Femoral fracture in an amputation stump.

Presentation: We report a case of a 60-year-old male with bilateral lower limb amputations who sustained a fracture to his right residual femur after weight bearing on his prosthetic leg during therapy. This was attributed to his raised body mass index that fell in the morbidly obese category. Diagnosis: Patient was referred for orthopaedic management and reviewed by the multidisciplinary prosthetic rehabilitation team following the fracture recovery. Treatment: He was deemed unsuitable for further prosthetic usage and ultimately referred to the national weight management service and the bariatric team. Discussion: Fracture of the residual limb in an amputee is not a common occurrence. There is a scarcity of information regarding fractures in an amputation stump of obese individuals. This is an unusual case which highlights the significance of body weight on lower limb prosthetic usage, impact on rehabilitation and the need to address weight management at an earlier stage post amputations.

Trends in measles cases in Bayelsa state, Nigeria: a five-year review of case-based surveillance data (2014-2018).

BACKGROUND: Measles is a vaccine preventable, highly transmissible viral infection that affects mostly children under five years. It has been ear marked for elimination and Nigeria adopted the measles elimination strategies of the World Health Organization (WHO) African region to reduce cases and deaths. This study was done to determine trends in measles cases in Bayelsa state, to describe cases in terms of person and place, identify gaps in the case-based surveillance data collection system and identify risk factors for measles infection. METHODS: We carried out a secondary data analysis of measles case-based surveillance data for the period of January 2014 to December 2018 obtained in Microsoft Excel from the State Ministry of Health. Cases were defined according to WHO standard case definitions. We calculated frequencies, proportions, estimated odds ratios (OR), 95% confidence intervals (CI) and multivariate analysis. RESULTS: A total of 449 cases of measles were reported. There were 245(54.6%) males and the most affected age group was 1-4 years with 288(64.1%) cases. Of all cases, 289(9.35%) were confirmed and 70 (48.27%) had received at least one dose of measles vaccine. There was an all-year transmission with increased cases in the 4th quarter of the year. Yenegoa local government area had the highest number of cases. Timeliness of specimen reaching the laboratory and the proportion of specimens received at the laboratory with results sent to the national level timely were below WHO recommended 80% respectively. Predictors of measles infection were, age less than 5 years (AOR: 0.57, 95% CI: 0.36-0.91) and residing in an urban area (AOR: 1.55, 95% CI:1.02-2.34). CONCLUSIONS: Measles infection occurred all-year round, with children less than 5 years being more affected. Measles case-based surveillance system showed high levels of case investigation with poor data quality and poor but improving indicators. Being less than 5 years was protective of measles while living in urban areas increased risk for infection. We recommended to the state government to prioritize immunization activities in the urban centers, start campaigns by the 4th quarter and continue to support measles surveillance activities and the federal government to strengthen regional laboratory capacities.

Trends and outcomes of non-traumatic major lower extremity amputations in an Irish tertiary referral hospital.

OBJECTIVES: Data on vascular patients following amputation in Ireland is lacking, limiting capability to plan services. This paper seeks to benchmark survival and rehabilitation outcomes among vascular patients in Ireland following lower extremity amputation (LEA), and compare subgroups of those undergoing transfemoral (TFA) or transtibial amputation (TTA). METHODS: A review was conducted of all patients who underwent non-traumatic TFA or TTA from 2000 to 2009 in a tertiary vascular surgery centre. Demographics, surgical data, perioperative outcomes, medium-term functional outcomes, and survival were assessed. RESULTS: One hundred and seventy-two patients (2:1 male: female) underwent 192 non-traumatic LEAs. Median age for TFA was 75 years and TTA 67 (p = 0.002). A percentage of 36.5% had undergone prior attempts at surgical revascularization, 25% had undergone prior distal amputation or debridement. Thirty-three (17%) required stump revision. Twenty-three (13.2%) died in hospital. Median survival for those who died in hospital was 17 days (0-367), versus 17 months (2-106) for those who survived to discharge. CONCLUSION: LEA for vascular pathology has significant morbidity and mortality, with long in-patient stays and short median survival; there is need to focus on improving quality of life in postoperative pathways.

