Biology subjects

Pierzynska-Mach, A.

Publications and source records attributed to Pierzynska-Mach, A..

5 recordsLinked to original sources

bioRxiv2023

A Compact and Effective Photon-Resolved Image Scanning Microscope

Fluorescence confocal laser-scanning microscopy (LSM) is one of the most popular tools for life science research. This popularity is expected to grow thanks to single-photon array detectors tailored for LSM. These detectors offer unique single-photon spatiotemporal information, opening new perspectives for gentle and quantitative super-resolution imaging. However, a flawless recording of this information poses significant challenges for the microscope data-acquisition system. Here, we present a data-acquisition module based on the digital frequency domain principle, able to record photons essential spatial and temporal features. We use this module to extend the capabilities of established imaging techniques based on single-photon avalanche diode (SPAD) array detectors, such as fluorescence lifetime image scanning microscopy. Furthermore, we use the module to introduce a robust multi-species approach encoding the fluorophores excitation spectra in the time domain. Lastly, we combine time-resolved stimulated emission depletion microscopy with image scanning microscopy, boosting spatial resolution. Our results demonstrate how a conventional fluorescence laser scanning microscope can transform into a simple, information-rich, super-resolved imaging system with the simple addition of a SPAD array detector with a tailored data acquisition system. We expected a blooming of advanced single-photon imaging techniques which effectively harness all the sample information encoded in each photon.

biophysics↗

bioRxiv2023

DEK oncoprotein participates in heterochromatin replication via SUMO-dependent nuclear bodies

The correct inheritance of chromatin structure is key for maintaining genome function and preventing cellular transformation. DEK, a conserved chromatin protein, has recognized tumor-promoting properties, its overexpression being associated with poor prognosis in various cancer types. At the cellular level, DEK displays pleiotropic functions, influencing differentiation, apoptosis, and stemness, but a characteristic oncogenic mechanism remains elusive. Here, we report the identification of DEK bodies, focal assemblies of DEK occurring at specific, yet unidentified sites of heterochromatin replication. In these bodies, DEK localizes in direct proximity to active replisomes suggesting a function in the early maturation of heterochromatin. A high-throughput siRNA screen identifies SUMO as a major regulator of DEK body formation, linking DEK to the SUMO network that controls chromatin states and cell fate. This work combines and refines our previous data on DEK as a factor essential for heterochromatin integrity and facilitating replication under stress and delineates an avenue of further study for unraveling DEKs contribution to cancer development.

cell biology↗

bioRxiv2023

Super-resolution microscopy reveals the nanoscale organization of the DEK protein cancer biomarker

The chromatin architectural factor DEK is involved in regulating the chromatin organization and is highly overexpressed in numerous forms of cancer. DEK is traditionally described as uniformly distributed within the nucleus by diffraction-limited microscopy studies, but super-resolution advent highlighted the formation of cluster-like DEK structures. Still, a characterization of the DEK protein cellular distribution and its role in cancer and cell proliferation is missing. In this work, we used single-molecule localization microscopy (SMLM) to investigate the nanoscale characteristics of DEK organization in normal-like and aggressive types of breast cancer cell lines, characterizing the number of localizations per cluster, as well as areas and density of clusters. We show how the cluster features correlate with the breast cell type and how the chromatin decompaction influences the DEK clusters in both cell lines. Our results suggest that the DEK density and nano-organization are preserved and are not influenced by protein overexpression itself or by chromatin compaction changes.

biophysics↗

bioRxiv2021

Cooled SPAD array detector for low light-dose fluorescence laser scanning microscopy

The single-photon timing and sensitivity performance and the imaging ability of asynchronous-readout single-photon avalanche diode (SPAD) array detectors have opened up enormous perspectives in fluorescence (lifetime) laser scanning microscopy (FLSM), such as super-resolution image scanning microscopy and high-information content fluorescence fluctuation spectroscopy (FFS). However, the strengths of these FLSM techniques depend on the many different characteristics of the detector, such as dark-noise, photon-detection efficiency, after-pulsing probability, and optical-cross talk, whose overall optimization is typically a trade-off between these characteristics. To mitigate this trade-off, we present a novel SPAD array detector with an active cooling system, which substantially reduces the dark-noise without significantly deteriorating any other detector characteristics. In particular, we show that lowering the temperature of the sensor to -15{degrees}C significantly improves the signal-to-noise ratio due to a 10-fold decrease in the dark-count rate compared to room temperature. As a result, for imaging, the laser power can be decreased by more than a factor of three, which is particularly beneficial for live-cell super-resolution imaging, as demonstrated in fixed and living cells expressing GFP-tagged proteins. For FFS, together with the benefit of the reduced laser power, we show that cooling the detector is necessary to remove artifacts in the correlation function, such as spurious negative correlations observed in the hot elements of the detector, i.e., elements whose dark-noise is substantially higher than the median value. Overall, this detector represents a further step towards the integration of SPAD array detectors in any FLSM system. SIGNIFICANCESingle-photon avalanche diode (SPAD) array detectors are revolutionizing fluorescence laser-scanning microscopy (FLSM). Thanks to their single-photon timing and sensitivity ability and their imaging faculty, a SPAD array detector transforms any FLSM into a super-resolution microscope, and opens a whole range of possibilities for the study of sample dynamics by means of fluorescence fluctuation spectroscopy (FFS). However, dark-noise can be a severe problem for both imaging and FFS. For imaging, the signal overcomes noise only for a relatively high illumination intensity, which can be detrimental for live-cell experiments. For FFS, the noise leads to artifacts in the correlation curves, potentially leading to wrong conclusions about the sample. We show that lowering the temperature of the detector to -15{degrees}C solves both problems

biophysics↗

bioRxiv2019

The oncoprotein DEK affects the outcome of PARP1/2 inhibition during replication stress

DNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

cancer biology↗