Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates
Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image,...
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| Veröffentlicht in: | Autophagy 2015-10, Vol.11 (10), p.1905-1916 |
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| container_title | Autophagy |
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| creator | Maulucci, Giuseppe Chiarpotto, Michela Papi, Massimiliano Samengo, Daniela Pani, Giovambattista De Spirito, Marco |
| description | Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5-6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates. |
| doi_str_mv | 10.1080/15548627.2015.1084455 |
| format | Article |
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Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5-6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates.</description><identifier>ISSN: 1554-8627</identifier><identifier>EISSN: 1554-8635</identifier><identifier>DOI: 10.1080/15548627.2015.1084455</identifier><identifier>PMID: 26506895</identifier><language>eng</language><publisher>United States: Taylor & Francis</publisher><subject>3-methyladenine ; autophagic flux ; autophagy ; Autophagy - physiology ; cathepsin B ; chloroquine ; Chloroquine - pharmacology ; Diagnostic Imaging - methods ; fluorescence microscopy ; Green Fluorescent Proteins - metabolism ; Humans ; Hydrogen-Ion Concentration ; LysoSensor ; Lysosomes - metabolism ; mechanistic target of rapamycin (serine/threonine kinase) ; microtubule-associated protein 1 light chain 3 ; Microtubule-Associated Proteins - metabolism ; mRFP-GFP-LC3 ; pH ratiometric sensor ; Phagosomes - pathology ; phosphatidylinositol 3-kinase catalytic subunit type 3 ; pHrodo red ; rapamycin ; starvation ; Toolbox</subject><ispartof>Autophagy, 2015-10, Vol.11 (10), p.1905-1916</ispartof><rights>2015 The Author(s). Published with license by Taylor & Francis Group, LLC © Giuseppe Maulucci, Michela Chiarpotto, Massimiliano Papi, Daniela Samengo, Giovambattista Pani, and Marco De Spirito 2015</rights><rights>2015 The Author(s). Published with license by Taylor & Francis Group, LLC 2015 The Author(s)</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c534t-a9e2640e6a046792a5bb48ca4cdf8628e8668eb0a5dc258c3fd9439279bf96c23</citedby><cites>FETCH-LOGICAL-c534t-a9e2640e6a046792a5bb48ca4cdf8628e8668eb0a5dc258c3fd9439279bf96c23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824579/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824579/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26506895$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Maulucci, Giuseppe</creatorcontrib><creatorcontrib>Chiarpotto, Michela</creatorcontrib><creatorcontrib>Papi, Massimiliano</creatorcontrib><creatorcontrib>Samengo, Daniela</creatorcontrib><creatorcontrib>Pani, Giovambattista</creatorcontrib><creatorcontrib>De Spirito, Marco</creatorcontrib><title>Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates</title><title>Autophagy</title><addtitle>Autophagy</addtitle><description>Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5-6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates.</description><subject>3-methyladenine</subject><subject>autophagic flux</subject><subject>autophagy</subject><subject>Autophagy - physiology</subject><subject>cathepsin B</subject><subject>chloroquine</subject><subject>Chloroquine - pharmacology</subject><subject>Diagnostic Imaging - methods</subject><subject>fluorescence microscopy</subject><subject>Green Fluorescent Proteins - metabolism</subject><subject>Humans</subject><subject>Hydrogen-Ion Concentration</subject><subject>LysoSensor</subject><subject>Lysosomes - metabolism</subject><subject>mechanistic target of rapamycin (serine/threonine kinase)</subject><subject>microtubule-associated protein 1 light chain 3</subject><subject>Microtubule-Associated Proteins - metabolism</subject><subject>mRFP-GFP-LC3</subject><subject>pH ratiometric sensor</subject><subject>Phagosomes - pathology</subject><subject>phosphatidylinositol 3-kinase catalytic subunit type 3</subject><subject>pHrodo