The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin
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{"title"=>"The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin", "type"=>"journal", "authors"=>[{"first_name"=>"Wenya", "last_name"=>"Hou", "scopus_author_id"=>"56888285000"}, {"first_name"=>"Maryam", "last_name"=>"Izadi", "scopus_author_id"=>"55921752300"}, {"first_name"=>"Sabine", "last_name"=>"Nemitz", "scopus_author_id"=>"57193750008"}, {"first_name"=>"Natja", "last_name"=>"Haag", "scopus_author_id"=>"8899970700"}, {"first_name"=>"Michael M.", "last_name"=>"Kessels", "scopus_author_id"=>"6603964964"}, {"first_name"=>"Britta", "last_name"=>"Qualmann", "scopus_author_id"=>"6603579179"}], "year"=>2015, "source"=>"PLoS Biology", "identifiers"=>{"issn"=>"15457885", "pmid"=>"26334624", "pui"=>"606211070", "scopus"=>"2-s2.0-84943166116", "doi"=>"10.1371/journal.pbio.1002233", "sgr"=>"84943166116"}, "id"=>"78c9d894-c38f-3ad6-88ff-df388b3d4253", "abstract"=>"Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer-binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca2+ and multiple, direct associations of the Ca2+ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl's functions. Cobl-induced dendritic branch initiation was preceded by Ca2+ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca2+/CaM modulates Cobl's actin binding properties and furthermore promotes Cobl's previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca2+/CaM and reveal that the Ca2+/CaM-controlled molecular mechanisms we discovered are crucial for Cobl's cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca2+/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca2+ signals steer and power branch initiation during early arborization of nerve cells-a key process in neuronal network formation.", "link"=>"http://www.mendeley.com/research/actin-nucleator-cobl-controlled-calcium-calmodulin", "reader_count"=>28, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>3, "Researcher"=>4, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>1, "Student > Master"=>5, "Lecturer"=>1, "Professor"=>4, "Unspecified"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>3, "Researcher"=>4, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>1, "Student > Master"=>5, "Lecturer"=>1, "Professor"=>4, "Unspecified"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>4, "Medicine and Dentistry"=>1, "Agricultural and Biological Sciences"=>19, "Neuroscience"=>2, "Unspecified"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Neuroscience"=>{"Neuroscience"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>19}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Unspecified"=>{"Unspecified"=>2}}, "reader_count_by_country"=>{"United States"=>1, "Germany"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2252118"], "description"=>"<p>(<b>A,B</b>) Immunoblot analyses of coprecipitation experiments using purified, immobilized CaM, purified TrxHis-Cobl<sup>54–450</sup> and GST-syndapin I demonstrate that Cobl/CaM complexes but not CaM alone specifically interact with syndapin I. (<b>A</b>) Direct interaction of Cobl<sup>54–450</sup> with immobilized CaM. (<b>B</b>) Syndapin I is present in Cobl/CaM complexes in a Cobl-dependent manner (i.e., does not associate with CaM). Thus, Cobl can directly and simultaneously associate with both CaM and syndapin I. (<b>C–F</b>) Liposome floatation assays evaluating the lipid association of Cobl, CaM, and syndapin I when combined. Note that TrxHis-Cobl<sup>54–450</sup> and GST-syndapin I float in the presence of both GST (<b>C</b>) and GST-CaM (<b>D</b>) and that in the latter case, CaM also floats together with Cobl and syndapin I to fraction 2. In contrast, in both Cobl/CaM combinations that either lack syndapin I (<b>E</b>; TrxHis-Cobl<sup>54–450</sup>/GST/GST-CaM) or instead of syndapin I full-length only contain the Cobl-binding SH3 domain of syndapin I, i.e., lacking the ability of syndapin I to directly bind to lipids (<b>F</b>), neither Cobl nor CaM floated (<b>E,F</b>). Grey arrow heads (<b>C,E,F</b>) mark protein bands that remain in bottom fractions, the maximum of which usually is in fraction 5, and black arrow heads (<b>C,D</b>) mark protein bands at floating liposome positions (density gradient fraction 2).