siRNA screening reveals that SNAP29 contributes to exosome release.

Cells release extracellular vesicles (EVs) of different sizes. Small EVs (< 200 nm) can originate from the fusion of multivesicular bodies with the plasma membrane, i.e. exosomes, and from budding of the plasma membrane, i.e. small ectosomes. To investigate the molecular machinery required for the release of small EVs, we developed a sensitive assay based on incorporation of radioactive cholesterol in EV membranes and used it in a siRNA screening. The screening showed that depletion of several SNARE proteins affected the release of small EVs. We focused on SNAP29, VAMP8, syntaxin 2, syntaxin 3 and syntaxin 18, the depletion of which reduced the release of small EVs. Importantly, this result was verified using gold standard techniques. SNAP29 depletion resulted in the largest effect and was further investigated. Immunoblotting analysis of small EVs showed that the release of several proteins considered to be associated with exosomes like syntenin, CD63 and Tsg101 was reduced, while the level of several proteins that have been shown to be released in ectosomes (annexins) or by secretory autophagy (LC3B and p62) was not affected by SNAP29 depletion. Moreover, these proteins appeared in different fractions when the EV samples were further separated by a density gradient. These results suggest that SNAP29 depletion mainly affects the secretion of exosomes. To investigate how SNAP29 affects exosome release, we used microscopy to study the distribution of MBVs using CD63 labelling and CD63-pHluorin to detect fusion events of MVBs with the plasma membrane. SNAP29 depletion caused a redistribution of CD63-labelled compartments but did not change the number of fusion events. Further experiments are therefore needed to fully understand the function of SNAP29. To conclude, we have developed a novel screening assay that has allowed us to identify several SNAREs involved in the release of small EVs.

Diacylglycerol kinase and phospholipase D inhibitors alter the cellular lipidome and endosomal sorting towards the Golgi apparatus.

The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.

The role of lipid species in membranes and cancer-related changes.

Several studies have demonstrated interactions between the two leaflets in membrane bilayers and the importance of specific lipid species for such interaction and membrane function. We here discuss these investigations with a focus on the sphingolipid and cholesterol-rich lipid membrane domains called lipid rafts, including the small flask-shaped invaginations called caveolae, and the importance of such membrane structures in cell biology and cancer. We discuss the possible interactions between the very long-chain sphingolipids in the outer leaflet of the plasma membrane and the phosphatidylserine species PS 18:0/18:1 in the inner leaflet and the importance of cholesterol for such interactions. We challenge the view that lipid rafts contain a large fraction of lipids with two saturated fatty acyl groups and argue that it is important in future studies of membrane models to use asymmetric membrane bilayers with lipid species commonly found in cellular membranes. We also discuss the need for more quantitative lipidomic studies in order to understand membrane function and structure in general, and the importance of lipid rafts in biological systems. Finally, we discuss cancer-related changes in lipid rafts and lipid composition, with a special focus on changes in glycosphingolipids and the possibility of using lipid therapy for cancer treatment.

Drug-Loaded Photosensitizer-Chitosan Nanoparticles for Combinatorial Chemo- and Photodynamic-Therapy of Cancer.

In this study we have developed biodegradable polymeric nanoparticles (NPs) containing the cytostatic drugs mertansine (MRT) or cabazitaxel (CBZ). The NPs are based on chitosan (CS) conjugate polymers synthesized with different amounts of the photosensitizer tetraphenylchlorin (TPC). These TPC-CS NPs have high loading capacity and strong drug retention due to π-π stacking interactions between the drugs and the aromatic photosensitizer groups of the polymers. CS polymers with 10% of the side chains containing TPC were found to be optimal in terms of drug loading capacity and NP stability. The TPC-CS NPs loaded with MRT or CBZ displayed higher cytotoxicity than the free form of these drugs in the breast cancer cell lines MDA-MB-231 and MDA-MB-468. Furthermore, light-induced photochemical activation of the NPs elicited a strong photodynamic therapy effect on these breast cancer cells. Biodistribution studies in mice showed that most of the TPC-CS NPs accumulated in liver and lungs, but they were also found to be localized in tumors derived from HCT-116 cells. These data suggest that the drug-loaded TPC-CS NPs have a potential in combinatory anticancer therapy and as contrast agents.

