B. van Deurs

Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives

A large number of plant and bacterial toxins with enzymatic activity on intracellular targets are now known. These toxins enter cells by first binding to cell surface receptors, then they are endocytosed and finally they become translocated into the cytosol from an intracellular compartment. In the case of the plant toxin ricin and the bacterial toxin Shiga toxin, this happens after retrograde transport through the Golgi apparatus and to the endoplasmic reticulum. The toxins are powerful tools to reveal new pathways in intracellular transport. Furthermore, knowledge about their action on cells can be used to combat infectious diseases where such toxins are involved, and a whole new field of research takes advantage of their ability to enter the cytosol for therapeutic purposes in connection with a variety of diseases. This review deals with the mechanisms of entry of ricin and Shiga toxin, and the attempts to use such toxins in medicine are discussed.

Delivery into cells: lessons learned from plant and bacterial toxins

A number of protein toxins of bacterial and plant origin have cytosolic targets, and knowledge about these toxins have provided us with essential information about mechanisms that can be used to gain access to the cytosol as well as detailed knowledge about endocytosis and intracellular sorting. Such toxins include those that have two moieties, one (the B-moiety) that binds to cell surface receptors and another (the A-moiety) with enzymatic activity that enters the cytosol, as well as molecules that only have the enzymatically active moiety and therefore are inefficient in cell entry. The toxins discussed in the present article include bacterial toxins such as Shiga toxin and diphtheria toxin, as well as plant toxins such as ricin and ribosome-inactivating proteins without a binding moiety, such as gelonin. Toxins with a binding moiety can be used as vectors to translocate epitopes, intact proteins, and even nucleotides into the cytosol. The toxins fall into two main groups when it comes to cytosolic entry. Some toxins enter from endosomes in response to low endosomal pH, whereas others, including Shiga toxin and ricin, are transported all the way to the Golgi apparatus and the ER before they are translocated to the cytosol. Plant proteins such as gelonin that are without a binding moiety are taken up only by fluid-phase endocytosis, and normally they have a low toxicity. However, they can be used to test for disruption of endosomal membranes leading to cytosolic access of internalized molecules. Similarly to toxins with a binding moiety they are highly toxic when reaching the cytosol, thereby providing the investigator with an efficient tool to study endosomal disruption and induced transport to the cytosol. In conclusion, the protein toxins are useful tools to study transport and cytosolic translocation, and they can be used as vectors for transport to the interior of the cell.

Increased vesicular transfer of horseradish peroxidase across cerebral endothelium, evoked by acute hypertension

SummaryAcute hypertension in rats was produced by intravenous infusion of metaraminol bitartrate (Aramine). The permeability to intravenously injected horseradish peroxidase (HRP) was increased across the cerebral arterioles, capillaries and venules. From the basement membranes of the vessel walls the protein tracer moved into the extracellular spaces of the adjacent neuropil. No endothelial cell damage was observed. The tight junctions between endothelial cells were intact and prevented intercellular movement of peroxidase. Many HRP-labeled vesicles within the endothelial cells or connected with the luminal or abluminal surface, occurred in segments of the microvasculature. Otherwise the endothelium was unchanged. Diffuse uptake of HRP into the cytoplasm of neurons and glial cells was not observed. The alphablocker phentolamine (Regitin) was given to a group of rats simultaneously to Aramine. The increase in blood pressure was thus prevented; furthermore, the permeability remained as under normal conditions. The Aramine, Regitin and HRP did not significantly influence the pH,pO2 andpCO2 of the arterial blood.It is concluded that acute hypertension increases the vesicular transport of HRP across the endothelium of cerebral arterioles, venules and capillaries that normally occurs to a small extent only after intravenous injection of the tracer.

Structural Aspects of Brain Barriers, with Special Reference to the Permeability of the Cerebral Endothelium and Choroidal Epithelium

Publisher Summary The endothelium of the cerebral microvasculature is, under normal conditions, tight to proteins and peptides, or conceivably, a very limited blood-to-brain (and brain-to-blood) transport by means of endothelial vesicles takes place in segments of the microvasculature. This barrier to lipid-insoluble macromolecules can be ascribed to the tight junctions among the endothelial cells. Uncertainties exist, however, with respect to the permeability properties of the cerebral endothelial junctions to ions and small lipid-insoluble molecules. Many experimental or pathological situations, for example, acute hypertension, cause an opening of the blood–brain barrier. One possible pathway across the cerebral endothelium for larger hydrophilic molecules appears to be vesicular transport comparable with that occurring in normal noncerebral tissue. This may be indicative of the presence of a labile pore system in the cerebral microvasculature, which can be reversibly opened, but normally is closed. Transendothelial channels may also be responsible for the extravasation. Additionally, intravenous administration of hypertonic solutions and long-term hypertension cause an opening of the tight junctions between the endothelial cells.

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.

