Biology subjects

Reglinski, K.

Publications and source records attributed to Reglinski, K..

4 recordsLinked to original sources

bioRxiv2023

Neural Network Informed Photon Filtering Reduces Artifacts in Fluorescence Correlation Spectroscopy Data

Fluorescence Correlation Spectroscopy (FCS) techniques are well-established tools to investigate molecular dynamics in confocal and super-resolution microscopy. In practice, users often need to handle a variety of sample or hardware-related artifacts, an example being peak artifacts created by bright, slow-moving clusters. Approaches to address peak artifacts exist, but measurements suffering from severe artifacts are typically non-analyzable. Here, we trained a 1-dimensional U-Net to automatically identify peak artifacts in fluorescence time-series and then analyzed the purified, non-artifactual fluctuations by time-series editing. We show that in samples with peak artifacts, the transit time and particle number distributions can be restored in simulations and validated the approach in two independent biological experiments. We propose that it is adaptable for other FCS artifacts, such as detector dropout, membrane movement, or photobleaching. In conclusion, this simulation-based, automated, open-source pipeline makes measurements analyzable which previously had to be discarded and extends every FCS users experimental toolbox.

biophysics↗

bioRxiv2023

A novel PEX14/PEX5 interface links peroxisomal protein import and receptor recycling

Newly synthesized peroxisomal proteins are recognized in the cytosol by the cycling receptor PEX5 and directed to a docking complex comprising PEX14 and PEX13 at the peroxisomal membrane. After cargo translocation, the unloaded PEX5 is recycled in an ATP-dependent manner. Receptor docking involves the WxxxF-motifs in the N-terminal domain (NTD) of PEX5 that are recognized by the N-terminal domain of PEX14. Here, we combine biochemical methods and NMR spectroscopy to identify a novel binding interface between human PEX5 and PEX14. The interaction involves the PEX5 C-terminal cargo-binding TPR domain and a conserved IPSWQI peptide motif in the C-terminal intrinsically disordered region of PEX14. The three-dimensional structure of the PEX14 IPSWQI peptide bound the PEX5 TPR domain, shows the PEX14 interaction is non-overlapping with PTS1 binding to the TPR domain. Notably, PEX14 IPSWQI motif binding to a hinge region in the TPR domain shows a more open supercoil of the TPR fold that resembles the apo conformation in the absence of PTS1 peptide. Mutation of binding site residues in PEX5 or PEX14 leads to a partial protein import defect and decrease of the steady-state-concentration of PEX5. This resembles the mutant phenotype of cells affected in receptor recycling, suggesting a role in this process.

biochemistry↗

bioRxiv2021

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics. SummaryThe peroxisomal import receptor PEX5 transports newly synthesized proteins from the cytosol to the peroxisomal matrix. Here the cytosolic diffusion and interaction dynamics of PEX5 are characterized by advanced microscopic spectroscopy methods, revealing a so far unknown interaction partner.

biophysics↗

bioRxiv2019

Object Detection Networks and Augmented Reality for Cellular Detection in Fluorescence Microscopy Acquisition and Analysis.

In this paper we demonstrate the application of object detection networks for the classification and localization of cells in fluorescence microscopy. We benchmark two leading object detection algorithms across multiple challenging 2-D microscopy datasets as well as develop and demonstrate an algorithm which can localize and image cells in 3-D, in real-time. Furthermore, we exploit the fast processing of these algorithms and develop a simple and effective Augmented Reality (AR) system for fluorescence microscopy systems. Object detection networks are well-known high performance networks famously applied to the task of identifying and localizing objects in photography images. Here we show their application and efficiency for localizing cells in fluorescence microscopy images. Object detection algorithms are typically trained on many thousands of images, which can be prohibitive within the biological sciences due to the cost of imaging and annotating large amounts of data. Through taking different cell types and assays as an example, we show that with some careful considerations it is possible to achieve very high performance with datasets with as few as 26 images present. Using our approach, it is possible for relatively non-skilled users to automate detection of cell classes with a variety of appearances and enable new avenues for automation of conventionally manual fluorescence microscopy acquisition pipelines.

bioinformatics↗