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Article ; Online: Quantifying Golgi Apparatus Fragmentation Using Imaging Flow Cytometry.

Wortzel, Inbal / Porat, Ziv

Methods in molecular biology (Clifton, N.J.)

2023 Volume 2635, Page(s) 173–184

Abstract: Unlike the common conception of the Golgi apparatus as a static organelle, it is, in fact, a dynamic structure, as well as a sensitive sensor for the cellular status. In response to various stimuli, the intact Golgi structure undergoes fragmentation. ... ...

Abstract	Unlike the common conception of the Golgi apparatus as a static organelle, it is, in fact, a dynamic structure, as well as a sensitive sensor for the cellular status. In response to various stimuli, the intact Golgi structure undergoes fragmentation. This fragmentation can yield either partial fragmentation, resulting in several separated chunks, or complete vesiculation of the organelle. These distinct morphologies form the basis of several methods for the quantification of the Golgi status. In this chapter, we describe our imaging flow cytometry-based method for quantifying changes in the Golgi architecture. This method has all the benefits of imaging flow cytometry-namely, it is rapid, high-throughput, and robust-while affording easy implementation and analysis capabilities.
MeSH term(s)	Humans ; Flow Cytometry ; Golgi Apparatus ; Organelles ; HeLa Cells
Language	English
Publishing date	2023-04-19
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-3020-4_10
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Applying imaging flow cytometry and immunofluorescence in studying the dynamic Golgi structure in cultured cells.

Wortzel, Inbal / Porat, Ziv / Seger, Rony / Maik-Rachline, Galia

STAR protocols

2022 Volume 3, Issue 2, Page(s) 101278

Abstract: ... execution of this protocol, please refer to Wortzel et al. (2021). ...

Abstract	The Golgi apparatus is subjected to fragmentation under several cellular processes such as mitosis. Here we describe two complementary approaches to analyze different Golgi morphological changes during its mitotic fragmentation, using classical immunofluorescence and imaging flow cytometry. Although fluorescent microscopy provides information on the exact Golgi architecture in distinct cells, the imaging flow cytometry combines the morphological data with the high-throughput quantification of flow cytometry. Taken together, both approaches provide robust and significant unbiased data analysis. For complete details on the use and execution of this protocol, please refer to Wortzel et al. (2021).
MeSH term(s)	Cells, Cultured ; Flow Cytometry/methods ; Fluorescent Antibody Technique ; Golgi Apparatus ; Microscopy, Fluorescence
Language	English
Publishing date	2022-04-05
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2022.101278
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Article ; Online: Alternative Splicing of MAPKs in the Regulation of Signaling Specificity.

Maik-Rachline, Galia / Wortzel, Inbal / Seger, Rony

Cells

2021 Volume 10, Issue 12

Abstract: The mitogen-activated protein kinase (MAPK) cascades transmit signals from extracellular stimuli to a variety of distinct cellular processes. The MAPKKs in each cascade specifically phosphorylate and activate their cognate MAPKs, indicating that this ... ...

Abstract	The mitogen-activated protein kinase (MAPK) cascades transmit signals from extracellular stimuli to a variety of distinct cellular processes. The MAPKKs in each cascade specifically phosphorylate and activate their cognate MAPKs, indicating that this step funnels various signals into a seemingly linear pathway. Still, the effects of these cascades vary significantly, depending on the identity of the extracellular signals, which gives rise to proper outcomes. Therefore, it is clear that the specificity of the signals transmitted through the cascades is tightly regulated in order to secure the desired cell fate. Indeed, many regulatory components or processes that extend the specificity of the cascades have been identified. Here, we focus on a less discussed mechanism, that is, the role of distinct components in each tier of the cascade in extending the signaling specificity. We cover the role of distinct genes, and the alternatively spliced isoforms of MAPKKs and MAPKs, in the signaling specificity. The alternatively spliced MEK1b and ERK1c, which form an independent signaling route, are used as the main example. Unlike MEK1/2 and ERK1/2, this route's functions are limited, including mainly the regulation of mitotic Golgi fragmentation. The unique roles of the alternatively spliced isoforms indicate that these components play an essential role in determining the proper cell fate in response to distinct stimulations.
MeSH term(s)	Alternative Splicing/genetics ; Golgi Apparatus ; Humans ; MAP Kinase Kinase 1/genetics ; Mitogen-Activated Protein Kinase 3/genetics ; Mitogen-Activated Protein Kinase Kinases/genetics ; Mitogen-Activated Protein Kinases/genetics ; Mitosis/genetics ; Phosphorylation ; Signal Transduction/genetics
Chemical Substances	MAPK3 protein, human (EC 2.7.11.24) ; Mitogen-Activated Protein Kinase 3 (EC 2.7.11.24) ; Mitogen-Activated Protein Kinases (EC 2.7.11.24) ; MAP Kinase Kinase 1 (EC 2.7.12.2) ; MAP2K1 protein, human (EC 2.7.12.2) ; Mitogen-Activated Protein Kinase Kinases (EC 2.7.12.2)
Language	English
Publishing date	2021-12-08
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2661518-6
ISSN	2073-4409 ; 2073-4409
ISSN (online)	2073-4409
ISSN	2073-4409
DOI	10.3390/cells10123466
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Article ; Online: Applying imaging flow cytometry and immunofluorescence in studying the dynamic Golgi structure in cultured cells