Observations on the blood-brain barrier in hypertensive rats, with particular reference to phagocytic pericytes

The endothelial and periendothelial parts of the blood-brain barrier in the rat have been studied by electron microscopy. Extravasation of horseradish peroxidase (HRP) occurred after Aramine-induced acute hypertension. It was exclusively due to a local vesicular transport. This was most prominent in arterioles, but also occurred in segments of capillaries and venules. The tight junctions between adjacent endothelial cells were never penetrated by HRP. Following extravasation, HRP reached the endothelial basement membrane, from where it often spread into the extracellular space of the neuropil. A phagocytic pericyte was observed, exhibiting a well-developed vacuolar apparatus and a pronounced uptake of HRP. It is suggested that the phagocytic pericytes in the rat brain represent “microglial” cells, and that they play an important role in the periendothelial part of the blood-brain barrier.

The use of a tannic acid-glutaraldehyde fixative to visualize gap and tight junctions.

Fixation of livers from mice with a mixture of tannic acid and glutaraldehyde resulted in an increased density of the cell periphery. Gap and tight junctions were well demonstrated by this method. The gap junctions were filled with a homogeneous, electron-dense precipitate. Faint electron-lucent lines spaced 100 A center-to-center crossed the gaps. In oblique sections of gap junctions an array of round particles was seen. The tight junctions appeared as membrane fusions preventing the passage of the tannic acid from the intercellular clefts to the bile canaliculi. Morphologically, the results obtained by using tannic acid as a tracer are similar to those obtained with lanthanum and ruthenium red, but chemically the tannic acid may act in a different way, thus representing an “alternative” tracer.

Vesicular transport of horseradish peroxidase from brain to blood in segments of the cerebral microvasculature in adult mice

The transfer of protein from the cerebral ventricles to the parenchymal bloodstream in mice was studied by electron microscopy. After perfusion with the protein tracer horseradish peroxidase (HRP; M.W. approx. 40,000) through the cerebral ventricles, the tracer penetrated the ependymal lining of the ventricles and was found in the extracellular space of the neuropil close to the ependyma. HRP was also seen in the vascular basement membrane and in endothelial vesicles opening at the abluminal endothelial surface, or situated within the endothelial cells in segments of the microvasculature (mostly small arterioles). In some of these segments HRP was also seen on the luminal surface of the endothelia and in surface-connected vesicles. The junctions connecting adjacent endothelial cells were never penetrated by HRP. It is concluded that vesicular transport of HRP across the endothelium of the cerebral microvasculature represents a possible mechanism for protein removal from brain extracellular space.

Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA

To investigate the role of clathrin in coated vesicle formation, a cell line with inducible expression of clathrin heavy chain (CHC) antisense RNA was produced. After 18 h of CHC antisense RNA expression, the internalization of transferrin was inhibited by 90%. Although the amount of CHC was reduced by only 10%, the frequency of clathrin-coated pits at the cell surface increased by a factor of 3–5, and clathrin-coated structures also accumulated on a pleiomorphic, multivesicular, endosomal compartment. Remarkably, the coated pits were connected to the cell surface by long, tubular necks wrapped by dynamin rings, and the level of dynamin in the CHC antisense RNA-expressing cells was up-regulated 10-fold. In contrast, the amount of several other proteins associated with clathrin coat formation was unaffected. Thus, this study demonstrates that CHC antisense RNA causes accumulation of clathrin-coated pits with dynamin rings around the neck in intact cells not transfected with dynamin mutants, suggesting the existence of a previously uncharacterized functional interplay between clathrin and dynamin.

Delivery of internalized ricin from endosomes to cisternal Golgi elements is a discontinuous, temperature-sensitive process.

Galactose-terminating membrane glycoproteins and glycolipids on two established human breast carcinoma cell lines were tagged at 4 degrees C with a ricin-horseradish peroxidase conjugate (Ri-HRP). The cells were then incubated for various periods of time at 37 or 18 degrees C. After fixation and diaminobenzidine cytochemistry, the compartments reached by Ri-HRP were studied by analyzing thin serial sections. In both cell types a highly pleomorphic endosomal system comprising vacuolar elements as well as smaller, sometimes branched, tubular elements (tubular endosomes) was revealed at both 37 and 18 degrees C. At 37 degrees C Ri-HRP was consistently observed in flattened cisterns of the Golgi region in 30-40% of the Golgi complexes examined after 30-60 min of incubation. However, no Ri-HRP reached such Golgi elements at 18 degrees C, even after incubation for 180 min. Moreover, at 18 degrees C the ability of ricin to inhibit protein synthesis was virtually abolished, whereas the effect of diphtheria toxin was reduced much less. Following incubation with a monovalent transferrin-HRP conjugate or with unconjugated HRP, no labeling of cisternal Golgi elements was detected. These data indicate that delivery of galactose-terminating membrane molecules from endosomes to the Golgi complex is a discontinuous, temperature-sensitive process and that this process may be required for optimal ricin A-chain translocation.