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A proteína Anexina A1 (ANXA1), sintetizada e liberada por fagócitos pela ação de glicocorticóides, é uma proteína anti-inflamatória, pois inibe o influxo de neutrófilos para o foco da inflamação, e induz os mecanismos de eferocitose em neutrófilos e macrófagos. Nosso grupo mostrou que a ANXA1 regula a expressão do receptor ativado por proliferadores de peroxissomos (PPAR) em macrófagos. Em continuidade, o presente trabalho investigou o mecanismo da ANXA1 sobre a expressão de PPARγ em macrófagos, e se este controle ocorre em demais leucócitos e tecido adiposo. Para tanto, macrófagos, neutrófilos peritoneais, linfócitos do baço, tecido adiposo epididimal foram obtidos de camundongos machos Balb/c selvagens (wild type, WT) ou geneticamente deficientes para ANXA1 (ANXA1-/-). Os resultados obtidos mostraram que a ANXA1 controla a expressão proteica e gênica de PPARγ em macrófagos, já que os níveis proteico (Western Blot, WB) e de RNAm (Real-time PCR) para PPARγ constitutivo, bem como induzidos pelos tratamentos in vitro com bezafibrato ou pioglitazona estavam reduzidos em macrófagos de animais ANXA1-/- em comparação com os níveis de macrófagos de animais WT, e o efeito parece ser dependente de CREB (WB), já que os níveis constitutivos deste fator de transcrição estavam maiores em macrófagos de animais ANXA1-/-. O tratamento in vitro com cicloheximida (CHX), um inibidor da síntese proteica, reduziu a expressão de PPARγ estimulada por bezafibrato ou LYSO-7 em macrófagos de animais WT, reforçando o papel da ANXA1 na expressão gênica de PPARγ. O FPR2 parece não estar envolvido no efeito, uma vez que o pré-tratamento de macrófagos com o antagonista de FPR2 (WRW4) não modificou a expressão de PPARγ em macrófagos de animais WT. O efeito modulador da ANXA1 ocorre em neutrófilos, mas não em tecido adiposo e linfócitos de animais ANXA1-/-. Ademais, a deficiência de ANXA1 não alterou a apoptose espontânea de neutrófilos. Em conjunto, os resultados obtidos mostram uma possível via adicional da ANXA1 sobre a resolução da inflamação, controlando a expressão de PPARγ em fagócitos. / Annexin A1 (ANXA1), is a protein synthetized and released by phagocytes due to the action of glucocorticoids, and an anti-inflammatory protein that inhibits neutrophil influx to site of inflammation and induces the mechanisms of efferocytosis in neutrophils and macrophages. Our group has already demonstrated that ANXA1 regulates the expression of peroxisome proliferator-activated receptor (PPAR) in macrophages. The present work aimed to investigate the role of ANXA1-dependent mechanisms on the expression of PPARγ in macrophages, and if said role also extends to other leukocytes and adipose tissue. For such, macrophages, peritoneal neutrophils, spleen lymphocytes, epididymal adipose tissue were obtained from male Balb/c wild type mice or from mice lacking ANXA1 genetically (ANXA-/-). Obtained results have demonstrated that ANXA1 regulates both proteic and genic expression of PPARγ in macrophages, as protein (Western Blotting, WB) and mRNA (Real-Time PCR) levels of constitutive PPARγ were reduced in macrophages from ANXA1-/- mice in comparison with the observed levels of macrophages from WT mice; the same is true for increased protein and mRNA levels as induced by in vitro treatments with bezafibrate or pioglitazone. This effect appears to be CREB-dependent (WB), as the constitutive levels of this transcription factor were found to be increased in macrophages from ANXA1-/- mice. In vitro treatment with cycloheximide (CHX), an inhibitor of proteic synthesis, reduced the bezafibrate or LYSO-7 (PPAR pan agonist, 10 µM / 2h) induced expression of PPARγ in WT mice, which further suggests a role for ANXA1 in PPARγ genic expression. FPR2 does not seem to be involved with these effects of ANXA1, as pre-treatment of macrophages from WT mice with an FPR2-antagonist (WRW4) did not alter expression of PPARγ. The modulating effect of ANXA1 can be verified in neutrophils of ANXA-/- mice, but not in adipocytes and lymphocytes from the same animals. Moreover, deficiency of ANXA1 did not affect spontaneous apoptosis of neutrophils. Altogether, the obtained results show the existence of a probable additional pathway with which ANXA1 promotes inflammation resolution, also controlling the expression of PPARγ in phagocytes.