Biological response and cytotoxicity induced by lipid nanocapsules.

BACKGROUND: Lipid nanocapsules (LNCs) are promising vehicles for drug delivery. However, since not much was known about cellular toxicity of these nanoparticles in themselves, we have here investigated the mechanisms involved in LNC-induced intoxication of the three breast cancer cell lines MCF-7, MDA-MD-231 and MDA-MB-468. The LNCs used were made of Labrafac™ Lipophile WL1349, Lipoid® S75 and Solutol® HS15. RESULTS: High resolution SIM microscopy showed that the DiD-labeled LNCs ended up in lysosomes close to the membrane. Empty LNCs, i.e. without encapsulated drug, induced not only increased lysosomal pH, but also acidification of the cytosol and a rapid inhibition of protein synthesis. The cytotoxicity of the LNCs were measured for up to 72 h of incubation using the MTT assay and ATP measurements in all three cell lines, and revealed that MDA-MB-468 was the most sensitive cell line and MCF-7 the least sensitive cell line to these LNCs. The LNCs induced generation of reactive free oxygen species and lipid peroxidation. Experiments with knock-down of kinases in the near-haploid cell line HAP1 indicated that the kinase HRI is essential for the observed phosphorylation of eIF2α. Nrf2 and ATF4 seem to play a protective role against the LNCs in MDA-MB-231 cells, as knock-down of these factors sensitizes the cells to the LNCs. This is in contrast to MCF-7 cells where the knock-down of these factors had a minor effect on the toxicity of the LNCs. Inhibitors of ferroptosis provided a large protection against LNC toxicity in MDA-MB-231 cells, but not in MCF-7 cells. CONCLUSIONS: High doses of LNCs showed a different degree of toxicity on the three cell lines studied, i.e. MCF-7, MDA-MD-231 and MDA-MB-468 and affected signaling factors and the cell fate differently in these cell lines.

Exogenous lysophospholipids with large head groups perturb clathrin-mediated endocytosis.

In this study, we have investigated how clathrin-dependent endocytosis is affected by exogenously added lysophospholipids (LPLs). Addition of LPLs with large head groups strongly inhibits transferrin (Tf) endocytosis in various cell lines, while LPLs with small head groups do not. Electron and total internal reflection fluorescence microscopy (EM and TIRF) reveal that treatment with lysophosphatidylinositol (LPI) with the fatty acyl group C18:0 leads to reduced numbers of invaginated clathrin-coated pits (CCPs) at the plasma membrane, fewer endocytic events per membrane area and increased lifetime of CCPs. Also, endocytosis of Tf becomes dependent on actin upon LPI treatment. Thus, our results demonstrate that one can regulate the kinetics and properties of clathrin-dependent endocytosis by addition of LPLs in a head group size- and fatty acyl-dependent manner. Furthermore, studies performed with optical tweezers show that less force is required to pull membrane tubules outwards from the plasma membrane when LPI is added to the cells. The results are in agreement with the notion that insertion of LPLs with large head groups creates a positive membrane curvature which might have a negative impact on events that require plasma membrane invagination, while it may facilitate membrane bending toward the cell exterior.

Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology.

