Mutant huntingtin inhibits clathrin-independent endocytosis and causes accumulation of cholesterol in vitro and in vivo.

We show that the mutant Huntington's disease (HD) protein (mhtt) specifically inhibits endocytosis in primary striatal neurons. Unexpectedly, mhtt does not inhibit clathrin-dependent endocytosis as was anticipated based on known interacting partners. Instead, inhibition occurs through a non-clathrin, caveolar-related pathway. Expression of mhtt inhibited internalization of BODIPY-lactosylceramide (LacCer), which is internalized by a caveolar-related mechanism. In contrast, endocytosis of Alexa Fluor 594-transferrin (Tfn) and epidermal growth factor, internalized through clathrin pathway, was unaffected by mhtt expression. Caveolin-1 (cav1), the major structural protein of caveolae binds cholesterol and is responsible for its trafficking inside cells. Mhtt interacts with cav-1 and caused a striking accumulation of intracellular cholesterol. Cholesterol accumulated in cultured neurons expressing mhtt in vitro and in brains of mhtt-expressing animals in vivo, and was observed after induction of mhtt expression in PC-12 cell lines. The accumulation occurred only when mhtt and cav1 were simultaneously expressed in cells. Knockdown of cav1 in mhtt-expressing neurons blocked cholesterol accumulation and restored LacCer endocytosis. Thus, mhtt and cav1 functionally interact to cause both cellular defects. These data provide the first direct link between mhtt and caveolar-related endocytosis and also suggest a possible mechanism for HD neurotoxicity where cholesterol homeostasis is perturbed.

Genetic association and cellular function of MC1R variant alleles in human pigmentation

We have examined MC1R variant allele frequencies in the general population of South East Queensland and in a collection of adolescent dizygotic and monozygotic twins and family members to define statistical associations with hair and skin color, freckling, and mole count. Results of these studies are consistent with a linear recessive allelic model with multiplicative penetrance in the inheritance of red hair. Four alleles, D84E, R151C, R160W, and D294H, are strongly associated with red hair and fair skin with multinomial regression analysis showing odds ratios of 63, 118, 50, and 94, respectively. An additional three low-penetrance alleles V60L, V92M, and R163Q have odds ratios 6, 5, and 2 relative to the wild-type allele. To address the cellular effects of MC1R variant alleles in signal transduction, we expressed these receptors in permanently transfected HEK293 cells. Measurement of receptor activity via induction of a cAMP-responsive luciferase reporter gene found that the R151C and R160W receptors were active in the presence of NDP-MSH ligand, but at much reduced levels compared with that seen with the wild-type receptor. The ability to stimulate phosphorylation of the cAMP response element binding protein (CREB) transcription factor was also apparent in all stimulated MC1R variant allele-expressing HEK293 cell extracts as assessed by immunoblotting. In contrast, human melanoma cell lines showed wide variation in the their ability to undergo cAMP-mediated CREB phosphorylation. Culture of human melanocytes of known MC1R genotype may provide the best experimental approach to examine the functional consequences for each MC1R variant allele. With this objective, we have established more than 300 melanocyte cell strains of defined MC1R genotype.

Molecular and cellular biology of pre-malignancy in the gastrointestinal tract

Important pathogenic alterations within established cancers are acquired during the premalignant stage. These genetic alterations can be grouped into specific neoplastic pathways that differ within and between anatomical sites. By understanding the mechanisms that determine the initiation and progression of each pathway, it will be possible to develop novel approaches to the diagnosis, prevention and treatment of cancer. This chapter outlines the principles underlying the molecular characterization of pre-malignant lesions, taking colorectal neoplasia as the main model.

Integrating spatial optimization and cellular automata for evaluating urban change

Urban growth and change presents numerous challenges for planners and policy makers. Effective and appropriate strategies for managing growth and change must address issues of social, environmental and economic sustainability. Doing so in practical terms is a difficult task given the uncertainty associated with likely growth trends not to mention the uncertainty associated with how social and environmental structures will respond to such change. An optimization based approach is developed for evaluating growth and change based upon spatial restrictions and impact thresholds. The spatial optimization model is integrated with a cellular automata growth simulation process. Application results are presented and discussed with respect to possible growth scenarios in south east Queensland, Australia.

Combining influence maps and cellular automata for reactive game agents

Agents make up an important part of game worlds, ranging from the characters and monsters that live in the world to the armies that the player controls. Despite their importance, agents in current games rarely display an awareness of their environment or react appropriately, which severely detracts from the believability of the game. Some games have included agents with a basic awareness of other agents, but they are still unaware of important game events or environmental conditions. This paper presents an agent design we have developed, which combines cellular automata for environmental modeling with influence maps for agent decision-making. The agents were implemented into a 3D game environment we have developed, the EmerGEnT system, and tuned through three experiments. The result is simple, flexible game agents that are able to respond to natural phenomena (e.g. rain or fire), while pursuing a goal.

Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin

Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120(ctn), also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

dEndocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wildtype Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and enclocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

Lipid rafts and caveolae as portals for endocytosis: New insights and common mechanisms

Clathrin-coated pits and caveolae are two of the most recognizable features of the plasma membrane of mammalian cells. While our understanding of the machinery regulating and driving clathrin-coated pit-mediated endocytosis has progressed dramatically, including the elucidation of the structure of individual components and partial in vitro reconstitution, the role of caveolae as alternative endocytic carriers still remains elusive 50 years after their discovery. However, recent work has started to provide new insights into endocytosis by caveolae and into apparently related pathways involving lipid raft domains. These pathways, distinguished by their exquisite sensitivity to cholesterol-sequestering agents, can involve caveolae but also exist in cells devoid of caveolins and caveolae. This review examines the current evidence for the involvement of rafts and caveolae in endocytosis and the molecular players involved in their regulation.

Semliki Forest virus and Kunjin virus RNA replicons elicit comparable cellular immunity but distinct humoral immunity

RNA replicons offer a number of qualities which make them attractive as vaccination vectors. Both alphavirus and flavivirus replicon vaccines have been investigated in preclinical models yet there has been little direct comparison of the two vector systems. To determine whether differences in the biology of the two vectors influence immunogenicity, we compared two prototypic replicon vectors based on Semliki Forest virus (SFV) (alphavirus) and Kunjin virus (KUN) (flavivirus). Both vectors when delivered as naked RNAs elicited comparable CD8+ T cell responses but the SFV vectors elicited greater humoral responses to an encoded cytoplasmic antigen beta-galactosidase. Studies in MHC class II-deficient mice revealed that neither vector could overcome the dependence of CD4+ T cell help in the development of humoral and cellular responses following immunization. These studies indicate that the distinct biology of the two replicon systems may differentially impact the adaptive immune response and this may need to be considered when designing vaccination strategies. (c) 2005 Elsevier Ltd. All rights reserved.