Recycling endosome-dependent and -independent mechanisms for IL-10 secretion in LPS-activated macrophages.

IL-10 is a key anti-inflammatory cytokine secreted by activated macrophages as a feedback control mechanism to prevent excessive inflammatory responses. Here, we define multiple intracellular trafficking pathways involved in the secretion of newly synthesized IL-10 from macrophages following TLR4 activation with LPS, and show how this relates to the previously defined trafficking pathways for IL-6 and TNF in macrophages simultaneously producing these proinflammatory cytokines. IL-10 exits the Golgi in multiple tubular carriers, including those dependent on p230GRIP. Some of the IL-10 is then delivered to recycling endosomes, where cytokine sorting may occur prior to its release. Another portion of the IL-10 is delivered to the cell surface in distinct vesicles colabeled for apoE. Thus, we show at least two post-Golgi pathways via which IL-10 is trafficked, ensuring its secretion from activated macrophages under different physiological conditions.

PCBs in sediments and the coastal food web near a local contaminant source in Saglek Bay, Labrador.

Polychlorinated biphenyls (PCBs) were measured in marine sediments and the coastal food web in Saglek Bay, Labrador, to investigate the influence of a local PCB source. Saglek Bay has been the site of a military radar station since the late 1950s and there was PCB-contaminated soil at a beach prior to cleanup in 1997-1999. PCB concentrations in marine sediments during 1997-1999 ranged from 0.24 to 62000 ng/g (dry weight) and decreased exponentially with distance from the contaminated beach. Given this gradient, spatial trends of PCBs in the food web were examined over four zones, according to distance from the contaminated beach: within 1.5 km--zone one, 1.5-4.5 km--zone two, 4.5-7.5 km--zone three, and greater than 7.5 km--zone four. PCB concentrations in a bottom-feeding fish (shorthorn sculpin, Myoxocephalus scorpius), decreased significantly from zone one to zone two, three, four, and distant Labrador reference sites. PCB concentrations in the eggs of a diving seabird (black guillemot, Cepphus grylle) were as high as 48000 ng/g during 1997-1999 and average concentrations in zones one and two were 84 and 13 times higher than in zone four. Marine invertebrates closely reflected the concentrations of PCBs in the associated sediment. In contrast to the benthic-based food web, anadromous arctic char (Salvelinus alpinus) showed no evidence of PCB accumulation from the contaminated sediments. Relatively high PCB concentrations were discovered in some great black-backed gulls (Larus marinus) and ringed seals (Phoca hispida) but appear to relate more to their high trophic level than sampling location. Those species that fed on or near the seabed and had limited foraging ranges were strongly influenced by the local contamination. Total PCB concentrations in the benthic-based food web were significantly higher than background levels for a distance of at least 7.5 km from the contaminated beach. This area is small in the context of widely distributed contamination from long-range transport but the area's high concentrations are comparable to levels associated with adverse effects elsewhere. Our findings should be useful to better assess the environmental impacts of PCB contamination at other coastal sites in the Arctic.

The relative influence of distant and local (DEW-line) PCB sources in the Canadian Arctic.

