Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer.

The adenovirus (Ad) penton base protein facilitates viral infection by binding cell surface integrins, triggering receptor-mediated endocytosis and mediating endosomal penetration. Given these multiple functions, recombinant penton base proteins have been utilized as non-viral vehicles for gene transfer by our lab and others. Although we have previously demonstrated that penton base-derived vectors undergo integrin-specific binding and cell entry, less than desirable levels of gene expression have led us to re-evaluate the recombinant penton base as an agent for gene delivery. To do so, we have examined here the intracellular trafficking of an Ad serotype 5 (Ad5) recombinant penton base protein (PB). Here, we not only observed that PB utilizes a similar, typical trafficking pathway of whole Ad, but also found that PB entered HeLa cells through pathways not yet identified as contributing to cell entry by the whole virus. We show by high-resolution confocal microscopy and biochemical methods that binding to alphav-integrins is a requirement for cell entry, but that early internalization stages did not substantially pass through clathrin-positive and early endosomal compartments. Moreover, a subpopulation of internalized protein localized with caveolin-positive compartments and Golgi markers, suggesting that a certain percentage of proteins pass through non-clathrin-mediated pathways. Similar to the virus, trafficking toward the nucleus was affected by disruption of microtubules and dynein. The majority of penton base molecules avoided the lysosome while facilitating early vesicle release of low molecular weight dextran molecules. In further support of a vesicle escape capacity, a subpopulation of internalized penton base appeared to enter the nucleus, as observed by high-resolution confocal microscopy and cell fractionation. As a confirmation of these findings, we demonstrate that a recombinant penton base facilitated cytosolic entry of an siRNA molecule as observed by RNA interference of a marker gene. Based on our findings here, we suggest that whereas soluble penton base proteins may enter cells through clathrin- and non-clathrin-mediated pathways, vesicle escape and nuclear delivery appear to be supported by a clathrin-mediated pathway. As our previous efforts have focused on utilizing recombinant penton base proteins as delivery agents for therapeutics, these findings allow us to evaluate the use of the penton base as a cell entry and intracellular trafficking agent, and may be of interest concerning the development of vectors for efficient delivery of therapeutics to cells.

Accumulation of catalytically active proteases in lacrimal gland acinar cell endosomes during chronic ex vivo muscarinic receptor stimulation.

Chronic muscarinic stimulation induces functional quiescence (Scand J Immunol 2003;58:550-65) and alters the traffic of immature cathepsin B (Exp Eye Res 2004;79:665-75) in lacrimal acinar cells. To test whether active proteases aberrantly accumulate in the endosomes, cell samples were cultured 20 h with and without 10-microm carbachol (CCh), incubated with [125I]-bovine serum albumin and then lysed and analysed by subcellular fractionation. CCh decreased total cysteine protease and cathepsin S activities in the isolated lysosome, redistributing them to early endocytic and biosynthetic compartments. CCh decreased [125I] accumulation in all compartments of cells loaded in the absence of protease inhibitors; the cysteine protease inhibitor, leupeptin, prevented the endosomal decrease but not the lysosomal decrease. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography demonstrated [125I]-labelled proteolytic products in endomembrane compartments of both control and CCh-stimulated cells, even in the presence of leupeptin, but analysis indicated that CCh increased the amount in endosomes. Two-dimensional fractionation analyses suggest that the CCh-induced redistributions result from blocks in traffic to the late endosome from both the early endosome and the trans-Golgi network. Therefore, we conjecture that chronic muscarinic acetylcholine receptor stimulation leads to aberrant proteolytic processing of autoantigens in endosomes, from whence previously cryptic epitopes may be secreted to the underlying interstitial space.

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland.