Exosomes are a type of extracellular vesicle released from cells after fusion of multivesicular bodies with the plasma membrane. These vesicles are often enriched in cholesterol, SM, glycosphingolipids, and phosphatidylserine. Lipids not only have a structural role in exosomal membranes but also are essential players in exosome formation and release to the extracellular environment. Our knowledge about the importance of lipids in exosome biology is increasing due to recent technological developments in lipidomics and a stronger focus on the biological functions of these molecules. Here, we review the available information about the lipid composition of exosomes. Special attention is given to ether lipids, a relatively unexplored type of lipids involved in membrane trafficking and abundant in some exosomes. Moreover, we discuss how the lipid composition of exosome preparations may provide useful information about their purity. Finally, we discuss the role of phosphoinositides, membrane phospholipids that help to regulate membrane dynamics, in exosome release and how this process may be linked to secretory autophagy. Knowledge about exosome lipid composition is important to understand the biology of these vesicles and to investigate possible medical applications.

Clathrin-independent endocytosis: an increasing degree of complexity.

This article aims at providing an update on the complexity of clathrin-independent endocytosis. It is now almost 30 years since we first wrote a review about its existence; at that time many people believed that with the exception of macropinocytosis, which will only be briefly mentioned in this review, all uptake could be accounted for by clathrin-dependent endocytosis. Now it is generally accepted that there are different clathrin-independent mechanisms, some of them regulated by ligands and membrane lipid composition. They can be both dynamin-dependent and -independent, meaning that the uptake cannot be accounted for by caveolae and other dynamin-dependent processes such as tubular structures that can be induced by toxins, e.g. Shiga toxin, or the fast endophilin mediated endocytosis recently described. Caveolae seem to be mostly quite stable structures with other functions than endocytosis, but evidence suggests that they may have cell-type dependent functions. Although several groups have been working on endocytic mechanisms for years, and new advanced methods have improved our ability to study mechanistic details, there are still a number of important questions we need to address, such as: How many endocytic mechanisms does a cell have? How quantitatively important are they? What about the complexity in polarized cells where clathrin-independent endocytosis is differentially regulated on the apical and basolateral poles? These questions are not easy to answer since one and the same molecule may contribute to more than one process, and manipulating one mechanism can affect another. Also, several inhibitors of endocytic processes commonly used turn out to be less specific than originally thought. We will here describe the current view of clathrin-independent endocytic processes and the challenges in studying them.

Benzyl alcohol induces a reversible fragmentation of the Golgi apparatus and inhibits membrane trafficking between endosomes and the trans-Golgi network.

Benzyl alcohol (BnOH) is widely used as a component of foods, cosmetics, household products and medical products. It is generally considered to be safe for human use, however, it has been connected to a number of adverse effects, including hypersensitivity reactions and neonatal deaths. BnOH is a membrane fluidizing agent that can affect membrane protein activity and cellular processes such as ligand binding to cell surface receptors, endocytosis and degradation of lysosomal cargo. In this study, we examined the effects of BnOH on intracellular transport using Shiga toxin (Stx), diphtheria toxin (DT) and ricin. BnOH caused reduced toxicity of all three toxins at BnOH concentrations that cause membrane fluidization. The reduced toxicity of Stx and ricin was mainly due to inhibition of retrograde transport between endosomes and the trans-Golgi network as BnOH had small effects on cell association and endocytosis of ricin and Stx. Strikingly, BnOH also induced a reversible fragmentation of the Golgi apparatus.

Interdigitation of long-chain sphingomyelin induces coupling of membrane leaflets in a cholesterol dependent manner.

It has been a long-standing question how the two leaflets in a lipid bilayer modulate each others' physical properties. In this paper, we discuss how this interaction may take place through interdigitation. We use atomistic molecular dynamics simulations to consider asymmetric lipid membrane models whose compositions are based on the lipidomics data determined for exosomes released by PC-3 prostate cancer cells. The simulations show interdigitation to be exceptionally strong for long-chain sphingomyelin (SM) molecules. In asymmetric membranes the amide-linked chain of SM is observed to extend deep into the opposing membrane leaflet. Interestingly, we find that the conformational order of the amide-linked SM chain increases the deeper it penetrates to the opposing leaflet. Analysis of this finding reveals that the amide-linked SM chain interacts favorably with the lipid chains in the opposite leaflet, and that cholesterol modulates the effect of SM interdigitation by influencing the conformational order of lipid hydrocarbon chains in the opposing (cytosolic) leaflet.

