Some of our ongoing projects
Hit one of the project buttons for more details
Project #1: DESIGN AND DEVELOPMENT OF FLOURESCENT PROTEIN BASED BIOSENSORS
Genetically encoded biosensors are indispensable tools for modern cell biology. Fluorescent protein-based live-cell imaging techniques have revolutionized our understanding of cellular processes. Almost any biological process can be investigated in detail with high spatial and temporal resolution using differently colored fluorescent protein variants. Nowadays, a plethora of genetic biosensors is available for more than 100 different targets including ions, metabolites, reactive oxygen, and reactive nitrogen species, temperature, pressure, signaling events, protein interactions and more. In the past, together with our collaboration partners, we have developed novel, or further refined existing Foerstner Resonance Energy Transfer (FRET)-based (Figure 1A), single FP based (Figure 1B) and differentially targeted biosensors (Figure 1C) for calcium, nitric oxide, ATP, potassium and pH levels. Prospective students are welcome to join us to develop novel and informative biosensors and imaging tools for (sub)cellular visualization of intracellular signaling events.
Figure 1: (A) Schematic of a classic FRET biosensor. A FRET-based sensor consists of two fluorescent proteins, a FRET donor (cyan FP) and a FRET acceptor (yellow FP). Both FPs are sandwich a substrate specific domain which is capable of binding the analyte of interest in a reversible manner (Calcium, ATP, etc.). In the presence of the analyte, the sensor domain of the construct undergoes a dramatic conformational rearrangement which permits the donor FP to transfer its energy to the acceptor which allows a ratiometric readout on the fluorescence microscope in real-time. (B) Schematic of a single FP based circularly permuted and ratiometric biosensor. This class of a sensor consists of only one fluorescent protein and two substrate specific domains which are capable of binding the analyte of interest in a reversible manner (Calcium, ATP, etc.). In the presence of the analyte, the sensor domains of the construct interact and lead to a dramatic conformational rearrangement of the fluorescent protein which in turn changes the spectral properties of the FP. This again allows a ratiometric readout on the fluorescence microscope in real-time. (C) Schematic of a differentially targeted FP to the outer mitochondrial membrane (left panel) and microtubule (right panel). Exploiting well-established and newly identified targeting peptides, we can direct genetic biosensors to specific subcellular locals within a cell or tissue. © Eroglu – 2016
Putative candidates joining this project will deal with:
• Literature screen, identification, and assessment of a putative sensor or targeting element
• In-silico design and computation of biosensors using bioinformatics for 3-D modeling and calculation on the protein level.
• Design and fabrication of DNA plasmids that includes standard molecular cloning approaches.
• Recombinant protein production, purification, and testing using fluorescent spectrophotometry.
• Cell culture methods for primary cells and various cancer cell lines.
• Purification of DNA-plasmids and transfection of cultured cells.
• Generation and application of recombinant viral vector systems including Adenoviral and Lentiviral systems.
• High-resolution fluorescence microscopy including Widefield and Confocal Microscopy.
• Biostatistical Analysis, data interpretation, and presentation.
Selected Literature for interested students:
Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo. Bischof et al, 2017, Nature Communications
Project #2: ADVANCED CHEMOGENETIC STRATEGIES FOR INVESTIGATING CELL DYNAMICS
Chemogenetic tools are engineered molecular systems that enable precise manipulation and control of cellular processes using specific small molecules. These tools are highly selective, allowing researchers to probe and dissect complex biological signaling networks with exceptional spatial and temporal precision. Today, a diverse array of chemogenetic tools is available to modulate various cellular processes. In our research, we have developed innovative tools, including a modified D-amino acid oxidase (mDAAO) and a novel pH-control system:
mDAAO Chemogenetic Tool: This tool allows us to manipulate intracellular hydrogen peroxide (H₂O₂) levels, a critical reactive oxygen species (ROS). By modulating H₂O₂, we can explore cellular pathways and processes linked to oxidative stress and redox signaling.
pH-Control Chemogenetic Tool: This tool generates protons, enabling precise modulation of the local pH environment. It provides a robust method for studying cellular events influenced by acidification, such as pH-dependent enzymatic activity.