</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534752, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g007", "stats"=>{"downloads"=>2, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Complexes_composed_of_the_three_components_syndapin_I_Cobl_and_CaM_form_and_associate_with_liposomes_/1534752", "title"=>"Complexes composed of the three components syndapin I, Cobl, and CaM form and associate with liposomes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252203", "https://ndownloader.figshare.com/files/2252204", "https://ndownloader.figshare.com/files/2252205", "https://ndownloader.figshare.com/files/2252206", "https://ndownloader.figshare.com/files/2252207", "https://ndownloader.figshare.com/files/2252208", "https://ndownloader.figshare.com/files/2252209", "https://ndownloader.figshare.com/files/2252210", "https://ndownloader.figshare.com/files/2252211", "https://ndownloader.figshare.com/files/2252213", "https://ndownloader.figshare.com/files/2252214", "https://ndownloader.figshare.com/files/2252215", "https://ndownloader.figshare.com/files/2252216", "https://ndownloader.figshare.com/files/2252217", "https://ndownloader.figshare.com/files/2252218", "https://ndownloader.figshare.com/files/2252219", "https://ndownloader.figshare.com/files/2252220", "https://ndownloader.figshare.com/files/2252221", "https://ndownloader.figshare.com/files/2252222", "https://ndownloader.figshare.com/files/2252224"], "description"=>"<div><p>Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer–binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca<sup>2+</sup> and multiple, direct associations of the Ca<sup>2+</sup> sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl’s functions. Cobl-induced dendritic branch initiation was preceded by Ca<sup>2+</sup> signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca<sup>2+</sup>/CaM modulates Cobl’s actin binding properties and furthermore promotes Cobl’s previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca<sup>2+</sup>/CaM and reveal that the Ca<sup>2+</sup>/CaM-controlled molecular mechanisms we discovered are crucial for Cobl’s cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca<sup>2+</sup>/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca<sup>2+</sup> signals steer and power branch initiation during early arborization of nerve cells—a key process in neuronal network formation.</p></div>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534829, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.1002233.s001", "https://dx.doi.org/10.1371/journal.pbio.1002233.s002", "https://dx.doi.org/10.1371/journal.pbio.1002233.s003", "https://dx.doi.org/10.1371/journal.pbio.1002233.s004", "https://dx.doi.org/10.1371/journal.pbio.1002233.s005", "https://dx.doi.org/10.1371/journal.pbio.1002233.s006", "https://dx.doi.org/10.1371/journal.pbio.1002233.s007", "https://dx.doi.org/10.1371/journal.pbio.1002233.s008", "https://dx.doi.org/10.1371/journal.pbio.1002233.s009", "https://dx.doi.org/10.1371/journal.pbio.1002233.s010", "https://dx.doi.org/10.1371/journal.pbio.1002233.s011", "https://dx.doi.org/10.1371/journal.pbio.1002233.s012", "https://dx.doi.org/10.1371/journal.pbio.1002233.s013", "https://dx.doi.org/10.1371/journal.pbio.1002233.s014", "https://dx.doi.org/10.1371/journal.pbio.1002233.s015", "https://dx.doi.org/10.1371/journal.pbio.1002233.s016", "https://dx.doi.org/10.1371/journal.pbio.1002233.s017", "https://dx.doi.org/10.1371/journal.pbio.1002233.s018", "https://dx.doi.org/10.1371/journal.pbio.1002233.s019", "https://dx.doi.org/10.1371/journal.pbio.1002233.s020"], "stats"=>{"downloads"=>11, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_Actin_Nucleator_Cobl_Is_Controlled_by_Calcium_and_Calmodulin_/1534829", "title"=>"The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252127"], "description"=>"<p>Transient Ca<sup>2+</sup> signals and CaM as a Ca<sup>2+</sup> sensor protein control the functions of Cobl in neurons. Newly identified components of the pathway of Cobl-mediated actin filament formation in early neuronal development are in green, the molecular mechanisms experimentally demonstrated to be critical for the role of Cobl in branch induction are written in black. Colors of arrows indicate whether the mechanism identified to be important for Cobl-mediated function represents an inhibition or a promotion of the respective molecular property of Cobl.