Identification of non-invasive miRNAs biomarkers for prostate cancer by deep sequencing analysis of urinary exosomes.

The aim of this study was to identify microRNAs in urinary exosomes that are differently expressed in prostate cancer patients and healthy donors. For this purpose, RNA was extracted from urinary exosomes from 20 prostate cancer patients and 9 healthy males and the microRNAs were analyzed by next generation sequencing. Interestingly, 5 microRNAs - miR-196a-5p, miR-34a-5p, miR-143-3p, miR-501-3p and miR-92a-1-5p - were significantly downregulated in exosomes from prostate cancer patients. Furthermore, RT-qPCR analysis of an independent cohort of 28 prostate cancer patients and 19 healthy males confirmed that miR-196a-5p and miR-501-3p were downregulated in prostate cancer samples. These results suggest that specific microRNAs in urinary exosomes might serve as non-invasive biomarkers for prostate cancer. In particular, miR-196a-5p and miR-501-3p are promising biomarkers that need to be further studied in large patient cohorts.

Cross-linking of glycosphingolipids at the plasma membrane: consequences for intracellular signaling and traffic.

Glycosphingolipids (GSLs) are predominantly found in the outer leaflet of the plasma membrane, where they play a role in important processes such as cell adhesion, migration and signaling. However, by which mechanisms GSLs regulate these processes remains elusive. In this study, we therefore took advantage of the fact that some GSLs also serve as receptors for certain protein toxins, which rely on receptor binding for internalization and intoxication. Here, we demonstrate that Shiga and cholera toxins, which both possess multivalent GSL-binding capacity, induce dissociation of the cytosolic cPLA2α-AnxA1 complex in HeLa and HMEC-1 cells. The dissociation is mediated through an increase in cytosolic calcium levels and activation of the tyrosine kinase Syk. Ricin, a protein toxin that does not cross-link surface molecules, has no effect on the same complex. Importantly, we find that antibody-mediated cross-linking of Gb3 and GM1, the GSL receptors for Shiga and cholera toxin, respectively, also induces dissociation. These data demonstrate that cross-linking of GSLs at the plasma membrane mediates the intracellular signaling events resulting in dissociation of the complex. After dissociation, cPLA2α and AnxA1 are translocated to intracellular membranes where they are known to function in regulating membrane transport processes. In conclusion, we have characterized a novel mechanism for cell surface-induced initiation of intracellular signaling and transport events.

PIKfyve inhibition increases exosome release and induces secretory autophagy.

Exosomes are vesicles released from cells by fusion of multivesicular bodies (MVBs) with the plasma membrane. This study aimed to investigate whether the phosphoinositide kinase PIKfyve affects this process. Our results show that in PC-3 cells inhibition of PIKfyve by apilimod or depletion by siRNA increased the secretion of the exosomal fraction. Moreover, quantitative electron microscopy analysis showed that cells treated with apilimod contained more MVBs per cell and more intraluminal vesicles per MVB. Interestingly, mass spectrometry analysis revealed a considerable enrichment of autophagy-related proteins (NBR1, p62, LC3, WIPI2) in exosomal fractions released by apilimod-treated cells, a result that was confirmed by immunoblotting. When the exosome preparations were investigated by electron microscopy a small population of p62-labelled electron dense structures was observed together with CD63-containing exosomes. The p62-positive structures were found in less dense fractions than exosomes in density gradients. Inside the cells, p62 and CD63 were found in the same MVB-like organelles. Finally, both the degradation of EGF and long-lived proteins were shown to be reduced by apilimod. In conclusion, inhibition of PIKfyve increases secretion of exosomes and induces secretory autophagy, showing that these pathways are closely linked. We suggest this is due to impaired fusion of lysosomes with both MVBs and autophagosomes, and possibly increased fusion of MVBs with autophagosomes, and that the cells respond by secreting the content of these organelles to maintain cellular homeostasis.

