Ima Avalos Vizcarra

ON/OFF and Beyond - A Boolean Model of Apoptosis

Apoptosis is regulated by several signaling pathways which are extensively linked by crosstalks. Boolean or logical modeling has become a promising approach to capture the qualitative behavior of such complex networks. Here we built a large-scale literature-based Boolean model of the central intrinsic and extrinsic apoptosis pathways as well as pathways connected with them. The model responds to several external stimuli such as Fas ligand, TNF-α, UV-B irradiation, interleukin-1β and insulin. Timescales and multi-value node logic were used and turned out to be indispensable to reproduce the behavior of the apoptotic network. The coherence of the model was experimentally validated. Thereby an UV-B dose-effect is shown for the first time in mouse hepatocytes. Analysis of the model revealed a tight regulation emerging from high connectivity and spanning crosstalks and a particular importance of feedback loops. An unexpected feedback from Smac release to RIP could further increase complex II formation. The introduced Boolean model provides a comprehensive and coherent description of the apoptosis network behavior. It gives new insights into the complex interplay of pro- and antiapoptotic factors and can be easily expanded to other signaling pathways.

Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field still needs to rely on a 50-y-old laborious and time-consuming method to assess the lifespan of yeast cells and to isolate differentially aged cells for microscopic snapshots via manual dissection of daughter cells from the larger mother cell. Here, we are unique in achieving continuous and high-resolution microscopic imaging of the entire replicative lifespan of single yeast cells. Our microfluidic dissection platform features an optically prealigned single focal plane and an integrated array of soft elastomer-based micropads, used together to allow for trapping of mother cells, removal of daughter cells, monitoring gradual changes in aging, and unprecedented microscopic imaging of the whole aging process. Using the platform, we found remarkable age-associated changes in phenotypes (e.g., that cells can show strikingly differential cell and vacuole morphologies at the moment of their deaths), indicating substantial heterogeneity in cell aging and death. We envision the microfluidic dissection platform to become a major tool in aging research.

Bistable expression of virulence genes in Salmonella leads to the formation of an antibiotic-tolerant subpopulation

The bistable expression of virulence genes in Salmonella allows a clonal population to hedge its bets: one subpopulation suffers a growth cost, but is tolerant to antibiotics.

Construction and use of a microfluidic dissection platform for long-term imaging of cellular processes in budding yeast

This protocol describes the production and operation of a microfluidic dissection platform for long-term, high-resolution imaging of budding yeast cells. At the core of this platform is an array of micropads that trap yeast cells in a single focal plane. Newly formed daughter cells are subsequently washed away by a continuous flow of fresh culture medium. In a typical experiment, 50–100 cells can be tracked during their entire replicative lifespan. Apart from aging-related research, the microfluidic platform can also be a valuable tool for other studies requiring the monitoring of single cells over time. Here we provide step-by-step instructions on how to fabricate the silicon wafer mold, how to produce and operate the microfluidic device and how to analyze the obtained data. Production of the microfluidic dissection platform and setting up an aging experiment takes ∼7 h.

Bacterial filamentation accelerates colonization of adhesive spots embedded in biopassive surfaces

Sessile bacteria adhere to engineered surfaces and host tissues and pose a substantial clinical and economical risk when growing into biofilms. Most engineered and biological interfaces are of chemically heterogeneous nature and provide adhesive islands for bacterial attachment and growth. To mimic either defects in a surface coating of biomedical implants or heterogeneities within mucosal layers (Peyers patches), we embedded micrometre-sized adhesive islands in a poly(ethylene glycol) biopassive background. We show experimentally and computationally that filamentation of Escherichia coli can significantly accelerate the bacterial surface colonization under physiological flow conditions. Filamentation can thus provide an advantage to a bacterial population to bridge non-adhesive distances exceeding 5 μm. Bacterial filamentation, caused by blocking of bacterial division, is common among bacterial species and can be triggered by environmental conditions or antibiotic treatment. While great awareness exists that the build-up of antibiotic resistance serves as intrinsic survival strategy, we show here that antibiotic treatment can actually promote surface colonization by triggering filamentation, which in turn prevents daughter cells from being washed away. Our combined microfabrication and computational approaches provide quantitative insights into mechanisms that enable biofouling of biopassive surfaces with embedded adhesive spots, even for spot distances that are multiples of the bacterial length.

