DrSTAR: Tracking real-time nanometer axial changes

Nawara, Tomasz J.; Dean, William F.; Mattheyses, Alexa L.

doi:10.1016/j.bpj.2023.01.019

Cited by 2 publications

(5 citation statements)

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“…To overcome this limitation we used STAR microscopy to parse out whether the observed increase in clathrin dynamics was caused by the surge in formation of productive clathrin-coated vesicles or nonproductive flat clathrin lattices ( Fig. 2A ) (Nawara et al, 2023; Nawara and Mattheyses, 2023; Nawara et al, 2022). STAR uses a ratiometric approach to recover axial Δz information, which requires the dual-tag (CLCa-iRFP713-EGFP).…”

Section: Resultsmentioning

confidence: 99%

“…STAR uses a ratiometric approach to recover axial Δz information, which requires the dual-tag (CLCa-iRFP713-EGFP). The bespoke MATLAB-based data processing pipeline DrSTAR was employed to process multiple cells and experimental replicates in a robust and high throughout manner (Nawara et al, 2023). Next CMEanalysis tracked clathrin events automatically based on their florescence signal allowing quantification of hundreds of clathrin events (Aguet et al, 2013).…”

Section: Resultsmentioning

confidence: 99%

“…We next asked whether the mechanism of CCV formation is altered in response to FSS. To do so, we quantified the initiation of CCV formation relative to clathrin arrival, reported as the time difference in when Δz and CLCa are detected (Nawara et al, 2023; Nawara et al, 2022). Multiple productive routes of clathrin-coated vesicle formation have been observed in living cells, which have been classified into three main models (Chen and Schmid, 2020; Nawara et al, 2022; Scott et al, 2018).…”

Section: Resultsmentioning

confidence: 99%

“…Raw STAR microscopy data was processed and Δz was calculated using a high-throughput custom-written MATLAB software – DrSTAR (Nawara et al, 2023). Detection, tracking and classification of endocytic events was achieved using CMEanalysis (Aguet et al, 2013), as previously described (Nawara et al, 2022).…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we used a total internal reflection fluorescence (TIRF)-based technique called simultaneous two-wavelength axial ratiometry (STAR) microscopy to define how clathrin-coated vesicles form in human umbilical vein endothelial cells (HUVECs) grown under static conditions (Nawara et al, 2022). STAR has the power to capture the dynamics and axial ( z ) distribution of clathrin in living cells with a nanoscale axial resolution (Nawara et al, 2023; Nawara and Mattheyses, 2023). Our previous research showed that clathrin dynamics are similar between HUVECs cultured under static conditions and non-endothelial green monkey kidney fibroblast-like (Cos-7) cells.…”

Section: Introductionmentioning

confidence: 99%

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

Nawara,

Sztul,

Mattheyses

2024

Preprint

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%