Cytotoxicity of Poly(Alkyl Cyanoacrylate) Nanoparticles.

Although nanotoxicology has become a large research field, assessment of cytotoxicity is often reduced to analysis of one cell line only. Cytotoxicity of nanoparticles is complex and should, preferentially, be evaluated in several cell lines with different methods and on multiple nanoparticle batches. Here we report the toxicity of poly(alkyl cyanoacrylate) nanoparticles in 12 different cell lines after synthesizing and analyzing 19 different nanoparticle batches and report that large variations were obtained when using different cell lines or various toxicity assays. Surprisingly, we found that nanoparticles with intermediate degradation rates were less toxic than particles that were degraded faster or more slowly in a cell-free system. The toxicity did not vary significantly with either the three different combinations of polyethylene glycol surfactants or with particle size (range 100-200 nm). No acute pro- or anti-inflammatory activity on cells in whole blood was observed.

LYST affects lysosome size and quantity, but not trafficking or degradation through autophagy or endocytosis.

Mutations in the large BEACH domain-containing protein LYST causes Chediak-Higashi syndrome. The diagnostic hallmark is enlarged lysosomes and lysosome-related organelles in various cell types. Dysfunctional secretion of enlarged lysosome-related organelles has been observed in cells with mutations in LYST, but the capacity of the enlarged lysosomes to degrade endogenous proteins has not been studied. Here, we show for the first time that small interfering RNA-depletion of LYST in human cell lines recapitulates the LYST mutant phenotype of enlarged lysosomes. We found no evidence for an effect of LYST depletion on autophagy or endocytic degradation. Autophagosomes are formed in normal size and quantities and are able to fuse to the enlarged lysosomes, leading to normal rates of degradation. Degradation of the epidermal growth factor receptor (EGFR) was similarly not affected, indicating that the enlarged lysosomes are fully functional in degrading endogenous proteins. Retrograde trafficking of toxins as well as the localization of transporters of lysosomal proteins, adaptor protein-3 (AP-3) and cation-independent mannose-6-phosphate receptor (CI-MPR), were all found to be unaffected by LYST. Quantitative analysis of the enlarged lysosomes shows that LYST depletion causes a reduction in vesicle quantity per cell, while the total enzymatic content and vesicular pH are unaffected, supporting a role for LYST in lysosomal fission and/or fusion events.

The ether lipid precursor hexadecylglycerol stimulates the release and changes the composition of exosomes derived from PC-3 cells.

Exosomes are vesicles released by cells after fusion of multivesicular bodies with the plasma membrane. In this study, we have investigated whether ether lipids affect the release of exosomes in PC-3 cells. To increase the cellular levels of ether lipids, the ether lipid precursor hexadecylglycerol was added to cells. Lipidomic analysis showed that this compound was in fact able to double the cellular levels of ether lipids in these cells. Furthermore, increased levels of ether lipids were also found in exosomes released by cells containing high levels of these lipids. Interestingly, as measured by nanoparticle tracking analysis, cells containing high levels of ether lipids released more exosomes than control cells, and these exosomes were similar in size to control exosomes. Moreover, silver staining and Western blot analyses showed that the protein composition of exosomes released in the presence of hexadecylglycerol was changed; the levels of some proteins were increased, and the levels of others were reduced. In conclusion, this study clearly shows that an increase in cellular ether lipids is associated with changes in the release and composition of exosomes.

Novel actions of 2-deoxy-D-glucose: protection against Shiga toxins and changes in cellular lipids.

