Tore Geir Iversen

Pathways followed by ricin and Shiga toxin into cells

Abstract. The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.

Cellular trafficking of quantum dot-ligand bioconjugates and their induction of changes in normal routing of unconjugated ligands.

Can quantum dots (Qdots) act as relevant intracellular probes to investigate routing of ligands in live cells? The intracellular trafficking of Qdots that were coupled to the plant toxin ricin, Shiga toxin, or the ligand transferrin (Tf) was studied by confocal fluorescence microscopy. The Tf:Qdots were internalized by clathrin-dependent endocytosis as fast as Tf, but their recycling was blocked. Unlike Shiga toxin, the Shiga:Qdot bioconjugate was not routed to the Golgi apparatus. The internalized ricin:Qdot bioconjugates localized to the same endosomes as ricin itself but could not be visualized in the Golgi apparatus. Importantly, we find that the endosomal accumulation of ricin:Qdots affects endosome-to-Golgi transport of both ricin and Shiga toxin: Transport of ricin was reduced whereas transport of Shiga toxin was increased. In conclusion, the data reveal that, although coupling of Qdots to a ligand does not necessarily change the endocytic pathway normally used by the ligands studied, it appears that the ligand-coupled Qdot nanoparticles can be arrested within endosomes and somehow perturb the normal endosomal sorting in cells. Thus, the results demonstrate that Qdots may have severe consequences on cell physiology.

Protein toxins from plants and bacteria: Probes for intracellular transport and tools in medicine

A number of protein toxins produced by bacteria and plants enter eukaryotic cells and inhibit protein synthesis enzymatically. These toxins include the plant toxin ricin and the bacterial toxin Shiga toxin, which we will focus on in this article. Although a threat to human health, toxins are valuable tools to discover and characterize cellular processes such as endocytosis and intracellular transport. Bacterial infections associated with toxin production are a problem worldwide. Increased knowledge about toxins is important to prevent and treat these diseases in an optimal way. Interestingly, toxins can be used for diagnosis and treatment of cancer.

Transport of Ricin from Endosomes to the Golgi Apparatus is Regulated by Rab6A and Rab6A

Ricin is transported from early endosomes and/or the recycling compartment to the trans‐Golgi network (TGN) and subsequently to the endoplasmic recticulum (ER) before it enters the cytosol and intoxicates cells. We have investigated the role of the Rab6 isoforms in retrograde transport of ricin using both oligo‐ and vector‐based RNAi assays. Ricin transport to the TGN was inhibited by the depletion of Rab6A when the Rab6A messenger RNA (mRNA) levels were reduced by more than 40% and less than 75%. However, when Rab6A mRNA was reduced by more than 75% and Rab6A′ mRNA was simultaneously up‐regulated, the inhibition of ricin sulfation was abolished, indicating that the up‐regulation of Rab6A′ may compensate for the loss of Rab6A function. In addition, we found that a near complete depletion of Rab6A′ gave approximately 40% reduction in ricin sulfation. The up‐regulation of Rab6A mRNA levels did not seem to compensate for the loss of Rab6A′ function. The depletion of both Rab6A and Rab6A′ gave a stronger inhibition of ricin sulfation than what was observed knocking down the two isoforms separately. In conclusion, both Rab6A and Rab6A′ seem to be involved in the transport of ricin from endosomes to the Golgi apparatus.

New metal-based nanoparticles for intravenous use: requirements for clinical success with focus on medical imaging

Animal studies have during the last years revealed a large potential for in vivo imaging with new metal-based nanoparticles and will certainly during the next years also continue to improve our understanding of basic biological processes. In the present article we discuss what is needed to bring such non-iron oxide particles into clinical imaging. For imaging agents it is essential to have a rapid clearance from blood so as to obtain low background signals and good images. The surface charge and hydrodynamic diameter of the nanoparticles in the presence of plasma proteins are important for their biodistribution, excretion. and a rapid clearance from blood. As discussed, some major challenges remain to be met regarding safety and metabolism issues. Measurements and optimization of the critical parameters will shorten the time needed for such particles to be accepted for widespread medical use.

Cholesterol Loading Induces a Block in the Exit of VSVG from the TGN

Recent work from our laboratory demonstrated that increased cellular cholesterol content affects the structure of the Golgi apparatus. We have now investigated the functional consequences of the cholesterol‐induced vesiculation of the Golgi apparatus and the role of actin for these changes. The results showed that cholesterol‐induced vesiculation and dispersion of the Golgi apparatus is a reversible process and that reversal can be inhibited by cytochalasin D, an actin‐disrupting reagent. Furthermore, electron microscopy revealed that jasplakinolide, which stabilizes actin filaments, prevented the dispersion, but not the vesiculation of the Golgi cisternae. Importantly, the different Golgi markers seemed to be separated even after vesiculation. To investigate whether transport through the different steps of the exocytic pathway was affected in cholesterol‐treated cells, we visualized ER to plasma membrane transport by using ts045‐VSVG‐GFP. In COS‐1 cells expressing ts045‐VSVG‐GFP increased cholesterol levels did not affect transport of VSVG into the vesiculated Golgi apparatus. However, increased levels of cholesterol resulted in retention of the nascent G protein in vesicles with the TGN‐marker TGN46. Biotinylation of cell surface molecules to quantify arrival of VSVG at the plasma membrane confirmed that cholesterol treatment inhibited export of the VSVG protein. In conclusion, the data show that transport of VSVG into/through a vesiculated Golgi is feasible, but that cholesterol loading inhibits exit of VSVG from the vesicles containing TGN markers. Furthermore, the data illustrate the importance of actin filaments for Golgi structure.

