Eric Carlemalm

WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells

BackgroundWnt proteins are important for developmental processes and certain diseases. WNT5A is a non-canonical Wnt protein that previously has been shown to play a role in the progression of malignant melanoma. High expression of WNT5A in melanoma tumors correlates to formation of distant metastasis and poor prognosis. This has partly been described by the findings that WNT5A expression in melanoma cell lines increases migration and invasion.MethodsMalignant melanoma cell lines were treated with rWNT5A or WNT5A siRNA, and mRNA versus protein levels of soluble mediators were measured using RT-PCR, cytokine bead array and ELISA. The induced signaling pathways were analyzed using inhibitors, Rho-GTPase pull down assays and western blot. Ultracentrifugation and electron microscopy was used to analyze microvesicles. Gene expression microarray data obtained from primary malignant melanomas was used to verify our data.ResultsWe show that WNT5A signaling induces a Ca2+-dependent release of exosomes containing the immunomodulatory and pro-angiogenic proteins IL-6, VEGF and MMP2 in melanoma cells. The process was independent of the transcriptional machinery and depletion of WNT5A reduced the levels of the exosome-derived proteins. The WNT5A induced exosomal secretion was neither affected by Tetanus toxin nor Brefeldin A, but was blocked by the calcium chelator Bapta, inhibited by a dominant negative version of the small Rho-GTPase Cdc42 and was accompanied by cytoskeletal reorganization. Co-cultures of melanoma/endothelial cells showed that depletion of WNT5A in melanoma cells decreased endothelial cell branching, while stimulation of endothelial cells with isolated rWNT5A-induced melanoma exosomes increased endothelial cell branching in vitro. Finally, gene expression data analysis of primary malignant melanomas revealed a correlation between WNT5A expression and the angiogenesis marker ESAM.ConclusionsThese data indicate that WNT5A has a broader function on tumor progression and metastatic spread than previously known; by inducing exosome-release of immunomodulatory and pro-angiogenic factors that enhance the immunosuppressive and angiogenic capacity of the tumors thus rendering them more aggressive and more prone to metastasize.

Ultrastructural and antigenic properties of neural stem cells and their progeny in adult rat subventricular zone

Neural stem cells (NSCs) in the subventricular zone (SVZ) continuously generate olfactory bulb interneurons in the adult rodent brain. Based on their ultrastructural and antigenic properties, NSCs, transient amplifying precursor cells, and neuroblasts (B, C, and A cells, respectively) have been distinguished in mouse SVZ. Here, we aimed to identify these cell types in rat SVZ ultrastructurally and at the light microscopy level, and to determine the antigenic properties of each cell type using gold and fluorescence immunolabeling. We found astrocytes with single cilia (NSCs, correspond to B cells) and neuroblasts (A cells). We also observed mitotic cells, ependymal cells, displaced ependymal cells, and mature astrocytes. In contrast, transient amplifying precursor cells (C cells) were not detected. The NSCs and neuroblasts had epidermal growth factor receptor (EGFR) and platelet‐derived growth factor receptor alpha (PDGFRα) expressed on the ciliary apparatus and were the only cell types incorporating the proliferation marker BrdU. Throughout mitosis, EGFR and PDGFRα were associated with the microtubule of the mitotic spindle. Ependymal and displaced ependymal cells also expressed EGFR and PDGFRα on their cilia but did not incorporate BrdU. Our findings indicate that the NSCs in adult rat SVZ give rise directly to neuroblasts. During mitosis, the NSCs disassemble the primary cilium and symmetrically distribute EGFR and PDGFRα among their progeny.

Low-temperature embedding procedures applied to chloroplasts

Pea and spinach chloroplasts were fixed with glutaraldehyde and dehydrated with ethylene glycol at 0°C. In some experiments dehydration was achieved by the resin itself. The material was infiltrated at temperatures between −35 and 0°C with mixtures of hydroxypropylmethacrylate and butylmethacrylate or a mixture of methacrylates and acrylates containing crosslinking agents. Polymerization was achieved, at the same temperature as the infiltration, using uv light and benzoin methyl ether as the catalyst. In uranyl acetate stained sections of specimens embedded at −35°C the thylakoid membranes appeared light between the strongly and evenly stained partition gaps. Dark spots of various shapes appeared in the middle of each thylakoid intercalated at irregular intervals by closely apposed membrane segments. In specimens embedded at −18 or 0°C the dark spots were replaced by a continuous dark layer identical to the partition gaps. The patterns seen in sections of embedded chloroplasts also appeared in specimens prepared by cryoultramicrotomy. The structural patterns described deviate markedly from those obtained when a conventional procedure is applied to the material mentioned.