Our ongoing efforts focus on developing novel chemogenetic tools to expand the scope of cellular modulation and visualization. By integrating these advanced tools with state-of-the-art imaging and biosensing technologies, we aim to uncover groundbreaking insights into intracellular signaling dynamics and regulatory networks.
Figure 2: Schematic overview of chemogenetic approaches in vitro and in vivo and in cancer therapy.
We invite prospective students and collaborators to join us in advancing the development of chemogenetic tools. Together, we can push the boundaries of cellular and molecular biology research by enabling the visualization and manipulation of dynamic signaling events at the (sub)cellular level.
Putative candidates joining this project will deal with:
Cell physiology of vascular cells and cancer cells with a particular focus on NO biosynthesis and ROS signaling.
Design and development of bicistronic constructs for simultaneous expression of multiple biosensors.
Multichromatic live cell imaging techniques using genetically encoded biosensors and chemical sensors for Ca2+, NO, and H₂O₂
Cell culture methods for primary cells and various cancer cell lines.
Application of recombinant viral vector systems including Adenoviral and Lentiviral systems for the generation of stable cell lines.
High-resolution confocal microscopy.
Biostatistical Analysis, data interpretation, and presentation.
Selected Literature for interested students:
Development of a Chemogenetic Approach to Manipulate Intracellular pH, Zaki et al, 2023, Journal of the American Chemical Society
Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting Erdoğan et al, 2021, FRBM
Project #3: MULTICHROMATIC IMAGING OF SIGNALING EVENTS IN INDIVIDUAL CELLS
Multiplex fluorescence imaging is a powerful approach with broad applications for in-vivo and single-cell analysis. We leverage spectral variants of well-characterized biosensors, which possess unique spectral properties and high selectivity and sensitivity for their specific analytes. This allows for the simultaneous imaging of multiple biosensors using standard fluorescence microscopy techniques.
In this project, we apply these techniques independently—imaging solely with biosensors or relying exclusively on chemogenetic tools—as well as in combination, enabling a comprehensive view of cellular signaling. Chemogenetic tools provide the ability to precisely control cellular processes by introducing engineered molecular systems that respond to specific small molecules. By combining these tools with multiplex fluorescence imaging, we can modulate specific intracellular pathways in real time and visualize the effects of this modulation simultaneously with biosensor activity. This flexible approach allows us to examine how different signaling pathways interact with each other and respond to various stimuli in a dynamic and highly controlled manner.
Figure 3: Multiparametric manipulation of local ROS levels and simultaneous imaging using different biosensors.
We welcome prospective students and collaborators to join us in advancing the integration of chemogenetic tools with cutting-edge imaging technologies. By working together, we can push the frontiers of cellular and molecular biology research, allowing for real-time observation and targeted manipulation of dynamic signaling events within individual cells, thereby providing unparalleled insights into the intricate regulation of cellular processes.
Putative candidates joining this project will deal with:
Cell physiology with a particular focus on ROS, RNS, and Energy metabolism in individual cells.
Multiparametric imaging techniques with genetic and chemical biosensors along with chemogenetic tools.
Agilent Seahorse XF Technology for real-time imaging of cellular energy metabolism.
Single cell imaging of ATP levels using genetic biosensors (ATeam).
Application of recombinant viral vector systems including Adeno viral and Lentiviral systems
High-resolution confocal microscopy.
Small interfering molecules including siRNA and shRNA.
Biostatistical Analysis, data interpretation and presentation
Selected Literature for interested students:
Genetically encoded biosensors unveil neuronal injury dynamics via multichromatic ATP and calcium imaging, Zaki et al, 2024, ACS Sensors.
Visualizing hydrogen peroxide and nitric oxide dynamics in endothelial cells using multispectral imaging under controlled oxygen conditions, Altun et al, 2024, FRBM
Chemogenetic generation of hydrogen peroxide in the heart induces severe cardiac dysfunction. Steinhorn et al, 2017, Nature Communications