A proteína Anexina A1 (ANXA1), sintetizada e liberada por fagócitos pela ação de glicocorticóides, é uma proteína anti-inflamatória, pois inibe o influxo de neutrófilos para o foco da inflamação, e induz os mecanismos de eferocitose em neutrófilos e macrófagos. Nosso grupo mostrou que a ANXA1 regula a expressão do receptor ativado por proliferadores de peroxissomos (PPAR) em macrófagos. Em continuidade, o presente trabalho investigou o mecanismo da ANXA1 sobre a expressão de PPARγ em macrófagos, e se este controle ocorre em demais leucócitos e tecido adiposo. Para tanto, macrófagos, neutrófilos peritoneais, linfócitos do baço, tecido adiposo epididimal foram obtidos de camundongos machos Balb/c selvagens (wild type, WT) ou geneticamente deficientes para ANXA1 (ANXA1-/-). Os resultados obtidos mostraram que a ANXA1 controla a expressão proteica e gênica de PPARγ em macrófagos, já que os níveis proteico (Western Blot, WB) e de RNAm (Real-time PCR) para PPARγ constitutivo, bem como induzidos pelos tratamentos in vitro com bezafibrato ou pioglitazona estavam reduzidos em macrófagos de animais ANXA1-/- em comparação com os níveis de macrófagos de animais WT, e o efeito parece ser dependente de CREB (WB), já que os níveis constitutivos deste fator de transcrição estavam maiores em macrófagos de animais ANXA1-/-. O tratamento in vitro com cicloheximida (CHX), um inibidor da síntese proteica, reduziu a expressão de PPARγ estimulada por bezafibrato ou LYSO-7 em macrófagos de animais WT, reforçando o papel da ANXA1 na expressão gênica de PPARγ. O FPR2 parece não estar envolvido no efeito, uma vez que o pré-tratamento de macrófagos com o antagonista de FPR2 (WRW4) não modificou a expressão de PPARγ em macrófagos de animais WT. O efeito modulador da ANXA1 ocorre em neutrófilos, mas não em tecido adiposo e linfócitos de animais ANXA1-/-. Ademais, a deficiência de ANXA1 não alterou a apoptose espontânea de neutrófilos. Em conjunto, os resultados obtidos mostram uma possível via adicional da ANXA1 sobre a resolução da inflamação, controlando a expressão de PPARγ em fagócitos. / Annexin A1 (ANXA1), is a protein synthetized and released by phagocytes due to the action of glucocorticoids, and an anti-inflammatory protein that inhibits neutrophil influx to site of inflammation and induces the mechanisms of efferocytosis in neutrophils and macrophages. Our group has already demonstrated that ANXA1 regulates the expression of peroxisome proliferator-activated receptor (PPAR) in macrophages. The present work aimed to investigate the role of ANXA1-dependent mechanisms on the expression of PPARγ in macrophages, and if said role also extends to other leukocytes and adipose tissue. For such, macrophages, peritoneal neutrophils, spleen lymphocytes, epididymal adipose tissue were obtained from male Balb/c wild type mice or from mice lacking ANXA1 genetically (ANXA-/-). Obtained results have demonstrated that ANXA1 regulates both proteic and genic expression of PPARγ in macrophages, as protein (Western Blotting, WB) and mRNA (Real-Time PCR) levels of constitutive PPARγ were reduced in macrophages from ANXA1-/- mice in comparison with the observed levels of macrophages from WT mice; the same is true for increased protein and mRNA levels as induced by in vitro treatments with bezafibrate or pioglitazone. This effect appears to be CREB-dependent (WB), as the constitutive levels of this transcription factor were found to be increased in macrophages from ANXA1-/- mice. In vitro treatment with cycloheximide (CHX), an inhibitor of proteic synthesis, reduced the bezafibrate or LYSO-7 (PPAR pan agonist, 10 µM / 2h) induced expression of PPARγ in WT mice, which further suggests a role for ANXA1 in PPARγ genic expression. FPR2 does not seem to be involved with these effects of ANXA1, as pre-treatment of macrophages from WT mice with an FPR2-antagonist (WRW4) did not alter expression of PPARγ. The modulating effect of ANXA1 can be verified in neutrophils of ANXA-/- mice, but not in adipocytes and lymphocytes from the same animals. Moreover, deficiency of ANXA1 did not affect spontaneous apoptosis of neutrophils. Altogether, the obtained results show the existence of a probable additional pathway with which ANXA1 promotes inflammation resolution, also controlling the expression of PPARγ in phagocytes.