Inbal Wortzel / Ziv Porat / Rony Seger / Galia Maik-Rachline

STAR Protocols, Vol 3, Iss 2, Pp 101278- (2022)

2022

Abstract: ... on the use and execution of this protocol, please refer to Wortzel et al. (2021). ...

Abstract	Summary: The Golgi apparatus is subjected to fragmentation under several cellular processes such as mitosis. Here we describe two complementary approaches to analyze different Golgi morphological changes during its mitotic fragmentation, using classical immunofluorescence and imaging flow cytometry. Although fluorescent microscopy provides information on the exact Golgi architecture in distinct cells, the imaging flow cytometry combines the morphological data with the high-throughput quantification of flow cytometry. Taken together, both approaches provide robust and significant unbiased data analysis.For complete details on the use and execution of this protocol, please refer to Wortzel et al. (2021).
Keywords	Cell Biology ; Flow Cytometry/Mass Cytometry ; Microscopy ; Science (General) ; Q1-390
Language	English
Publishing date	2022-06-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Alternative Splicing of MAPKs in the Regulation of Signaling Specificity

Galia Maik-Rachline / Inbal Wortzel / Rony Seger

Cells, Vol 10, Iss 3466, p

2021 Volume 3466

Abstract	The mitogen-activated protein kinase (MAPK) cascades transmit signals from extracellular stimuli to a variety of distinct cellular processes. The MAPKKs in each cascade specifically phosphorylate and activate their cognate MAPKs, indicating that this step funnels various signals into a seemingly linear pathway. Still, the effects of these cascades vary significantly, depending on the identity of the extracellular signals, which gives rise to proper outcomes. Therefore, it is clear that the specificity of the signals transmitted through the cascades is tightly regulated in order to secure the desired cell fate. Indeed, many regulatory components or processes that extend the specificity of the cascades have been identified. Here, we focus on a less discussed mechanism, that is, the role of distinct components in each tier of the cascade in extending the signaling specificity. We cover the role of distinct genes, and the alternatively spliced isoforms of MAPKKs and MAPKs, in the signaling specificity. The alternatively spliced MEK1b and ERK1c, which form an independent signaling route, are used as the main example. Unlike MEK1/2 and ERK1/2, this route’s functions are limited, including mainly the regulation of mitotic Golgi fragmentation. The unique roles of the alternatively spliced isoforms indicate that these components play an essential role in determining the proper cell fate in response to distinct stimulations.
Keywords	MAPK ; alternative splicing ; ERK ; JNK ; p38 ; ERK1c ; Biology (General) ; QH301-705.5
Subject code	570 ; 572
Language	English
Publishing date	2021-12-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Mitotic HOOK3 phosphorylation by ERK1c drives microtubule-dependent Golgi destabilization and fragmentation.

Wortzel, Inbal / Maik-Rachline, Galia / Yadav, Suresh Singh / Hanoch, Tamar / Seger, Rony

iScience

2021 Volume 24, Issue 6, Page(s) 102670

Abstract: ERK1c is an alternatively spliced isoform of ERK1 that specifically regulates mitotic Golgi fragmentation, which allows division of the Golgi during mitosis. We have previously shown that ERK1c translocates to the Golgi during mitosis where it is ... ...