Although adenovirus (Ad) exhibits tropism for epithelial cells, little is known about the cellular effects of adenoviral binding and internalization on epithelial functions. Here, we examine its effects on the secretory acinar epithelial cells of the lacrimal gland, responsible for stimulated release of tear proteins into ocular fluid. Exposure of reconstituted rabbit lacrimal acini to replication-defective Ad for 16-18 h under conditions that resulted in >80% transduction efficiency did not alter cytoskeletal filament or biosynthetic/endosomal membrane compartment organization. Transduction specifically altered the organization of the stimulated secretory pathway, eliciting major dispersal of rab3D immunofluorescence from apical stores normally associated with mature secretory vesicles. Biochemical studies revealed that this dispersal was not associated with altered rab3D expression nor its release from cellular membranes. Ultraviolet (UV)-inactivated Ad elicited similar dispersal of rab3D immunofluorescence. In acini exposed to replication-defective or UV-inactivated Ad, carbachol-stimulated release of bulk protein and beta-hexosaminidase were significantly (P< or =0.05) inhibited to an extent proportional to the loss of rab3D-enriched mature secretory vesicles associated with these treatments. We propose that the altered secretory compartment organization and function caused by Ad reflects changes in the normal maturation of secretory vesicles, and that these changes are caused by exposure to the Ad capsid.

Biochemical changes contributing to functional quiescence in lacrimal gland acinar cells after chronic ex vivo exposure to a muscarinic agonist.

Profound secretory dysfunction can be associated with relatively modest lymphocytic infiltration of the lacrimal and salivary glands of Sjögren's syndrome (SjS) patients. SjS patients' sera contain autoantibodies to M3 muscarinic acetylcholine receptors (MAChR) that have variously been reported to have agonistic and antagonistic effects. We sought to identify consequences of chronic agonist stimulation by maintaining acinar cells from rabbit lacrimal glands for 20 h in the presence or absence of 10 microM carbachol (CCh). Exposure to CCh diminished the cells' ability to elevate cytosolic Ca2+ and secrete beta-hexosaminidase in response to acute stimulation with 100 microM CCh, but it enhanced their secretory responses to phenylephrine and ionomycin. Secretory vesicles appeared normal by electron microscopy, but confocal fluorescence microscopy revealed depletion of the secretory vesicle membrane marker, rab3D, and decreased ability to recruit secretory transport vesicles in response to acute 100 microM CCh. Additionally, the apical cortical actin cytoskeleton was disrupted and diminished compared to the basal-lateral cortical network. Subcellular fractionation analyses revealed that total membrane phase protein content was increased. The contents of beta-hexosaminidase and MAChR relative to total protein were not significantly altered, and MAChR abundance in the plasma membrane fraction was increased as the result of redistribution from endomembrane pools. However, relative cellular contents of the heterotrimeric guanosine triphosphate (GTP)-binding proteins, Gq and G11, were decreased. Additional biochemical changes included decreased contents of 47 kDa Gs and Gi3, protein kinase Calpha and rab3D and polymeric immunoglobulin (Ig) receptors; internalization of Na,K-ATPase from the plasma membranes to endomembrane compartments and decreased content of beta-hexosaminidase in the lysosomes. The observations demonstrate that chronic exposure to a MAChR agonist induces refractoriness to optimal stimulation, without causing receptor downregulation, by downregulating postreceptor-signalling mediators and effectors. The cells' secretory mechanisms for IgA and electrolytes also appear to be impaired, as does their ability to properly sort proteins to the lysosomes.

Etk/Bmx activation modulates barrier function in epithelial cells.

Etk/Bmx is a member of the Tec family of cytoplasmic non-receptor tyrosine kinases known to express in epithelial cells. We demonstrate herein that Etk activation in stably Etk-transfected epithelial Pa-4 cells resulted in a consistently increased transepithelial resistance (TER). After 24 h of hypoxic (1% O(2)) exposure, the TER and equivalent active ion transport rate (I(eq)) were reduced to <5% of the normoxia control in Pa-4 cells, whereas both TER and I(eq) were maintained at comparable and 60% levels, respectively, relative to their normoxic controls in cells with Etk activation. Moreover, Pa-4 cells exhibited an abundant actin stress fiber network with a diffuse distribution of beta-catenin at the cell periphery. By contrast, Etk-activated cells displayed a redistribution of actin to an exclusively peripheral network, with a discrete band of beta-catenin also concentrated at the cell periphery, and an altered occludin distribution profile. On the basis of these findings, we propose that Etk may be a novel regulator of epithelial junctions during physiological and pathophysiological conditions.