red</subject><subject>rapamycin</subject><subject>starvation</subject><subject>Toolbox</subject><issn>1554-8627</issn><issn>1554-8635</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>0YH</sourceid><sourceid>EIF</sourceid><recordid>eNp9UctOHDEQtKKgQIBPIJpjLkM8fo3nghIhCJGQEBI55WD1eOzFyGsvtodk_z5e7bJKcsipW93VVaUuhM46fN5hiT91nDMpSH9OcMc3I8Y4f4OONvNWCsrf7nvSH6L3OT9hTIUcyDt0SATHteVH6Mf9DKG4AsW9mAYC-HV2uYm2gbnE1SMsnG6sn38147rRMdiowTerm9Yt6yos_kG6UExamslBMfkEHVjw2Zzu6jH6fn31cHnT3t59_Xb55bbVnLLSwmCIYNgIwEz0AwE-jkxqYHqy1bw0UghpRgx80oRLTe00MDqQfhjtIDShx-hiy7uax6qtTSgJvFql6jGtVQSn_t4E96gW8UUxSRjvh0rwcUeQ4vNsclFLl7XxHoKJc1ZdTyThPaO0QvkWqlPMORm7l-mw2gSjXoNRm2DULph69-FPj_ur1yQq4PMW4OqP0xJ-xuQnVWDtY7IJgnZZ0f9r_AZ7-Z-t</recordid><startdate>20151003</startdate><enddate>20151003</enddate><creator>Maulucci, Giuseppe</creator><creator>Chiarpotto, Michela</creator><creator>Papi, Massimiliano</creator><creator>Samengo, Daniela</creator><creator>Pani, Giovambattista</creator><creator>De Spirito, Marco</creator><general>Taylor & Francis</general><scope>0YH</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20151003</creationdate><title>Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates</title><author>Maulucci, Giuseppe ; Chiarpotto, Michela ; Papi, Massimiliano ; Samengo, Daniela ; Pani, Giovambattista ; De Spirito, Marco</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c534t-a9e2640e6a046792a5bb48ca4cdf8628e8668eb0a5dc258c3fd9439279bf96c23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>3-methyladenine</topic><topic>autophagic flux</topic><topic>autophagy</topic><topic>Autophagy - physiology</topic><topic>cathepsin B</topic><topic>chloroquine</topic><topic>Chloroquine - pharmacology</topic><topic>Diagnostic Imaging - methods</topic><topic>fluorescence microscopy</topic><topic>Green Fluorescent Proteins - metabolism</topic><topic>Humans</topic><topic>Hydrogen-Ion Concentration</topic><topic>LysoSensor</topic><topic>Lysosomes - metabolism</topic><topic>mechanistic target of rapamycin (serine/threonine kinase)</topic><topic>microtubule-associated protein 1 light chain 3</topic><topic>Microtubule-Associated Proteins - metabolism</topic><topic>mRFP-GFP-LC3</topic><topic>pH ratiometric sensor</topic><topic>Phagosomes - pathology</topic><topic>phosphatidylinositol 3-kinase catalytic subunit type 3</topic><topic>pHrodo red</topic><topic>rapamycin</topic><topic>starvation</topic><topic>Toolbox</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Maulucci, Giuseppe</creatorcontrib><creatorcontrib>Chiarpotto, Michela</creatorcontrib><creatorcontrib>Papi, Massimiliano</creatorcontrib><creatorcontrib>Samengo, Daniela</creatorcontrib><creatorcontrib>Pani, Giovambattista</creatorcontrib><creatorcontrib>De Spirito, Marco</creatorcontrib><collection>Taylor & Francis Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Autophagy</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Maulucci, Giuseppe</au><au>Chiarpotto, Michela</au><au>Papi, Massimiliano</au><au>Samengo, Daniela</au><au>Pani, Giovambattista</au><au>De Spirito, Marco</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates</atitle><jtitle>Autophagy</jtitle><addtitle>Autophagy</addtitle><date>2015-10-03</date><risdate>2015</risdate><volume>11</volume><issue>10</issue><spage>1905</spage><epage>1916</epage><pages>1905-1916</pages><issn>1554-8627</issn><eissn>1554-8635</eissn><abstract>Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5-6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates.</abstract><cop>United States</cop><pub>Taylor & Francis</pub><pmid>26506895</pmid><doi>10.1080/15548627.2015.1084455</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central |
| subjects | 3-methyladenine autophagic flux autophagy Autophagy - physiology cathepsin B chloroquine Chloroquine - pharmacology Diagnostic Imaging - methods fluorescence microscopy Green Fluorescent Proteins - metabolism Humans Hydrogen-Ion Concentration LysoSensor Lysosomes - metabolism mechanistic target of rapamycin (serine/threonine kinase) microtubule-associated protein 1 light chain 3 Microtubule-Associated Proteins - metabolism mRFP-GFP-LC3 pH ratiometric sensor Phagosomes - pathology phosphatidylinositol 3-kinase catalytic subunit type 3 pHrodo red rapamycin starvation Toolbox |
| title | Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates |
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