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The formation of biomimetic lipid membranes has the potential to provide insights into cellular lipid membrane dynamics. The construction of such membranes necessitates not only the utilization of appropriate lipids, but also physiologically relevant substrate/support materials. The substrate materials employed have been shown to have demonstrable effects on the behavior of the overlying lipid membrane, and thus must be studied before use as a model cushion support. To our knowledge, we report the formation and investigation of a novel actin protein-supported lipid membrane. Specifically, inner leaflet lateral mobility of globular actin-supported DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers, deposited via the Langmuir-Blodgett/Langmuir Schaefer methodology, was investigated by z-scan fluorescence correlation spectroscopy across a temperature range of 20-44°C. The actin substrate was found to decrease the diffusion coefficient when compared to an identical membrane supported on glass. The depression of the diffusion coefficient occurred across all measured temperatures. These results indicated that the actin substrate exerted a direct effect on the fluidity of the lipid membrane and highlighted the fact that the choice of substrate/support is critical in studies of model lipid membranes., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Acute ischemic kidney injury results in marked increases in local and systemic cytokine levels. IL-1alpha, IL-6, and TNF-alpha orchestrate various inflammatory reactions influencing endothelial permeability by altering cell-to-cell and cell-to-extracellular matrix attachments. To explore the role of actin and the regulatory proteins RhoA and cofilin in this process, microvascular endothelial cells (MS1) were exposed to individual cytokines or a cytokine cocktail. Within minutes, a marked, time-dependent redistribution of the actin cytoskeleton occurred with the formation of long, dense F-actin basal stress fibers. The concentration of F-actin, normalized to nuclear staining, significantly increased compared with untreated cells (up 20%, P < or = 0.05). Western blot analysis of MS1 lysates incubated with the cytokine cocktail for 4 h showed an increase in phosphorylated/inactive cofilin (up 25 +/- 15%, P < or = 0.05) and RhoA activation (up to 227 +/- 26% increase, P < or = 0.05) compared with untreated cells. Decreasing RhoA levels using small interfering RNA blocked the effect of cytokines on stress fiber organization. Treatment with Y-27632, an inhibitor of the RhoA effector p160-ROCK, decreased levels of phosphorylated cofilin and reduced stress fiber fluorescence by 22%. In cells treated with Y-27632 followed by treatment with the cytokine cocktail, stress fiber levels were similar to control cells and cofilin phosphorylation was 55% of control levels. Taken together, these studies demonstrate cytokine stimulation of RhoA, which in turn leads to cofilin phosphorylation and formation of numerous basal actin stress fibers. These results suggest cytokines signal through the Rho-ROCK pathway, but also through another pathway to affect actin dynamics.

The introduction of two-photon microscopy, along with the development of new fluorescent probes and innovative computer software, has advanced the study of intracellular and intercellular processes in the tissues of living organisms. Researchers can now determine the distribution, behavior, and interactions of labeled chemical probes and proteins in live kidney tissue in real time without fixation artifacts. Chemical probes, such as fluorescently labeled dextrans, have extended our understanding of dynamic events with subcellular resolution. To accomplish expression of specific proteins in vivo, cDNAs of fluorescently labeled proteins have been cloned into adenovirus vectors and infused by micropuncture to induce proximal tubule cell infection and protein expression. The localization and intensity of the expressed fluorescent proteins can be observed repeatedly at different time points allowing for enhanced quantitative analysis while limiting animal use. Optical sections of images acquired with the two-photon microscope can be 3-D reconstructed and quantified with Metamorph, Voxx, and Amira software programs.

