Changes in cell morphology due to plasma membrane wounding by acoustic cavitation.

Acoustic cavitation-mediated wounding (i.e., sonoporation) has great potential to improve medical and laboratory applications requiring intracellular uptake of exogenous molecules; however, the field lacks detailed understanding of cavitation-induced morphologic changes in cells and their relative importance.

Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin-containing focal adhesions.

Actin-myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30-40% higher than serum-free cultures.

A novel bioassay for detecting GPCR heterodimerization: transactivation of beta 2 adrenergic receptor by bradykinin receptor.

Many G-protein-coupled receptors (GPCRs) have been shown to form heteromeric complexes primarily by biochemical methods, including competitive radioligand binding assays or measurements of changes in second-messenger concentration in lysed cells. These results are often cell line specific, and the expression of other cell surface proteins makes it difficult to detect potential functional consequences of GPCR interaction. Here, 2-electrode voltage clamping in Xenopus oocytes was used as a bioassay to explore heterodimerization of bradykinin type 2 receptor (Bk2R) and beta 2 adrenergic receptor (beta(2)AR), using chloride channels as outputs for receptor activation.

Serine palmitoyltransferase subunit 1 is present in the endoplasmic reticulum, nucleus and focal adhesions, and functions in cell morphology.

Serine palmitoyltransferase (SPT) has been localized to the endoplasmic reticulum (ER) by subcellular fractionation and enzymatic assays, and fluorescence microscopy of epitope-tagged SPT; however, our studies have suggested that SPT subunit 1 might be present also in focal adhesions and the nucleus. These additional locations have been confirmed by confocal microscopy using HEK293 and HeLa cells, and for focal adhesions by the demonstration that SPT1 co-immunoprecipitates with vinculin, a focal adhesion marker protein. The focal adhesion localization of SPT1 is associated with cell morphology, and possibly cell migration, because it is seen in most cells before they reach confluence but disappears when they become confluent, and is restored by a standard scratch-wound healing assay.

Sharpening the edges of understanding the structure/function of the LPA1 receptor: expression in cancer and mechanisms of regulation.

Since the molecular cloning of the vzg-1/Edg-2/LPA1 gene, studies have attempted to characterize LPA1 receptor functionality into a single categorical role, different from the other Edg-family LPA receptors. The desire to categorize LPA1 function has highlighted its complexity and demonstrated that the LPA1 receptor does not have one absolute function throughout every system. The central nervous system is highly enriched in the LPA1 receptor, suggesting an integral role in neuronal processes.

A case report of familial periodic paralysis.

A 20-year-old male was brought to the hospital with the complaints of severe weakness and inability to move the limbs of 12 hours duration. For the last 2 years he had the same episodes with spontaneous recovery. Family history strongly suggested involvement of other members of the family.

Metabolic labeling and immunoprecipitation of Drosophila proteins.

The genetics of Drosophila is a powerful tool in the analysis of mutants and mutant proteins. Cultures of cells derived from wild-type or mutant flies can be pulse labeled to biosynthetically label the proteins made by the cells. Immunoprecipitation (and subcellular fractionation) are used to characterize the expression of specific proteins.

Different mechanisms regulate lysophosphatidic acid (LPA)-dependent versus phorbol ester-dependent internalization of the LPA1 receptor.

Lysophosphatidic acid (LPA) stimulates cells by activation of five G-protein-coupled receptors, termed LPA 1-5. The LPA 1 receptor is the most widely expressed and is a major regulator of cell migration. In this study, we show that phorbol ester (PMA)-induced internalization of the LPA(1) receptor requires clathrin AP-2 complexes, protein kinase C, and a distal dileucine motif (amino acids 352 and 353) in the cytoplasmic tail but not beta-arrestin.

Lysophosphatidic acid decreases the nuclear localization and cellular abundance of the p53 tumor suppressor in A549 lung carcinoma cells.

Lysophosphatidic acid (LPA) is a bioactive lipid that promotes cancer cell proliferation and motility through activation of cell surface G protein-coupled receptors. Here, we provide the first evidence that LPA reduces the cellular abundance of the tumor suppressor p53 in A549 lung carcinoma cells, which express endogenous LPA receptors. The LPA effect depends on increased proteasomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription.

Mechanism of intracellular delivery by acoustic cavitation.

Using conditions different from conventional medical imaging or laboratory cell lysis, ultrasound has recently been shown to reversibly increase plasma membrane permeability to drugs, proteins and DNA in living cells and animals independently of cell or drug type, suggesting a ubiquitous mechanism of action. To determine the mechanism of these effects, we examined cells exposed to ultrasound by flow cytometry coupled with electron and fluorescence microscopies. The results show that cavitation generated by ultrasound facilitates cellular incorporation of macromolecules up to 28 nm in radius through repairable micron-scale disruptions in the plasma membrane with lifetimes >1 min, which is a period similar to the kinetics of membrane repair after mechanical wounding.