Robotically controlled microprey to resolve initial attack modes preceding phagocytosis

The behavior of phagocytes to capture intruders is tracked using remotely rotated and translated nanoparticles. Phagocytes, predatory cells of the immune system, continuously probe their cellular microenvironment on the hunt for invaders. This requires prey recognition followed by the formation of physical contacts sufficiently stable for pickup. Although immune cells must apply physical forces to pick up their microbial prey, little is known about their hunting behavior preceding phagocytosis because of a lack of appropriate technologies. To study phagocyte hunting behavior in which the adhesive bonds by which the prey holds on to surfaces must be broken, we exploited the use of microrobotic probes to mimic bacteria. We simulate different hunting scenarios by confronting single macrophages with prey-mimicking micromagnets using a 5–degree of freedom magnetic tweezers system (5D-MTS). The energy landscape that guided the translational and rotational movement of these microparticles was dynamically adjusted to explore how translational and rotational resistive forces regulate the modes of macrophage attacks. For translational resistive prey, distinct push-pull attacks were observed. For rod-shaped, nonresistive prey, which mimic free-floating pathogens, cells co-aligned their prey with their long axis to facilitate pickup. Increasing the rotational trap stiffness to mimic resistive or surface-bound prey disrupts this realignment process. At stiffness levels on the order of 105 piconewton nanometer radian−1, macrophages failed to realign their prey, inhibiting uptake. Our 5D-MTS was used as a proof-of-concept study to probe the translational and rotational attack modes of phagocytes with high spatial and temporal resolution, although the system can also be used for a variety of other mechanobiology studies at length scales ranging from single cells to organ-on-a-chip devices.

How type 1 fimbriae help Escherichia coli to evade extracellular antibiotics

To survive antibiotics, bacteria use two different strategies: counteracting antibiotic effects by expression of resistance genes or evading their effects e.g. by persisting inside host cells. Since bacterial adhesins provide access to the shielded, intracellular niche and the adhesin type 1 fimbriae increases bacterial survival chances inside macrophages, we asked if fimbriae also influenced survival by antibiotic evasion. Combined gentamicin survival assays, flow cytometry, single cell microscopy and kinetic modeling of dose response curves showed that type 1 fimbriae increased the adhesion and internalization by macrophages. This was caused by strongly decreased off-rates and affected the number of intracellular bacteria but not the macrophage viability and morphology. Fimbriae thus promote antibiotic evasion which is particularly relevant in the context of chronic infections.

Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria

Bacterial adhesion and biofilm growth can cause severe biomaterial-related infections and failure of medical implants. To assess the antifouling properties of engineered coatings, advanced approaches are needed for in situ monitoring of bacterial viability and growth kinetics as the bacteria colonize a surface. Here, we present an optimized protocol for optical real-time quantification of bacterial viability. To stain living bacteria, we replaced the commonly used fluorescent dye SYTO® 9 with endogenously expressed eGFP, as SYTO® 9 inhibited bacterial growth. With the addition of nontoxic concentrations of propidium iodide (PI) to the culture medium, the fraction of live and dead bacteria could be continuously monitored by fluorescence microscopy as demonstrated here using GFP expressing Escherichia coli as model organism. The viability of bacteria was thereby monitored on untreated and bioactive dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC)-coated glass substrates over several hours. Pre-adsorption of the antimicrobial surfaces with serum proteins, which mimics typical protein adsorption to biomaterial surfaces upon contact with host body fluids, completely blocked the antimicrobial activity of the DMOAC surfaces as we observed the recovery of bacterial growth. Hence, this optimized eGFP/PI viability assay provides a protocol for unperturbed in situ monitoring of bacterial viability and colonization on engineered biomaterial surfaces with single-bacteria sensitivity under physiologically relevant conditions.

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 μm). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

Stretchable Silver Nanowire Microelectrodes for Combined Mechanical and Electrical Stimulation of Cells

The use of stretchable electrodes interfaced with the human body has enabled a new frontier in biomedical engineering, and the miniaturization of such electrodes can allow for a more precise spatial control to monitor or stimulate tissues. The understanding of the response of cells or tissues to combined electromechanical stimulation, as made possible by stretchable electrodes, is essential to improve medical devices and therapies. Cheap to produce and easy to use platforms for in vitro cell studies are thus urgently needed. This study reports the successful implementation of silver nanowires (AgNWs) into an integrated miniaturized electromechanical stimulator, which is compatible with cell culture. The innovative steps include a lithography-based lift-off method to micropattern AgNWs onto an elastic silicone membrane. These stretchable microelectrodes are then integrated into a microfluidic device for cell culture, which enables the synchronous electromechanical stimulation of cells. In a proof-of-concept study, it is furthermore shown that fibroblasts respond uniquely to mechanical stretching, electrical stimulation, and combined electromechanical stimulations in terms of cell alignment and morphology, as well as by producing the extracellular matrix protein collagen. This proof-of-concept study illustrates the functionality and usability of these stretchable AgNWs microelectrodes for either basic research or future biomedical applications.