</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534761, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g011", "stats"=>{"downloads"=>0, "page_views"=>22, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_signaling_pathway_and_mechanisms_revealed_to_control_Cobl_functions_/1534761", "title"=>"The signaling pathway and mechanisms revealed to control Cobl functions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252126"], "description"=>"<p>(<b>A</b>) Scheme of Cobl as in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.g005\" target=\"_blank\">Fig 5</a> with a schematic representation of further pieces of Cobl positively tested for CaM association (green lines; for immunoblottings of coprecipitation analyses see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s015\" target=\"_blank\">S14 Fig</a>), an overview of the identified CaM binding interfaces (black lines; grey lines, binding not addressed individually) and Cobl mutants lacking CaM interfaces (blue lines with grey deletions). (<b>B</b>) Coprecipitation experiments with immobilized CaM and GFP-Cobl deletion mutant GFP- Cobl<sup>∆48–299,714–1175</sup> (Cobl<sup>∆CaM-N,C</sup>) in comparison to wild-type GFP-Cobl showing that Cobl<sup>∆CaM-N,C</sup> lacking all identified CaM binding areas did not associate with immobilized Ca<sup>2+</sup>-activated CaM but remained in the supernatant. (<b>C–L</b>) Functional analyses of Cobl<sup>∆CaM-N,C</sup>, Cobl<sup>∆CaM-N</sup>, Cobl<sup>∆CaM-C</sup>, Cobl<sup>∆CaM-C</sup>’, Cobl<sup>∆CaM-C</sup>”, and Cobl<sup>∆CaM-C</sup>”‘ mutants for their ability to elicit Cobl-mediated dendrite and dendritic branch formation upon overexpression in primary hippocampal neurons transfected at DIV4. Representative images of neurons transfected as indicated, fixed 2 d after transfection and immunostained for MAP2. In images displaying several neurons, transfected cells are marked by arrows. Bar, 10 μm. Quantitative analyses normalized to GFP controls show that both dendrite (<b>K</b>) and dendritic branch formation (<b>L</b>) require both the N-terminal and the C-terminal CaM interaction areas. Data are mean ± SEM. GFP, <i>n</i> = 206; GFP-Cobl, <i>n</i> = 187; Cobl<sup>∆CaM-N,C</sup>, <i>n</i> = 91, GFP-Cobl<sup>∆CaM-N</sup>, <i>n</i> = 98; Cobl<sup>∆CaM-C</sup>, <i>n</i> = 117; Cobl<sup>∆CaM-C</sup>’, <i>n</i> = 52, Cobl<sup>∆CaM-C</sup>”, <i>n</i> = 63, Cobl<sup>∆CaM-C”‘</sup>, <i>n</i> = 64. Statistical significances were tested using one-way ANOVA with Tukey’s post-test. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. (<b>M–U</b>) Parasagittal cerebellar slices (250 μm; DIV2) prepared from postnatal day 10 (P10) mice showing individual Purkinje cells transfected as indicated. Bar, 20 μm. (<b>V</b>) Quantification of dendritic branch points of Purkinje cells in cerebellar slice cultures showed that coexpression of Cobl RNAi/GFP-Cobl*<sup>∆CaM-N,C</sup>, Cobl RNAi/GFP-Cobl*<sup>∆CaM-N</sup>, Cobl RNAi/GFP-Cobl*<sup>∆CaM-C</sup>, Cobl RNAi/GFP-Cobl*<sup>∆CaM-C</sup>’, Cobl RNAi/GFP-Cobl*<sup>∆CaM-C</sup>”, and Cobl RNAi/GFP-Cobl*<sup>∆CaM-C</sup>”‘ failed to rescue the reduction of branch points observed upon Cobl RNAi, whereas re-expressing full-length Cobl (GFP-Cobl*) did rescue the Cobl loss-of-function phenotype (Cobl*, RNAi-resistant Cobl (amino acid sequence unchanged)). Data are mean ± SEM. Scrambled RNAi<sup>GFP</sup>, <i>n</i> = 50; Cobl RNAi<sup>GFP</sup>, <i>n</i> = 21; Cobl RNAi/GFP-Cobl*, <i>n</i> = 16; Cobl RNAi/GFP-Cobl*<sup>∆CaM-N,C</sup>, <i>n</i> = 7; Cobl RNAi/GFP-Cobl*<sup>∆CaM-N</sup>, <i>n</i> = 18; Cobl RNAi/GFP-Cobl*<sup>∆CaM-C</sup>, <i>n</i> = 10; Cobl RNAi/GFP-Cobl*<sup>∆CaM-C</sup>’, <i>n</i> = 11; Cobl RNAi/GFP-Cobl*<sup>∆CaM-C</sup>”, <i>n</i> = 3 and Cobl RNAi/GFP-Cobl*<sup>∆CaM-C</sup>”‘, <i>n</i> = 16 cells. For data underlying <b>K, L,</b> and <b>V,</b> see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s001\" target=\"_blank\">S1 Data</a>. Statistical significances were tested using one-way ANOVA with Tukey’s post-test (comparisons to scrambled RNAi controls are shown above columns; all others as marked by lines). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534760, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g010", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cobl_functions_in_dendritogenesis_rely_on_CaM_association_with_both_the_Cobl_N_and_C_terminus_/1534760", "title"=>"Cobl functions in dendritogenesis rely on CaM association with both the Cobl N- and C-terminus.