scholarly journals Divergent modes for cargo-mediated control of clathrin-coated pit dynamics

2013 ◽  
Vol 24 (11) ◽  
pp. 1725-1734 ◽  
Author(s):  
Amanda L. Soohoo ◽  
Manojkumar A. Puthenveedu
Keyword(s):  
Quantitative Analysis ◽  
Fluorescence Microscopy ◽  
Total Internal Reflection ◽  
Live Cells ◽  
Coated Pits ◽  
Microscopy Imaging ◽  
Cargo Proteins ◽  
Μ Opioid Receptor ◽  
Signaling Receptors ◽  
G Protein Coupled

Clathrin-mediated endocytosis has long been viewed as a process driven by core endocytic proteins, with internalized cargo proteins being passive. In contrast, an emerging view suggests that signaling receptor cargo may actively control its fate by regulating the dynamics of clathrin-coated pits (CCPs) that mediate their internalization. Despite its physiological implications, very little is known about such “cargo-mediated regulation” of CCPs by signaling receptors. Here, using multicolor total internal reflection fluorescence microscopy imaging and quantitative analysis in live cells, we show that the μ-opioid receptor, a physiologically relevant G protein–coupled signaling receptor, delays the dynamics of CCPs in which it is localized. This delay is mediated by the interactions of two critical leucines on the receptor cytoplasmic tail. Unlike the previously known mechanism of cargo-mediated regulation, these residues regulate the lifetimes of dynamin, a key component of CCP scission. These results identify a novel means for selectively controlling the endocytosis of distinct cargo that share common trafficking components and indicate that CCP regulation by signaling receptors can operate via divergent modes.

Visualizing membrane trafficking using total internal reflection fluorescence microscopy

2003 ◽  
Vol 31 (4) ◽  
pp. 819-823 ◽  
Author(s):  
V. Beaumont
Keyword(s):  
Fluorescence Microscopy ◽  
Membrane Trafficking ◽  
Cell Biology ◽  
Total Internal Reflection ◽  
Optical Resolution ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Live Cells ◽  
Excitable Cells ◽  
Cellular Processes

There is a dizzying array of fluorescent probes now commercially available to monitor cellular processes, and advances in molecular biology have highlighted the ease with which proteins can now be labelled with fluorophores without loss of functionality. This has led to an explosion in the popularity of fluorescence microscopy techniques. One such specialized technique, total internal reflection fluorescence microscopy (TIR-FM), is ideally suited to gaining insight into events occurring at, or close to, the plasma membrane of live cells with excellent optical resolution. In the last few years, the application of TIR-FM to membrane trafficking events in both non-excitable and excitable cells has been an area of notable expansion and fruition. This review gives a brief overview of that literature, with emphasis on the study of the regulation of exocytosis and endocytosis in excitable cells using TIR-FM. Finally, recent applications of TIR-FM to the study of cellular processes at the molecular level are discussed briefly, providing promise that the future of TIR-FM in cell biology will only get brighter.

Quantitative Analysis of Biofilm Formed on Vascular Prostheses by Staphylococcus Epidermidis with Different ica and aap Genetic Status

2013 ◽  
Vol 36 (2) ◽  
pp. 105-112 ◽  
Author(s):  
Joanna Golus ◽  
Magdalena Stankevic ◽  
Rafal Sawicki ◽  
Renata Los ◽  
Anna Malm ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Fluorescence Microscopy ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Bacterial Colonization ◽  
Formation Mechanisms ◽  
Microscopy Imaging ◽  
Vascular Prostheses ◽  
Coverage Ratio ◽  
Genetic Status

Objectives This study aims to examine biofilm formed on vascular prostheses by Staphylococcus epidermidis with different ica and aap genetic status, and to evaluate the effect of antibiotic-modified prostheses on bacterial colonization. Methods Biofilm formation was determined using fluorescence microscopy imaging. Quantitative analysis was conducted using the biofilm coverage ratio (BCR) calculations. Results Our investigations prove that the BCR method with fluorescent dye enabled an accurate assessment of biofilm coverage and comparison of the obtained results. The ica+ aap+ strains formed a biofilm on all of the examined vascular prostheses. Uni-Graft® modified with covalently immobilized amikacin was effective in preventing bacterial adherence. Conclusions Molecular biology techniques combined with phenotype studies give a broad insight into biofilm formation mechanisms. On the other hand, fluorescence microscopy imaging along with BCR calculations are reliable and simple tools to quantitatively estimate biofilm formation, as well as the effectiveness of antimicrobial prosthesis modification.

scholarly journals Loss of PTEN promotes formation of signaling-capable clathrin-coated pits

2017 ◽  
Author(s):  
Luciana K Rosselli-Murai ◽  
Joel A Yates ◽  
Sei Yoshida ◽  
Julia Bourg ◽  
Kenneth K.Y. Ho ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Growth Factor Receptor ◽  
Vesicular Trafficking ◽  
Coated Pits ◽  
Automated Tracking ◽  
Tensin Homolog ◽  
Specific Subset ◽  
Pten Deletion ◽  
Epidermal Growth ◽  
Signaling Receptors

AbstractDefective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs), the fundamental unit of clathrin-mediated endocytosis, display highly heterogeneous dynamics on the plasma membrane where they can take from 20 seconds to over a minute to form cytosolic coated-vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3), which is dephosphorylated by phosphatase tensin homolog (PTEN), is a potent tumorigenic signaling lipid that is present in excess in many types of cancers. Using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we find PTEN and EGF bound EGFR are enriched in a distinct subset of short-lived CCPs that corresponded with clathrin-dependent EGF-induced signaling. By deleting PTEN using CRISPR-Cas9 and reconstituting PTEN, we demonstrate that PTEN plays a role in the regulation of CCP dynamics; this appears to recapitulate CCP dynamics in highly metastatic PTEN-deleted cancer cells where we find a larger proportion of short-lived CCPs and higher initiation density compared to the normal cells. Furthermore, increased PI(3,4,5)P3 results in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Our findings provide strong evidence for the existence of short-lived ‘signaling-capable’ CCPs. Altogether, these findings demonstrate the importance of PTEN and PI(3,4,5)P3 in regulating CCP dynamics and assign a new function to PTEN as a modulator of signaling-capable CCPs.

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