The microtubule cytoskeleton is regulated by a group of microtubule associated proteins that are specifically targeted to growing microtubule plus ends. These protein are called plus-end tracking proteins or +TIPs. The highly conserved autonomous +TIP family of End Binding (EB) proteins controls the recruitment of most other +TIPs to microtubule ends and is a central regulator at microtubule ends. The interaction of EBs with microtubules and its partners is regulated by posttranslational phosphorylation events to control the multiple different functions microtubules have in the cell. In budding yeast the EB family is represented by the single homologue Bim1. Interestingly, in a proteomics screen for Bim1/EB1 binding partners several protein kinases were uncovered. Many of the kinases or their complex partners contain structural features observed in known Bim1/EB1 partners suggesting that they could represent novel Bim1/EB1 partners phospho-regulating microtubule function. Preliminary analysis of several kinases has confirmed the surprising interaction with Bim1/EB1. In addition, colocalization of microtubule tips with several kinases could be observed, and finally in vitro a purified kinase complex could phosphorylate recombinant +TIPs. The aim of this research proposal is to unravel the mechanistic and functional link between the microtubule plus ends and the identified kinases in budding yeast. I will determine the molecular mechanisms responsible for the interaction by generating mutant yeast strains and using purified proteins in vitro. I will study the temporal and spatial regulation of the interactions by cell cycle controlled experiments and fluorescent microscopy techniques. I aim to understand the cellular role of the interactions by studying microtubule dynamics, cell morphology and any cellular processes the kinases have previously been linked to, using mutant cells. Finally, I will determine if there is a level of conservation in higher eukaryotes by probing the interaction between +TIPs and the identified kinases in mammalian cells.

Alterations in cAMP signaling have been linked to many human disorders underscoring their significance in health and disease. cAMP is a ubiquitous small diffusible second messenger mediating the action of several hormones and mechanisms involved in signaling specificity remain ill-defined. cAMP compartmentalization (i.e., microdomains) emerged in the field as an established mechanism to attain signaling specificity. However, current computational models indicate diffusion needs to be restricted locally in order to reach cAMP levels consistent with efficient localized effectors’ activation. Validation of these models awaits the identification of bona fide cAMP compartments, the generation of compartment-specific cAMP actuator tools and their application in live cell-imaging approaches to dissect details of cAMP diffusivity and function. Dr Altschuler’s laboratory has identified a new sub-membrane compartment required for cAMP-dependent cell proliferation and developed novel chemo-optogenetic actuators tools to address some of these issues. Utilizing targeted actuators with spectrally compatible immobilized sensors in combination with fast-speed time-lapse FLIM approaches developed in Dr. Jalink’s lab, we intend to assess diffusivity properties of cAMP generated in distinct microdomains and provide new mechanistic details of local cAMP dynamics in signaling. The common long-standing interest of Dr. Altschuler and Dr. Jalink to understand the spatial and temporal regulation of cAMP-dependent signaling events in combination to the complementary skills of both groups holds promise for a productive synergistic collaboration.

MHC class I and II molecules are critical directors of the adaptive immune response in many pathological conditions, like cancer, infection, stem cell and organ transplantation, and auto-immune disease. The expression of MHC molecules is tightly controlled. They are generally regulated during cross-talk of immune cells via soluble proteins such as IFN-gamma (upregulation) or IL-10 (downregulation). However, also tumors such as melanoma can secrete IL-10 to prevent anti-tumor responses. While various factors secreted by immune cells are defined as controlling MHC expression, an unbiased systemic analysis of the secretome including proteins secreted by non-immune cells has not been considered. Here I propose to screen the full human secretome for proteins controlling the expression of MHC molecules. My approach is feasible as in preliminary experiments I already identified a novel secreted factor, KIAA0527, that manipulates MHC expression on dendritic cells. After hit definition and validation, the potential clinical relevance of the secreted proteins will be analyzed using publicly available expression arrays and subsequent primary tumor and tissue material. The potential clinical applicability will be investigated using in vitro multi-cellular assays supplemented with the secreted proteins. As a long-term goal beyond the current proposal, signalling elements in the target cells downstream of the secreted proteins will be identified. The characterization of new secreted proteins manipulating MHC expression will provide insights into novel immune-editing mechanisms including control in peripheral tissues and tumors. In addition, new soluble proteins may have clinical implications as immunomodulatory tools. These are potentially applicable for the treatment of a wide variety of diseases, from infection to cancer.

A cell is often likened to a bowl of spaghetti, in which each chromosome reflects a single entity of pasta. Remarkably though, the different chromosomes are not intermingled, as each chromosome is confined to its own region. What actually keeps the chromosomes apart? And what purpose does this serve? Molecular machines known as condensin play a central role in these processes. We have discovered how cells activate condensin such that it can correct structure our chromosomes.

A common experience when reading a book is being absorbed by the story that is told. This process is known as transportation into a narrative. Transportation makes the narrative world appear more real and increases attachment to and strong feelings for the characters in the story. However, transportation also reduces critical thinking. This raises the question whether transportation hurts in situations that require critical thinking. The research described in this proposal will address this question with respect to decision making in court, a process that ought to be rational. It will be investigated whether the experience of being transported while reading fictive criminal case files influences the processing of information and decision making. Because subtle differences in the use of language (e.g., grammatical aspect, direct vs. indirect speech, active vs. passive voice) are known to influence transportation, these linguistic cues will be manipulated across case files. The fact that these cues vary across real police reports contributes to the external validity of this project. Both laymen and professionals working in the legal system will participate in the proposed studies. This makes the results practically relevant for jury as well as bench trials. Also, this allows for the investigation of the role of prior knowledge, experiences and expectations. The proposed research uses corpus and context analyses, eyetracking, and memory and judgment tasks. Because of this combination of methods, all aspects of the decision making process can be studied. An important innovative aspect of this project is that it combines research in the areas of (psycho)linguistics and cognitive psychology to explore topics in the forensic field. The results will contribute to a better understanding of the influence of subtle variations in language on the processing and evaluating of police reports. This is necessary to improve legal decision making and promote justice.