ABSTRACT Infection with the hepatitis B virus (HBV) is still a major global health problem with an estimated 6% of the world’s population chronically infected with the virus. Chronic infection with HBV subgenotype A1, which is hyperendemic to southern Africa, is associated with a particularly high incidence of liver cancer and cirrhosis. Understanding HBV replication and developing effective HBV treatment to prevent liver cancer remain important medical priorities. Although there is a preventative vaccine for HBV, efficacy of currently available treatment of established infection is limited. Exploiting the RNA interference (RNAi) pathway through the use of small interfering (siRNA) and short hairpin RNA (shRNA) is an attractive new approach for the development gene therapies against HBV infection. Our laboratory has designed and demonstrated the efficacy both in vitro and in vivo of several shRNAs designed to target the X open reading frame (ORF) of HBV. Thus, the objective of this study was to construct a replication competent plasmid vector of the A1 subgenotype, a reporter plasmid vector of HBV and to assess the efficacy of RNAi effecters against these vectors both in vitro and in vivo. The first HBV replication competent vector, pCR-HBVA1 1.3x, containing the sequence of an HBV subgenotype A1 isolate, was successfully constructed by generating a greater than genome length sequence of HBV, that starts just upstream of endogenous HBV basic core promoter (BCP) and ends just downstream of the unique HBV polyadenylation (pA) site. Human hepatoma (Huh7) cells transfected with this plasmid secreted HBV surface antigen (HBsAg) into Abstract viii culture supernatants. In the murine hydrodynamic injection model of HBV replication, serum HBsAg, hepatitis B e antigen (HBeAg) and viral particle levels as well as relative surface and core mRNA levels were shown to be significantly elevated as compared to mock-injected mice. The second HBV vector, pCH-FLuc, was successfully generated by replacing the surface ORF with the sequence encoding Firefly Luciferase. The ability of pCH-FLuc to express Firefly Luciferase was demonstrated in a liver cell line (Huh7 cells). Co-transfection of the reporter plasmid, pCH-FLuc, with shRNAs targeted to HBV caused a significant reduction in Luciferase expression. Co-transfection/injection of the pCR-HBVa1 1.3x with shRNAs caused significant inhibition in the level of viral antigens (HBsAg, HBeAg and hepatitis B core antigen (HBcAg) as well as relative surface and core mRNA levels. This was observed both in vitro and in vivo. Our results demonstrate the potential this model allows for the study of HBV replication as well as the assessment of potential therapeutic strategies in a regionally significant subgenotype of HBV.