Ischemia and sepsis lead to endothelial cell damage, resulting in compromised microvascular flow in many organs. Much remains to be determined regarding the intracellular structural events that lead to endothelial cell dysfunction. To investigate potential actin cytoskeletal-related mechanisms, ATP depletion was induced in mouse pancreatic microvascular endothelial cells (MS1). Fluorescent imaging and biochemical studies demonstrated a rapid and progressive increase in F-actin along with a decrease in G-actin at 60 min. Confocal microscopic analysis showed ATP depletion resulted in destruction of actin stress fibers and accumulation of F-actin aggregates. We hypothesized these actin alterations were secondary to dephosphorylation/activation of actin-depolymerizing factor (ADF)/cofilin proteins. Cofilin, the predominant isoform expressed in MS1 cells, was rapidly dephosphorylated/activated during ATP depletion. To directly investigate the role of cofilin activation on the actin cytoskeleton during ischemia, MS1 cells were infected with adenoviruses containing the cDNAs for wild-type Xenopus laevis ADF/cofilin green fluorescent protein [XAC(wt)-GFP], GFP, and the constitutively active and inactive isoforms XAC(S3A)-GFP and XAC(S3E)-GFP. The rate and extent of cortical actin destruction and actin aggregate formation were increased in ATP-depleted XAC(wt)-GFP- and XAC(S3A)-GFP-expressing cells, whereas increased actin stress fibers were observed in XAC(S3E)-GFP-expressing cells. To investigate the upstream signaling pathway of ADF/cofilin, LIM kinase 1-GFP (LIMK1-GFP) was expressed in MS1 cells. Cells expressing LIMK1-GFP protein had higher levels of phosphorylated ADF/cofilin, increased stress fibers, and delayed F-actin cytoskeleton destruction during ATP depletion. These results strongly support the importance of cofilin regulation in ischemia-induced endothelial cell actin cytoskeleton alterations leading to cell damage and microvascular dysfunction.

In vivo fluorescence imaging, using confocal or multiphoton microscopes, provides a powerful method to analyze kidney function in experimental animals. In this review, the preparation used for physiological studies in rats is described. A variety of fluorescent probes are available to study glomerular permeability, renal blood flow, peritubular capillary permeability, cell ion concentrations, tubule transport properties, and the functional status of renal cells. We have recently used micropuncture techniques and an adenovirus vector to accomplish gene transfer into kidney tubule and endothelial cells; this new methodology will allow the dynamic study of fluorescently-labeled proteins. Two examples of the use of two-photon fluorescence microscopy to study renal pathophysiology, namely polycystic kidney disease and renal ischemia, are presented. Software is available to quantify data collected from in vivo imaging experiments and to construct 3-dimensional images of renal structures. Two-photon or confocal microscopy offers many opportunities for a better understanding of kidney function in health and disease., (2006 S. Karger AG, Basel.)

Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the virally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any fluorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology.

Ischemic-induced cell injury results in rapid duration-dependent actin-depolymerizing factor (ADF)/cofilin-mediated disruption of the apical microvilli microfilament cores. Because intestinal microvillar microfilaments are bound and stabilized in the terminal web by the actin-binding protein tropomyosin, we questioned whether a protective effect of tropomyosin localization to the terminal web of the proximal tubule microfilament cores is disrupted during ischemic injury. With tropomyosin-specific antibodies, we examined rat cortical sections under physiological conditions and following ischemic injury by confocal microscopy. In addition, Western blot analysis of cortical extracts and urine was undertaken. Our studies demonstrated the presence of tropomyosin isoforms in the proximal tubule microvillar terminal web under physiological conditions and their dissociation in response to 25 min of ischemic injury. This correlated with the excretion of tropomyosin-containing plasma membrane vesicles in urine from ischemic rats. In addition, we noted increased tropomyosin Triton X-100 solubility following ischemia in cortical extracts. These studies suggest tropomyosin binds to and stabilizes the microvillar microfilament core in the terminal web under physiological conditions. With the onset of ischemic injury, we propose that tropomyosin dissociates from the microfilament core providing access to microfilaments in the terminal web for F-actin binding, severing and depolymerizing actions of ADF/cofilin proteins.