Changes in cytoskeletal organization in polyoma middle T antigen-transformed fibroblasts: involvement of protein phosphatase 2A and src tyrosine kinases.

The major transforming activity of polyomavirus, middle T antigen, targets several cellular regulatory effectors including protein phosphatase 2A and src tyrosine kinases. Although transformed cells exhibit profound morphological changes, little is known about how middle T antigen-induced changes in the cellular regulatory environment specifically affect the cytoskeleton. We have investigated these changes in 10T(1/2) mouse fibroblasts transformed with polyoma middle T antigen. Immunofluorescence microscopy revealed that expression of middle T antigen (Pym T cells) depleted the stable (acetylated) microtubule array and increased the sensitivity of dynamic (tyrosinated) microtubules to nocodazole-induced disassembly. These effects were associated with a modest but statistically significant (P

Identification of a novel taxol-sensitive kinase activity associated with the cytoskeleton.

The microtubule-targeted drug, taxol, enhances assembly of alphabeta tubulin dimers into microtubules. Recent work has established that taxol also elicits diverse effects on intracellular signaling. In-gel kinase assays with myelin basic protein as substrate revealed that taxol treatment significantly (P

Protein phosphatase inhibitors alter cellular microtubules and reduce carbachol-dependent protein secretion in lacrimal acini.

PURPOSE: To further understand the regulation of microtubules and their function in the lacrimal gland, we investigated the effects of two serine/threonine phosphatase inhibitors, okadaic acid (300 nM-1 microM) and calyculin A (20-100 nM), on microtubules and stimulated secretion in lacrimal acini. METHODS: Primary rabbit lacrimal acini cultured for two days were utilized. Microtubule structure was probed using biochemical analysis and confocal fluorescence microscopy. Carbachol-stimulated and basal protein secretion were determined by measurement of released protein or, for pulse-chase studies, [(35)S]-protein. RESULTS: Biochemical analysis and confocal fluorescence microscopy showed that both inhibitors caused a major loss of cellular microtubules and also of acetylated (stable) microtubules. However, calyculin A was more potent than okadaic acid in causing microtubule loss. Because changes in microtubules can partially impair stimulated protein secretion in lacrimal acini, the effects of inhibitors on protein secretion were also evaluated. Both inhibitors caused a comparable dose-dependent and significant (p

Increased protein phosphorylation of cytoplasmic dynein results in impaired motor function.

Inhibition of serine/threonine protein phosphatases in rat hepatocytes by okadaic acid and microcystin increased the phosphorylation of several components of the cytoplasmic dynein complex. UV light/vanadate cleavage and Western blot analysis revealed that two of these components with molecular masses of approx. 400 kDa and 74 kDa were dynein heavy- and intermediate-chains respectively. This increased phosphorylation resulted in inhibition of dynein ATPase activity, and reduced motor-dependent avidity of endosomal/lysosomal membranes for microtubules.

Taxol inhibits endosomal-lysosomal membrane trafficking at two distinct steps in CV-1 cells.

Although taxol inhibits membrane trafficking, the nature of this inhibition has not been well defined. In this study, we define the effects of taxol on endocytosis in CV-1 cells using density gradient centrifugation of membranes over sorbitol density gradients. After taxol treatment, resident endosomal enzymes and the epidermal growth factor (EGF) receptor (EGFR) showed significant (P 500 nm) vesicles found in controls. These data demonstrate that disruption of endocytic events by taxol includes the early accumulation of protein and endocytic markers in endosomes and the later accumulation in a dense compartment that we propose is a subdomain of the lysosomes.

Microtubule-dependent vesicle transport: modulation of channel and transporter activity in liver and kidney.