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252087"], "description"=>"<p>(<b>A,B</b>) Individual frames (maximum intensity projections [MIPs]) of 3-D-time-lapse recordings of dendritic, locally restricted Ca<sup>2+</sup> signals in developing primary hippocampal neurons visualized by GCaMP5G (marked by}) and F-actin accumulation visualized using LifeAct-RFP (transfection, days in vitro (DIV)6; imaging, DIV7). Local increase of intracellular Ca<sup>2+</sup> levels correlates with subsequent protrusion (arrows mark initiation sites) formation (marked by green *) from a previously existing F-actin–rich site (<b>A</b>) and from a site that newly acquired F-actin accumulation after a Ca<sup>2+</sup> signal (<b>B</b>). Static protrusions are marked by yellow I. Bars in <b>A</b> and <b>B</b>, 5 μm. (<b>C</b>) Example images (MIPs) of 3-D-time-lapse recording (shown are 0–2:30 and 6:00–15:30 min:s) of GFP-Cobl reveal that Cobl is dynamically enriched at distinct sites within dendrites in immature neurons undergoing dendritogenesis. Initiation of dynamic, dendritic protrusions (marked by green *) often is preceded by Cobl accumulation (white arrows). Retraction events are marked by red ° and static phases with yellow I. Dendrite branch induction is a dynamic process with often several protrusive attempts until a dendritic branch is firmly established and strongly elongated. Three consecutive initiations of a protrusion from the same site are shown. Data (see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s002\" target=\"_blank\">S1 Fig</a> for original data of the GFP channel) are shown in a color-coded manner (heat map purple to white, see legend for color coding). Bar, 5 μm. Please also see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s017\" target=\"_blank\">S1 Movie</a>. (<b>D</b>) Quantitative analyses of GFP-Cobl enrichment at dendritic protrusion initiation sites 30 s prior to protrusion initiation in comparison to adjacent control regions of interest (ROIs). Errors represent standard error of the mean (SEM). Statistical significances, one-way ANOVA with Tukey’s post test. GFP-Cobl, <i>n</i> = 25; GFP, <i>n</i> = 18. ***<i>p</i> < 0.001. For underlying data, see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s001\" target=\"_blank\">S1 Data</a>. (<b>E</b>) Simultaneous recordings of GFP-Cobl and LifeAct-RFP show that initiation of dendritic protrusions coincides with Cobl accumulation followed by F-actin buildup at the dendritic base (arrows). Labeling as in <b>C</b>. Bar, 2 μm. Please also see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s018\" target=\"_blank\">S2 Movie</a>. (<b>F</b>) Anti-Cobl and anti-MAP2-stained hippocampal neuron at DIV6 transfected with LifeAct-GFP to mark putative sites of dendritic branch induction show colocalization of Cobl with F-actin at actin-rich sites protruding from the dendrites (arrow). Bar, 1 μm.</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534724, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g001", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dendrite_branching_coincides_with_local_Ca_2_signals_and_Cobl_accumulation_leading_to_bursts_of_F_actin_formation_at_the_base_of_initiating_dendritic_protrusions_/1534724", "title"=>"Dendrite branching coincides with local Ca<sup>2+</sup> signals and Cobl accumulation leading to bursts of F-actin formation at the base of initiating dendritic protrusions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252120"], "description"=>"<p>(<b>A</b>) Coimmunoprecipitations of GFP-Cobl<sup>1-408</sup>/Flag-syndapin I (Flag-Sdp I) and GFP-Cobl<sup>1-408∆CaM</sup>/Flag-syndapin I complexes from HEK293 cells under Ca<sup>2+</sup>-free conditions (−), 2 μM Ca<sup>2+</sup> (+) and 2 μM Ca<sup>2+</sup> with subsequent incubation with EGTA (2 μM Ca<sup>2+</sup>/1 mM EGTA, [+]). The syndapin I interaction with Cobl is promoted upon activation of Ca<sup>2+</sup> signaling (2 μM Ca<sup>2+</sup>) (indicated by the green upright arrowhead). For further confirmation of the Ca<sup>2+</sup>/CaM association-dependent increase of the syndapin I interaction, also see coimmunoprecipitations under alternative conditions shown in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s013\" target=\"_blank\">S12 