Biology subjects

Dobrzynski, M.

Publications and source records attributed to Dobrzynski, M..

10 recordsLinked to original sources

bioRxiv2025

Real-time feedback control microscopy for automation of optogenetic targeting

Optogenetics has revolutionized our ability to study cellular signaling by enabling the precise control of cellular functions with light. Most classical implementations rely on fixed or manually updated illumination patterns, limiting their ability to accommodate living systems that move, change shape or rapidly adapt their signaling states. Here, we present an experimental platform for Feedback Adaptive Real-time Optogenetics (FARO) that combines automated image segmentation, tracking, feature extraction, and adaptive hardware control to dynamically adjust optogenetic stimulation based on live cell behavior. By continuously analyzing biosensor signals, FARO updates illumination patterns in real time across biological scales; from maintaining stimulation on specific subcellular regions, to selectively activating single cells in deforming tissue. This fully automated, Python-based framework is built on open standards for data management and microscope control and data handling, supporting large-scale experiments and long-term timelapse studies, compatible with different microscope hardware. Eliminating the need for human intervention to reposition light patterns or select target cells enables reproducible, systematic and high-throughput interrogation of spatiotemporal signaling. We show how automated and adaptive optogenetic perturbations are a powerful tool to study how local signaling events shape cellular behavior, from subcellular dynamics and single-cell migration up to emergent tissue-level processes.

cell biology↗

bioRxiv2025

Tracking Coordinated Cellular Dynamics in Time-Lapse Microscopy with ARCOS.px

Subcellular structures like actin waves, podosomes and focal adhesions exhibit dynamic organization that traditional tracking methods struggle to capture. We developed ARCOS.px, an open-source Python package with an interactive napari plugin, to identify and track spatial clusters in microscopy time-lapse images. Unlike conventional approaches that focus on individual particles or bulk intensity changes, ARCOS.px tracks spatial relationships, coordination, and lineage evolution over time. We demonstrate the method by tracking podosome-like structures and RhoA activity waves triggered by growth factors or optogenetics. We revealed how various drugs affect the stability, mobility, and interactions of podosome-like structures in REF52 cells. Additionally, we discovered that actin waves in these cells are followed by active RhoA and Myosin, suggesting feedback mechanisms that recruit active RhoA at their trailing edge. ARCOS.px can be applied to diverse research contexts requiring the tracking of discontinuous yet dynamically associated regions.

cell biology↗

bioRxiv2024

Convpaint - Universal framework for interactive pixel classification using pretrained neural networks

We develop Convpaint, a universal computational framework for interactive pixel classification. Convpaint utilizes pretrained convolutional neural networks (CNNs) or vision transformers (ViTs) for feature extraction and enables easy segmentation across a wide variety of tasks. Available within the Python-based napari software ecosystem, Convpaint integrates seamlessly with other plugins into image processing pipelines, which we demonstrate with three workflows across different data modalities.

bioinformatics↗

bioRxiv2023

GTPase activating protein DLC1 spatio-temporally regulates Rho signaling

Tightly regulated spatio-temporal Rho GTPase activity patterns regulate morphogenetic processes such as cell migration. Emerging evidence suggests that binding of Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) to the cytoskeleton or adhesions mediate feedback regulation to spatio-temporal Rho GTPase activation. To explore such feedback regulation, we study the Rho specific GAP Deleted in Liver Cancer 1 (DLC1) which binds to focal adhesions (FAs) through mechanosensitive interactions. Using a FRET biosensor, we show that DLC1 loss of function leads to global increase in Rho activity and contractility throughout the cell without affecting a striking lamellar RhoA activity band in fibroblasts. Although RhoA activity may appear steady at the population level, individual molecules are continually activated by GEFs, inactivated by GAPs, and diffusing between subcellular regions, a dynamic turnover referred to as "signaling flux". To directly measure this flux, we built a genetic circuit combining an optogenetic actuator to control Rho activity with a Rho activity biosensor. In spreading cells at steady state, optogenetic manipulation of Rho activity reveals that DLC1 controls the rate of Rho activation rather than duration, both at FAs and at the plasma membrane (PM). Local and reversible optogenetic control of contractility shows that DLC1 associates/dissociates with FAs during their reinforcement/relaxation. This might provide positive feedback that locally increases the rate of Rho activation at FAs that experience local tension to facilitate FA disassembly. Our results indicate that DLC1 operates both at the PM and at FAs to regulate global Rho activity levels at steady state, or to amplify local Rho activity at FAs experiencing a strong mechanical input, presumably to induce robust FA disassembly. This provides new insights in the complexity of spatio-temporal Rho GTPase signaling.

