Biology subjects

Zanacchi, F. C.

Publications and source records attributed to Zanacchi, F. C..

3 recordsLinked to original sources

bioRxiv2022

Correlation Single Molecule Localization Microscopy (corrSMLM) Detects Fortunate Molecules for Better Localization Precision and High Signal-to-Noise Ratio

Single-molecule localization microscopy can decipher fine details that are otherwise not possible using diffraction-limited microscopy. Often the reconstructed super-resolved image contains unwanted noise, random background and is prone to false detections. This cause spurious data that necessitates several trials, multiple experimentations, and repeated preparation of specimens. Moreover, this is not suitable for experiments that require time-lapse imaging and real-time microscopy. To overcome these limitations, we propose a technique (corrSMLM) that can recognize and detect fortunate molecules (molecules with long fluorescence cycles) from the recorded data. The technique uses correlation between two or more consecutive frames to extract fortunate molecules that blink for longer than the standard blinking time. Accordingly, strongly-correlated spots (single molecule signatures) are compared in consecutive frames, followed by data integration (mean centroid position and the total number of photons) and estimation of critical parameters (position and localization precision). The technique addresses two major problems that plague SMLM : (1) random noise due to false detection that contributes to strong background, and (2) poor localization precision offered by standard SMLM techniques. On the brighter side, corrSMLM allows only fortunate molecules contribute to the super-resolved image, thereby suppressing the background and improving localization precision by a factor of 2-4 times as compared to standard SMLM. To substantiate, corrSMLM is used for imaging fixed cell samples (Dendra2-Actin and Dendra2-Tubulin transfected NIH3T3 cells). Results show multi-fold reduction in noise and localization precision with a marked improvement in overall resolution and SBR. We anticipate corrSMLM to improve overall image quality and offer a better understanding of single molecule dynamics in cell biology.

biophysics↗

bioRxiv2022

Strategies and experimental tips for optimized quantitative single-molecule studies of membrane and membrane-associated proteins

The study of stoichiometry and supra-molecular organization of membrane (and membrane-associated) proteins plays a key role in understanding membrane structure and function. Single-molecule localization techniques (SML), besides providing imaging at unprecedented resolution, also offer quantitative tools such as stepwise photobleaching (SP) experiments and quantitative single-molecule localization (qSMLM). SML is becoming widely present in imaging core facilities but addressing biological problems by molecular counting experiments still remains not straightforward since experimental approaches for sample preparation require particular attention. We will focus on the experimental aspects that may prevent successful quantitative SML experiments of membrane-associated proteins. Depending on the specific experiment, to avoid artifacts and to miscount, fine-tuning of the expression levels and proper staining procedures are required, as well as optimized protocols and controls for counting. The work aims to highlight the crucial aspects that must be faced when quantitative single-molecule experiments are performed, helping to match the gap between sample preparation and the application of quantitative fluorescence microscopy techniques.

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↗