Abstract	ERK1c is an alternatively spliced isoform of ERK1 that specifically regulates mitotic Golgi fragmentation, which allows division of the Golgi during mitosis. We have previously shown that ERK1c translocates to the Golgi during mitosis where it is activated by a resident MEK1b to induce Golgi fragmentation. However, the mechanism of ERK1c functions in the Golgi remained obscure. Here, we searched for ERK1c substrates and identified HOOK3 as a mediator of ERK1c-induced mitotic Golgi fragmentation, which requires a second phosphorylation by AuroraA for its function. In cycling cells, HOOK3 interacts with microtubules (MTs) and links them to the Golgi. Early in mitosis, HOOK3 is phosphorylated by ERK1c and later by AuroraA, resulting in HOOK3 detachment from the MTs, and elevated interaction with GM130. This detachment modulates Golgi stability and allows fragmentation of the Golgi. This study demonstrates a novel mechanism of Golgi apparatus destabilization early in mitosis to allow mitotic progression.
Language	English
Publishing date	2021-05-31
Publishing country	United States
Document type	Journal Article
ISSN	2589-0042
ISSN (online)	2589-0042
DOI	10.1016/j.isci.2021.102670
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Article ; Online: Exosome-Mediated Metastasis: Communication from a Distance.

Wortzel, Inbal / Dror, Shani / Kenific, Candia M / Lyden, David

Developmental cell

2019 Volume 49, Issue 3, Page(s) 347–360

Abstract: Metastasis, a critical phase of tumor progression, remains a primary challenge in treating cancer and a major cause of cancer mortality. Cell-cell communication via extracellular vesicles (exosomes and microvesicles) between primary tumor cells and the ... ...

Abstract	Metastasis, a critical phase of tumor progression, remains a primary challenge in treating cancer and a major cause of cancer mortality. Cell-cell communication via extracellular vesicles (exosomes and microvesicles) between primary tumor cells and the microenvironment of distant organs is crucial for pre-metastatic niche (PMN) formation and metastasis. Here, we review work on the contribution of exosome cargo to cancer progression, the role of exosomes in PMN establishment, and the function of exosomes in organotropic metastasis. We also describe the clinical utility of exosomes.
MeSH term(s)	Animals ; Cell Communication ; Disease Progression ; Exosomes/metabolism ; Exosomes/pathology ; Fibroblasts/metabolism ; Humans ; Neoplasm Invasiveness/pathology ; Neoplasm Metastasis/pathology ; Neoplasms/metabolism ; Neoplasms/pathology ; Neovascularization, Pathologic/pathology ; Tumor Microenvironment
Language	English
Publishing date	2019-05-04
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	2054967-2
ISSN	1878-1551 ; 1534-5807
ISSN (online)	1878-1551
ISSN	1534-5807
DOI	10.1016/j.devcel.2019.04.011
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Article ; Online: Mitotic HOOK3 phosphorylation by ERK1c drives microtubule-dependent Golgi destabilization and fragmentation

Inbal Wortzel / Galia Maik-Rachline / Suresh Singh Yadav / Tamar Hanoch / Rony Seger

iScience, Vol 24, Iss 6, Pp 102670- (2021)

2021

Abstract: Summary: ERK1c is an alternatively spliced isoform of ERK1 that specifically regulates mitotic Golgi fragmentation, which allows division of the Golgi during mitosis. We have previously shown that ERK1c translocates to the Golgi during mitosis where it ... ...