cell biology↗

bioRxiv2022

Automatic detection of spatio-temporal signalling patterns in cell collectives

An increasing experimental evidence points to physiological importance of space-time correlations in signalling of cell collectives. From wound healing to epithelial homeostasis to morphogenesis, coordinated activation of bio-molecules between cells allows the collectives to perform more complex tasks and better tackle environmental challenges. To understand this information exchange and to advance new theories of emergent phenomena, we created ARCOS, a computational method to detect and quantify collective signalling. We demonstrate ARCOS on cell and organism collectives with space-time correlations on different scales in 2D and 3D. We make a new observation that oncogenic mutations in the MAPK/ERK and PIK3CA/Akt pathways of MCF10A epithelial cells induce ERK activity waves with different size, duration, and frequency. The open-source implementations of ARCOS are available as R and Python packages, and as a plugin for napari image viewer to interactively quantify collective phenomena without prior programming experience.

cell biology↗

bioRxiv2021

Optogenetic actuator/biosensor circuits for large-scale interrogation of ERK dynamics identify sources of MAPK signaling robustness.

Combining single-cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single-cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK-RAF and the ERK-RSK2-SOS negative feedbacks operate simultaneously to regulate ERK dynamics. Bypassing the RSK2-mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2-dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2-mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs and its perturbation can enhance the efficiency of MAPK inhibitors.

systems biology↗

bioRxiv2020

Spatio-temporal Control of ERK Pulse Frequency Coordinates Fate Decisions during Mammary Acinar Morphogenesis

The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the average frequency of non-periodic ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated ERK waves emerge across multiple cells of an acinus, correlating with high and low ERK pulse frequency in outer surviving and inner dying cells respectively. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Acini harboring the PIK3CA H1047R mutation, commonly observed in breast cancer, display increased ERK pulse frequency, inner cell survival and loss of lumen formation. Thus, fate decisions during acinar morphogenesis are fine-tuned by different spatio-temporal coordination modalities of ERK pulse frequency.

cell biology↗

bioRxiv2020

CODEX, a neural network approach to explore signaling dynamics landscapes

Fluorescent biosensors routinely yield thousands of single-cell, heterogeneous, multi-dimensional signaling trajectories that are difficult to mine for relevant information. We present CODEX, an approach based on artificial neural networks to guide exploration of time-series datasets and to identify motifs in dynamic signaling states.

cell biology↗

bioRxiv2020

Collective ERK/Akt activity waves orchestrate epithelial homeostasis by driving apoptosis-induced survival

Cell death events continuously challenge epithelial barrier function, yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of Extracellular Signal-Regulated Kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. This requires Epidermal Growth Factor Receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4h. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense environmental insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to environmental insults.

cell biology↗

bioRxiv2019

Temporal perturbation of Erk dynamics reveals network architecture of FGF2-MAPK signaling

Stimulation of PC-12 cells with epidermal (EGF) versus nerve (NGF) growth factors (GFs) biases the distribution between transient and sustained single-cell ERK activity states, and between proliferation and differentiation fates within a cell population. We report that fibroblast GF (FGF2) evokes a distinct behavior that consists of a gradually changing population distribution of transient/sustained ERK signaling states in response to increasing inputs in a dose response. Temporally-controlled GF perturbations of MAPK signaling dynamics applied using microfluidics reveals that this wider mix of ERK states emerges through the combination of an intracellular feedback, and competition of FGF2 binding to FGF receptors (FGFR) and heparan-sulfate proteoglycans (HSPGs) co-receptors. We show that the latter experimental modality is instructive for model selection using a Bayesian parameter inference. Our results provide novel insights into how different receptor tyrosine kinase (RTK) systems differentially wire the MAPK network to fine tune fate decisions at the cell population-level.\n\nMicrofluidics, Erk Signaling Dynamics, Mechanistic Modelling, Parameter Estimation, Cell Fate Determination

systems biology↗