A requirement for membrane cholesterol in the beta-arrestin- and clathrin-dependent endocytosis of LPA1 lysophosphatidic acid receptors.

Lysophosphatidic acid (LPA) stimulates heterotrimeric G protein signaling by activating three closely related receptors, termed LPA(1), LPA(2) and LPA(3). Here we show that in addition to promoting LPA(1) signaling, membrane cholesterol is essential for the association of LPA(1) with beta-arrestin, which leads to signal attenuation and clathrin-dependent endocytosis of LPA(1). Reduction of clathrin heavy chain expression, using small interfering RNAs, inhibited LPA(1) endocytosis.

Protein binding properties of surface-modified porous polyethylene membranes.

In this study, we quantified the adsorption of immunoglobulin G (IgG) protein onto several polyelectrolyte-modified sintered porous polyethylene (PPE) membranes. The polymer surfaces had both cationic and anionic charges obtained via the adsorption of polyethylenimine (PEI) and polyacrylic acid (PAA), respectively, onto plasma-activated PPE. The amount of IgG adsorption was determined by measuring the gamma radiation emitted by [125I]-IgG radio labeled protein.

Protein kinase A inhibits lysophosphatidic acid induction of serum response factor via alterations in the actin cytoskeleton.

Lysophosphatidic acid (LPA; 1-acyl-2-hydroxy-sn-glycero-3-phosphate) is a lipid growth factor that stimulates the proliferation of ovarian cancer cells. Recent studies indicate that elevation of cellular cAMP levels inhibits ovarian epithelial cancer cell growth. In this study, we investigated the effects of elevating cellular cAMP levels on LPA stimulation of OVCAR-3 ovarian cancer cell growth and on LPA stimulation of the serum response factor (SRF) transcription factor.

Agonist-induced endocytosis of lysophosphatidic acid-coupled LPA1/EDG-2 receptors via a dynamin2- and Rab5-dependent pathway.

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that exerts a pleiotropic range of effects on cells through activation of three closely related G-protein-coupled receptors termed LPA1/EDG-2, LPA2/EDG-4 and LPA3/EDG-7. Of these receptors, the LPA1 receptor is the most widely expressed. In this study, we investigated the agonist-induced endocytosis of the human LPA1 receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells.

Transfer of M2 muscarinic acetylcholine receptors to clathrin-derived early endosomes following clathrin-independent endocytosis.

Upon agonist stimulation, many G protein-coupled receptors such as beta(2)-adrenergic receptors are internalized via beta-arrestin- and clathrin-dependent mechanisms, whereas others, like M(2) muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M(2) mAChR endocytosis, we investigated the post-endocytic trafficking of M(2) mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M(2) mAChR internalization. Here, we report that M(2) mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, Galpha(q) Q209L.

Polyneuritis cranialis following herpes zoster.

Herpes zoster is a common clinical condition involving cranial nerves. We encountered 3 cases in which multiple cranial nerves were involved besides the commoner ones. All the three cases were treated with acyclovir and oral steroids.

Separation of membrane trafficking and actin remodeling functions of ARF6 with an effector domain mutant.

The ADP-ribosylation factor 6 (ARF6) GTPase has a dual function in cells, regulating membrane traffic and organizing cortical actin. ARF6 activation is required for recycling of the endosomal membrane back to the plasma membrane (PM) and also for ruffling at the PM induced by Rac. Additionally, ARF6 at the PM induces the formation of actin-containing protrusions.

ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements.

The ARF6 GTPase regulates a novel endosomal-plasma membrane recycling pathway and influences cortical actin remodeling. Here we examined the relationship between ARF6 and Rac1, a Rho family GTPase, implicated in cortical actin rearrangements. Endogenous Rac1 colocalized with ARF6 at the plasma membrane and on the ARF6 recycling endosome in untransfected HeLa and primary human fibroblast cells.

ASAP1, a phospholipid-dependent arf GTPase-activating protein that associates with and is phosphorylated by Src.

Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4, 5-biphosphate (PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b.

Localization of endogenous ARF6 to sites of cortical actin rearrangement and involvement of ARF6 in cell spreading.

To study the function of the endogenous ARF6 GTP binding protein in cells, we generated an antibody which specifically recognizes ARF6, and not the other ARF proteins. Using this antibody, ARF6 was detected in all mouse organs tested and in a variety of cultured cell lines including RBL, MDCK, NRK, BHK, COS, and HeLa cells. In NRK cells, by immunofluorescence, ARF6 localized to the plasma membrane, especially at regions exhibiting membrane ruffling, and was also concentrated in a fine punctate distribution in the juxtanuclear region.