardiovascular+mortality+in+general+population+cohorts:+a+collaborative+meta-analysis.">Association of estimated glomerular filtration rate and albuminuria with all-cause and cardiovascular mortality in general population cohorts: a collaborative meta-analysis.</a> Lancet. 375 (9731), 2073-2081 (2010).</li><li>Faul, C., Asanuma, K., Yanagida-Asanuma, E., Kim, K., Mundel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+up:+regulation+of+podocyte+structure+and+function+by+components+of+the+actin+cytoskeleton.">Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton.</a> Trends in Cell Biology. 17 (9), 428-437 (2007).</li><li>Saleem, M. 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=Co-localization+of+nephrin,+podocin,+and+the+actin+cytoskeleton+-+Evidence+for+a+role+in+podocyte+foot+process+formation.">Co-localization of nephrin, podocin, and the actin cytoskeleton - Evidence for a role in podocyte foot process formation.</a> American Journal of Pathology. 161 (4), 1459-1466 (2002).</li><li>Kestila, 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=Positionally+cloned+gene+for+a+novel+glomerular+protein+-+nephrin+-+is+mutated+in+congenital+nephrotic+syndrome.">Positionally cloned gene for a novel glomerular protein - nephrin - is mutated in congenital nephrotic syndrome.</a> Molecular Cell. 1 (4), 575-582 (1998).</li><li>Huber, T. B., 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=Podocin-mediated+recruitment+of+nephrin+into+lipid+rafts+is+required+for+efficient+nephrin+signaling.">Podocin-mediated recruitment of nephrin into lipid rafts is required for efficient nephrin signaling.</a> Journal of the American Society of Nephrology. 14, 8 (2003).</li><li>Boute, N., 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=NPHS2,+encoding+the+glomerular+protein+podocin,+is+mutated+in+autosomal+recessive+steroid-resistant+nephrotic+syndrome.">NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.</a> Nature Genetics. 24 (4), 349-354 (2000).</li><li>Galbraith, C. G., Galbraith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+at+a+glance.">Super-resolution microscopy at a glance.</a> Journal of Cell Science. 124 (10), 1607-1611 (2011).</li><li>Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+reveals+that+synaptotagmin+remains+clustered+after+synaptic+vesicle+exocytosis.">STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.</a> Nature. 440 (7086), 935-939 (2006).</li><li>van de Linde, S., 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=Direct+stochastic+optical+reconstruction+microscopy+with+standard+fluorescent+probes.">Direct stochastic optical reconstruction microscopy with standard fluorescent probes.</a> Nature Protocols. 6 (7), 991-1009 (2011).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nature Methods. 3 (10), 793-795 (2006).</li><li>Testa, I., 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=Multicolor+fluorescence+nanoscopy+in+fixed+and+living+cells+by+exciting+conventional+fluorophores+with+a+single+wavelength.">Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength.</a> Biophysical Journal. 99 (8), 2686-2694 (2010).</li><li>Wassie, A. T., Zhao, Y., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+microscopy:+principles+and+uses+in+biological+research.">Expansion microscopy: principles and uses in biological research.</a> Nature Methods. 16 (1), 33-41 (2019).</li><li>Tillberg, P. 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=Protein-retention+expansion+microscopy+of+cells+and+tissues+labeled+using+standard+fluorescent+proteins+and+antibodies.">Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies.</a> Nature Biotechnology. 34 (9), 987-992 (2016).</li><li>Truckenbrodt, S., Sommer, C., Rizzoli, S. O., Danzl, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+optimization+in+X10+expansion+microscopy.">A practical guide to optimization in X10 expansion microscopy.</a> Nature Protocols. 14 (3), 832-863 (2019).</li><li>Truckenbrodt, S., 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=X10+expansion+microscopy+enables+25-nm+resolution+on+conventional+microscopes.">X10 expansion microscopy enables 25-nm resolution on conventional microscopes.</a> Embo Reports. 19 (9), (2018).</li><li>Chang, J. B., 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=Iterative+expansion+microscopy.">Iterative expansion microscopy.</a> Nature Methods. 14 (6), 593-599 (2017).</li><li>Chozinski, T. J., 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=nanoscale+optical+imaging+of+mouse+and+human+kidney+via+expansion+microscopy.">nanoscale optical imaging of mouse and human kidney via expansion microscopy.</a> Scientific Reports. 8 (1), 10396 (2018).</li><li>Richter, K. N., 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=Glyoxal+as+an+alternative+fixative+to+formaldehyde+in+immunostaining+and+super-resolution+microscopy.">Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.</a> The EMBO Journal. 37 (1), 139-159 (2018).</li><li>Rinschen, M. 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=Quantitative+deep+mapping+of+the+cultured+podocyte+proteome+uncovers+shifts+in+proteostatic+mechanisms+during+differentiation.">Quantitative deep mapping of the cultured podocyte proteome uncovers shifts in proteostatic mechanisms during differentiation.</a> American Journal of Physiology-Cell Physiology. 311 (3), 404-417 (2016).</li><li>Asano, S. 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=Expansion+microscopy:+protocols+for+imaging+proteins+and+RNA+in+cells+and+tissues.">Expansion microscopy: protocols for imaging proteins and RNA in cells and tissues.</a> Current Protocols in Cell Biology. 80 (1), 56 (2018).</li><li>Wen, G., 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=Evaluation+of+direct+grafting+strategies+via+trivalent+anchoring+for+enabling+lipid+membrane+and+cytoskeleton+staining+in+expansion+microscopy.">Evaluation of direct grafting strategies via trivalent anchoring for enabling lipid membrane and cytoskeleton staining in expansion microscopy.</a> ACS Nano. 14 (7), 7860-7867 (2020).</li><li>Chen, F., 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=Nanoscale+imaging+of+RNA+with+expansion+microscopy.">Nanoscale imaging of RNA with expansion microscopy.</a> Nature Methods. 13 (8), 679-684 (2016).</li><li>Gambarotto, D., 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=Imaging+cellular+ultrastructures+using+expansion+microscopy+(U-ExM).">Imaging cellular ultrastructures using expansion microscopy (U-ExM).</a> Nature Methods. 16 (1), 71-74 (2019).</li><li>Zhao, Y., 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=Nanoscale+imaging+of+clinical+specimens+using+pathology-optimized+expansion+microscopy.">Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.</a> Nature Biotechnology. 35 (8), 757-764 (2017).</li></ol>

