Clostridium botulinum C2 toxin is internalized by clathrin‐ and Rho‐dependent mechanisms
Clostridium botulinum C2 toxin is an ADP‐ribosyltransferase, causing depolymerization of the actin cytoskeleton in eukaryotic cells. The C2 toxin is a binary toxin consisting of the enzymatic subunit C2I and the binding subunit C2II. Proteolytical activation of the binding subunit triggers the forma...
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| Veröffentlicht in: | Cellular microbiology 2010-12, Vol.12 (12), p.1809-1820 |
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| creator | Pust, Sascha Barth, Holger Sandvig, Kirsten |
| description | Clostridium botulinum C2 toxin is an ADP‐ribosyltransferase, causing depolymerization of the actin cytoskeleton in eukaryotic cells. The C2 toxin is a binary toxin consisting of the enzymatic subunit C2I and the binding subunit C2II. Proteolytical activation of the binding subunit triggers the formation of heptameric structures (C2IIa), which bind to cellular receptors. C2I is able to bind to C2IIa oligomers, and it has been suggested that the whole complex is internalized by a raft‐dependent mechanism. Here we analysed by which mechanism C2 toxin is endocytosed. In HeLa cells expressing a dominant‐negative dynamin mutant, cytotoxicity and C2 toxin uptake were blocked. Furthermore, siRNA‐mediated knockdown of flotillins or inhibition of Arf6 function, proteins suggested to be involved in dynamin‐independent endocytosis, did not affect C2 toxicity. Knockdown of caveolin did not inhibit endocytosis of C2 toxin, whereas inhibition of clathrin function reduced the uptake of C2 toxin and delayed the cytotoxic effect. Finally, we found evidence for a Rho‐mediated uptake of C2 toxin. In conclusion, C2 toxin is endocytosed by dynamin‐dependent mechanisms and we provide evidence for involvement of clathrin and Rho. |
| doi_str_mv | 10.1111/j.1462-5822.2010.01512.x |
| format | Article |
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The C2 toxin is a binary toxin consisting of the enzymatic subunit C2I and the binding subunit C2II. Proteolytical activation of the binding subunit triggers the formation of heptameric structures (C2IIa), which bind to cellular receptors. C2I is able to bind to C2IIa oligomers, and it has been suggested that the whole complex is internalized by a raft‐dependent mechanism. Here we analysed by which mechanism C2 toxin is endocytosed. In HeLa cells expressing a dominant‐negative dynamin mutant, cytotoxicity and C2 toxin uptake were blocked. Furthermore, siRNA‐mediated knockdown of flotillins or inhibition of Arf6 function, proteins suggested to be involved in dynamin‐independent endocytosis, did not affect C2 toxicity. Knockdown of caveolin did not inhibit endocytosis of C2 toxin, whereas inhibition of clathrin function reduced the uptake of C2 toxin and delayed the cytotoxic effect. Finally, we found evidence for a Rho‐mediated uptake of C2 toxin. In conclusion, C2 toxin is endocytosed by dynamin‐dependent mechanisms and we provide evidence for involvement of clathrin and Rho.</description><identifier>ISSN: 1462-5814</identifier><identifier>EISSN: 1462-5822</identifier><identifier>DOI: 10.1111/j.1462-5822.2010.01512.x</identifier><identifier>PMID: 20690924</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>Actin ; ADP-Ribosylation Factors - antagonists & inhibitors ; ADP-Ribosylation Factors - metabolism ; Botulinum Toxins - metabolism ; C2 toxin ; Caveolin ; Clathrin ; Clathrin - metabolism ; Clostridium botulinum ; Clostridium botulinum - pathogenicity ; Cytoskeleton ; Cytotoxicity ; Depolymerization ; Dynamin ; Dynamins - deficiency ; Dynamins - metabolism ; Endocytosis ; Gene Deletion ; Gene Silencing ; HeLa Cells ; Humans ; Membrane Proteins - antagonists & inhibitors ; Membrane Proteins - metabolism ; NAD(P) super(+)-arginine ADP-ribosyltransferase ; rho GTP-Binding Proteins - metabolism ; Toxicity</subject><ispartof>Cellular microbiology, 2010-12, Vol.12 (12), p.1809-1820</ispartof><rights>2010 Blackwell Publishing Ltd</rights><rights>2010 Blackwell Publishing Ltd.