Projects Judith Klumperman
Research interest and highlights
My research aims to understand how cells organize their intra- and extracellular communication and how genetic mutations lead to cellular disorganization and disease. The focus of my work is the endo-lysosomal system and associated diseases.
Many genetic diseases find their basis in a defect in membrane trafficking, which is required for cell functioning in general and the biogenesis of organelles. Membrane traffic requires protein sorting, vesicle formation and vesicle fusion. These steps must be tightly regulated and need specific sets of machinery proteins, including Rabs, coat components and SNAREs. My group, together with many fantastic international collaborators, has contributed to the functional characterization of various machinery proteins, including SNAREs adaptor proteins and COP coat complexes.
The secretory pathway
Most newly synthesized proteins must leave the endoplasmic reticulum (ER) to travel to their final cellular destination. By a combination of immuno-electron microscopy (IEM) and electron tomography, we found that ER exit is mediated by complex, COPII-coated tubulo-vesicles, which fuse with the so-called vesicular tubular clusters (VTCs). We discovered that VTCs are the major protein concentration station in the secretory pathway of professional secretory cells and postulated the ‘concentration-by-exclusion’ model of secretory protein transport. As a spin-off, we found that VTCs regulate the level of the Alzheimer-associated proteins Presenilin-I and amyloid precursor protein (APP) that is targeted to the plasma membrane. ‘Concentration-by-exclusion’ proceeds in the Golgi complex. The mode of transport through the Golgi complex, however, remains debated. My group was the first to demonstrate that COPI-coated vesicles budding from the Golgi cisternae contain Golgi enzymes, but are depleted of forward moving cargo, providing a key argument for the ‘cisternal maturation’ model of intra-Golgi transport.
Lysosomes are the primary degradative compartments of the cell. They degrade extracellular material internalized by endocytosis and intracellular components by autophagy. To date, more than 50 diseases are known with a primary defect in lysosomal functioning. My group has studied how mannose 6-phosphate receptors (MPRs) mediate transport of lysosomal enzymes to endosomes and lysosomes. We discovered a bi-layered Hrs/clathrin-coated membrane sub-domain on endosomes that regulates the sorting of growth factor receptors for down-regulation in lysosomes. We also defined a novel, AP3-mediated pathway by which lysosomal membrane proteins travel from tubular sorting endosomes to lysosomes. These studies clarified the cellular role of AP3 as causative gene for Hermansky-Pudlak syndrome II (a pigmentation bleeding disorder), but also implicated early endosomes in lysosomal protein trafficking and revealed the existence of a tubular sorting endosome. Our most recent finding is the discovery of a clathrin-independent pathway that directly transports lysosomal membrane proteins from the trans Golgi network to late endosomes. This pathway operates in parallel to the MPR pathway, which implicates that the supply of lysosomal enzymes and lysosmal membrane proteins can be regulated independently.
During my career I have specialized in the application of immuno-electron microscopy (IEM) to biomedical questions. I chair the Cell Microscopy Center of the University Medical Centre Utrecht, The Netherlands, and coordinated the CLEM workpackage within the ESFRI initiative EuroBioimaging that aims to set up an European network for microscopy infrastructure. The recently correlative light-electron microscopy (CLEM) developed in my group literally bridges the gap between live-cell imaging and IEM.
We will further characterize the molecular mechanisms that underlie the MPR-independent transport pathway for lysosomal membrane proteins and establish its physiological relevance. In addition we study the role of components of the HOPS and CORVET complex in lysosome biogenesis, two endo-lysosomal based tethering complexes that are increasingly recognized as causative genes for myopathies, neurological and multisystem disorders. In addition to these fundamental studies, we focus on diseases in which the endo-lysosomal system is primarily or secondary affected. These include cancer, especially breast cancer, and neurodegenerative disorders, like Alzheimer’s disease. Finally, we continuously seek to improve our correlative microscopy methods.
Morphological analyses of human diseases, especially cancer
(Ann De Mazière, George Posthuma, Suzanne van Dijk)
By the application of different electron microscopy (EM) techniques, we study at high resolution aspects of various cancer-related subjects. The currently applied EM techniques are conventional EM of plastic sections, immunogold labeling of ultrathin cryosections (immunoEM), 3D electron tomography, and correlative microscopy by means of SLEM (Section Light and Electron microscopy). Following the specific needs of projects, modifications of established EM techniques are developed and implemented. Examples of current projects are:
-The role of Akt/PKB in autophagy in PTEN-deficient cancer cell lines and tissues. According to the current hypothesis, phosphorylated Akt/PKB activates mTOR, which suppresses autophagy. In cells deficient for the PTEN phosphatase, where Akt/PKB is constitutively activated, inhibition of Akt/PKB releases autophagy induction. Upregulated autophagy may then cause a growth inhibition, or be a target where cancer cells can be hit in their energy and resource supply. For the quantitative detection of autophagic vacuoles (phagophores, autophagosomes and autolysosomes) in distinct conditions we use EM of plastic sections.