Soil PCB contamination has been delineated at 18 of 21 Distant Early Warning Line (DEW-line) stations being cleaned up by the Canadian Department of National Defence (DND). As a result, detailed surface soil delineation data has been reported for contamination exceeding 1 microg/g (dw total Aroclor), which is the remedial criteria for PCB contaminated soil under the DEW-line cleanup project. The results of this delineation work has allowed us to estimate the mass of PCB contained in surface soil at these sites and to quantify the DEW-line as a source of PCBs to both local and Arctic wide contamination. Our analysis of DEW-line cleanup delineation reports suggests that pre-cleanup surface soils (top 10 cm) with over 1 microg/g PCB constituted a source of PCBs that ranged from 0.8 to 43 kg with a mean of 18 kg. The total mass of PCB at all 18 sites was 119 kg. Previous studies have described a "halo-effect" that surrounds DEW-line sites, whereby PCB signatures in soil and plants up to 10 km from source areas were attributed to the local source. At Cambridge Bay (CAM-M), Nunavut, our inventory of PCB sources and redistribution suggests that up to 3.4 kg of PCB were exported from the site to the surrounding tundra prior to cleanup. The primary mechanism of transportation appears to be wind borne particulate. Potential vapour phase emissions of PCB from contaminated soil at DEW-line sites appears to have been negligible.

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein.

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH(2)-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH(2)-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers.

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno-electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230GRIP fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230GRIP-transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230GRIP binds to a specific population of vesicles distinct from those labelled for beta-COP or gamma-adaptin. The GFP-p230GRIP fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulo-vesicular carriers.

A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells.

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface. We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodomain of the interleukin-2alpha (IL-2alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK(1) epithelial cells. Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin. Truncation mutants unable to bind beta-catenin were correctly targeted, showing, contrary to current understanding, that beta-catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine-mediated targeting is maintained in LLC-PK(1) cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line. These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.

Endocytosis of uncleaved tumor necrosis factor-alpha in macrophages.

Activated monocytes and macrophages secrete the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is produced as a 26 kd transmembrane protein that is cleaved to release a 17 kd soluble protein. TNF-alpha in both forms is biologically active. The intracellular trafficking of membrane-associated TNF-alpha in lipopolysaccharide-activated mouse macrophages was assessed after treatment with the metalloprotease inhibitor BB-3103, which prevents the cleavage of pro-TNF-alpha. Immunoprecipitation and immunofluorescence studies showed sustained expression of cell-associated TNF-alpha in the presence of the inhibitor. Cell immunoreactivity and surface biotinylation revealed that uncleaved TNF-alpha accumulated on the cell surface and was endocytosed, appearing in intracellular vesicles. Perturbation of post-Golgi traffic blocked the surface expression of 26 kd TNF-alpha. Tracking a bolus of TNF-alpha over time in cycloheximide-treated cells confirmed that uncleaved TNF-alpha is first transported to the cell surface and subsequently endocytosed. Vesicular structures immunoreactive for TNF-alpha were identified as endosomes by double labeling. The secretory and membrane-associated endocytic trafficking of TNF-alpha provides a mechanism for modulating the quantity of biologically active 26 kd TNF-alpha expressed on macrophages, allowing regulation of paracrine and autocrine responses.

Localization and post-Golgi trafficking of tumor necrosis factor-alpha in macrophages.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine secreted by activated macrophages. In this study, we examined the intracellular distribution and trafficking of TNF-alpha. Immunofluorescence and immunogold localization demonstrated that in lipopolysaccharide (LPS)-stimulated RAW264 macrophages, the greatest concentration of TNF-alpha is found in the perinuclear Golgi complex. Staining of the Golgi complex appeared 20 min after activation of cells and persisted for 2-12 h, and TNF-alpha appeared on the cell surface only transiently during this time. The rate of disappearance of Golgi staining correlated with the release of the cleaved, mature TNF-alpha into the medium. Pulse chase labeling and subcellular fractionation studies indicated that both 26-kDa and 17-kDa forms of TNF-alpha may be present at the level of the Golgi complex. Post-Golgi trafficking of TNF-alpha was modulated by agents that disrupt the cytoskeleton. Interferon-gamma (IFN-gamma), which primes macrophages for TNF-alpha-dependent cellular cytotoxicity, potentiated the effect of LPS by sustaining enhanced intracellular pools of TNF-alpha and also promoted redistribution of TNF-alpha into post-Golgi vesicular compartments. We propose that the primary pool of biologically active TNF-alpha in activated macrophages is held in the Golgi complex and that the cytokine is recruited directly from this intracellular pool for release in response to tumor cells or pathogens.

Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics.

E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles.

Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with beta-COP, gamma-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins. beta-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with beta-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.

GAIP, a Galphai-3-binding protein, is associated with Golgi-derived vesicles and protein trafficking.

Proteins of the regulators of G protein signaling (RGS) family bind to Galpha subunits to downregulate their signaling in a variety of systems. Galpha-interacting protein (GAIP) is a mammalian RGS protein that shows high affinity for the activated state of Galphai-3, a protein known to regulate post-Golgi trafficking of secreted proteins in kidney epithelial cells. This study aimed to localize GAIP in epithelial cells and to investigate its potential role in the regulation of membrane trafficking. LLC-PK1 cells were stably transfected with a c-myc-tagged GAIP cDNA. In the transfected and untransfected cells, GAIP was found in the cytosol and on cell membranes. Immunogold labeling showed that membrane-bound GAIP was localized on budding vesicles around Golgi stacks. When an in vitro assay was used to generate vesicles from isolated rat liver and Madin-Darby canine kidney cell Golgi membranes, GAIP was found to be concentrated in fractions of newly budded Golgi vesicles. Finally, the constitutive trafficking and secretion of sulfated proteoglycans was measured in cell lines overexpressing GAIP. We show evidence for GAIP regulation of secretory trafficking before the level of the trans-Golgi network but not in post-Golgi secretion. The location and functional effects of GAIP overlap only partially with those of Galphai-3 and suggest multiple roles for GAIP in epithelial cells.

Vesicle budding on Golgi membranes: regulation by G proteins and myosin motors.

One of the main functions of the Golgi complex is to generate transport vesicles for the post-Golgi trafficking of proteins in secretory pathways. Many different populations of vesicles are distinguished by unique sets of structural and regulatory proteins which participate in vesicle budding and fusion. Monomeric and heterotrimeric G proteins regulate vesicle budding and secretory traffic into and out of the Golgi complex. An inventory of G protein alpha subunits associated with Golgi membranes highlights their diverse involvement and potential for coupling Golgi trafficking, through various signal transduction pathways, to cell growth or other more specialized cell functions. Cytoskeletal proteins are now also known to associate specifically with the Golgi complex and Golgi-derived vesicles. Amongst these, conventional and unconventional myosins are recruited to vesicle membranes. Several roles in vesicle budding and vesicle trafficking can be proposed for these actin-based motors.

Budding roles for myosin II on the Golgi.

Myosin II--conventional myosin--has been typecast in muscle-man roles. While members of the Schwarzenegger clan from skeletal muscle have grabbed the limelight, myosin II motors in nonmuscle cells labour away in many varied and subtle roles. Recent findings show that nonmuscle myosin II, along with other myosins and cytoskeletal proteins, assembles on Golgi membranes. Nonmuscle myosin II associates transiently with membranes of the trans-Golgi network during the budding of a subpopulation of transport vesicles. The exact role of myosin II in vesicular trafficking is not yet understood, but its participation heralds a novel role for actin-based motors in vesicle budding.

Fluid-phase markers in the basolateral endocytic pathway accumulate in response to the actin assembly-promoting drug Jasplakinolide.

To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize actin in intact polarized Madin-Darby canine kidney (MDCK) cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a polarized manner, with a marked effect on fluid-phase uptake from the basolateral surface but not from the apical surface of polarized MDCK cells. The early uptake of FITC-dextran and HRP was increased more than twofold in JAS-treated cells. At later times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumulated endosomes were similar to late endosomes but they were not colabeled for other late endosome markers, such as rab7 or mannose-6-phosphate receptor. JAS altered transport in the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell morphology, inducing membrane bunching at the apical pole of MDCK cells. Although other studies have implicated actin in endocytosis at the apical cell surface, our results provide novel evidence that filamentous actin is also involved in the endocytosis of fluid-phase markers from the basolateral membrane of polarized cells.