RNA-binding proteins play a central role in the post-transcriptional regulation of gene expression; however, little is known about the endogenous transcripts to which they bind. Here, I have used the ultra-violet cross-linking and immuno-precipitation (CLIP) technique to identify RNA targets directly bound to two RNA-binding proteins: Acinus and hnRNP A1. Acinus (apoptotic chromatin condensation inducer in the nucleus) contains a region that is homologous to the RNA binding domain of the Drosophila splicing regulator sex-lethal, and a serine and arginine rich region similar to that seen in the SR family of proteins, which function extensively in splicing. Furthermore it is a component of the multi-protein spliceosome complex, and I have demonstrated it can directly bind polyadenylated RNA. I have shown that Acinus displays a diffuse nuclear localisation pattern, however, overexpression of an epitope-tagged protein results in its accumulation in enlarged nuclear speckles. Together these results suggest a role in pre-mRNA splicing. Acinus is cleaved during apoptosis by caspase-3, resulting in a truncated protein with chromatin condensation inducing activity (Sahara et al., 1999). Accordingly, I have demonstrated that overexpression of epitope-tagged Acinus results in an increased number of cells exhibiting an apoptotic phenotype. The proteolytic fragment contains the RNA binding region, and to determine if the role of Acinus in apoptosis is mediated by RNA interactions I utilised CLIP to identify in vivo RNA targets. I have identified several mRNA targets of Acinus and found that the binding sites in those mRNA targets predominantly map to constitutively expressed exons. This is in agreement with the exon junction complex, of which Acinus is a component, being deposited on mRNAs after splicing. These results may indicate that Acinus is a core RNA binding factor of the exon junction complex. To complement this approach, I also performed CLIP with a known alternative splicing regulator, hnRNP A1. In this manner, the binding site preferences could be compared between the two proteins. As expected, the majority of hnRNP A1 binding sites are located in introns, corresponding with their identified role of antagonizing pre-mRNA splicing by binding intronic splicing elements. Interestingly, a number of the CLIP tags are located in, or adjacent to, alternatively spliced events suggesting a role for hnRNP A1 in the regulation of alternative splicing of these specific pre-mRNAs. In addition to pre-mRNA splicing hnRNP A1 also functions in the cellular stress response. Upon environmental stresses it relocates to the cytoplasm and accumulates in cytoplasmic foci known as stress granules (Guil et al., 2006). Here I show some of the targets identified by CLIP are regulated by hnRNP A1 in times of cellular stress. In summary, I have identified two novel subsets of RNAs, bound by Acinus or hnRNP A1 in vivo. I have shown these proteins exhibit distinct binding preferences, which correspond to their biological function. This work is consistent with hnRNP A1 acting as an alternative splicing regulator, and provides evidence for a dual role of Acinus in mRNA splicing and apoptosis. This study also demonstrates the power of the CLIP technique, as identification of in vivo RNA targets allows greater understanding of the mechanisms by which RNA-binding proteins exert their regulatory control.

Student Number : 0009716H - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Positive charges play an important role in the active site of a variety of proteins including alpha class GST. The presence of Arg15 at the subunit interface between the H- and Gsite of hGST A1-1 was shown to be important for the catalytic function of the enzyme, thus providing an electrostatic potential in the G-site. This electrostatic potential favours ionisation and activation of GSH bound to hGST A1-1. Although much is known regarding the catalytic function of GSTs, little is known of the effect of Arg15 on the binding of glutathione conjugates and nonsubstrate ligands. Others (Matulis and Lovrien, 1998) have pointed out that binding of the nonsubstrate ligand ANS is accomplished via ion pair formation with a charged residue such as Arg. Therefore, in order to evaluate the importance of the positive charge provided by the side chain of Arg15 in the active site of hGST A1-1 protein engineering techniques were employed to generate an R15L variant. The variant does not display a detectable difference in the secondary or tertiary structural content relative to the wild