ERC FUNDED PROJECTS

"The megadalton cytoplasmic dynein complex, whose motor subunit is encoded by a single gene, provides the major microtubule minus end-directed motility in cells and is essential for a wide range of processes, ranging from the transport of proteins, RNA, and membrane vesicles to nuclear migration and cell division. To achieve this stunning functional diversity, cytoplasmic dynein is subject to tight regulation by co-factors that modulate localization, interaction with cargo, and motor activity. At present, our knowledge of the underlying mechanisms remains limited. An overarching goal of this proposal is to gain an understanding of how interactions with diverse adaptor proteins regulate dynein function in space and time. We choose the nematode C. elegans as our model system, because it will enable us to study the biology of dynein regulation in the broad context of a metazoan organism. The nematode’s versatile genetic tools, its biochemical tractability, and the powerful molecular replacement technologies available, this makes for a uniquely attractive experimental system to address the mechanisms employed by dynein regulators through a combination of biochemical, proteomic, and cell biological assays. Specifically, we propose to use a biochemical reconstitution approach to obtain a detailed molecular picture of how dynein is targeted to the mitotic kinetochore; we will perform a forward genetic and proteomic screen to expand the so-far limited inventory of metazoan dynein interactors, whose functional characterization will shed light on known dynein-dependent processes and lead to novel unanticipated lines of research into dynein regulation; we will dissect the function and regulation of the most important dynein co-factor, the multi-subunit dynactin complex; and finally we will strive to establish a novel C. elegans model for human neurodegenerative disease, based on pathogenic point mutations in a dynactin subunit."

1 367 466 €

Start date: 2014-03-01, End date: 2019-02-28

"Communication between immune cells is crucial for regulating the magnitude and quality of immune responses. A newly uncovered means of intercellular communication involves transfer of small cell-derived vesicles. I recently discovered that vesicles released by immune cells are enriched for small noncoding RNAs, which may act as regulatory RNAs that can influence gene expression in vesicle-targeted cells. Furthermore, remarkable parallels emerged between RNAs abundantly present in cell-derived vesicles and a group of host RNAs specifically incorporated into retroviruses. These shared RNAs may underlie the formation or function of both cell-derived vesicles and retroviruses. Until now, mechanisms behind selective incorporation of small RNAs into cell-derived vesicles and their function in vesicle-targeted cells are poorly understood. Aim of INTERCOM: To resolve how the exchange of small RNAs via cell-derived vesicles contributes to intercellular communication between immune cells. Key objectives: 1. To determine the diversity and plasticity of the RNA content of vesicle subpopulations released by immune cells. 2. To explain functional differences between immune cell vesicle populations based on their RNA contents. 3. To determine the function of structural RNAs shared by immune cell-derived vesicles and retroviruses. Tools in virology research will be used in combination with several high-end technologies, which were uniquely adapted in my lab for vesicle-related research. These include a high-resolution flow cytometric method suited to analyze individual nano-sized vesicles, RNA deep sequencing with previously developed data analysis methods, and super-resolution microscopic imaging. The proposed work advances our understanding of communication processes in the immune system. This knowledge can be applied in defining vesicle RNA-based biomarkers for immune-related diseases and in designing genetically engineered cell-derived vesicles for therapeutic application."

1 499 806 €

Start date: 2013-11-01, End date: 2018-10-31

My goal is to elucidate how structurally closely-related proteins are selectively partitioned in distinct membrane domains that allow localization, clustering and segregation of specific cellular activities. Although many of the mechanisms that govern membrane organization are increasingly well understood, such as lipid ‘rafts’ or protein-anchoring to the cortical cytoskeleton, these mechanisms are not sufficiently specific to account for the partitioning of closely homologous proteins in separate membrane domains. I believe that the observed highly selective membrane partitioning can only be explained by the combined action of protein-protein and protein-lipid interactions and thermodynamic properties of the membrane (length, charge, degree of hydrophobicity). I aim to gain a full understanding of how homologous proteins partition in distinct functional membrane domains by studying SNARE proteins as a model system. Different SNAREs partition in different domains with different degrees of overlap in the plasma membrane where they catalyze the final membrane fusion steps of various exocytotic pathways. I will employ quantitative super-resolution microscopy to study the effects of selective (biochemical and genetic) perturbations on SNARE partitioning in both precisely controllable artificial membranes and in PC12 cells. This will allow me to elucidate the mechanisms, contributions and interplay of individual membrane clustering mechanisms in SNARE domain organization. I then plan to demonstrate that the mechanisms of SNARE partitioning explain the membrane organization of other (homologous) proteins as well. My ultimate ambitious goal is to generate a complete model of how proteins are organized in biological membranes. I anticipate that my findings will uncover new and general mechanisms of membrane organization and, since membranes are involved in almost all cellular processes, my work may have impact on virtually all areas of the health and life sciences.

1 500 000 €

Start date: 2013-12-01, End date: 2018-11-30

Not all cells in bacterial populations exhibit exactly the same phenotype, even though they grow in the same environment and are genetically identical. One of the main driving forces of phenotypic variation is stochasticity, or noise, in gene expression. Possible molecular origins contributing to noise in protein synthesis are stochastic fluctuations in the biochemical reactions of gene expression itself, namely transcription and translation. The driving hypothesis of this application is that the human pathogen Streptococcus pneumoniae utilizes noisy gene expression to successfully colonize and invade its host. To test this supposition, the total amount of noise in key regulatory networks for virulence factor production will be quantified. Using natural and synthetic bistable switches as highly sensitive probes for noise, in combination with state-of-the-art single-cell imaging, microfluidics and direct transcriptome sequencing, the molecular mechanisms underlying noise generation in S. pneumoniae will be determined. By constructing strains with altered levels of phenotypic variation, the importance of noisy gene expression in S. pneumoniae pathogenesis will be tested. S. pneumoniae is a leading cause of bacterial pneumoniae, meningitis, and sepsis worldwide. The molecular mechanisms that cause switching of S. pneumoniae to its virulent states are barely understood, although it becomes increasingly clear that noise-driven phenotypic variation plays an important role in pneumococcal pathogenesis. Therefore, understanding the molecular origins of phenotypic variation in S. pneumoniae might not only provide novel fundamental insights in gene expression, but also result in the identification of new anti-pneumococcal targets.

1 498 846 €

Start date: 2013-11-01, End date: 2018-10-31