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5182-3f34a0ebbcf2b23369f4c58356622c51363c315bba2eb7303d9a8bcba3d3faa13</citedby></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.1462-5822.2010.01512.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fj.1462-5822.2010.01512.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1417,1433,27924,27925,45574,45575,46409,46833</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20690924$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Pust, Sascha</creatorcontrib><creatorcontrib>Barth, Holger</creatorcontrib><creatorcontrib>Sandvig, Kirsten</creatorcontrib><title>Clostridium botulinum C2 toxin is internalized by clathrin‐ and Rho‐dependent mechanisms</title><title>Cellular microbiology</title><addtitle>Cell Microbiol</addtitle><description>Clostridium botulinum C2 toxin is an ADP‐ribosyltransferase, causing depolymerization of the actin cytoskeleton in eukaryotic cells. The C2 toxin is a binary toxin consisting of the enzymatic subunit C2I and the binding subunit C2II. Proteolytical activation of the binding subunit triggers the formation of heptameric structures (C2IIa), which bind to cellular receptors. C2I is able to bind to C2IIa oligomers, and it has been suggested that the whole complex is internalized by a raft‐dependent mechanism. Here we analysed by which mechanism C2 toxin is endocytosed. In HeLa cells expressing a dominant‐negative dynamin mutant, cytotoxicity and C2 toxin uptake were blocked. Furthermore, siRNA‐mediated knockdown of flotillins or inhibition of Arf6 function, proteins suggested to be involved in dynamin‐independent endocytosis, did not affect C2 toxicity. Knockdown of caveolin did not inhibit endocytosis of C2 toxin, whereas inhibition of clathrin function reduced the uptake of C2 toxin and delayed the cytotoxic effect. Finally, we found evidence for a Rho‐mediated uptake of C2 toxin. In conclusion, C2 toxin is endocytosed by dynamin‐dependent mechanisms and we provide evidence for involvement of clathrin and Rho.</description><subject>Actin</subject><subject>ADP-Ribosylation Factors - antagonists & inhibitors</subject><subject>ADP-Ribosylation Factors - metabolism</subject><subject>Botulinum Toxins - metabolism</subject><subject>C2 toxin</subject><subject>Caveolin</subject><subject>Clathrin</subject><subject>Clathrin - metabolism</subject><subject>Clostridium botulinum</subject><subject>Clostridium botulinum - pathogenicity</subject><subject>Cytoskeleton</subject><subject>Cytotoxicity</subject><subject>Depolymerization</subject><subject>Dynamin</subject><subject>Dynamins - deficiency</subject><subject>Dynamins - metabolism</subject><subject>Endocytosis</subject><subject>Gene Deletion</subject><subject>Gene Silencing</subject><subject>HeLa Cells</subject><subject>Humans</subject><subject>Membrane Proteins - antagonists & inhibitors</subject><subject>Membrane Proteins - metabolism</subject><subject>NAD(P) super(+)-arginine ADP-ribosyltransferase</subject><subject>rho GTP-Binding Proteins - metabolism</subject><subject>Toxicity</subject><issn>1462-5814</issn><issn>1462-5822</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkU1OwzAQhS0EolC4AlhsWCX4J3GSBQsU8VOpCAnoDsmyE4c6SpwSJ6JlxRE4IyfBoaULvPGT55sZ-T0AIEY-duei9HHAiBfGhPgEuVeEQ0z85Q442BZ2txoHI3BobYkQZhHG-2BEEEtQQoID8JJWje1aneu-hrLp-kobp1ICu2apDdQWatOp1ohKf6gcyhXMKtHNW22-P7-gMDl8nDdO5mqhTK5MB2uVzYXRtrZHYK8QlVXHm3sMZjfXz-mdN324naRXUy8LcUw8WtBAICVlVhBJKGVJEWRhTEPGCHEIZTSjOJRSECUjimieiFhmUtCcFkJgOgbn67mLtnnrle14rW2mqkoY1fSWxxHBxK0KHXn2jyybfvic5VHE4oAyxhx0soF6WaucL1pdi3bF_1xzwOUaeNeVWm3rGPEhHV7ywXg-hMCHdPhvOnzJ0_vJoFz_6bq_EA0Xr622fPbkSIpwgsM4QvQHIAeNkw</recordid><startdate>201012</startdate><enddate>201012</enddate><creator>Pust, Sascha</creator><creator>Barth, Holger</creator><creator>Sandvig, Kirsten</creator><general>Blackwell Publishing Ltd</general><general>Hindawi