Biology subjects

Nawara, T. J.

Publications and source records attributed to Nawara, T. J..

3 recordsLinked to original sources

bioRxiv2025

The role of actin dynamics in vesicle formation during clathrin mediated endocytosis

Clathrin-mediated endocytosis (CME) is an important internalization route for macromolecules, lipids, and membrane receptors in eukaryotic cells. During CME, the plasma membrane invaginates and pinches off forming a clathrin coated vesicle. We previously identified heterogeneity in this process with clathrin coated vesicles forming though multiple routes including simultaneous clathrin accumulation and membrane invagination (constant curvature; CCM) as well as membrane bending after accumulation of flat clathrin (flat to curved; FTC). The architectural dynamics of vesicle formation could be influenced by osmotic or confining pressure, membrane stiffness, fluid force, or cytoskeletal arrangement. Whether these biophysical factors regulate the heterogeneity of vesicle formation dynamics is not well understood. To address this, we investigated the interconnected roles of actin and membrane tension in CME using simultaneous two-wavelength axial ratiometry (STAR) microscopy with nanometer-scale axial resolution. First, we treated Cos-7 cells with latrunculin A (LatA) to inhibit actin polymerization and found the total number of clathrin coated vesicles increased significantly, short-lifetime curved events especially. The proportion of vesicles formed following the FTC model was reduced, the membrane curved sooner after clathrin recruitment, and vesicles were less stable in the x-y plane compared to control. Next, we disrupted actin branching by inhibiting Arp2/3 with CK-869. We found an increased delay between membrane invagination and clathrin recruitment, reduced number of curved events, increased vesicle stability and an increase in the FTC model compared to control. As loss of actin filaments also reduces membrane tension, we treated Cos7 with high osmolality to decrease membrane tension and observed similar result with LatA treated group except vesicle stability stayed unchanged. This suggested the increased curved events in LatA groups may result from reduced membrane tension. We conclude actin polymerization promotes FTC while actin branching promotes vesicle formation though the CCM.

cell biology↗

bioRxiv2024

Fluidic shear stress alters clathrin dynamics and vesicle formation in endothelial cells

Endothelial cells (ECs) experience a variety of highly dynamic mechanical stresses. Among others, cyclic stretch and increased plasma membrane tension inhibit clathrin-mediated endocytosis (CME) in non-ECs cells. How ECs overcome such unfavorable, from biophysical perspective, conditions and maintain CME remains elusive. Previously, we have used simultaneous two-wavelength axial ratiometry (STAR) microscopy to show that endocytic dynamics are similar between statically cultured human umbilical vein endothelial cells (HUVECs) and fibroblast-like Cos-7 cells. Here we asked whether biophysical stresses generated by blood flow could favor one mechanism of clathrin-coated vesicle formation to overcome environment present in vasculature. We used our data processing platform - DrSTAR - to examine if clathrin dynamics are altered in HUVECs grown under fluidic sheer stress (FSS). Surprisingly, we found that FSS led to an increase in clathrin dynamics. In HUVECs grown under FSS we observed a 2.3-fold increase in clathrin-coated vesicle formation and a 1.9-fold increase in non-productive flat clathrin lattices compared to cells grown in static conditions. The curvature-positive events had significantly delayed curvature initiation in flow-stimulated cells, highlighting a shift toward flat-to-curved clathrin transitions in vesicle formation. Overall, our findings indicate that clathrin dynamics and CCV formation can be modulated by the local physiological environment and represents an important regulatory mechanism. SignificanceTargeted nanomedicine holds a great promise of improved drug bioavailability and specificity. While some cargoes must cross the blood-tissue barrier, the understanding of endocytic pathways in the context of vasculature is limited, which is an obstacle to targeted nanomedicine delivery. In this pilot study we show that the physiological local vascular environment must be considered in the context of internalization of growth factors, membrane proteins, therapeutics, or pathogens. Studies in non-ECs or ECs not cultured under fluidic shear stress do not properly recapitulate clathrin dynamics and will lead to incorrect conclusions.

cell biology↗

bioRxiv2023

OOPS: Object-Oriented Polarization Software for analysis of fluorescence polarization microscopy images

Most essential cellular functions are performed by proteins assembled into larger complexes. Fluorescence Polarization Microscopy (FPM) is a powerful technique that goes beyond traditional imaging methods by allowing researchers to measure not only the localization of proteins within cells, but also their orientation or alignment within complexes or cellular structures. FPM can be easily integrated into standard widefield microscopes with the addition of a polarization modulator. However, the extensive image processing and analysis required to interpret the data have limited its widespread adoption. To overcome these challenges and enhance accessibility, we introduce OOPS (Object-Oriented Polarization Software), a MATLAB-based analysis tool tailored for FPM data. This work highlights the distinctive features of our software, which empower researchers to efficiently manage large datasets; detect and analyze individual structures; conduct population assessments based on morphology, intensity, and polarization-specific parameters; and create publication-quality visualizations, all within a user-friendly graphical interface. Importantly, OOPS is adaptable to various sample types, labeling techniques, and imaging setups, facilitating in-depth analysis of diverse polarization-sensitive samples. Here, we demonstrate the power and versatility of our approach by applying OOPS to FPM images of both punctate and filamentous structures. OOPS is freely available under the GNU GPL 3.0 license and can be downloaded at https://github.com/Mattheyses-Lab/OOPS.

cell biology↗