Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

A di-leucine sequence and a cluster of acidic amino acids are required for dynamic retention in the endosomal recycling compartment of fibroblasts.

Insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase, is dynamically retained within the endosomal compartment of fibroblasts. The characteristics of this dynamic retention are rapid internalization from the plasma membrane and slow recycling back to the cell surface. These specialized trafficking kinetics result in <15% of IRAP on the cell surface at steady state, compared with 35% of the transferrin receptor, another transmembrane protein that traffics between endosomes and the cell surface. Here we demonstrate that a 29-amino acid region of IRAP's cytoplasmic domain (residues 56--84) is necessary and sufficient to promote trafficking characteristic of IRAP. A di-leucine sequence and a cluster of acidic amino acids within this region are essential elements of the motif that slows IRAP recycling. Rapid internalization requires any two of three distinct motifs: M(15,16), DED(64--66), and LL(76,77). The DED and LL sequences are part of the motif that regulates recycling, demonstrating that this motif is bifunctional. In this study we used horseradish peroxidase quenching of fluorescence to demonstrate that IRAP is dynamically retained within the transferrin receptor-containing general endosomal recycling compartment. Therefore, our data demonstrate that motifs similar to those that determine targeting among distinct membrane compartments can also regulate the rate of transport of proteins from endosomal compartments. We propose a model for dynamic retention in which IRAP is transported from the general endosomal recycling compartment in specialized, slowly budding recycling vesicles that are distinct from those that mediate rapid recycling back to the surface (e.g., transferrin receptor-containing transport vesicles). It is likely that the dynamic retention of IRAP is an example of a general mechanism for regulating the distribution of proteins between the surface and interior of cells.

Insulin-regulated release from the endosomal recycling compartment is regulated by budding of specialized vesicles.

In several cell types, specific membrane proteins are retained intracellularly and rapidly redistributed to the surface in response to stimulation. In fat and muscle, the GLUT4 glucose transporter is dynamically retained because it is rapidly internalized and slowly recycled to the plasma membrane. Insulin increases the recycling of GLUT4, resulting in a net translocation to the surface. We have shown that fibroblasts also have an insulin-regulated recycling mechanism. Here we show that GLUT4 is retained within the transferrin receptor-containing general endosomal recycling compartment in Chinese hamster ovary (CHO) cells rather than being segregated to a specialized, GLUT4-recycling compartment. With the use of total internal reflection microscopy, we demonstrate that the TR and GLUT4 are transported from the pericentriolar recycling compartment in separate vesicles. These data provide the first functional evidence for the formation of distinct classes of vesicles from the recycling compartment. We propose that GLUT4 is dynamically retained within the endosomal recycling compartment in CHO cells because it is concentrated in vesicles that form more slowly than those that transport TR. In 3T3-L1 adipocytes, cells that naturally express GLUT4, we find that GLUT4 is partially segregated to a separate compartment that is inaccessible to the TR. We present a model for the formation of this specialized compartment in fat cells, based on the general mechanism described in CHO cells, which may explain the increased retention of GLUT4 and its insulin-induced translocation in fat cells.

Exploiting the lacZ reporter gene for quantitative analysis of disseminated tumor growth within the brain: use of the lacZ gene product as a tumor antigen, for evaluation of antigenic modulation, and to facilitate image analysis of tumor growth in situ.

We extend use of the lacZ reporter gene for tumor biology. Intracerebral growth of 9L/lacZ, a gliosarcoma cell line that stably expresses lacZ, was evaluated in syngeneic rats. The reporter gene product, Escherichia coli-derived beta-galactosidase (beta-gal), was detected histochemically on tissue sections. This permits visualization of disseminated tumor and, as shown here, facilitates image analysis. We show that the beta-gal marker protein itself can serve as a tumor antigen in appropriate contexts. Quantitative image analysis of tumor areas is used to show that immunization with beta-gal protects against tumor growth. Abnormal beta-gal- areas are easily detected, facilitating study of antigenic modulation. The tumor studied did not escape through this mechanism. All abnormal beta-gal- areas examined were shown to reflect accumulation of inflammatory or reactive cells, not tumor. Taken together, these findings show several ways in which the lacZ reporter gene can be exploited to facilitate quantitative analysis of disseminated tumor growth within the brain. They draw attention to the growing appreciation that tumor antigens need not be cell surface molecules.

Effects of gamma-interferon on major histocompatibility complex antigen expression and lymphocytic infiltration in the 9L gliosarcoma brain tumor model: implications for strategies of immunotherapy.