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...

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&#39;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 cytoskeleton2,3. Mutations in the nephrin-encoding gene NPHS1 lead to nephrotic syndrome of the Finnish type4.
One of nephrin&#39;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 microscopes14,15,16.
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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrylamide &#62;99%</td>
			<td>Sigma-Aldrich</td>
			<td>A3553-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>6-((Acryloyl)amino)hexanoic acid, succinimidyl ester, Acryloyl-X, SE</td>
			<td>invitrogen</td>
			<td>A-20770</td>
			<td>store up to 4 months</td>
		</tr>
		<tr>
			<td>APS</td>
			<td>Sigma-Aldrich</td>
			<td>A3678-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Deckgl&#228;ser (cover glasses)</td>
			<td>Engelbrecht</td>
			<td>K12432</td>
			<td>24x32mm #1.0</td>
		</tr>
		<tr>
			<td>Diamont cutter</td>
			<td>VWR</td>
			<td>201-0392</td>
			<td>for cutting the cover slips</td>
		</tr>
		<tr>
			<td>Guanidine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>G3272-100G</td>
			<td>8M Stock can be kept at RT</td>
		</tr>
		<tr>
			<td>Marten hair paintbrush</td>
			<td>Leon Hardy</td>
			<td>3 (770)</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Menzel&#34; Deckgl&#228;ser (cover glasses)</td>
			<td>Thermo Fischer&#160;</td>
			<td>15654786</td>
			<td>24x24mm #1.5</td>
		</tr>
		<tr>
			<td>N,N`-Methylenbisacrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>M7256-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Objekttr&#228;ger UniMark</td>
			<td>Marienfeld</td>
			<td>703010</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>New England Biolabs</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acrylate</td>
			<td>Sigma-Aldrich</td>
			<td>408220</td>
			<td>check purity&#160;</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining chamber</td>
			<td></td>
			<td></td>
			<td>produced at the university&#39;s workshop</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>ROTH</td>
			<td>2367.1</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Well glass bottom plates</td>
			<td>Cellvis</td>
			<td>P06-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Actin-ExM 546&#160;</td>
			<td>chrometra</td>
			<td>non-available</td>
			<td>1:40</td>
		</tr>
		<tr>
			<td>Anti Podocin produced in rabbit</td>
			<td>Sigma</td>
			<td>P-0372-200UL</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Donkey anti guinea-pig CF633</td>
			<td>Sigma</td>
			<td>SAB4600129-50UL</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Goat anti rabbit 488</td>
			<td>Life Technologies</td>
			<td>A11034</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Guinea pig anti nephrin</td>
			<td>Origene</td>
			<td>BP5030</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Visiview</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AXIO Observer Z1</td>
			<td>Zeiss</td>
			<td>non-available</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging of podocytic proteins nephrin, actin, and podocin with expansion microscopy

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&#39;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.