Abstract	Summary: ERK1c is an alternatively spliced isoform of ERK1 that specifically regulates mitotic Golgi fragmentation, which allows division of the Golgi during mitosis. We have previously shown that ERK1c translocates to the Golgi during mitosis where it is activated by a resident MEK1b to induce Golgi fragmentation. However, the mechanism of ERK1c functions in the Golgi remained obscure. Here, we searched for ERK1c substrates and identified HOOK3 as a mediator of ERK1c-induced mitotic Golgi fragmentation, which requires a second phosphorylation by AuroraA for its function. In cycling cells, HOOK3 interacts with microtubules (MTs) and links them to the Golgi. Early in mitosis, HOOK3 is phosphorylated by ERK1c and later by AuroraA, resulting in HOOK3 detachment from the MTs, and elevated interaction with GM130. This detachment modulates Golgi stability and allows fragmentation of the Golgi. This study demonstrates a novel mechanism of Golgi apparatus destabilization early in mitosis to allow mitotic progression.
Keywords	cell biology ; functional aspects of cell biology ; Science ; Q
Subject code	571
Language	English
Publishing date	2021-06-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: High Throughput Analysis of Golgi Structure by Imaging Flow Cytometry.

Wortzel, Inbal / Koifman, Gabriela / Rotter, Varda / Seger, Rony / Porat, Ziv

Scientific reports

2017 Volume 7, Issue 1, Page(s) 788

Abstract: The Golgi apparatus is a dynamic organelle, which regulates the vesicular trafficking. While cellular trafficking requires active changes of the Golgi membranes, these are not accompanied by changes in the general Golgi's structure. However, cellular ... ...

Abstract	The Golgi apparatus is a dynamic organelle, which regulates the vesicular trafficking. While cellular trafficking requires active changes of the Golgi membranes, these are not accompanied by changes in the general Golgi's structure. However, cellular processes such as mitosis, apoptosis and migration require fragmentation of the Golgi complex. Currently, these changes are most commonly studied by basic immunofluorescence and quantified by manual and subjective classification of the Golgi structure in 100-500 stained cells. Several other high-throughput methods exist as well, but those are either complicated or do not provide enough morphological information. Therefore, a simple and informative high content methodology should be beneficial for the study of Golgi architecture. Here we describe the use of high-throughput imaging flow cytometry for quantification of Golgi fragmentation, which provides a simple way to analyze the changes in an automated, quantitative and non-biased manner. Furthermore, it provides a rapid and accurate way to analyze more than 50,000 cells per sample. Our results demonstrate that this method is robust and statistically powerful, thus, providing a much-needed analytical tool for future studies on Golgi dynamics, and can be adapted to other experimental systems.
MeSH term(s)	Animals ; Biomarkers ; COS Cells ; Cercopithecus aethiops ; Flow Cytometry ; Golgi Apparatus/drug effects ; Golgi Apparatus/metabolism ; HeLa Cells ; High-Throughput Screening Assays ; Humans ; Mice ; Mitosis ; Neoplasms/metabolism ; Neoplasms/pathology
Chemical Substances	Biomarkers
Language	English
Publishing date	2017-04-11
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-017-00909-y
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Article ; Online: High Throughput Analysis of Golgi Structure by Imaging Flow Cytometry

Inbal Wortzel / Gabriela Koifman / Varda Rotter / Rony Seger / Ziv Porat

Scientific Reports, Vol 7, Iss 1, Pp 1-

2017 Volume 11

Abstract: Abstract The Golgi apparatus is a dynamic organelle, which regulates the vesicular trafficking. While cellular trafficking requires active changes of the Golgi membranes, these are not accompanied by changes in the general Golgi’s structure. However, ... ...

Abstract	Abstract The Golgi apparatus is a dynamic organelle, which regulates the vesicular trafficking. While cellular trafficking requires active changes of the Golgi membranes, these are not accompanied by changes in the general Golgi’s structure. However, cellular processes such as mitosis, apoptosis and migration require fragmentation of the Golgi complex. Currently, these changes are most commonly studied by basic immunofluorescence and quantified by manual and subjective classification of the Golgi structure in 100–500 stained cells. Several other high-throughput methods exist as well, but those are either complicated or do not provide enough morphological information. Therefore, a simple and informative high content methodology should be beneficial for the study of Golgi architecture. Here we describe the use of high-throughput imaging flow cytometry for quantification of Golgi fragmentation, which provides a simple way to analyze the changes in an automated, quantitative and non-biased manner. Furthermore, it provides a rapid and accurate way to analyze more than 50,000 cells per sample. Our results demonstrate that this method is robust and statistically powerful, thus, providing a much-needed analytical tool for future studies on Golgi dynamics, and can be adapted to other experimental systems.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2017-04-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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