Maria Lyngaas Torgersen

Clathrin-independent endocytosis: From nonexisting to an extreme degree of complexity

Today it is generally accepted that there are several endocytic mechanisms, both the clathrin-dependent one and mechanisms which operate without clathrin and with different requirements when it comes to dynamin, small GTP-binding proteins of the Rho family and specific lipids. It should be noted that clathrin-independent endocytosis can occur even when the cholesterol level in the membrane has been reduced to so low levels that caveolae are gone and clathrin-coated membrane areas are flat. Although new investigators in the field take it for granted that there is a multitude of entry mechanisms, it has taken a long time for this to become accepted. However, more work needs to be done, because one can still ask the question: How many endocytic mechanisms does a cell have, what are their function, and how are they regulated? This article describes some of the history of endocytosis research and attempts to give an overview of the complexity of the mechanisms and their regulation.

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.

Pathways followed by protein toxins into cells.

A number of protein toxins have an enzymatically active part, which is able to modify a cytosolic target. Some of these toxins, for instance ricin, Shiga toxin and cholera toxin, which we will focus on in this article, exert their effect on cells by first binding to the cell surface, then they are endocytosed, and subsequently they are transported retrogradely all the way to the ER before translocation of the enzymatically active part to the cytosol. Thus, studies of these toxins can provide information about pathways of intracellular transport. Retrograde transport to the Golgi and the ER seems to be dependent not only on different Rab and SNARE proteins, but also on cytosolic calcium, phosphatidylinositol 3-kinase and cholesterol. Comparison of the three toxins reveals differences indicating the presence of more than one pathway between early endosomes and the Golgi apparatus or, alternatively, that transport of different toxin-receptor complexes present in a certain subcompartment is differentially regulated.

Efficient endosome-to-Golgi transport of Shiga toxin is dependent on dynamin and clathrin

It has previously been shown that Shiga toxin, despite being bound to a glycolipid receptor, can be efficiently endocytosed from clathrin-coated pits. However, clathrin-independent endocytosis is also responsible for a proportion of the toxin uptake in some cells. After endocytosis the toxin can be transported in retrograde fashion to the Golgi apparatus and the endoplasmic reticulum, and then to the cytosol, where it exerts its toxic effect by inactivating ribosomes. In order to investigate the role of dynamin and clathrin in endosome-to-Golgi transport of Shiga toxin, we have used HeLa dynK44A and BHK antisense clathrin heavy chain (CHC) cells that, in an inducible manner, express mutant dynamin or CHC antisense RNA, respectively. In these cell lines, one can study the role of dynamin and clathrin on endosome-to-Golgi transport because they, as shown here, still internalize Shiga toxin when dynamin- and clathrin-dependent endocytosis is blocked. Butyric acid has been shown to sensitize A431 cells to Shiga toxin by increasing the proportion of cell-associated toxin that is transported to the Golgi apparatus and the endoplasmic reticulum. Here, we find that, in HeLa and BHK cells also, butyric acid also increased toxin transport to the Golgi apparatus and sensitized the cells to Shiga toxin. We have therefore studied the role of dynamin and clathrin in both untreated and butyric-acid-treated cells by measuring the sulfation of a modified Shiga B fragment. Our results indicate that endosome-to-Golgi transport of Shiga toxin is dependent on functional dynamin in both untreated cells and in cells treated with butyric acid. Interestingly, the regulation of Shiga toxin transport in untreated and butyric-acid-treated cells differs when it comes to the role of clathrin, because only cells that are sensitized to Shiga toxin with butyric acid need functional clathrin for endosome-to-Golgi transport.

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.

Caveolae: Stable Membrane Domains with a Potential for Internalization

The role of caveolae in endocytosis is hotly debated. Here, we argue that most caveolae are stable microdomains at the cell surface. Only a small fraction of caveolae is constitutively internalized, leading to a quantitatively minor uptake of ligands and receptors. In addition, we suggest that a more pronounced downregulation of caveolae from the plasma membrane can occur, presumably stimulated by receptor cross‐linking and clustering in caveolae. Finally, we propose that future studies dealing with internalization of caveolae should actually document such internalization and include kinetic data.