Uptake of ricinB-quantum dot nanoparticles by a macropinocytosis-like mechanism

BackgroundThere is a huge effort in developing ligand-mediated targeting of nanoparticles to diseased cells and tissue. The plant toxin ricin has been shown to enter cells by utilizing both dynamin-dependent and -independent endocytic pathways. Thus, it is a representative ligand for addressing the important issue of whether even a relatively small ligand-nanoparticle conjugate can gain access to the same endocytic pathways as the free ligand.ResultsHere we present a systematic study concerning the internalization mechanism of ricinB:Quantum dot (QD) nanoparticle conjugates in HeLa cells. Contrary to uptake of ricin itself, we found that internalization of ricinB:QDs was inhibited in HeLa cells expressing dominant-negative dynamin. Both clathrin-, Rho-dependent uptake as well as a specific form of macropinocytosis involve dynamin. However, the ricinB:QD uptake was not affected by siRNA-mediated knockdown of clathrin or inhibition of Rho-dependent uptake caused by treating cells with the Clostridium C3 transferase. RicinB:QD uptake was significantly reduced by cholesterol depletion with methyl-β-cyclodextrin and by inhibitors of actin polymerization such as cytochalasin D. Finally, we found that uptake of ricinB:QDs was blocked by the amiloride analog EIPA, an inhibitor of macropinocytosis. Upon entry, the ricinB:QDs co-localized with dextran, a marker for fluid-phase uptake. Thus, internalization of ricinB:QDs in HeLa cells critically relies on a dynamin-dependent macropinocytosis-like mechanism.ConclusionsOur results demonstrate that internalization of a ligand-nanoparticle conjugate can be dependent on other endocytic mechanisms than those used by the free ligand, highlighting the challenges of using ligand-mediated targeting of nanoparticles-based drug delivery vehicles to cells of diseased tissues.

Cell-Penetrating Peptides: Possibilities and Challenges for Drug Delivery in Vitro and in Vivo

In this review, we discuss how cell-penetrating peptides (CPPs) might get access to their intracellular targets. We specifically focus on the challenge of deciding whether the positively-charged CPPs are just bound to the negatively-charged cell surface and subsequently endocytosed or actually transported into the cytosol, either by direct plasma membrane penetration or after endocytosis. This discussion includes comments about pitfalls when using pharmacological inhibitors in such studies. The possibility of exploiting CPPs as carriers for the delivery of drugs of different sizes in vitro is discussed, as is the use of CPPs as carriers for therapeutic drugs or contrast agents in vivo. We conclude that in many cases, more studies are needed to demonstrate conclusively whether increased delivery of a substance attached to CPPs is due to a membrane-penetrating property or whether the increase is a consequence of just changing the charge of the substance to be delivered. Finally, the expected dose needed for the use of such conjugates in vivo is discussed, including aspects to consider in order to bring potential products into clinical use.

Development of nanoparticles for clinical use

During recent years, there has been much interest in using nanoparticles (NPs) for delivery of therapeutic drugs or contrast agents. Many interesting in vitro and animal studies have been reported [1–3], and some NP-based products are already in clinical use. Iron-oxide containing NPs have been used as iron supplement or contrast agents in MRI for more than 20 years [4,5]. Albumin and liposomal-based products containing anticancer drugs have also been available for several years, and new NP-based products are in clinical trials [2,6]. The discrepancy between the large number of papers published about NPs and other drug candidates versus the few new drugs entering the market is heavily debated, and important discussions regarding what is needed to bring new products to the market have been published [6–9]. No doubt, there are many challenges to overcome before NPs may become common tools in clinical practice.

Comment on "short ligands affect modes of QD uptake and elimination in human cells".

’ In a recent study, Al-Hajaj et al. presented interesting data for uptake and elimination of four different quantum dots (QDs) labeled with various short ligands. The inhibition of cellular uptake by using an inhibitor of an amino acid transporter (X-Ag cysteine transporter) was interpreted as evidence for the direct uptake of one of these QDs (QD-Cys) via this transporter into cytosol. The effects obtained using an inhibitor or activator of P-glycoprotein were interpreted as evidence for elimination of QDs from cytosol (their Figure 1). It is very surprising and interesting if a QD with hydrodynamic diameter of 8 10 nm (measured in absence of proteins) should be able to pass through an amino acid transporter. Thus, it is important that all necessary control experiments are performed to draw the conclusion that QDs can enter cytosol via this route.Whether QDs are present in cytosol or contained within intracellular organelles is a very important issue regarding the intracellular targets that can be reached. In our opinion, the following information is lacking in order to draw the conclusions shown in Figure 1: (a) Confocal microscopy or electron microscopy should demonstrate the presence of QDs in cytosol and also changes in amount of cytosolic QDs after the use of inhibitors/activator. Confocal microscopy data are shown only for two of the QDs studied and not for the QD-Cys stated to end up in cytosol. Moreover, the confocal pictures shown indicate that all internalized QDs are localized within intracellular organelles, that is, possibly endosomes/lysosomes. (b) Control experiments should be performed using the inhibitor of the amino acid transporter and the three other QDs studied. For such studies, it is essential to know if the QDs are entering the cells or if they are just adsorbed to the cells. If there is adsorption during incubation at 37 C and an inhibitor reduces this adsorption, this would appear as decreased uptake. It is not possible to discriminate between these possibilities for the analyses performed. The upper picture of Figure 3C in the study by Hajaj et al. shows particles that seem to be adsorbed to the cell only. There aremany pitfalls in studying cellular uptake of nanoparticles. We have recently discussed endocytic uptake of nanoparticles and provided a toolbox for studies of cellular uptake and intracellular localization to guide nanoparticle scientists and avoiding wrong conclusions.