Microtubule-based vesicle transport driven by kinesin and cytoplasmic dynein motor proteins facilitates several membrane-trafficking steps including elements of endocytosis and exocytosis in many different cell types. Most early studies on the role of microtubule-dependent vesicle transport in membrane trafficking focused either on neurons or on simple cell lines. More recently, other work has considered the role of microtubule-based vesicle transport in other physiological systems, including kidney and liver. Investigation of the role of microtubule-based vesicle transport in membrane trafficking in cells of the kidney and liver suggests a major role for microtubule-based vesicle transport in the rapid and directed movement of ion channels and transporters to and from the apical plasma membranes, events essential for kidney and liver function and homeostasis. This review discusses the evidence supporting a role for microtubule-based vesicle transport and the motor proteins, kinesin and cytoplasmic dynein, in different aspects of membrane trafficking in cells of the kidney and liver, with emphasis on those functions such as maintenance of ion channel and transporter composition in apical membranes that are specialized functions of these organs. Evidence that defects in microtubule-based transport contribute to diseases of the kidney and liver is also discussed.

Molecular identification of a role for tyrosine 167 in the function of the human intestinal proton- coupled dipeptide transporter (hPepT1).

hPepT1 is a proton-coupled peptide transporter that mediates the absorption of di- and tripeptides. Here we show that tyrosine 167 (Y167) in transmembrane domain 5 (TMD5) of this 12-transmembrane spanning protein contributes to its transport function. We identified this particular amino acid by a computer model of the arrangement of the TMDs of hPepT1 and investigated its role by site-directed mutagenesis and dipeptide uptake studies. [3H]Gly-sar uptake in cells transiently transfected with Y167A-hPepT1 was abolished completely, even though the level of Y167A-hPepT1 expression by Western blot analysis and cell surface expression by immunofluorescence microscopy was similar to those of the wild type. Therefore, mutation affected transport function, but apparently not the steady-state protein level or trafficking of the transporter to the plasma membrane. Moreover, mutation of Y167 into phenylalanine, serine, or histidine all abolished gly-sar uptake in transfected HEK 293 cells. Taken together, these findings suggest that Y167 plays an essential role in hPepT1 function, perhaps due to the unique chemistry of its phenolic side chain.

Microtubules facilitate the stimulated secretion of beta-hexosaminidase in lacrimal acinar cells.

Stimulation of lacrimal acini with secretagogues such as carbachol initiates movement and fusion of acinar secretory vesicles with the apical plasma membrane, resulting in release of protein into the nascent tear fluid. Using rabbit lacrimal acini reconstituted in vitro from isolated cells, we have investigated the organization of the apical cytoskeleton and its role in stimulated secretion. Confocal microscopy revealed a microtubule array emanating from the apical region of the acini; the apical region was also enriched in microfilaments and (gamma)-tubulin. Cytokeratin-based intermediate filaments were apically concentrated, and also detected at the cell periphery. Neither confocal microscopy nor biochemical analysis revealed any reorganization of lumenal microfilaments or microtubules which might accompany carbachol-stimulated release of secretory proteins. However, major changes in the acinar microtubule array induced by taxol or nocodazole were correlated with inhibition of carbachol-dependent release of the secreted protein, beta-hexosaminidase. Major changes in lumenal microfilaments induced by jasplakinolide or cytochalasin D did not inhibit the carbachol-dependent release of beta-hexosaminidase; rather, release of beta-hexosaminidase from jasplakinolide- or cytochalasin D-treated carbachol-stimulated acini was markedly increased relative to the release from untreated stimulated acini. Our findings demonstrate that microtubules play a major role in stimulated lacrimal secretion, and suggest a contributory role for microfilaments.

A novel taxol-induced vimentin phosphorylation and stabilization revealed by studies on stable microtubules and vimentin intermediate filaments.