-The role of an endothelial-cell-specific secreted protein, EGFL7, in blood vessel formation during development. Effects of EGFL7 knockdown are investigated in the zebrafish and mouse model.
-Mycobacterium marinum infection. By means of EM of plastic sections and immunoEM, subcellular compartments invaded by the bacteria are characterized.
Trafficking of lysosomal membrane proteins and the function of the mammalian HOPS complex in lysosome biogenesis and function (Maaike Pols, Romain Galmes)
The lysosomal membrane proteins LAMP-1 and LAMP-2 make up over 50% of the proteins present in the lysosomal limiting membrane. They reach the lysosome by either direct or indirect trafficking from the TGN. The indirect pathway involves passaging over the plasma membrane and subsequent endocytosis, while the direct TGN to endosome pathways are less well understood. Here, we study the direct trafficking route of LAMPs from the TGN to the endosomal system and the protein machinery involved in this process.
The HOPS (homotypic fusion and protein sorting) and CORVET (Class c core vacuole/endosomes transport) complexes are tethering complexes consisting of 6 Vps (vacuolar protein sorting) proteins that play an important role in the fusion at the endolysosomal system. These complexes have been well studied in yeast, where the HOPS complex is responsible for all fusion events at the late endosome/lysosome (vacuole) and the CORVET complex at the early/late endosome. However, little is known about the function of their mammalian homologues. In this study, we aim to unravel the role of the HOPS specific subunits, Vps39 and Vps41 (Maaike Pols), and common or CORVET specific subunit, Vps33B and Vps8 respectively (Romain Galmes), in lysosome biogenesis and function in human cell lines.
The role of mammalian CORVET and HOPS components in intracellular trafficking in polarized cells (Caspar Jonker)
HOPS (homotypic fusion and protein sorting) and CORVET (Class C core vacuole/endosomes transport) are protein complexes that play an important role in intracellular trafficking within the endolysosomal system. These structurally and functionally equivalent hexameric complexes contain four common and two specific Vps (vacuolar proteins sorting) subunits. The HOPS and CORVET complex have been extensively studied in yeast, where they are involved in early/late endosomal and vacuolar membrane tethering. In mammalian cells however the functions of these complexes are less clear. Moreover, in the case of the CORVET complex its existence in mammalian cells is yet to be shown. We aim to understand the function of these complexes in mammalian cells by studying the involvement of the HOPS/ CORVET subunit vps18 and the CORVET specific subunit vps8 in intracellular trafficking events.
Most cells in eukaryotic organisms are polarized in the sense that they display cellular asymmetry. Epithelial cells fall into this category and harbor spatially asymmetric plasma membranes. To maintain cellular asymmetry many intracellular trafficking mechanisms differ from non-polarized cells. For this reason we believe that the HOPS and CORVET components may function differently in polarized cells. Therefore we aim to analyze the function of HOPS and CORVET components in various polarized and non-polarized mammalian cell lines.
The role of the endo-lysosomal system in desmosome-mediated epithelial cancer cell metastasis (Prajakta Gosavi)
A common cause of mortality in cancer patients is metastasis, the spreading of cancer cells from one organ or tissue to another, and the subsequent development of secondary tumors. For epithelial cancers a key event in the onset of metastasis is dissolution of cell-cell contacts. Recently, an increasing number of studies indicated that loss of desmosomes, a specialized type of junctional complex mediating cell-cell contact, leads to tumorigenesis. Interestingly, desmosome down-regulation occurs in an early stage of tumor development and invasion, which makes it an interesting target for therapeutics. The cellular mechanisms that underlie desmosome down-regulation have remained poorly understood, but several recent studies have indicated a role for the endo-lysosomal system. Interestingly, the endo-lysosomal system of cancer cells often differs from normal cells by lysosome size and numbers, localization, Cathepsin expression and enzymatic activity. However, the functional interrelationship between neoplastic transformation, the endo-lysosomal system and the assembly of cell-cell junctions is not understood. The aim of my project is to identify the role of the endo-lysosomal system in the regulation of cell-cell adhesion and neoplastic transformation of epithelial cells, with focus on desmosome turnover and functioning.