Effects of gamma-interferon (IFN-gamma) on immune parameters in the 9L gliosarcoma model were examined. IFN-gamma increased class I major histocompatibility complex (MHC) expression in 9L cells in vitro. In vivo, intratumor injections of IFN-gamma led to increased numbers of inflammatory cells within the tumor and class II+ mononuclear phagocytes at its periphery, and increased MHC class I or II expression by endothelial and ependymal cells. Class I expression in 9L cells themselves was not increased. This suggests that there may be inhibition of class I induction in vivo for certain cell types, for which immunotherapies based on non-MHC restricted mechanisms may be more effective.

Characterization of the insulin-regulated endocytic recycling mechanism in 3T3-L1 adipocytes using a novel reporter molecule.

The endocytic trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) are regulated by insulin. We have used a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor (vpTR) to characterize IRAP-like trafficking in 3T3-L1 adipocytes. Our data demonstrate that the cytoplasmic domain of IRAP is sufficient to target vpTR to the insulin-regulated, slow recycling pathway in adipocytes and that the dynamic retention of vpTR is dependent on a di-leucine motif. Our kinetic analysis demonstrates that vpTR recycles as a single kinetic pool and that vpTR is very efficiently sorted from endosomes to the insulin-regulated recycling pathway. An implication of these findings is that the key step in the dynamic retention of vpTR occurs within the early endosomal system. We have previously shown that vpTR is trafficked by an insulin-regulated pathway in Chinese hamster ovary cells (Johnson, A. O., Subtil, A., Petrush, R., Kobylarz, K., Keller, S., and Mc Graw, T. E. (1998) J. Biol. Chem. 273, 17968-17977). The behavior of vpTR in Chinese hamster ovary cells is similar to its behavior in 3T3-L1 adipocytes. The main difference is that insulin has a larger effect on the trafficking of vpTR in the adipocytes. We concluded that the insulin-regulated slow recycling endocytic mechanism is expressed in many different cell types and therefore is not a unique characteristic of cells that express GLUT4.

Demonstration of insulin-responsive trafficking of GLUT4 and vpTR in fibroblasts.

Insulin-responsive trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) in adipose and muscle cells is well established. Insulin regulation of GLUT4 trafficking in these cells underlies the role that adipose tissue and muscle play in the maintenance of whole body glucose homeostasis. GLUT4 is expressed in a very limited number of tissues, most highly in adipose and muscle, while IRAP is expressed in many tissues. IRAP's physiological role in any of the tissues in which it is expressed, however, is unknown. The fact that IRAP, which traffics by the same insulin-regulated pathway as GLUT4, is expressed in 'non-insulin responsive' tissues raises the question of whether these other cell types also have a specialized insulin-regulated trafficking pathway. The existence of an insulin-responsive pathway in other cell types would allow regulation of IRAP activity at the plasma membrane as a potentially important physiological function of insulin. To address this question we use reporter molecules for both GLUT4 and IRAP trafficking to measure insulin-stimulated translocation in undifferentiated cells by quantitative fluorescence microscopy. One reporter (vpTR), a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor, has been previously characterized. The other is a GLUT4 construct with an exofacial HA epitope and a C-terminal GFP. By comparing these reporters to the transferrin receptor, a marker for general endocytic trafficking, we demonstrate the existence of a specialized, insulin-regulated trafficking pathway in two undifferentiated cell types, neither of which normally express GLUT4. The magnitude of translocation in these undifferentiated cells (approximately threefold) is similar to that reported for the translocation of GLUT4 in muscle cells. Thus, undifferentiated cells have the necessary retention and translocation machinery for an insulin response that is large enough to be physiologically important.

Acute cholesterol depletion inhibits clathrin-coated pit budding.

Many biologically important macromolecules are internalized into cells by clathrin-coated pit endocytosis. The mechanism of clathrin-coated pit budding has been investigated intensively, and considerable progress has been made in characterizing the proteins involved in internalization. Membrane lipid composition and the lateral organization of lipids and proteins within membranes are believed to play an important role in the regulation of membrane-trafficking processes. Here we report that membrane cholesterol plays a critical role in clathrin-coated pit internalization. We show that acute cholesterol depletion, using beta-methyl-cyclodextrin, specifically reduces the rate of internalization of transferrin receptor by more than 85%, without affecting intracellular receptor trafficking back to the cell surface. The effect on endocytosis is attributable to a failure of coated pits to detach from the plasma membrane, as visualized by using a green fluorescent protein-clathrin conjugate in living cells. Ultrastructural studies indicate that acute cholesterol depletion causes accumulation of flat-coated membranes and a corresponding decrease in deep-coated pits, consistent with the possibility that flat clathrin lattices are direct precursors of indented pits and endocytic vesicles in intact cells. We conclude that clathrin is unable to induce curvature in the membrane depleted of cholesterol.