Thomas Braulke, University Medical Center Hamburg-Eppendorf, Dept. Biochemistry, Children's Hospital

Subunit interactions of the Golgi-resident GlcNac-1-phosphotransferase complex

Mucolipidoses II and II are inherited lysosomal storage disorders which are caused by defects in the Golgi-localized GlcNAc-1-phosphotransferase complex. This enzyme complex is composed of six subunits, α2β2γ2, playing a key role in the generation of mannose 6-phosphate recognition marker on lysosomal hydrolases which are essential for their efficient transport to lysosomes. The membrane-bound α- and β-subunits of the GlcNAc-1-phosphotransferase are catalytically active whereas the function of the soluble γ-subunit is unclear. The aim of this project is to investigate the interactions of the γ-subunit with α- and/or β-subunits and to define structural requirements for protein binding. The functional significance of these interactions will be analyzed in patient or knock-out cells lacking the γ-subunit.  Furthermore, the vesicular transport of the three subunits from the ER to the Golgi-apparatus will be examined.

 

Stephan Storch, University Medical Center Hamburg-Eppendorf, Dept. Biochemistry, Children's Hospital

Sorting and signal-mediated trafficking of the neuronal ceroid lipofuscinosis-related CLN7 membrane protein

The variant form of late infantile neuronal ceroid lipofuscinosis caused by mutations in the CLN7 gene belongs to a group of autosomal recessive inherited neurodegenerative disorders of childhood, which are characterized by lysosomal storage and the selective damage and loss of neurons. The CLN7 gene product is a ubiquitously expressed multispanning lysosomal membrane protein of unknown function. In this project we want to investigate the lysosomal targeting of CLN7 and the importance of potential cytosolic sorting signals in CLN7 and their interacting partners for sequential sorting between subcellular compartments. Methods include expression analyses, interaction studies with clathrin adaptor subunits and hemicomplexes and modern imaging techniques.

 

Paul Saftig and Michael Schwake, Institute of Biochemistry, University of Kiel

LIMP-2: A fascinating lysosomal membrane protein with multiple functions. Analysis of its role in late endocytotic compartments.

Mutations in the human gene encoding the lysosomal integral membrane protein LIMP-2 are responsible for the Action Myo­clonus-Renal Failure Syndrome (AMRF), a fatal autosomal-recessive dis­order characterized by focal glomerulosclerosis, progressive myoclonus epilepsy and ataxia, asso­ciated with accumulation of a yet undefined storage material in the central nervous system. Similar to the AMRF phenotype, LIMP-2-deficient mice also exhibit intra­cellular inclusions in cerebral and cerebellar cortex, glomerular changes and ataxia. We have shown recently that LIMP-2 is the receptor for the mannose-6-phosphate (M6P) independent transport of β-glucocerebrosidase (β-GC) to the lysosome. This enzyme hydrolyses the β-glycosyl linkage of glucosylceramide and is defective in patients with the most common lysosomal storage disorder Gaucher disease. Our research focuses on the impact of LIMP-2 on trans­port processes within (neuronal) cells, to determine the stoichiometry and the structure of the LIMP-2/β-GC complex and to identify additional interaction partners.

 

Tobias Spielmann, Bernhard Nocht Institute for Tropical Medicine

Decoding the molecular mechanisms of endocytic processes in the human malaria parasite Plasmodium falciparum

The pathology of malaria is caused by the development of Plasmodium parasites in human red blood cells. In this part of the life cycle, the parasite invades red blood cells, wherein it grows and multiplies to produce new invasive stages. After host cell rupture, these stages invade new red blood cells to continue the cycle. During its development in red blood cells the parasite ingests most of the host cell cytoplasm (predominantly hemoglobin) and transports the internalised material to a food vacuole for digestion. This prominent process is essential for the parasite to provide nutrients and to create space for growth. Knowledge about this process so far relied predominantly on electron microscopy and inhibitor studies but the actual uptake of host cell cytosol has never been observed in real time. The aim of this project is to visualise the ingestion of host cell cytosol and to shed light on the underlying molecular mechanisms.