To understand how protein phosphorylation modulates cytoskeletal organization, we used immunofluorescence microscopy to examine the effects of okadaic acid, a serine/threonine protein phosphatase inhibitor, and taxol, a microtubule-stabilizing agent, on stable (acetylated and detyrosinated) microtubules, vimentin intermediate filaments and other cytoskeletal elements in CV-1 cells. Okadaic acid caused major changes in both stable microtubules and vimentin intermediate filaments, but through independent mechanisms. At 300 nM, okadaic acid caused apparent fragmentation and loss of stable microtubules which was not prevented by prior exposure to K252a. In contrast, major reorganization of vimentin intermediate filaments elicited at 750 nM okadaic acid was prevented by prior exposure to K252a. Taxol pretreatment blocked the effects of okadaic acid on stable microtubules and vimentin intermediate filaments. Recent reports have revealed that taxol can activate cellular signal transduction pathways in addition to its known ability to promote microtubule stabilization, so the possibility that taxol-induced resistance of vimentin intermediate filaments to okadaic acid was through a microtubule-independent mechanism involving direct phosphorylation of intermediate filament proteins was explored. Vimentin immunoprecipitation from cytoskeletal extracts from 32P-labeled cells revealed that taxol (4 microM, 1 or 2 hours) caused about a 2-fold increase in vimentin phosphorylation. This phosphorylation was recovered exclusively in cytoskeletal vimentin, in contrast to the increased phosphorylation of soluble and cytoskeletal vimentin caused by exposure to 750 nM okadaic acid. Phosphorylation of soluble and cytoskeletal vimentin from cells exposed to taxol (4 microM, 1 hour) then okadaic acid (750 nM, 1 hour) was comparable to taxol-treatment alone. These findings demonstrate a novel new activity of taxol, induction of vimentin phosphorylation, that may impact on vimentin organization and stability.

Sjögren's autoimmunity: how perturbation of recognition in endomembrane traffic may provoke pathological recognition at the cell surface.

CD4 T cell antigen recognition requires presentation by major histocompatibility complex Class II molecules (MHC II). B cell surface immunoglobulins recognize antigens independently of MHC II, but activation typically requires CD4 cell cytokines as accessory signals. Plasma membrane-endomembrane traffic in lacrimal gland acinar cells, targets of autoimmune activity in Sjögren's syndrome, may satisfy both requirements. The Golgi protein galactosyltransferase and the lysosomal proteins cathepsin B and cathepsin D appear at the plasma membranes during sustained secretomotor stimulation. The RNA transcription termination factor La, a frequent target of Sjögren's autoantibodies, appears in the acinar cell cytoplasm and plasma membranes during viral infection and during in vitro exposure to cytokines. MHC II cycle through endomembrane compartments which contain La, galactosyltransferase, cathepsin B and cathepsin D and which are sites of proteolysis. This traffic may permit trilateral interactions in which B cells recognize autoantigens at the surface membranes, CD4 T cells recognize peptides presented by MHC II, B cells provide accessory signals to CD4 T cells, and CD4 T cells provide cytokines that activate B cells. Acinar cells stimulate lymphocyte proliferation in autologous mixed cell reactions, confirming that they are capable of provoking autoimmune responses.

Transferrin receptor recycling in rat hepatocytes is regulated by protein phosphatase 2A, possibly through effects on microtubule-dependent transport.

To understand the regulation of receptor-mediated endocytosis in hepatocytes, we have used two specific inhibitors of serine-threonine protein phosphatases (PP), microcystin (MCYST) and okadaic acid (OKA) as probes to alter protein phosphorylation in hepatocytes. We have then examined the impact of these changes on the specific binding and uptake of transferrin (Tf) in hepatocytes. The measurement of PP activity in hepatocyte lysates showed that OKA and MCYST shared a common inhibition of protein phosphatase 2A (PP2A). Our results showed that both OKA (250 nmol/L) and MCYST (500 nmol/L) significantly reduced Tf uptake at steady state (P < or = .05). The measurement of Tf internalization after 15 minutes in protein phosphatase inhibitor-pretreated cells revealed that the initial uptake was also significantly reduced. Binding studies showed that pretreatment with either of the phosphatase inhibitors did not result in significant changes in the K(d) for Tf binding to transferrin receptor (TfR). Additionally, no significant changes in the number of TfR in the plasma membrane were observed in phosphatase inhibitor-pretreated cells. The treatment of hepatocytes with nocodazole (NOC), which results in microtubule disassembly and inhibition of microtubule-based vesicle transport, caused comparable reductions in initial and steady state levels of transferrin accumulation. The changes in transferrin accumulation by both phosphatase inhibitors and nocodazole were accompanied by redistribution of the microtubule-anchored Golgi apparatus and lysosomal network from the perinuclear region to the cell periphery. Our data show that the regulation of Tf uptake by receptor-mediated endocytosis is mediated by PP2A and additionally may occur through regulation of microtubule-based vesicle transport.