Fig</a>. (<b>B,C</b>) Quantitative evaluations of coimmunoprecipitated syndapin I normalized to immunoprecipitated Cobl and displayed as mean percental difference ± SEM from the respective Ca<sup>2+</sup>-free conditions show that the syndapin I interaction is reversibly promoted by increasing calcium. Note that syndapin I coimmunoprecipitation by the CaM binding-deficient Cobl mutant Cobl<sup>1-408∆CaM</sup> is insensitive to changes of calcium levels. The observed regulation of Cobl/syndapin I complex formation thus requires the CaM binding interface of Cobl. GFP-Cobl<sup>1-408</sup>/Flag-Sdp I +/− Ca<sup>2+</sup>, <i>n</i> = 3 each; with Ca<sup>2+</sup>/EGTA, <i>n</i> = 2. GFP-Cobl<sup>1-408∆CaM</sup>/Flag-Sdp I +/− Ca<sup>2+</sup>, <i>n</i> = 3 each; with Ca<sup>2+</sup>/EGTA, <i>n</i> = 2. Statistical significances were calculated by one-way ANOVA with Tukey’s post-test. *<i>p</i> < 0.05. (<b>D,E</b>) Coimmunoprecipitations from rat brain lysates (<b>D</b>) demonstrate the promotion of endogenous Cobl/syndapin I complexes upon calcium addition (highlighted by green upward arrowhead; <b>E,</b> quantitation; <i>n</i> = 2. For data underlying <b>B, C,</b> and <b>E,</b> see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s001\" target=\"_blank\">S1 Data</a>.</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534754, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g008", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Ca_2_CaM_association_with_the_Cobl_Homology_domain_of_Cobl_promotes_Cobl_syndapin_I_complex_formation_/1534754", "title"=>"Ca<sup>2+</sup>/CaM association with the Cobl Homology domain of Cobl promotes Cobl/syndapin I complex formation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252101"], "description"=>"<p>(<b>A</b>) MIPs from 3-D-time-lapse recordings of GFP-Cobl and mCherry-CaM in a dendrite of a primary hippocampal neuron transfected at DIV6 and imaged at DIV7 show that episodes of Cobl accumulation at distinct dendritic sites as well as the induction of protrusions from such sites are accompanied by accumulations of CaM at the same sites (data shown as heat map of fluorescence intensities from purple to white, see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.g001\" target=\"_blank\">Fig 1</a> for legend of color coding; see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s005\" target=\"_blank\">S4 Fig</a> for original data of both channels and for merged images). Bar, 2 μm. Please also see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s019\" target=\"_blank\">S3 Movie</a>. (<b>B</b>) MIPs from 3-D-time-lapse recordings of the calcium sensor GCaMP5G and mCherry-Cobl reveal that local rises in calcium levels coincide with subsequent induction of Cobl accumulation and dendritic protrusions arising from Cobl-enriched sites (arrows). Bar, 5 μm. Inset is a high-intensity image of the protrusion. Cobl and CaM accumulations, respectively, at sites giving rise to protrusion are marked by arrows. Green <b>*</b>, protrusion that was initiated or grew when compared to previous image; (green <b>*</b>), minor, aborted protrusion; red <b>°</b>, protrusion that shrunk compared to previous image; yellow I, static protrusion.</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534735, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g003", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Correlation_of_Cobl_and_CaM_dynamics_and_Ca_2_signals_at_forming_dendritic_protrusions_/1534735", "title"=>"Correlation of Cobl and CaM dynamics and Ca<sup>2+</sup> signals at forming dendritic protrusions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252095"], "description"=>"<p>(<b>A</b>) Yeast-2-hybrid verification of hit#3737 encoding CaM<sup>∆1–32</sup> using BD-Cobl<sup>1001–1337</sup> as bait and isolated and retransformed plasmid AD-3737 as prey. Reporter gene activities manifest in growth on quadruple dropout media (-Ade/-His/-Leu/-Trp). Growth on-Leu/-Trp medium confirms coexpression of both plasmids. (<b>B</b>) Coprecipitation analyses with immobilized CaM and GFP-Cobl<sup>1001–1337</sup> and GFP-Cobl (arrowheads mark bands) expressed in HEK293 cells verify that full-length CaM (under 500 μM Ca<sup>2+</sup>) binds to Cobl. (<b>C,D</b>) Visualization of the Cobl/CaM interaction in intact COS-7 cells by