α‐Latrotoxin‐induced transmitter release in feline oesophageal smooth muscle: focus on nitric oxide and vasoactive intestinal peptide

The effects of α‐latrotoxin (αLTX) on muscle tone, resting membrane potential, cyclic nucleotide content, and ultrastructure were examined in feline oesophageal smooth muscle, including the lower oesophageal sphincter (LOS). In circular smooth muscle strips from LOS developing active tone, αLTX (1 nm) induced a 94±3% (n=16) relaxation. Intermittent treatment with αLTX for 4 h abolished the response. Pretreatment with NG‐nitro‐l‐arginine (l‐NOARG; 0.1 mm) attenuated the relaxation. In carbachol‐contracted circular smooth muscle strips from the LOS and oesophageal body (OB), αLTX induced a 95±5% (n=6) and 73±9% (n=8) relaxation, respectively. The relaxations were attenuated by l‐NOARG, and in LOS strips, the relaxation was abolished by the combination of l‐NOARG and vasoactive intestinal peptide (VIP)‐antiserum (1:25). At resting tension in circular smooth muscle strips from the OB, αLTX induced a scopolamine sensitive contraction in the presence of l‐NOARG. In circular LOS and OB preparations, αLTX changed the resting membrane potential from −49±2 mV to −59±3 mV (n=4), and −62±2 mV to −71±3 mV (n=4), respectively. The αLTX‐induced relaxation of LOS and OB muscle was associated with a 138% and 72% increase in cyclic GMP levels, respectively. No changes in cyclic AMP levels were observed. Ultrastructural analysis of LOS and OB revealed a rich supply of nerve profiles containing small synaptic and large dense core vesicles. αLTX treatment resulted in a loss of both types of vesicle. These results suggest that αLTX induces relaxation of oesophageal circular smooth muscle associated with NO‐generation and transmitter release from synaptic vesicles. Beside NO, VIP seems to be involved in the relaxant effects of αLTX on the LOS. In addition, αLTX may have contractile effects by release of acetylcholine.

Subcellular distribution of spermidine/spermine N1-acetyltransferase

The subcellular distribution of the polyamine catabolic enzyme spermidine/spermine N1‐acetyltransferase (SSAT) was studied in L56Br‐C1 cells treated with 10 μM N1,N11‐diethylnorspermine (DENSPM) for 24 h. Cells were fractioned into three subcellular fractions. A particulate fraction containing the mitochondria was denoted as the mitochondrial fraction. After DENSPM treatment, an increase in SSAT activity was mainly found in the mitochondrial fraction. Western blot analysis showed an increased level of the SSAT protein in the mitochondrial fraction compared to the cytosolic fraction. Immunofluorescence microscopy and immunogold labeling transmission electron microscopy also showed a mitochondrial association of SSAT. Transmission electron microscopy revealed that the endoplasmic reticulum was devoid of ribosomes in DENSPM‐treated cells, in contrast to control cells that contained ample ribosomes. An increased SSAT activity in connection with the mitochondria may be part of the mechanism of DENSPM‐induced apoptosis.

Crystallization and preliminary X-ray investigation of the Escherichia coli molecular chaperone cpn60 (GroEL)

The Escherichia coli molecular chaperone, cpn60 (GroEL), has been purified from an overproducing E. coli strain and crystallized. Of the two crystal forms that were obtained, one was found to be suitable for crystallographic and structural studies at low resolution. Preliminary X‐ray investigation of the crystals show unit cell dimensions: a = 143.3, b = 154.6 and c = 265 Å, with α = 82°, β = 95° and γ = 107°. The space group is P1 and the crystals diffract to a maximum of 7 Å when using CuKα X‐rays from a rotating anode. Both electron microscopy and non‐denaturing electrophoretic analysis of redissolved cpn60 crystals show that cpn60 crystallizes in the native oligomeric form. Comparison between the dimensions of oligomeric cpn60 and the crystallographic unit cell volume suggests that the unit cell contains two oligomeric cpn60 molecules. The V M value for two cpn60 molecules per unit cell is 3.5 Å3/Da, corresponding to a water content of 65%. Electrophoretic analysis under denaturing conditions shows that the cpn60 in crystals is heterogeneous, and this probably explains the limited resolution of the diffraction data.

The response to electrons and developing conditions of two photographic films

Dose-response curves were determined for two 35 mm roll-films exposed to electrons and developed in various ways. The influence of exposure and emulsion type on the graininess of the developed material also was investigated. The results obtained were used to optimize the recording of electron microscopic images of sectioned bacteria.