Cholinergic stimulation of lacrimal acinar cells promotes redistribution of membrane-associated kinesin and the secretory protein, beta-hexosaminidase, and increases kinesin motor activity.

The role of the microtubule-based motor, kinesin, in membrane trafficking has been investigated in resting and stimulated acinar cells from rabbit lacrimal gland, a cholinergically controlled secretory tissue. Microtubule-dependent motors from extracts of control and carbachol-treated acini were isolated by microtubule-affinity purification and their activity was determined using a video-enhanced differential interference contrast microscopy assay for microtubule gliding. The observation that carbachol treatment resulted in a 2.2-fold stimulation of the frequency of GTP-dependent microtubule gliding in fractions isolated by microtubule-affinity purification and GTP release suggested that kinesin was a target of carbachol-induced stimulation. Resolution of membranes from resting cells by fractionation on a sorbitol density gradient followed by partitioning analysis in a dextran-polyethyleneglycol two-phase system revealed that membrane-associated kinesin codistributed with Golgi-derived membranes, a post-Golgi secretory compartment designated Hex1, membranes from a trans Golgi network-like compartment, endoplasmic reticulum and a group of putative lysosomal membranes containing cathepsin B. Comparable fractionation of carbachol-treated acini showed that stimulation caused redistributions of membrane-associated kinesin, the secretory enzyme beta-hexosaminidase, and galactosyltransferase that appeared to reflect both a reorganization within the Golgi complex and a return of material to the Golgi complex from the secretory pathway. Our findings that carbachol promotes activation of lacrimal acinar kinesin as well as major shifts in kinesin-membrane association within the secretory pathway suggests that kinesin plays a major role in secretory vesicle assembly, apical secretion, and/or secretory vesicle membrane recycling in the lacrimal gland.

Involvement of phosphorylation in doxorubicin-mediated myofibril degeneration. An immunofluorescence microscopy analysis.

Loss of myofilaments has been observed in both adaptive cardiac responses (i.e., hypertrophy) as well as in chemotheraputic use of antineoplastic drugs with cardiotoxic side effects (i.e., doxorubicin). An understanding of the degenerative process is a prerequisite for determining approaches to limit the cardiomyopathic changes associated with chronic heart disease or long-term chemotheraputic treatments. However, little is known about the specific events and molecular changes that initiate the degenerative process. To study this process, neonatal rat cardiomyocytes were treated with doxorubicin, which induced rapid and widespread thin-filament degeneration as observed by fluorescence confocal microscopy. Which demonstrated deterioration of sarcomeric thin-filament structure. Changes in the spontaneous beating of cardiomyocytes corresponding with myofibrillar degeneration were apparent using differential interference contrast video microscopy. After finding induction of kinase activity by doxorubicin in cultured cardiomyocytes, the protective effects of specific inhibitors of kinase activity were assessed for their ability to inhibit doxorubicin-induced myofibrillar break-down. Doxorubicin-induced changes appeared similar to the degeneration observed after treatment with a protein kinase activator (phorbol 12-myristate 13-acetate) or a serine-threonine protein phosphatase inhibitor (okadaic acid). Collectively, these results indicate that activation of protein kinase is an important event in the initiation of myofibrillar degeneration by doxorubicin. Further analyses of myofibrillar proteins with respect to biochemical modifications will be necessary to determine if phosphorylation events transmit signal(s) to initiate degeneration.