Ischemic injury induces actin cytoskeleton disruption and aggregation, but mechanisms affecting these changes remain unclear. To determine the role of actin-depolymerizing factor (ADF)/ cofilin participation in ischemic-induced actin cytoskeletal breakdown, we utilized porcine kidney cultured cells, LLC-PK(A4.8), and adenovirus containing wild-type (wt), constitutively active, and inactive Xenopus ADF/cofilin linked to green fluorescence protein [XAC(wt)-GFP] in an ATP depletion model. High adenoviral infectivity (70%) in LLC-PK(A4.8) cells resulted in linearly increasing XAC(wt)-GFP and phosphorylated (p)XAC(wt)-GFP (inactive) expression. ATP depletion rapidly induced dephosphorylation, and, therefore, activation, of endogenous pcofilin as well as pXAC(wt)-GFP in conjunction with the formation of fluorescent XAC(wt)-GFP/actin aggregates and rods. No significant actin cytoskeletal alterations occurred with short-term ATP depletion of LLC-PK(A4.8) cells expressing GFP or the constitutively inactive mutant XAC(S3E)-GFP, but cells expressing the constitutively active mutant demonstrated nearly instantaneous actin disruption with aggregate and rod formation. Confocal image three-dimensional volume reconstructions of normal and ATP-depleted LLC-PK(A4.8) cells demonstrated that 25 min of ATP depletion induced a rapid increase in XAC(wt)-GFP apical and basal signal in addition to XAC-GFP/actin aggregate formation. These data demonstrate XAC(wt)-GFP participates in ischemia-induced actin cytoskeletal alterations and determines the rate and extent of these ATP depletion-induced cellular alterations.

Breakdown of proximal tubule cell apical membrane microvilli is an early-occurring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is known that actin cytoskeleton alterations play a critical role in this cellular process. Our laboratory previously demonstrated that ischemia-induced cell injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [(ADF); Schwartz, N, Hosford M, Sandoval RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol Renal Fluid Electrolyte Physiol 276: F544-F551, 1999]. Therefore, we postulated that ischemia-induced ADF relocalization from the cytoplasm to the apical microvillar microfilament core was an early event occurring before F-actin alterations. To directly investigate this hypothesis, we examined the intracellular localization of ADF in ischemic rat cortical tissues by immunofluorescence and quantified the concentration of ADF in brush-border membrane vesicles prepared from ischemic rat kidneys by using Western blot techniques. Within 5 min of the induction of ischemia, ADF relocalized to the apical membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depolymerization of microvillar F-actin to G-actin was documented by using colocalization studies for G- and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain before microvillar destruction. These data further suggest that ADF plays a critical role in microvillar microfilament destruction and apical membrane damage during ischemia.

A vast array of actin binding proteins (ABPs), together with intracellular signaling molecules, modulates the spatiotemporal distribution of actin filaments in eukaryotic cells. To investigate the complex regulation of actin organization in plant cells, we designed experiments to reconstitute actin-ABP interactions in vitro with purified components. Because vertebrate skeletal [alpha]-actin has distinct and unpredictable binding affinity for nonvertebrate ABPs, it is essential that these in vitro studies be performed with purified plant actin. Here, we report the development of a new method for isolating functional actin from maize pollen. The addition of large amounts of recombinant profilin to pollen extracts facilitated the depolymerization of actin filaments and the formation of a profilin-actin complex. The profilin-actin complex was then isolated by affinity chromatography on poly-L-proline-Sepharose, and actin was selectively eluted with a salt wash. Pollen actin was further purified by one cycle of polymerization and depolymerization. The recovery of functional actin by this rapid and convenient procedure was substantial; the average yield was 6 mg of actin from 10 g of pollen. We undertook an initial physicochemical characterization of this native pollen actin. Under physiological conditions, pollen actin polymerized with kinetics similar in quality to those for vertebrate [alpha]-actin and had a critical concentration for assembly of 0.6 [mu]M. Moreover, pollen actin interacted specifically and in a characteristic fashion with several ABPs. Tradescantia cells were microinjected and used as an experimental system to study the behavior of pollen actin in vivo. We demonstrated that purified pollen actin ameliorated the effects of injecting excess profilin into live stamen hair cells.