Live‐Cell Assays to Identify Regulators of ER‐to‐Golgi Trafficking
We applied fluorescence microscopy‐based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)‐to‐Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi‐to‐ER relocalization of GalT‐CF...
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| Veröffentlicht in: | Traffic (Copenhagen, Denmark) Denmark), 2012-03, Vol.13 (3), p.416-432 |
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| creator | Lisauskas, Tautvydas Matula, Petr Claas, Christoph Reusing, Susanne Wiemann, Stefan Erfle, Holger Lehmann, Lars Fischer, Peter Eils, Roland Rohr, Karl Storrie, Brian Starkuviene, Vytaute |
| description | We applied fluorescence microscopy‐based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)‐to‐Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi‐to‐ER relocalization of GalT‐CFP (β1,4‐galactosyltransferase I‐cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash‐out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER‐to‐Golgi trafficking. Nine of them interfered with the rate of BFA‐induced redistribution of GalT‐CFP from the Golgi complex to the ER, six of them interfered with GalT‐CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash‐out and six of them were positive effectors in both assays. Notably, our live‐cell approach captures regulator function in ER‐to‐Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens. |
| doi_str_mv | 10.1111/j.1600-0854.2011.01318.x |
| format | Article |
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We first validated an automated procedure to identify factors which influence Golgi‐to‐ER relocalization of GalT‐CFP (β1,4‐galactosyltransferase I‐cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash‐out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER‐to‐Golgi trafficking. Nine of them interfered with the rate of BFA‐induced redistribution of GalT‐CFP from the Golgi complex to the ER, six of them interfered with GalT‐CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash‐out and six of them were positive effectors in both assays. Notably, our live‐cell approach captures regulator function in ER‐to‐Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens.</description><identifier>ISSN: 1398-9219</identifier><identifier>EISSN: 1600-0854</identifier><identifier>DOI: 10.1111/j.1600-0854.2011.01318.x</identifier><identifier>PMID: 22132776</identifier><language>eng</language><publisher>England: Wiley Subscription Services, Inc</publisher><subject>Animals ; BFA ; Biological Assay - methods ; Cells, Cultured ; Endoplasmic Reticulum - metabolism ; ER‐to‐Golgi trafficking ; GalT ; Golgi Apparatus - metabolism ; GOT1B ; Kidney - cytology ; Microscopy, Fluorescence ; Protein Transport ; Rats ; SACM1L ; USE1 ; YIPF</subject><ispartof>Traffic (Copenhagen, Denmark), 2012-03, Vol.13 (3), p.416-432</ispartof><rights>2011 John Wiley & Sons A/S</rights><rights>2011 John Wiley & Sons A/S.</rights><rights>2012 John Wiley & Sons A/S</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Fj.1600-0854.2011.01318.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fj.1600-0854.2011.01318.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>230,314,780,784,885,1417,1433,27924,27925,45574,45575,46409,46833</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22132776$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lisauskas, Tautvydas</creatorcontrib><creatorcontrib>Matula, Petr</creatorcontrib><creatorcontrib>Claas, Christoph</creatorcontrib><creatorcontrib>Reusing, Susanne</creatorcontrib><creatorcontrib>Wiemann, Stefan</creatorcontrib><creatorcontrib>Erfle, Holger</creatorcontrib><creatorcontrib>Lehmann, Lars</creatorcontrib><creatorcontrib>Fischer, Peter</creatorcontrib><creatorcontrib>Eils, Roland</creatorcontrib><creatorcontrib>Rohr, Karl</creatorcontrib><creatorcontrib>Storrie, Brian</creatorcontrib><creatorcontrib>Starkuviene, Vytaute</creatorcontrib><title>Live‐Cell Assays to Identify Regulators of ER‐to‐Golgi Trafficking</title><title>Traffic (Copenhagen, Denmark)</title><addtitle>Traffic</addtitle><description>We applied fluorescence microscopy‐based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)‐to‐Golgi trafficking and/or Golgi complex maintenance. 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Moreover, we show that our assays can be extended to RNAi and chemical screens.