Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Chen, Ji; Lou, Xuelin

doi:10.3791/54466

Cited by 6 publications

(6 citation statements)

References 48 publications

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“…Secretory cells may utilize such local PI(4,5)P 2 -mediated modulation at vesicle docking and release sites to regulate secretion, without significantly changing other PI(4,5)P 2 -mediated cellular functions. Direct imaging studies showed a heterogeneous PI(4,5)P 2 distribution in intact live cells ( Ji and Lou, 2016 ; Ji et al, 2015 ), although its nanometer-scale patterns are diverse in fixed samples ranging from dense nano-domains ( van den Bogaart et al, 2011 ), microdomains ( Ji et al, 2015 ; Fujita et al, 2009 ), or even distribution ( van Rheenen et al, 2005 ). About 20% of dense core vesicles reside in PI(4,5)P 2 -enriched regions on the fixed cell PM ( Aoyagi et al, 2005 ), and this number increases to 50% when probed with PH PLCδ4 -GFP ( Kabachinski et al, 2014 ).…”

Section: Resultsmentioning

confidence: 99%

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Vesicle Docking Is a Key Target of Local PI(4,5)P2 Metabolism in the Secretory Pathway of INS-1 Cells

2017

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SUMMARY Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) signaling is transient and spatially confined in live cells. How this pattern of signaling regulates transmitter release and hormone secretion has not been addressed. We devised an optogenetic approach to control PI(4,5)P2 levels in time and space in insulin-secreting cells. Combining this approach with total internal reflection fluorescence microscopy, we examined individual vesicle-trafficking steps. Unlike long-term PI(4,5)P2 perturbations, rapid and cell-wide PI(4,5)P2 reduction in the plasma membrane (PM) strongly inhibits secretion and intracellular Ca2+ concentration ([Ca2+]i) responses, but not sytaxin1a clustering. Interestingly, local PI(4,5)P2 reduction selectively at vesicle docking sites causes remarkable vesicle undocking from the PM without affecting [Ca2+]i. These results highlight a key role of local PI(4,5)P2 in vesicle tethering and docking, coordinated with its role in priming and fusion. Thus, different spatiotemporal PI(4,5)P2 signaling regulates distinct steps of vesicle trafficking, and vesicle docking may be a key target of local PI(4,5)P2 signaling in vivo.

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Section: Resultsmentioning

confidence: 99%

“…We used a Nikon Ti-E Eclipse inverted-microscope-based imaging system under the control of NIS-Elements AR software as described previously ( Ji et al, 2015 ; Ji and Lou, 2016 ). All experiments were performed at 35°C–37°C; imaging buffers and procedures are described in the Supplemental Information .…”

Section: Methodsmentioning

confidence: 99%

Vesicle Docking Is a Key Target of Local PI(4,5)P2 Metabolism in the Secretory Pathway of INS-1 Cells

2017

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“…Even though PH domain is the best option to visualize PtdIns(4,5)P2, not all of the lipids could be visualized using these methods. For instance, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 might bind with other proteins, thus rendering them incapable of binding with the PH probes or antibodies, which could lead to inaccurate detection (20).…”

Section: Discussionmentioning

confidence: 99%

A Comparative Study to Visualize Ptdins (4,5) P2 and Ptdins (3,4,5) P3 in MDA-MB-231 Breast Cancer Cell Line

et al. 2022

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Background: Phosphatidylinositol 3,4,4,5) P3) and Phosphatidylinositol 4,5trisphosphate (PtdIns(4,5) P2] form an insignificant amount of phospholipids but play important roles in controlling membrane-bound signalling. Little attention has been given to visualize and monitor changes or differences in the local generation of PtdIns(4,5) P2 and PtdIns(3,4,5) P3 in the cell membranes of MDA-MB-231 breast cancer cell lines. Methods: PLCδ1-PH-GFP and Btk-PH-GFP were used as biosensors to detected PtdIns(4,5) P2 and PtdIns(3,4,5)P3 respectively. These biosensors and antibodies were transfected, immuostained and then visualized by confocal microscopy on different cell surfaces. Results: Our results showed that PLCδ1-PH-GFP/mCherry was localized at the cell membrane, while Btk-PH-GFP/mCherry was sometimes localized at the cell membrane but there was also a large amount of fluorescence present in the cytosol and nucleus. Our results also showed that the cells that expressed low levels of Btk-PH-GFP the fluorescence was predominantly localised to the cell membrane. While the cells that expressed high levels of Btk-PH-GFP the fluorescence was localization in the cytosol and cell membrane. Our results demonstrated that both anti-PtdIns(4,5)P2 and anti-PtdIns(3,4,5)P3 antibodies were localized everywhere in cell. Conclusions: Our results suggest that PLCδ1-PH-GFP and Btk-PH-GFP/mCherry have more specificity, reliability, suitability and accuracy than antibodies in binding with and detecting PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and in studying the molecular dynamics of phospholipids in live and fixed cells.

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“…More direct evidence for PI(4)P and PI(4,5)P 2 microdomains is becoming available with development of adequate tools and the use of super resolution microscopy. Such an approach revealed punctate localization of PI(4,5)P 2 at the PM . PIs levels at MCSs are not static and dynamically change during cell stimulation.…”

Section: Introductionmentioning

confidence: 98%

Lipids at membrane contact sites: cell signaling and ion transport

et al. 2017

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Communication between organelles is essential to coordinate cellular functions and the cell's response to physiological and pathological stimuli. Organellar communication occurs at membrane contact sites (MCSs), where the endoplasmic reticulum (ER) membrane is tethered to cellular organelle membranes by specific tether proteins and where lipid transfer proteins and cell signaling proteins are located. MCSs have many cellular functions and are the sites of lipid and ion transfer between organelles and generation of second messengers. This review discusses several aspects of MCSs in the context of lipid transfer, formation of lipid domains, generation of Ca and cAMP second messengers, and regulation of ion transporters by lipids.

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Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Cited by 6 publications

References 48 publications

Vesicle Docking Is a Key Target of Local PI(4,5)P2 Metabolism in the Secretory Pathway of INS-1 Cells

Vesicle Docking Is a Key Target of Local PI(4,5)P2 Metabolism in the Secretory Pathway of INS-1 Cells

A Comparative Study to Visualize Ptdins (4,5) P2 and Ptdins (3,4,5) P3 in MDA-MB-231 Breast Cancer Cell Line

Lipids at membrane contact sites: cell signaling and ion transport

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