recruitment of GFP-Cobl<sup>1001–1337</sup> (<b>C</b>) and GFP-Cobl (<b>D</b>) to mitochondrially anchored mCherry-CaM (Mito-mCherry-CaM). Examples of successful CaM/Cobl complex reconstitution are marked with arrowheads. Bars in <b>C and D</b>, 10 μm. For establishment of mitochondrial targeting of Mito-mCherry-CaM and GFP-control of the Cobl recruitment and specificity control experiments see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s003\" target=\"_blank\">S2 Fig</a>. (<b>E,F</b>) Anti-Cobl and anti-CaM immunostaining of parasagittal sections of the hippocampus (<b>E)</b> and cerebellum (<b>F</b>) at high magnifications demonstrate that Cobl and CaM are enriched in the same cells in the CA3 and CA4 regions (<b>E</b>; examples marked with short arrows) and in Purkinje cells of the cerebellum including their dendritic arbor (<b>F</b>; long arrows). Lower panels, enlargements of boxed areas. DG, dentate gyrus; ML, Molecular Layer; PCL, Purkinje Cell Layer; GCL, Granule Cell Layer. Bars, 50 μm. (<b>G</b>) Coprecipitation analyses with immobilized CaM and brain lysates demonstrating that the Cobl/CaM interaction is Ca<sup>2+</sup>dependent and that CaM complexes with endogenous Cobl (arrowhead marks bands) are formed at all Ca<sup>2+</sup> concentrations tested. (<b>H</b>) Coimmunoprecipitation of endogenous Cobl (arrowhead marks band) with anti-CaM antibodies. For characterization of anti-CaM antibodies in immunoprecipitations and immunoblotting please see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s004\" target=\"_blank\">S3 Fig</a>).</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534729, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g002", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cobl_is_a_calmodulin_binding_protein_/1534729", "title"=>"Cobl is a calmodulin-binding protein.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252111"], "description"=>"<p>(<b>A</b>) Scheme of Cobl showing proline-rich domains (green), WH2 domains (red), Cobl Homology domain (blue frame), and syndapin-binding KrRAPpPP-motifs (yellow). Deletion mutants binding to CaM are in green, those not binding in red. Black lines, (independent) CaM-binding interfaces (grey lines, binding not addressed individually). (<b>B–D</b>) Coprecipitation experiments with immobilized CaM and GFP-Cobl deletion mutants in presence and absence of 500 μM Ca<sup>2+</sup>-identifying CaM binding sites within the N-terminus (<b>B</b>), the C-terminal half (<b>C</b>) and the WH2 domain-containing C-terminus (<b>D</b>). (<b>E–G</b>) Immunoprecipitations of GFP-Cobl<sup>1001–1337</sup>, GFP-Cobl<sup>1176–1337</sup> and GFP-Cobl<sup>1206–1337</sup> all show an increase of coimmunoprecipitation of endogenous actin upon Ca<sup>2+</sup> addition. Quantitative data (right panels in <b>E</b> and <b>F</b>) are mean percental differences±SEM from the respective Ca<sup>2+</sup>-free conditions. GFP-Cobl<sup>1001–1337</sup>, <i>n</i> = 4; GFP-Cobl<sup>1176–1337</sup>, Ca<sup>2+</sup>, <i>n</i> = 5; Persistence upon subsequent addition of 5 mM EGTA (Ca<sup>2+</sup>/EGTA), <i>n</i> = 3. (<b>H</b>) Quantitative coimmunoprecipitations demonstrating that actin association by a combination of the first WH2 domain and the CaM interface is inhibited upon Ca<sup>2+</sup> addition at both 500 μM (<i>n</i> = 6) and 2 μM Ca<sup>2+</sup> (<i>n</i> = 4). (<b>I</b>) In vitro reconstitution of Cobl<sup>1001–1224</sup>/CaM/actin complexes with purified proteins (GST-Cobl<sup>1001–1224</sup> immobilized) demonstrating the Ca<sup>2+</sup> and TrxHis-CaM dependence of the observed suppression of the actin binding of the first WH2 domain of Cobl (for input of purified proteins see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s012\" target=\"_blank\">S11 Fig</a>). Right panel, quantitative analyses, <i>n</i> = 3 each. (<b>J</b>) Reversibility of the actin binding to GFP-Cobl<sup>1001–1224</sup> suppressed by Ca<sup>2+</sup> via subsequent addition of EGTA (2 μM Ca<sup>2+</sup>/1 mM EGTA). 2 μM Ca<sup>2+</sup>, <i>n</i> = 4; 2 μM Ca<sup>2+</sup>/1 mM EGTA, <i>n</i> = 2. For data underlying <b>E, F,</b> and <b>H–J,</b> see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s001\" target=\"_blank\">S1 Data</a>. Statistical significances in all panels, one-way ANOVA with Tukey’s post-test. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534745, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g005", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cobl_has_multiple_CaM_binding_sites_and_Ca_2_CaM_controls_the_actin_binding_activity_of_Cobl_8217_s_WH2_domains_/1534745", "title"=>"Cobl has multiple CaM binding sites and Ca<sup>2+</sup>/CaM controls the actin binding activity of Cobl’s WH2 domains.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252110"], "description"=>"<p>(<b>A–H</b>) End-point analysis of CaM inhibitor effects in primary hippocampal neurons (DIV4+2). (<b>A–F</b>) Hippocampal neurons transfected as indicated at DIV4 and treated with 0.1% (final) DMSO (<b>A,D</b>), CaM inhibitor W7 (<b>B,E</b>) and CaM inhibitor CGS9343B (both in 0.1% DMSO final) (<b>C,F</b>), respectively, 30 h after transfection. Cells were processed for anti-MAP2 immunostaining 2 d after transfection. Bar, 20 μm. (<b>G,H</b>) Quantitative examinations of dendrite number (<b>G</b>) and dendritic branch points (<b>H</b>) normalized to the GFP+DMSO controls of each assay. Note that Cobl-mediated dendrite formation is suppressed by the CaM inhibitors W7 and CGS9343B. Data represent mean ± SEM. GFP+DMSO, <i>n</i> = 197; GFP+W7, <i>n</i> = 120; GFP+CGS9343B, <i>n</i> = 169; GFP-Cobl+DMSO, <i>n</i> = 101; GFP-Cobl+W7, <i>n</i> = 53; GFP-Cobl+CGS93943B, <i>n</i> = 80. Statistical significances (to control, marked above column; between other conditions, indicated by lines) were determined using one-way ANOVA with Tukey’s post-test. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. (<b>I</b>) 3-D-time-lapse imaging of GFP-Cobl dynamics and neuronal morphogenesis visualized by PM-mCherry before and after incubation with the CaM inhibitor CGS9343B. Shown is a selection (times as indicated) of MIPs recorded by spinning disc microscopy. Green <b>*</b>, protrusion that was initiated or grew when compared to previous image (examples numbered to allow for tracking); red <b>°</b>, protrusion that shrunk compared to previous image; yellow I, static protrusion. Note that whereas most neuronal structures were dynamic, CGS9343B addition (red line; 11:20 min:s) impeded this morphological dynamics and neuronal structures largely were static until the end of recording. Bar, 5 μm. Please also see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s020\" target=\"_blank\">S4 Movie</a>. (<b>J</b>) Quantitation of the frequencies of protrusion initiation before and after addition of DMSO and DMSO+inhibitor (CGS9343B), respectively. DMSO, <i>n</i> = 12; DMSO+inhibitor, <i>n</i> = 10 dendrite sections. For data underlying <b>G, H,</b> and <b>J</b> see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s001\" target=\"_blank\">S1 Data</a>. Statistical significances, one-way ANOVA with Tukey’s post-test. ***<i>p</i> < 0.001.</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534744, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g004", "stats"=>{"downloads"=>2, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cobl_mediated_dendritogenesis_is_suppressed_by_CaM_inhibition_/1534744", "title"=>"Cobl-mediated dendritogenesis is suppressed by CaM inhibition.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252122"], "description"=>"<p>(<b>A</b>) Colocalization of GFP-Cobl and Flag-mCherry-Sdp I at discrete sites within the dendritic arbor of a DIV7 hippocampal neuron. Lower panels represent magnifications of boxed areas. Bars, 10 μm. (<b>B</b>) Time-lapse spinning disc MIPs of a dendritic protrusion (green <b>*</b>) emanating from a Cobl and syndapin I-enriched site (arrows). Data is shown as heat map of fluorescence intensities from purple to white, see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.g001\" target=\"_blank\">Fig 1</a> for legend of color coding; for original fluorescence data and colocalization analyses, see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s014\" target=\"_blank\">S13 Fig</a>. Bar, 5 μm. (<b>C</b>) Localization of endogenous Cobl at dendritic sites marked by accumulations of anti-syndapin I immunosignals and of F-actin (detected by phalloidin). Bar, 1 μm.