</description><subject>Animals</subject><subject>BFA</subject><subject>Biological Assay - methods</subject><subject>Cells, Cultured</subject><subject>Endoplasmic Reticulum - metabolism</subject><subject>ER‐to‐Golgi trafficking</subject><subject>GalT</subject><subject>Golgi Apparatus - metabolism</subject><subject>GOT1B</subject><subject>Kidney - cytology</subject><subject>Microscopy, Fluorescence</subject><subject>Protein Transport</subject><subject>Rats</subject><subject>SACM1L</subject><subject>USE1</subject><subject>YIPF</subject><issn>1398-9219</issn><issn>1600-0854</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkc1O6zAQhS0EAm7hFVAkFqyS67GdOFmAVFX8SZWQqu4t23GKSxqXOAG64xF4Rp4E58KtAC_GI82n0ZlzEIoAJxDe32UCGcYxzlOWEAyQYKCQJy876HA72A09LfK4IFAcoD_eLzHGJGVsHx0QApRwnh2im6l9Mu-vbxNT19HYe7nxUeei29I0na020cws-lp2rvWRq6LLWUA7F8q1qxc2mreyqqx-sM3iCO1Vsvbm-OsfofnV5XxyE0_vrm8n42m8pgXP4zI3BDJGcEk4ZorkFdWMM12BSkupFaR5lUldKNClwiUDrCQutVQqBUo5HaGLz7XrXq1MqYPMVtZi3dqVbDfCSSt-Thp7LxbuSVAejAs2jdDZ14LWPfbGd2JlvQ7Xy8a43otgFzDMizSQp7_IpevbJhwngGdZzhjnLFAn3wVtlfy3OADnn8Czrc1mOwcshijFUgyJiSExMUQp_kUpXsR8Nh46-gHTQ5Sg</recordid><startdate>201203</startdate><enddate>201203</enddate><creator>Lisauskas, Tautvydas</creator><creator>Matula, Petr</creator><creator>Claas, Christoph</creator><creator>Reusing, Susanne</creator><creator>Wiemann, Stefan</creator><creator>Erfle, Holger</creator><creator>Lehmann, Lars</creator><creator>Fischer, Peter</creator><creator>Eils, Roland</creator><creator>Rohr, Karl</creator><creator>Storrie, Brian</creator><creator>Starkuviene, Vytaute</creator><general>Wiley Subscription Services, Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7QP</scope><scope>7TK</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>201203</creationdate><title>Live‐Cell Assays to Identify Regulators of ER‐to‐Golgi Trafficking</title><author>Lisauskas, Tautvydas ; Matula, Petr ; Claas, Christoph ; Reusing, Susanne ; Wiemann, Stefan ; Erfle, Holger ; Lehmann, Lars ; Fischer, Peter ; Eils, Roland ; Rohr, Karl ; Storrie, Brian ; Starkuviene, Vytaute</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p3978-d8e216420d2704b28f3c474cf1b5dacb158f6ac9b1cdb0d410ba0dcabb513373</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Animals</topic><topic>BFA</topic><topic>Biological Assay - methods</topic><topic>Cells, Cultured</topic><topic>Endoplasmic Reticulum - metabolism</topic><topic>ER‐to‐Golgi trafficking</topic><topic>GalT</topic><topic>Golgi Apparatus - metabolism</topic><topic>GOT1B</topic><topic>Kidney - cytology</topic><topic>Microscopy, Fluorescence</topic><topic>Protein Transport</topic><topic>Rats</topic><topic>SACM1L</topic><topic>USE1</topic><topic>YIPF</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lisauskas, Tautvydas</creatorcontrib><creatorcontrib>Matula, Petr</creatorcontrib><creatorcontrib>Claas, Christoph</creatorcontrib><creatorcontrib>Reusing, Susanne</creatorcontrib><creatorcontrib>Wiemann, Stefan</creatorcontrib><creatorcontrib>Erfle, Holger</creatorcontrib><creatorcontrib>Lehmann, Lars</creatorcontrib><creatorcontrib>Fischer, Peter</creatorcontrib><creatorcontrib>Eils, Roland</creatorcontrib><creatorcontrib>Rohr, Karl</creatorcontrib><creatorcontrib>Storrie, Brian</creatorcontrib><creatorcontrib>Starkuviene, Vytaute</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Traffic (Copenhagen, Denmark)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lisauskas, Tautvydas</au><au>Matula, Petr</au><au>Claas, Christoph</au><au>Reusing, Susanne</au><au>Wiemann, Stefan</au><au>Erfle, Holger</au><au>Lehmann, Lars</au><au>Fischer, Peter</au><au>Eils, Roland</au><au>Rohr, Karl</au><au>Storrie, Brian</au><au>Starkuviene, Vytaute</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Live‐Cell Assays to Identify Regulators of ER‐to‐Golgi Trafficking</atitle><jtitle>Traffic (Copenhagen, Denmark)</jtitle><addtitle>Traffic</addtitle><date>2012-03</date><risdate>2012</risdate><volume>13</volume><issue>3</issue><spage>416</spage><epage>432</epage><pages>416-432</pages><issn>1398-9219</issn><eissn>1600-0854</eissn><abstract>We applied fluorescence microscopy‐based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)‐to‐Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi‐to‐ER relocalization of GalT‐CFP (β1,4‐galactosyltransferase I‐cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash‐out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER‐to‐Golgi trafficking. Nine of them interfered with the rate of BFA‐induced redistribution of GalT‐CFP from the Golgi complex to the ER, six of them interfered with GalT‐CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash‐out and six of them were positive effectors in both assays. Notably, our live‐cell approach captures regulator function in ER‐to‐Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. 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| subjects | Animals BFA Biological Assay - methods Cells, Cultured Endoplasmic Reticulum - metabolism ER‐to‐Golgi trafficking GalT Golgi Apparatus - metabolism GOT1B Kidney - cytology Microscopy, Fluorescence Protein Transport Rats SACM1L USE1 YIPF |
| title | Live‐Cell Assays to Identify Regulators of ER‐to‐Golgi Trafficking |
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