Endocytosis and retrograde transport of Shiga toxin

Shiga toxin belongs to the group of bacterial and plant toxins that act on cells by binding to cell surface receptors via a binding-moiety, then the toxins are endocytosed and transported retrogradely to the Golgi apparatus and the endoplasmic reticulum (ER) before an enzymatically active moiety enters the cytosol and exerts the toxic effect. In the case of Shiga toxin, similarly to plant toxins such as ricin and viscumin, the toxin removes one adenine from the 28S RNA of the 60S subunit of the ribosome and thereby inhibits protein synthesis. This ribotoxic effect is in some cells followed by apoptosis. In this article we focus on new discoveries concerning endocytosis and retrograde transport of Shiga toxin to the Golgi, the ER and the cytosol.

Targeting autophagy potentiates the apoptotic effect of histone deacetylase inhibitors in t(8;21) AML cells.

The role of autophagy during leukemia treatment is unclear. On the one hand, autophagy might be induced as a prosurvival response to therapy, thereby reducing treatment efficiency. On the other hand, autophagy may contribute to degradation of fusion oncoproteins, as recently demonstrated for promyelocytic leukemia-retinoic acid receptor α and breakpoint cluster region-abelson, thereby facilitating leukemia treatment. Here, we investigated these opposing roles of autophagy in t(8;21) acute myeloid leukemia (AML) cells, which express the most frequently occurring AML fusion oncoprotein, AML1-eight-twenty-one (ETO). We demonstrate that autophagy is induced by AML1-ETO-targeting drugs, such as the histone deacetylase inhibitors (HDACis) valproic acid (VPA) and vorinostat. Furthermore, we show that autophagy does not mediate degradation of AML1-ETO but rather has a prosurvival role in AML cells, as inhibition of autophagy significantly reduced the viability and colony-forming ability of HDACi-treated AML cells. Combined treatment with HDACis and autophagy inhibitors such as chloroquine (CQ) led to a massive accumulation of ubiquitinated proteins that correlated with increased cell death. Finally, we show that VPA induced autophagy in t(8;21) AML patient cells, and combined treatment with CQ enhanced cell death. Because VPA and CQ are well-tolerated drugs, combinatorial therapy with VPA and CQ could represent an attractive treatment option for AML1-ETO-positive leukemia.

Protein Kinase Cδ Is Activated by Shiga Toxin and Regulates Its Transport

Protein kinase C (PKC) isozymes regulate different vesicular trafficking steps in the recycling or degradative pathways. However, a possible role of these kinases in the retrograde pathway from endosomes to the Golgi complex has previously not been investigated. We report here the involvement of a specific PKC isozyme, PKCδ, in the intracellular transport of the glycolipid-binding Shiga toxin (Stx), which utilizes the retrograde pathway to intoxicate cells. Upon binding to cells, Stx was shown to specifically activate PKCδ and not PKCα. The involvement of PKCδ and PKCα in the retrograde transport of Stx was then monitored biochemically and by immunofluorescence after inhibition or depletion of the isozymes. PKCδ, but not PKCα, was shown to selectively regulate the endosome-to-Golgi transport of StxB. Upon inhibition or knockdown of PKCδ, StxB molecules colocalized less with giantin and more with EEA1, indicating that the molecules were accumulated in endosomes, unable to reach the Golgi complex. The inhibition of Golgi transport of Stx was reflected by a strong reduction in the toxic effect, demonstrating that transport of Stx to the cytosol is dependent on PKCδ activity. These results are in agreement with our previous data, which show that Stx is able to stimulate its own transport.

The A-subunit of surface-bound Shiga toxin stimulates clathrin-dependent uptake of the toxin

Shiga toxin can be internalized by clathrin‐dependent endocytosis in different cell lines, although it binds specifically to the glycosphingolipid Gb3. It has been demonstrated previously that the toxin can induce recruitment of the toxin–receptor complex to clathrin‐coated pits, but whether this process is concentration‐dependent or which part of the toxin molecule is involved in this process, have so far been unresolved issues. In this article, we show that the rate of Shiga toxin uptake is dependent on the toxin concentration in several cell lines [HEp‐2, HeLa, Vero and baby hamster kidney (BHK)], and that the increased rate observed at higher concentrations is strictly dependent on the presence of the A‐subunit of cell surface‐bound toxin. Surface‐bound B‐subunit has no stimulatory effect. Furthermore, this increase in toxin endocytosis is dependent on functional clathrin, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain, thereby blocking clathrin‐dependent endocytosis. By immunofluorescence, we show that there is an increased colocalization between Alexa‐labeled Shiga toxin and Cy5‐labeled transferrin in HeLa cells upon addition of unlabeled toxin. In conclusion, the data indicate that the Shiga toxin A‐subunit of cell surface‐bound toxin stimulates clathrin‐dependent uptake of the toxin. Possible explanations for this phenomenon are discussed.