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

Abstract

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 integrity [1][2][3][4][5] . A cortical actin belt is thought to be important for the maintenance of stable junctions 1,2,4,5 . In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions [2][3][4][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 structures 5 .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-β-actin plasmid and grown to confluence on glass coverslips. Timelapse 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. Video LinkThe video component of this article can be found at https://www.jove.com/video/3187/ Protocol 1. Transfection of HUVEC with GFP-actin 1. Various methods can be used to transfect HUVEC. Our lab uses the Nucleofector system (Lonza, Basel Switzerland) outlined below. In general, work quickly to improve cell viability and transfection efficiency. Each transfection requires 5 x 10 5 HUVEC that will be seeded onto two glass coverslips (Corning No. 1, 22 x 50 mm). The Nucleofector combines ...

Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

“…Experiments were performed as previously described17, 18, 39 using a Nikon Eclipse TE2000U inverted microscope system (Nikon Instruments, Melville, NY). Briefly, HUVEC expressing GFP‐actin, with or without co‐expression of mCherryRnd3, were perfused with warm (37°C) albumin–physiological salt solution (APSS; NaCl, 120 mmol/L; KCl, 4.7 mmol/L; CaCl 2 ·2H 2 O, 2 mmol/L; MgSO 4 ·7H 2 O, 1.2 mmol/L; NaH 2 PO 4 , 1.2 mmol/L; Na pyruvate, 2 mmol/L; glucose, 5 mmol/L; EDTA, 0.02 mmol/L; MOPS, 3 mmol/L; purified BSA 1 g/100 mL; pH 7.4).…”

Dynamics between actin and the VE-cadherin/catenin complex

“…The circumferential junction-associated actin bundles are characteristic for intact endothelium in vivo, while stress fibers preferentially appear when endothelium becomes activated e.g., under inflammatory conditions and wound healing; conditions that are generally associated with cell proliferation and migration. 1,9,37,44 Actin networks mostly appear, like in other cells, as lammellipodia at the leading edge of migrating cells 11,45,46 and as junctionassociated intermittent lamellipodia (JAIL) at established cell junctions. 11 Those networks are of critical importance in controlling endothelial barrier function and remodeling (for details, see below).…”

Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics

“…Images were acquired every 6 seconds and cells were observed under basal conditions, after treatment with 1 U/ml thrombin × 15 minutes and then for 20–30 minutes after administration of 1μM S1P. Images were analyzed using ImageJ and kymographic analysis was performed using the Multiple Kymograph pluggin to assess membrane dynamics as described previously (Doggett and Breslin, 2011; Ott and Lippincott-Schwartz, 2012; Yi et al, 2011). …”