type hGST A1-1 protein. The catalytic activity of the R15L variant is, however, substantially reduced indicating local tertiary structural changes at and possibly near the active site. In the presence of ANS the fluorescence spectra of the variant R15L protein had a significantly lower intensity relative to wild type protein indicating either increased exposure of hydrophobic surface area at the Hsite and/or reduced number of ANS molecules bound (i.e., reduced affinity). Although ANS is displaced by GSO3 - in the wild type protein, no such displacement is observed for the R15L variant protein. Isothermal titration calorimetry experiments for GSO3 - and ANS binding to R15L indicated that the variant protein binds GSO3 - more tightly (higher affinity) than ANS (lower affinity). The above information, taken together, argues in favour of both molecules (ANS and GSO3 -) occupying the active site of R15L simultaneously. Despite the fact that there is no experimental crystal structure of hGST A1-1 complexed with either of the ligands, the placement of ANS was argued (Dirr et al., 2005) to be at the H-site and of GSO3 - at the G-site of the protein. The binding of ANS is both enthalpically and entropically favourable over the temperature range investigated (5 to 25°C) indicating expulsion of water molecules from the active site as well as the lack of a localised C-terminal #1;9 helix. The hydrophobic character of both the ligand and the site which accommodates it (H-site) contribute favourably towards the reaction enthalpy. ANS is conformationaly more constrained than GSO3 - when found free in solution, thus exhibiting more favourable conformational entropy change upon binding to R15L variant. The binding of GSO3 - is enthalpically favourable but entropically opposed. The expulsion of water molecules from the G-site, which accommodates GSO3 -, is insufficient to compensate for the loss of conformational entropy of both the ligand and C-terminal #1;9 helix, which becomes localised upon binding. However, favourable enthalpic contributions arise from the formation of hydrogen bonds, salt links and van der Waals contacts between the R15L variant and the ligand and the C-terminal #1;9 helix. Arg15 forms a salt link with Glu104 of the neighbouring domain and therefore provided an ideal opportunity to evaluate the role of Arg15 and its impact on interdomain stability. Conformational stability and unfolding studies of the R15L variant, monitored via fluorescence, indicated that the variant deviates from the model of a two-state transition. There are no stable thermodynamic intermediate(s) observed, but the increase in the mvalue is an indicator of a transition state which is more stable against urea relative to the wild type protein. Kinetically (Wallace et al., 1998b), the wild type hGST A1-1 follows a three-state unfolding transition where the kinetic intermediate is defined as having undergone conformational changes at the domain-domain interface and the C-terminal region. The substitution of Arg15 with Leu may have impacted on the packing of the native form of the variant R15L relative to the wild type GST A1-1, thus rendering the R15L protein more stable against urea. Arg15, therefore, may have an important role at the interdomain interface of GSTs.

Diabetes is a complex disease characterised by hyperglycaemia, which often leads to microvascular complications including diabetic nephropathy and cardiomyopathy. In this thesis, I have investigated the role of Annexin-A1 (ANXA1), an endogenous anti-inflammatory peptide, in two experimental murine models of diabetes caused by streptozotocin (STZ) or high-fat high-sugar diet (HFD), which mimic type-1 (T1DM) and type-2 diabetes (T2DM) respectively. I have also investigated the levels of ANXA1 in patients with either T1DM or T2DM. Patients with T1DM have increased plasma ANXA1 levels. In a murine models of type 1 diabetes loss of endogenous ANXA1 aggravates both cardiac and renal dysfunction in mice. Specifically, I have shown that key mediators of the MAPK pathway (p38, JNK and ERK1/2) are constitutively activated in ANXA1-/- mice, and activation of these pathways is exacerbated in diabetic ANXA1-/- mice. Administration of human recombinant (hr) ANXA1 did not alter the diabetic phenotype in diabetic WT mice, but attenuated the cardiac and renal dysfunction caused by STZ. Interestingly, late administration of ANXA1 (after significant cardiac and renal dysfunction had already developed) halted the progression of both cardiac and renal dysfunction. Patients with T2DM have increased