2-Deoxy-D-glucose (2DG) is a structural analogue of glucose with well-established applications as an inhibitor of glycolysis and N-glycosylation. Importantly, 2DG has been shown to improve the efficacy of several cancer chemotherapeutic agents in vivo and thus it is in clinical studies in combination with chemotherapy and radiotherapy. However, although 2DG has been demonstrated to modulate many cellular functions, including autophagy, apoptosis and cell cycle control, little is known about the effects of 2DG on intracellular transport, which is of great importance when predicting the effects of 2DG on therapeutic agents. In addition to proteins, lipids play important roles in cellular signalling and in controlling cellular trafficking. We have, in the present study, investigated the effects of 2DG on cellular lipid composition and by use of protein toxins we have studied 2DG-mediated changes in intracellular trafficking. By quantifying more than 200 individual lipid species from 17 different lipid classes, we have found that 2DG treatment changes the levels and/or species composition of several lipids, such as phosphatidylinositol (PI), diacylglycerol (DAG), cholesteryl ester (CE), ceramide (Cer) and lysophospho-lipids. Moreover, 2DG becomes incorporated into the carbohydrate moiety of glycosphingolipids (GSLs). In addition, we have discovered that 2DG protects cells against Shiga toxins (Stxs) and inhibits release of the cytotoxic StxA1 moiety in the endoplasmic reticulum (ER). The data indicate that the 2DG-induced protection against Stx is independent of inhibition of glycolysis or N-glycosylation, but rather mediated via the depletion of Ca(2+) from cellular reservoirs by 2DG. In conclusion, our results reveal novel actions of 2DG on cellular lipids and Stx toxicity.

The ERM proteins ezrin and moesin regulate retrograde Shiga toxin transport.

The ERM proteins (ezrin, radixin and moesin) are known for connecting the actin cytoskeleton to the plasma membrane. They have been found to associate with lipid rafts as well as to be important for endosomal sorting and receptor signaling. However, little is known about the role of ERM proteins in retrograde transport and lipid homeostasis. In this study, we show that ezrin and moesin are important for efficient cell surface association of Shiga toxin (Stx) as well as for its retrograde transport. Furthermore, we show that depletion of these proteins influences endosomal dynamics and seems to enhance Stx transport toward lysosomes. We also show that knockdown of Vps11, a subunit of the HOPS complex, leads to increased retrograde Stx transport and reverses the inhibiting effect of ezrin and moesin knockdown. Importantly, retrograde transport of the plant toxin ricin, which binds to both glycolipids and glycoproteins with a terminal galactose, seems to be unaffected by ezrin and moesin depletion.

Flotillin depletion affects ErbB protein levels in different human breast cancer cells.

The ErbB3 receptor is an important regulator of cell growth and carcinogenesis. Among breast cancer patients, up to 50-70% have ErbB3 overexpression and 20-30% show overexpressed or amplified ErbB2. ErbB3 has also been implicated in the development of resistance to several drugs used against cancers driven by ErbB1 or ErbB2. One of the main challenges in ErbB-targeting therapy is to inactivate signaling mediated by ErbB2-ErbB3 oncogenic receptor complexes. We analyzed the regulatory role of flotillins on ErbB3 levels and ErbB2-ErbB3 complexes in SKBR3, MCF7 and MDA-MB-134-VI human breast cancer cells. Recently, we described a mechanism for interfering with ErbB2 signaling in breast cancer and demonstrated a molecular complex of flotillin scaffolding proteins with ErbB2 and Hsp90. In the present study, flotillins were found to be in a molecular complex with ErbB3, even in cells without the presence of ErbB2 or other ErbB receptors. Depletion of either flotillin-1 or flotillin-2 resulted in downregulation of ErbB3 and a selective reduction of ErbB2-ErbB3 receptor complexes. Moreover, flotillin-2 depletion resulted in reduced activation of Akt and MAPK signaling cascades, and as a functional consequence of flotillin depletion, breast cancer cells showed an impaired cell migration. Altogether, we provide data demonstrating a novel and functional role of flotillins in the regulation of ErbB protein levels and stabilization of ErbB2-ErbB3 receptor complexes. Thus, flotillins are crucial regulators for oncogenic ErbB function and potential targets for cancer treatment.