Quantitative image analysis tool to study the plasma membrane localization of proteins and cortical actin in neuroendocrine cells

Kurps, Julia; Broeke, Jurjen H.; Cijsouw, Tony; Kompatscher, Andreas; Weering, Jan R.T. van; Wit, Heidi de

doi:10.1016/j.jneumeth.2014.07.022

Cited by 10 publications

(10 citation statements)

References 27 publications

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“…To test the role of the cortical F-actin network in docking and secretion in Munc18-1 wild-type (WT) and KO MCCs, we first assessed the F-actin network using Rhodamine-phalloidin labeling. Labeling was quantified using the automated image analysis tool PlasMACC (Kurps et al, 2014) in WT and KO cells, and KO cells overexpressing WT MUNC18-1. Fig.…”

Section: F-actin and Stx1 Phenotypes Are Munc18-1 Specific And Not Exmentioning

confidence: 99%

“…An additional zoom factor of 5 was applied and the images were acquired with a frame size of 1024×1024 pixels. The analysis of the images was primarily executed with PlasMACC, which is implemented as a plugin in the image analysis software Fiji (Kurps et al, 2014). The intensity of fluorescent signals was determined at the PM.…”

Section: Immunocytochemistry and Confocal Imagingmentioning

confidence: 99%

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MUNC18-1 regulates the submembrane F-actin network, independently of syntaxin1 targeting, via hydrophobicity in β-sheet 10

Pons-Vizcarra

Kurps

Tawfik

et al. 2019

Journal of Cell Science

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MUNC18-1 (also known as STXBP1) is an essential protein for docking and fusion of secretory vesicles. Mouse chromaffin cells (MCCs) lacking MUNC18-1 show impaired secretory vesicle docking, but also mistargeting of SNARE protein syntaxin1 and an abnormally dense submembrane F-actin network. Here, we tested the contribution of both these phenomena to docking and secretion defects in MUNC18-1-deficient MCCs. We show that an abnormal F-actin network and syntaxin1 targeting defects are not observed in Snap25-or Syt1-knockout (KO) MCCs, which are also secretion deficient. We identified a MUNC18-1 mutant (V263T in β-sheet 10) that fully restores syntaxin1 targeting but not F-actin abnormalities in Munc18-1-KO cells. MUNC18-2 and-3 (also known as STXBP2 and STXBP3, respectively), which lack the hydrophobic residue at position 263, also did not restore a normal F-actin network in Munc18-1-KO cells. However, these proteins did restore the normal F-actin network when a hydrophobic residue was introduced at the corresponding position. Munc18-1-KO MCCs expressing MUNC18-1(V263T) showed normal vesicle docking and exocytosis. These results demonstrate that MUNC18-1 regulates the F-actin network independently of syntaxin1 targeting via hydrophobicity in β-sheet 10. The abnormally dense F-actin network in Munc18-1-deficient cells is not a rate-limiting barrier in secretory vesicle docking or fusion. This article has an associated First Person interview with the first author of the paper.

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Section: F-actin and Stx1 Phenotypes Are Munc18-1 Specific And Not Exmentioning

confidence: 99%

Section: Immunocytochemistry and Confocal Imagingmentioning

confidence: 99%

MUNC18-1 regulates the submembrane F-actin network, independently of syntaxin1 targeting, via hydrophobicity in β-sheet 10

Pons-Vizcarra

Kurps

Tawfik

et al. 2019

Journal of Cell Science

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“…Imaging of immune stained cells is one of the principal techniques used to quantify membrane expression patterns [26][27][28] . In comparison to biochemical techniques, IF is appealing because the proteins can be detected directly, in a non-destructive fashion.…”

Section: Discussionmentioning

confidence: 99%

Imaging analysis to quantitate the Interplay of membrane and cytoplasm protein dynamics

Kislev

Egozi

Benayahu

2021

Preprint

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Plasma membrane proteins are extremely important in cell signaling and cellular functions. Protein expression and localization alter in response to various signals in a way that is dependent on cell type and niche. Compartmental quantification of the expression of particular proteins is a very useful means of understanding their role in cellular processes. Immunofluorescence staining is frequently used to investigate the distribution of proteins of interest. Here, we present an imaging method for quantifying the membrane to cytoplasm ratio (MCR) of proteins analyzed at single-cell resolution. This technique provides a robust quantification of membrane proteins and contributes new insights into membrane expression dynamics. We have developed a protocol that uses immunostaining to assess protein expression according to the fluorescent cellular distribution and to compute the MCR. The method was applied to measure the MCR of glucose transporter 4 (GLUT4) in response to insulin in 3T3-L1 cells, an in-vitro model for adipocyte function and adipogenesis. The results revealed informative changes in the subcellular localization of GLUT4 following insulin induction. MCR analysis is a powerful imaging tool that can be generally applied to membrane proteins to provide a rapid and efficient quantitative analysis of protein distribution and sub-cellular processes in cells.

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“…The apoptotic cells appear overt bright spots [ 42 ]. In order to determine the fluorescence intensity in the brain regions, florescence intensities on the staining of the region of interest (ROI) were measured on a total of 30 larvae with software Image J (National Institutes of Health, Bethesda, MD, USA) [ 43 ]. Different brain regions were evaluated on account of surrounding landmarks [ 44 ].…”

Section: Methodsmentioning

confidence: 99%

Developmental Neurotoxicity of Methamidophos in the Embryo-Larval Stages of Zebrafish

Gao

Dong

et al. 2016

IJERPH

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Methamidophos is a representative organophosphate insecticide. The knowledge of its developmental neurotoxicity is limited, especially for zebrafish in the early stages of their life. Four hour post-fertilization (hpf) zebrafish embryos were exposed to several environmentally relevant concentrations of methamidophos (0, 25, and 500 μg/L) for up to 72 hpf. Locomotor behavior was then studied in the zebrafish larvae at this timepoint. Acridine orange (AO) staining was carried out in the zebrafish larvae, and the mRNA levels of genes associated with neural development (mbp and syn2a) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The number of escape responders for mechanical stimulation was significantly decreased in exposed groups. AO staining showed noticeable signs of apoptosis mainly in the brain. In addition, the mRNA levels of mbp and syn2a were both significantly down-regulated in exposed groups. Our study provides the first evidence that methamidophos exposure can cause developmental neurotoxicity in the early stages of zebrafish life, which may be caused by the effect of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain.

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Quantitative image analysis tool to study the plasma membrane localization of proteins and cortical actin in neuroendocrine cells

Cited by 10 publications

References 27 publications

MUNC18-1 regulates the submembrane F-actin network, independently of syntaxin1 targeting, via hydrophobicity in β-sheet 10

MUNC18-1 regulates the submembrane F-actin network, independently of syntaxin1 targeting, via hydrophobicity in β-sheet 10

Imaging analysis to quantitate the Interplay of membrane and cytoplasm protein dynamics

Developmental Neurotoxicity of Methamidophos in the Embryo-Larval Stages of Zebrafish

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