The GFP-&beta;-actin plasmid was generously provided by Dr. Wayne Orr, LSUHSC-S Department of Pathology, and was amplified in the laboratory of Dr. Becky Worthylake, LSUHSC-NO Department of Pharmacology. This work was supported by grants from the National Institutes of Health (P20 RR-018766) and the American Heart Association (05835386N).

<ol>
	<li>Spindler, V., Schlegel, N., Waschke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+GTPases+in+control+of+microvascular+permeability.">Role of GTPases in control of microvascular permeability.</a> Cardiovasc. Res. 87, 243-253 (2010).</li><li>Wojciak-Stothard, B., Ridley, 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=Rho+GTPases+and+the+regulation+of+endothelial+permeability.">Rho GTPases and the regulation of endothelial permeability.</a> Vascul. Pharmacol. 39, 187-199 (2002).</li><li>Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+pathways+in+enhanced+microvascular+permeability.">Signal transduction pathways in enhanced microvascular permeability.</a> Microcirculation. 7, 395-403 (2000).</li><li>Birukov, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+GTPases+in+mechanosensitive+regulation+of+endothelial+barrier.">Small GTPases in mechanosensitive regulation of endothelial barrier.</a> Microvasc. Res. 77, 46-52 (2009).</li><li>Duran, W. N., Sanchez, F. A., Breslin, J. W., Tuma, R. F., Duran, W. 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P., Herzog, V., Kirfel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+ruffles+in+cell+migration:+indicators+of+inefficient+lamellipodia+adhesion+and+compartments+of+actin+filament+reorganization.">Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization.</a> Exp. Cell. Res. 302, 83-95 (2005).</li><li>Hotulainen, P., Lappalainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fibers+are+generated+by+two+distinct+actin+assembly+mechanisms+in+motile+cells.">Stress fibers are generated by two distinct actin assembly mechanisms in motile cells.</a> J. Cell. 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No conflicts of interest declared.

The imaging of GFP-actin in live endothelial cells enables a detailed analysis of the dynamics of the actin cytoskeleton in response to inflammatory stimuli. This method may also be useful to build upon previous findings showing remodeling of the cytoskeleton in response to physical forces like shear stress11. In addition, this method allows a detailed assessment of the contribution of actin cytoskeletal dynamics to various endothelial cell activities, including migration, mitosis, formation of intercellular junctions and junctional maturation, and maintenance of barrier function. 
In the data shown, the behavior of the endothelial actin cytoskeleton can be observed before and after treatment with thrombin. Local lamellipodia all along the edges of endothelial cells were observed forming and regressing over time in both nonconfluent and confluent cell monolayers. Treatment with thrombin briefly interrupted lamellipodia formation and turnover. Thrombin also caused the cells to contract slightly, in agreement with previous reports that thrombin causes actin stress fiber formation and increased centripetal tension development in endothelial cells12-14. However, from live cell imaging studies such as this, the origin of the stress fibers can now be determined. In HUVEC, most of the stress fibers originate at the cell periphery and resemble transverse arc fibers in migrating cells8, 9. Another strength of this method over using fixed cells is that the number of stress fibers can be quantified in individual cells before and after thrombin treatment, eliminating selection bias between experimental groups. 
With this protocol we evaluate dynamic motion of the cell edge and actin stress fibers. To understand actin monomer dynamics in endothelial cells, more advanced techniques such as fluorescence recovery after photobleaching (FRAP) or fluorescence speckle microscopy (FSM) can be applied15, 16. In addition, because microvascular endothelial cells may represent a better model of microvascular barrier function, optimization of transfection protocols to effectively express GFP-actin in microvascular endothelial cells represents a logical future direction.
In summary, imaging of live endothelial cells expressing GFP-actin represents a powerful tool to determine how the endothelial cell actin cytoskeleton responds to various types of stimuli. Studies using tightly confluent endothelial monolayers will help determine the roles of dynamic structures such as actin-rich lamellipodia and transverse arc stress fibers in endothelial barrier function. In addition, live cell imaging of endothelial cells expressing GFP-actin or other fusion proteins that allow visualization of other subcellular structures will provide detailed spatiotemporal information needed to understand the signaling and structural mechanisms that determine barrier integrity.

