Bacterial infections are increasingly difficult to treat due to the worldwide increase in antibiotic resistance. The development of new antibiotics is notoriously difficult and expensive. The majority of research is based on either in vitro target inhibition assays or in vivo random screening assays. In the former the developed antibiotics fail because they cannot pass the bacterial envelope or are promiscuous and in the latter the antibiotics often fail because they are toxic to eukaryotes. Target specific assays in living bacteria are much more cost effective because the chance of toxicity is lower as the target has been chosen carefully and positive compounds can pass the envelope. Recently, it is appreciated that the penicillin binding proteins that synthesize the peptidoglycan layer collaborate with and are activated by other proteins. These protein complexes are interesting novel targets for antibiotic development. Unfortunately, no assays exist that measure the interaction of these proteins in the periplasm. By developing a Fluorescence Energy Transfer (FRET) assay in the periplasm, like we did for the cytoplasm, we fill this omission in the toolbox. In addition, the FRET assay will be the first assay that is able to measure proteins interactions in the periplasm in living cells. The obtained information on localization and interactions as a function of the bacterial division cycle will be, in combination with the data available for the cytoplasm, used to build a computer model on the molecular mechanisms of bacterial envelope growth that can be used to identify antibiotic targets.

A watertight fence. The wall of our blood vessels form the border between blood and tissue. In case of an inflammation, white blood cells move through the wall to fight the inflammation. An important question is how the wall remains intact and watertight, while at the same time it allows cells to pass. This proposal aim to understand this process by combining microscopy with a physiologically relevant model system. Microscopy will provide a unique and detailed view and it will allow us to better understand the role of the vessel wall as a watertight fence.

Fluorescent proteins (FPs) have revolutionized molecular cell biology and the more recent development of photoswitchable fluorescent proteins (psFPs) holds promise for even further boosts in this dynamic scientific field. The first (currently available) generation psFPs is limited in number and with respect to their photophysical characteristics. Here we propose to engineer a second generation of psFP with enhanced properties. In conjunction with parallel development of advanced optical imaging methodologies, these new psFPs will allow novel avenues for quantitative (multi-color) single-molecule microscopy in live cells. Specifically, we propose to i) develop psFP for enhanced fluorescence fluctuation microscopy (FFS) extending the capability of this method from the 1-500 nM range to the physiologically relevant 0.5-10 µM range in quantifying in situ molecular concentration, diffusion and aggregation; ii) develop psFPs for very fast (sub second), non-destructive, sensitive and quantitative determination of molecular proximity by Förster Resonance Energy Transfer Microscopy; and iii) develop psFP with increased quantum yields and optical switching behavior for super resolution microscopy of psFP-labeled proteins. After successful development, the psFPs will be used to label and study the localization, mobility and molecular interactions of proteins involved in G-protein signaling.

The Amsterdam Science Park Study Group is a local community of life scientists that share their expertise, organize training and promote good practices in data-related topics such as data analysis, programming and data management. Engaging early-career researchers through peer-to-peer mentoring and training is a valuable approach to foster a cultural change toward a more Open Science. This proposal aims to expand our current activities and to upscale our community to increase the impact of the Amsterdam Science Park Study Group on Open Science practices in the life sciences.

Long-lived, tissue-resident stem cells are crucial for maintaining the integrity and function of all organs in the adult human body. Under normal physiological conditions their proliferation is tightly controlled, ensuring a balance between self-renewal and terminal differentiation. Disruption of this precarious equilibrium can have dramatic consequences, ranging from cancer formation to degenerative diseases and aging. It is thus of critical importance that we gain a better understanding of stem cell activity and control. Stem cell division is regulated by dedicated self-renewal signals from the local microenvironment or ?niche?. However, in higher vertebrates the location and identity of the niche are virtually unexplored and it remains an experimental challenge to manipulate stem cell populations in situ. Therefore, the overall aim of this proposal is to probe the interaction between stem cells and their niche in a complex mammalian tissue, using the mouse mammary gland as a model system. Because so-called Wnt proteins act as self-renewal factors in multiple contexts and because Wnt-responsive stem cells exist in the adult mammary epithelium, I hypothesize that Wnt-secreting cells constitute a mammary stem cell niche. Based on this idea I will meet the following key objectives: 1. Establish a genetic pipeline that allows selective manipulation of Wnt-secreting cells in the mammary gland. 2. Obliterate or hyperactivate the mammary stem-cell niche. 3. Test how the local loss or gain of self-renewal factor signaling affects the plasticity and developmental fate of Wnt-responsive mammary stem cells. To this end, I will identify COnserved Mammary Enhancers (COMEs) and use these sequences as tissue-specific, genetic control elements to drive suicide-gene expression or Wnt overexpression in Wnt-secreting cells. Next, I will track the effects on Wnt-responsive stem cell behavior using my recently developed lineage-tracing model. Together, these experiments will reveal how stem cells respond when communication with the niche is disrupted.