 

Martin Aepfelbacher, University Medical Center Hamburg-Eppendorf, Institite of Microbiology and Virology

Intracellular complexes and transport during bacterial phagocytosis

Unique intracellular signalling complexes are assembled at the contact site of pathogenic bacteria with host cells during bacterial invasion and phagocytosis. We study the transport of a regulator of the Rho GTPase family (CDC42GAP) to the bacteria loaded phagosome in endothelial and immune cells. CDC42GAP is bound to exocytic vesicles and controls function of the exocyst during phagocytosis by unknown mechanisms. In addition, we also investigate the intracellular transport of phagolysosomes by kinesin motor proteins and RabGTPases. To study these events a combination of microbiological, biochemical and cell biological techniques is applied.

 

Markus Glatzel, University Medical Center Hamburg-Eppendorf, Institute of Neuropathology

Exosomal transport of prions

Prion diseases are fatal neurodegenerative disorders which are transmissible. An essential part of the infectious agent, termed the prion, is composed of an abnormal isoform of the host-encoded cellular prion-protein termed PrPSc. The molecular details underlying PrPSc formation and the mechanisms involved in the spread of infectious prions within affected hosts and from donor to host are poorly understood. Recently, exosomes have been shown to mobilize PrPSc as well as prion infectivity. Moreover retroviral infection increases exosome formation and virions have been shown to contain PrPSc. The project focus on the investigation of the role of exosomes in secretion of cellular- and disease-associated forms of the prion protein.

 

Kerstin Kutsche, University Medical Center Hamburg-Eppendorf, Institute of Human Genetics

OCRL-mediated vesicular transport

Mutations in the OCRL gene cause the oculocerebrorenal syndrome of Lowe (LS), an X-linked disorder characterized by congenital cataracts, selective proximal tubulopathy, and mental retardation. The OCRL protein is a catalytically active phosphoinositide 5-phosphatase with phosphatidylinositol 4,5-bisphosphate as the preferred substrate. OCRL binds to a variety of proteins implicated in vesicular trafficking processes, such as Rab GTPases, APPL1, clathrin, and AP-2. Recently, OCRL has been implicated in membrane trafficking from endosomes to the Golgi apparatus as well as in early steps of the endocytic pathway. We used siRNA-mediated depletion of OCRL to study the role of this 5-phosphatase in various vesicle-mediated transport mechanisms. We could show that OCRL is involved in trafficking of the mannose 6-phosphate receptor, but not in that of the epidermal growth factor and transferrin receptor. The aim of this project is to understand the function of OCRL and its binding partners in mannose 6-phosphate receptor trafficking by applying a combination of biochemical and cell biology techniques.

Hans-Jürgen Kreienkamp, University Medical Center Hamburg-Eppendorf, Institute of Human Genetics

Regulated sorting of plasma membrane receptors through the Golgi-associated protein PIST

PIST/GOPC is a Golgi protein which has been implicated in in the sorting and intracellular transport of various receptor proteins. PIST is tethered to the trans-Golgi network through interactions with the GTPase Rab6 and syntaxin-6; in neurons it is present in somatic TGN and in Golgi outposts. Live cell imaging and biochemical assays indicate that PIST is stationary at these sites and does not participate in vesicular transport to the plasma membrane. We will analyze the relevance of PIST for targeting of receptors to postsynaptic sites in neurons. In particular, we will determine how the distribution of PIST-associated membrane proteins neuroligin-2 and stargazin is altered in the absence of PIST, and how PIST contributes to the regulation of membrane protein availibility at the cell surface. These experiments will contribute to our understanding of synapse formation and synaptic plasticity.

 

Joerg Heeren, Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf

Regulation of lipoprotein uptake into liver and brown adipose tissue

Abnormalities in lipid and lipoprotein metabolism are associated with the development of atherosclerosis and the metabolic syndrome. The uptake and intracellular processing of lipoproteins into liver and brown adipose tissue directly influence plasma lipoprotein levels. These processes are mediated by lipoprotein receptors such as CD36, LDL receptor and the LDL receptor-related protein 1 (LRP1) in dependence on intracellular adaptor proteins. In this project we want to investigate how lipoprotein receptor function and lipid uptake processes are regulated with the focus on endosomal trafficking in hepatocytes and brown adipocytes.