plasma ANXA1 levels. HFD-fed ANXA1-/- mice have a more severe diabetic phenotype compared to HFD-fed WT mice. Therapeutic administration of hrANXA1 prevented the development of a diabetic phenotype. Specifically, I have shown that the insulin signalling pathway is further perturbed in diabetic mice resulting in severe insulin resistance, and that these signalling abnormalities were prevented by therapeutic administration of hrANXA1. In addition, loss of endogenous ANXA1 aggravates both cardiac and renal dysfunction in mice with experimental T2DM. The GTPase RhoA is constituently activated in ANXA1-/- mice leading downstream activation of MYPT1. Feeding a HFD also activated the small GTPase RhoA, leading to increased MYPT1 activity, which could be attenuated with treatment with hrANXA1. Mice subjected to HFD for 12 weeks had a more 'leaky' blood brain barrier (BBB), which is further exacerbated in ANXA1-/- mice fed a HFD. Compared to mice fed a chow diet, mice fed a HFD had an augmented CD4+ T-cell profile; with a clear decline in CD4+FoxP3+ (anti-inflammatory) and increase in CD4+RORgt+ (pro-inflammatory) cells. Administration of hrANXA1 to mice fed on HFD restored BBB integrity and CD4+ T-cells profile similar to mice fed on normal chow diet. Mice fed a HFD also had more activated CD4+ T-cells, which adhered more readily and transmigrated through a brain endothelial mono-layer ex vivo. In contrast, administration of hrANXA1 to mice fed on HFD reduced re-activity of CD4+ T-cells, reducing the number of adherent CD4+ T-cells to the brain endothelial mono-layer.

Neutrophil extravasation into tissues is an essential process required for the inflammatory response. Upon receiving an inflammatory cue, neutrophils begin accumulating on the luminal surface of the endothelium. Neutrophil recruitment is initiated by selectin-mediated tethering and rolling of neutrophils along the endothelial monolayer, followed by integrin-mediated firm adhesion. Adherent neutrophils then traverse the endothelium in a process known as transendothelial migration. The events mediating the rolling and adhesion steps are well characterised, but research into the molecular mechanisms regulating transendothelial migration is an area of intense focus. A previous study conducted in our laboratory found that the activation of endothelial extracellular signal-regulated kinase (ERK) 1/2 was required for neutrophil transmigration. Furthermore, it was found that endothelial ERK was activated in response to a soluble protein produced by fMLP- or IL-8-stimulated neutrophils. In the present study, the soluble ERK-activating neutrophil protein was identified as annexin A1, which was selected as a possible candidate following mass spectrometry analysis of proteins secreted from activated neutrophils. Annexin A1 antibodies (Abs) were found to block endothelial ERK activation induced by conditioned medium harvested from stimulated neutrophils. Annexin A1 Abs were additionally able to inhibit neutrophil transmigration across human umbilical vein endothelial cell (HUVEC) monolayers in an in vitro transmigration assay. Following the purification of recombinant annexin A1, it was demonstrated that it could activate endothelial ERK in a similar manner to neutrophil conditioned medium. Upon further investigation, ERK activation was found to be induced by a truncated form of annexin A1 present in the protein preparation rather than the full length protein. Calpain I, a calcium dependent protease that is activated upon neutrophil stimulation and is known to cleave annexin A1 within the N-terminal domain, was shown to process full length inactive recombinant annexin A1 into an unidentified product that could activate endothelial ERK. A calpain I inhibitor was also found to prevent stimulated neutrophils from secreting an ERK-activating protein, thus further suggesting a role for calpain I in this process. As full length annexin A1 has been reported to signal through the formyl peptide receptor (FPR) family, a pan-FPR antagonist was incubated with endothelial cells and was found to inhibit ERK activation induced by neutrophil conditioned medium, indicating that pro-inflammatory annexin A1 is also a FPR ligand. Endothelial projections termed “transmigratory cups” form around neutrophils during extravasation, of which ICAM-1 is a major component. Using an assay that examined transmigratory cups during neutrophil transmigration, it was found that annexin A1 Abs could inhibit neutrophil adhesion and transmigration through HUVEC monolayers by interfering with transmigratory