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...

Watch this Scientific Journal Video about Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy at JoVE.com

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

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

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte ...

This expansion microscopic protocol enables the investigator to visualize glomerular proteins with nanoscale resolution on a conventional microscope. The main advantage of expansion microscopy is that it is accessible to a large research community. Begin by making spacers for the gelation chamber.Cut number one and number 1.5 glass cover slips into five millimeter stripes, using a diamond knife. Place a glass slide into a staining chamber and position the number 1.5 cover slip stripes on the glass slide, so that they form a 2.5 centimeter square. Pipette a droplet of double distilled water into the corners of the square to adhere the glass cover slip stripes to each other and to the glass slide.Wait for approximately 20 minutes so that the number 1.5 cover slips are stably attached. Pipette a droplet of water on each number 1.5 cover slip stripe. Place four number one glass cover slip stripes onto the number 1.5 cover slips.For the gelation chamber lid, wrap a cover glass with paraffin film, avoiding any folds or dirt on the film. For anchoring treatment, remove PBS from the immune stained cells and add 250 microliters of anchoring buffer per well directly onto the glass cover slip. Incubate the plate for three hours at room temperature.Keep the substrate in the dark using a box. After the incubation, remove the anchoring buffer and wash the cover slip once with 1.5 milliliters of PBS per well. To stain actin fibers, thaw an EXM compatible phalloidin solution and incubate it for 45 minutes at room temperature.Meanwhile, dissolve sodium acrylate in double distilled water using a stirring device and prepare the monomer solution on ice. Remove phalloidin from the cells and wash them with 1.5 milliliters of PBS, twice, at room temperature. Leave 1.5 milliliters of PBS within the well after the last wash.Use forceps and a cannula to lift the cover glass slip from the six well plate and place it into the gelation chamber. Add APS to the gelling solution and vortex briefly. Pipette 200 microliters of gelling solution on the sample and cautiously close the chamber, avoiding air bubbles within the gel.Add water to the staining chamber, and incubate the staining chamber for at least one hour at 37 degrees Celsius to polymerize the gel. Take the staining chamber out of the incubator. To open the gelation chamber lid, introduce a razor blade between the lid and the spacer, and remove the lid cautiously.Remove the spacers with the razor blade and cut off all extra gel. Use a paint brush to detach the edges of the gel. Put the slide with the gel and cover glass into a dish filled with PBS.Then remove the detached cover glass from the gel by shaking it gently. Put the slide below the gel to attach the gel to the slide. With the gel on the slide, divide the gel into small pieces using the razor blade.Gently push one piece of gel into a well of a six well plate with a glass bottom and unfold it with a paintbrush. Use the paintbrush to keep the gel moisturized with a small amount of PBS. Using an inverted microscope, take overview images with low numerical aperture for pre-expansion images.Dilute Proteinase K to four units per milliliter in digestion buffer to make the digestion solution. Add 500 microliters of the digestion solution to each well, and immerse the gel within the solution. Let it digest overnight at room temperature with the lid closed to keep the samples in the dark.Remove the digestion solution with a pipette and discard it. Add one milliliter of double distilled water and incubate the immersed gel for 10 minutes at room temperature. Remove the water and add one milliliter of fresh, double distilled water.Continue exchanging water every 10 minutes until a plateau of expansion is reached, causing the gel to become optically clear. Remove the water from the gel and immediately start microscopy. Using an inverted microscope, use an air objective at low magnification to find cells in the pre-expansion state.Switch to 40 X and 63 X objectives for better resolution. Excite with the wavelength of interest and take the image via the camera. This EXM protocol enables expansion of up to four fold.To determine the expansion factor it is essential to image cells before and after expansion. Insufficient anchoring and homogenization may lead to distortions and ruptures of cells. Representative examples of ruptured cells are shown here.This method can be used to investigate the colocalization of F-actin and actin adapter proteins, such as podocin and nephrin. Podocin is depicted in green, actin in blue, and nephrin in red. White areas indicate colocalization.The fluorescent signal intensity and abundance are key for successful EXM. Efficient anchoring of the fluorescent dyes via ACX is of critical importance for this protocol. In our hands solubilized ACX is good for up to three to four months.

imaging-podocytic-proteins-nephrin-actin-podocin-with-expansion

Research

JoVE Journal

Biochemistry

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein&#39;s solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Here, we characterize protein structure and interaction sites in living cells using a protein footprinting technique termed in-cell fast photochemical oxidation of proteins (IC-FPOP).

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.