Distinct localization of renin and GLUT-4 in juxtaglomerular cells of mouse kidney.

The insulin-responsive glucose transporter, GLUT-4, is found primarily in adipocytes and skeletal muscle cells, where it is sequestered in a specialized recycling compartment, from which it can be recruited to the cell surface following insulin stimulation. Lower levels of GLUT-4 are also expressed in other tissues, including the kidney, where it is present particularly in cells of the afferent arteriole and juxtaglomerular apparatus (JGA). The exact nature of GLUT-4-containing compartments and their relationship to other regulated trafficking pathways in different cells are not yet well defined. The trafficking of GLUT-4 has been studied in different cells with regulated secretory pathways, and a recent study shows that, in cardiomyocytes, GLUT-4 is sorted and packaged into multiple regulated pathways (J. W. Slot, G. Garruti, S. Martin, V. Oorschot, G. Pshuma, E. W. Kraegen, R. Laybutt, G. Thibault, and D. E. James. J. Cell Biol. 137: 1243-1254, 1997). In the kidney, cells of the JGA synthesize and secrete their major product, renin, via a well-established, regulated, secretory pathway. These cells also express GLUT-4 and thus offer the potential to directly compare the localization and trafficking of GLUT-4 and renin in a unique cell type. The present study was undertaken to investigate the intracellular distribution of GLUT-4 in mouse kidney cortex and to determine whether GLUT-4 and renin are trafficked in the same or in separate regulated pathways. Ultrathin cryosections of mouse kidney were labeled by the immunogold technique and viewed by electron microscopy, demonstrating the distribution of GLUT-4 in cells of the JGA, afferent arteriole, and distal tubule. In granular cells of the JGA, renin was localized in secretory granules of the regulated secretory pathway, whereas GLUT-4 labeling in the same cells was found in a distinct tubulovesicular compartment located adjacent to the trans-Golgi network. We show that granular cells have separate, morphologically distinct compartments for the sequestration of renin and GLUT-4, providing evidence that there may be distinct pathways for the sorting and trafficking of these two proteins.

Myosin II is associated with Golgi membranes: identification of p200 as nonmuscle myosin II on Golgi-derived vesicles.

A variety of peripheral membrane proteins associate dynamically with Golgi membranes during the budding and trafficking of transport vesicles in eukaryotic cells. A monoclonal antibody (AD7) raised against Golgi membranes recognizes a peripheral membrane protein, p200, which associates with vesicles budding off the trans-Golgi network (TGN). Based on preliminary findings, a potential association between p200 and myosin on Golgi membranes was investigated. Immunofluorescence staining of cultured cells under a variety of fixation conditions was carried out using an antibody raised against chick brush border nonmuscle myosin II. We show that, in addition to being found in the cytoplasm or associated with stress fibres, nonmuscle myosin II is also specifically localized on Golgi membranes. Myosin II was also detected on Golgi membranes by immunoblotting and by immunogold labeling at the electron microscopy level where it was found to be concentrated on Golgi-derived vesicles. The association of myosin II with Golgi membranes is dynamic and was found to be enhanced following activation of G proteins. Myosin II staining of Golgi membranes was also disrupted by brefeldin A (BFA). Colocalization of the AD7 and myosin II antibodies at the light and electron microscopy levels led us to investigate the nature of the 200 kDa protein recognized by both antibodies. The 200 kDa protein immunoprecipiated by the AD7 antibody was isolated from MDCK cells and used for microsequencing. Amino acid sequence data enabled us to identify p200 as the heavy chain of nonmuscle myosin IIA. In addition, an extra protein (240 kDa) recognized by the AD7 antibody specifically in extracts of HeLa cells, was sequenced and identified as another actin-binding protein, filamin. These results show that nonmuscle myosin II is associated with Golgi membranes and that the vesicle-associated protein p200, is itself a heavy chain of myosin II.