Limited</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7QL</scope><scope>7T7</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>7U7</scope></search><sort><creationdate>201012</creationdate><title>Clostridium botulinum C2 toxin is internalized by clathrin‐ and Rho‐dependent mechanisms</title><author>Pust, Sascha ; Barth, Holger ; Sandvig, Kirsten</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5182-3f34a0ebbcf2b23369f4c58356622c51363c315bba2eb7303d9a8bcba3d3faa13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Actin</topic><topic>ADP-Ribosylation Factors - antagonists & inhibitors</topic><topic>ADP-Ribosylation Factors - metabolism</topic><topic>Botulinum Toxins - metabolism</topic><topic>C2 toxin</topic><topic>Caveolin</topic><topic>Clathrin</topic><topic>Clathrin - metabolism</topic><topic>Clostridium botulinum</topic><topic>Clostridium botulinum - pathogenicity</topic><topic>Cytoskeleton</topic><topic>Cytotoxicity</topic><topic>Depolymerization</topic><topic>Dynamin</topic><topic>Dynamins - deficiency</topic><topic>Dynamins - metabolism</topic><topic>Endocytosis</topic><topic>Gene Deletion</topic><topic>Gene Silencing</topic><topic>HeLa Cells</topic><topic>Humans</topic><topic>Membrane Proteins - antagonists & inhibitors</topic><topic>Membrane Proteins - metabolism</topic><topic>NAD(P) super(+)-arginine ADP-ribosyltransferase</topic><topic>rho GTP-Binding Proteins - metabolism</topic><topic>Toxicity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pust, Sascha</creatorcontrib><creatorcontrib>Barth, Holger</creatorcontrib><creatorcontrib>Sandvig, Kirsten</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Toxicology Abstracts</collection><jtitle>Cellular microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Pust, Sascha</au><au>Barth, Holger</au><au>Sandvig, Kirsten</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Clostridium botulinum C2 toxin is internalized by clathrin‐ and Rho‐dependent mechanisms</atitle><jtitle>Cellular microbiology</jtitle><addtitle>Cell Microbiol</addtitle><date>2010-12</date><risdate>2010</risdate><volume>12</volume><issue>12</issue><spage>1809</spage><epage>1820</epage><pages>1809-1820</pages><issn>1462-5814</issn><eissn>1462-5822</eissn><abstract>Clostridium botulinum C2 toxin is an ADP‐ribosyltransferase, causing depolymerization of the actin cytoskeleton in eukaryotic cells. The C2 toxin is a binary toxin consisting of the enzymatic subunit C2I and the binding subunit C2II. Proteolytical activation of the binding subunit triggers the formation of heptameric structures (C2IIa), which bind to cellular receptors. C2I is able to bind to C2IIa oligomers, and it has been suggested that the whole complex is internalized by a raft‐dependent mechanism. Here we analysed by which mechanism C2 toxin is endocytosed. In HeLa cells expressing a dominant‐negative dynamin mutant, cytotoxicity and C2 toxin uptake were blocked. Furthermore, siRNA‐mediated knockdown of flotillins or inhibition of Arf6 function, proteins suggested to be involved in dynamin‐independent endocytosis, did not affect C2 toxicity. Knockdown of caveolin did not inhibit endocytosis of C2 toxin, whereas inhibition of clathrin function reduced the uptake of C2 toxin and delayed the cytotoxic effect. Finally, we found evidence for a Rho‐mediated uptake of C2 toxin. 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| subjects | Actin ADP-Ribosylation Factors - antagonists & inhibitors ADP-Ribosylation Factors - metabolism Botulinum Toxins - metabolism C2 toxin Caveolin Clathrin Clathrin - metabolism Clostridium botulinum Clostridium botulinum - pathogenicity Cytoskeleton Cytotoxicity Depolymerization Dynamin Dynamins - deficiency Dynamins - metabolism Endocytosis Gene Deletion Gene Silencing HeLa Cells Humans Membrane Proteins - antagonists & inhibitors Membrane Proteins - metabolism NAD(P) super(+)-arginine ADP-ribosyltransferase rho GTP-Binding Proteins - metabolism Toxicity |
| title | Clostridium botulinum C2 toxin is internalized by clathrin‐ and Rho‐dependent mechanisms |
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