cup formation around neutrophils, as shown by monitoring ICAM-1 during the process. Quantification of transmigrating neutrophils highlighted that the majority of neutrophils were emigrating via a transcellular pathway, which is in opposition to many in vitro studies where paracellular transmigration predominates. The results generated from this study identified a novel pro-inflammatory role for annexin A1 in neutrophil transendothelial migration. Preliminary experiments suggested that the pro-inflammatory annexin A1 responsible for endothelial ERK activation was a truncated form. Calpain I appears to be a likely candidate responsible for the generation of this uncharacterised, truncated annexin A1 product, however further experiments are required to confirm this hypothesis. Pro-inflammatory annexin A1 represents a new target for the treatment of inflammatory disorders. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374554 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Zugl.: Berlin, Freie Univ., Diss., 2007

Abstract The role of vacuolar H+-ATPase (V-ATPase) in exocytic and endocytic membrane transport processes was studied by using its specific inhibitor, bafilomycin A1 (Baf A1), as a tool. On the exocytic pathway, both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER) were inhibited by Baf A1. Furthermore, p58/ERGIC-53, which normally cycles between the ER, the intermediate compartment (IC), and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive 80-nm Golgi (COPI) vesicles was reduced, suggesting that the drug inhibits the vesicle-mediated retrieval of the protein from post-ER compartments. The small GTPase rab1p was efficiently recruited to the tubules, accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of anterogradely transported proteins, indicating that they participate in retrograde transport. Interestingly, acidic lumenal pH could only be detected in the more central pre-Golgi elements. The forward (anterograde) transport of newly synthesized Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV) glycoproteins from the ER to the cis-Golgi was largely unaffected by Baf A1. However, maturation processes occurring in the trans-Golgi were inhibited, and the amounts of viral glycoproteins appearing at the cell surface were reduced. Newly synthesized VSV glycoprotein accumulated into rab1p-positive Golgi membranes in the presence of Baf A1, indicating that the transport from cis-Golgi was affected. Furthermore, O-glycosylation of the expressed CD8 chimeras and lectin cytochemistry experiments indicate that Baf A1 affects the transport from cis-Golgi. Instead, Baf A1 did not affect the transport of viral glycoproteins from the trans-Golgi network to the cell surface. We propose, that anterograde intra-Golgi traffic may be affected indirectly by Baf A1, as it inhibits retrograde vesicle-mediated transport and thus cisternal maturation. Baf A1 inhibited the entry of SFV into BHK-21 cells. Thus, V-ATPase was responsible for the acidification of the endosomes needed for virus entry. In cells infected with VSV and subsequently treated with Baf A1, virus particles were found to be accumulated in tubular membrane structures, which also contained endocytosed BSA-gold. Neither VSV nor BSA-gold particles were detected in lysosomal glycoprotein (lgp) 120-positive lysosomes, however. Thus, secreted and further endocytosed virus particles accumulate into tubulated endocytic organelles, apparently early endosomes, in Baf A1-treated cells. We conclude that the transport from endosomes to lysosomes is inhibited by Baf A1. The bulk of rab7 GTPase, which participates in vesicle fusion to late endosomes, was localized to the ruffled border (RB) membrane of bone-resorbing osteoclast. This indicates that the membrane has some characteristics of late endosomal membranes and that endocytic membrane transport is oriented towards the RB. Consistently, both endocytosed lumenal horseradish peroxidase and receptor-bound transferrin were delivered to the RB. The delivery of membrane-associated transferrin to the RB further indicates that the RB has some endosomal characteristics and suggests that the endocytic pathway contributes to the maintenance of functional RB. The endocytic pathway could act in balancing the membrane traffic associated with transcytosis from the RB to the basal plasma membrane. Endocytic processes in osteoclasts appeared to be very sensitive to Baf A1. Thus, blocking of the endocytic membrane traffic towards the RB could explain the inactivation of cells by low concentrations of the drug.