<table border="1">
<tbody>
	<tr>
		<td colspan="4">1. Ringer 5x Stock</td>
	</tr>
	<tr>
		<td>Chemical</td>
		<td>Company</td>
		<td>Catalog Number</td>
		<td>Amount</td>
	</tr>
	<tr>
		<td>Sodium Chloride</td>
		<td>EMD</td>
		<td>SX0420-3</td>
		<td>35 g</td>
	</tr>
	<tr>
		<td>Potassium Chloride</td>
		<td>J.T. Baker</td>
		<td>3040</td>
		<td>1.75 g</td>
	</tr>
	<tr>
		<td>Calcium Chloride</td>
		<td>Sigma</td>
		<td>C-3881</td>
		<td>1.47 g</td>
	</tr>
	<tr>
		<td>Magnesium Sulfate</td>
		<td>Sigma</td>
		<td>M-9397</td>
		<td>1.44 g</td>
	</tr>
	<tr>
		<td>Sterile Filtered Water</td>
		<td>N/A</td>
		<td>N/A</td>
		<td>Bring to 1 L</td>
	</tr>
	<tr>
		<td colspan="4">Sterile filter into autoclaved bottles and stores at 4&deg;C</td>
	</tr>
	<tr>
		<td colspan="4">&nbsp;</td>
	</tr>
	<tr>
		<td colspan="4">2. MOPS buffer</td>
	</tr>
	<tr>
		<td>Chemical</td>
		<td>Company</td>
		<td>Catalog Number</td>
		<td>Amount </td>
	</tr>
	<tr>
		<td>MOPS</td>
		<td>Sigma</td>
		<td>M3183</td>
		<td>125.6 g</td>
	</tr>
	<tr>
		<td>Sterile Filtered Water</td>
		<td>N/A</td>
		<td>N/A</td>
		<td>Bring to 1 L</td>
	</tr>
	<tr>
		<td colspan="4">Sterile filter into autoclaved bottles and stores at 4&deg;C</td>
	</tr>
	<tr>
		<td colspan="4">&nbsp;</td>
	</tr>
	<tr>
		<td colspan="4">3. Albumin Physiological Salt Solution (APSS)</td>
	</tr>
	<tr>
		<td>Chemical</td>
		<td>Company</td>
		<td>Catalog Number</td>
		<td>Amount </td>
	</tr>
	<tr>
		<td>Ringer stock (5x)</td>
		<td>N/A</td>
		<td>N/A</td>
		<td>200 mL </td>
	</tr>
	<tr>
		<td>Mops Buffer</td>
		<td>N/A</td>
		<td>N/A</td>
		<td>5 mL </td>
	</tr>
	<tr>
		<td>Sodium Phosphate</td>
		<td>Sigma</td>
		<td>S-9638</td>
		<td>0.168 g</td>
	</tr>
	<tr>
		<td>Sodium Pyruvate</td>
		<td>Sigma</td>
		<td>P5280</td>
		<td>0.22 g</td>
	</tr>
	<tr>
		<td>EDTA sodium salt</td>
		<td>Sigma</td>
		<td>ED2SS</td>
		<td>0.0074 g</td>
	</tr>
	<tr>
		<td>Glucose</td>
		<td>Sigma</td>
		<td>G7528</td>
		<td>0.901 g</td>
	</tr>
	<tr>
		<td>Albumin, Bovine</td>
		<td>USB</td>
		<td>10856</td>
		<td>10 g</td>
	</tr>
	<tr>
		<td>Sterile Filtered Water</td>
		<td>N/A</td>
		<td>N/A</td>
		<td>Bring to 1 L</td>
	</tr>
	<tr>
		<td colspan="4">Adjust pH to 7.4 at 37&deg;C, then sterile filter into autoclaved bottles and store at 4&deg;C.</td>
	</tr>
	<tr>
		<td colspan="4">&nbsp;</td>
	</tr>
	<tr>
		<td colspan="4">4. 0.9% Saline</td>
	</tr>
	<tr>
		<td>Chemical</td>
		<td>Company</td>
		<td>Catalog Number</td>
		<td>Amount </td>
	</tr>
	<tr>
		<td>Sodium Chloride</td>
		<td>EMD</td>
		<td>SX0420-3</td>
		<td>9 g</td>
	</tr>
	<tr>
		<td>Sterile Filtered Water</td>
		<td>N/A</td>
		<td>N/A</td>
		<td>Bring to 1 L</td>
	</tr>
	<tr>
		<td colspan="4">Sterile filter into autoclaved bottles and stores at 4&deg;C</td>
	</tr>
	<tr>
		<td colspan="4">&nbsp;</td>
	</tr>
	<tr>
		<td colspan="4">5. 1.5 % Gelatin Solution</td>
	</tr>
	<tr>
		<td>Gelatin from porcine skin</td>
		<td>Sigma</td>
		<td>G2500</td>
		<td>15 g</td>
	</tr>
	<tr>
		<td>0.9% Saline</td>
		<td>N/A</td>
		<td>N/A</td>
		<td>Bring to 1 L</td>
	</tr>
	<tr>
		<td colspan="4">Warm the solution to 37&deg;C to dissolve gelatin sufficiently. While still warm, sterile filter into autoclaved bottles and store at 4&deg;C</td>
	</tr>	
</tbody>
</table>

study of the actin cytoskeleton in live endothelial cells expressing gfp-actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Watch this Scientific Journal Video about Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin at JoVE.com

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

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The Cytoskeleton I: Actin and Microfilaments

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26.9K Views.  Louisiana State University Health Sciences Center. Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.