</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534756, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g009", "stats"=>{"downloads"=>1, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Syndapin_I_and_Cobl_localize_to_sites_of_dendritic_branch_initiation_/1534756", "title"=>"Syndapin I and Cobl localize to sites of dendritic branch initiation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/2252117"], "description"=>"<p>(<b>A,B</b>) CaM complex formation with Cobl in intact cells demonstrated by GFP-Cobl<sup>106–324</sup> recruitment to mitochondria coated with mCherry-CaM (Mito-mCherry-CaM) (<b>A</b>; arrowheads mark examples) but not to those with mCherry alone (<b>B</b>; Mito-mCherry control). Bars, 10 μm. (<b>C</b>) Coimmunoprecipitation experiments with Flag-mCherry-CaM (FlagC-CaM) and GFP-Cobl<sup>106–324</sup> expressed in HEK293 cells further confirming the interaction in vivo. Red arrowheads mark GFP-Cobl<sup>106–324</sup> coimmunoprecipitated with FlagC-CaM (see middle lane of middle panel). Bands of sizes smaller than 57kD in lysates (GFP-Cobl106-324) are degradation products. (<b>D</b>) Immunoblot analyses of density gradient fractions (F1, top; F6, bottom) from floatation assays with liposomes made of Folch fraction I. TrxHis-Cobl<sup>54–450</sup> was incubated with and without liposomes. Note that TrxHis-Cobl<sup>54–450</sup> has a specific membrane-binding activity and floats together with the liposomes to density gradient fraction 2. (<b>E</b>) TrxHis-Cobl<sup>54–450</sup> floatation assays with and without calcium revealing that Cobl’s membrane-binding ability is Ca<sup>2+</sup>insensitive. (<b>F</b>) Floatation assays examining the ability of TrxHis-Cobl<sup>54–450</sup> to bind to liposomes in the presence of CaM. TrxHis and GST were used to control for putative unspecific interactions. Note that presence of Ca<sup>2+</sup>/CaM strongly and specifically suppresses the lipid association of TrxHis-Cobl<sup>54–450</sup>, and that both proteins remain at the bottom of the gradients. Black arrow heads in <b>D–F</b> mark protein bands at floating liposome positions (density gradient fraction 2), and grey arrow heads mark protein bands that remain in bottom fractions, the maximum of which usually is in fraction 5.</p>", "links"=>[], "tags"=>["wh", "Cobl regulation", "power branch initiation", "nucleates actin", "Actin filaments", "rescue experiments", "actin nucleator", "CaM binding", "dendritic protrusion sites", "WASP Homology 2", "Mechanistic studies", "Calmodulin Actin nucleation triggers", "CaM accumulations", "shape cells", "CaM inhibitor studies", "Actin Nucleator Cobl", "tissue slices", "network formation", "novel class", "Cobl mutants", "Overexpression analyses", "CaM activity", "actin filament formation"], "article_id"=>1534751, "categories"=>["Biological Sciences"], "users"=>["Wenya Hou", "Maryam Izadi", "Sabine Nemitz", "Natja Haag", "Michael M. Kessels", "Britta Qualmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002233.g006", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Ca_2_CaM_associates_with_the_Cobl_Homology_domain_in_vivo_and_this_association_suppresses_membrane_binding_of_the_Cobl_Homology_domain_/1534751", "title"=>"Ca<sup>2+</sup>/CaM associates with the Cobl Homology domain in vivo, and this association suppresses membrane binding of the Cobl Homology domain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-09-03 14:47:34"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"15", "full-text"=>"8", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"8", "cited-by"=>"0", "year"=>"2019", "month"=>"1"}
  • {"unique-ip"=>"10", "full-text"=>"11", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"5"}
  • {"unique-ip"=>"5", "full-text"=>"4", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"6"}
  • {"unique-ip"=>"17", "full-text"=>"12", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2018", "month"=>"4"}
  • {"unique-ip"=>"16", "full-text"=>"9", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"8", "cited-by"=>"0", "year"=>"2018", "month"=>"7"}
  • {"unique-ip"=>"18", "full-text"=>"15", "pdf"=>"9", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"8"}
  • {"unique-ip"=>"13", "full-text"=>"11", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2018", "month"=>"9"}
  • {"unique-ip"=>"17", "full-text"=>"18", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"10"}
  • {"unique-ip"=>"23", "full-text"=>"25", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"11"}
  • {"unique-ip"=>"12", "full-text"=>"16", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"19", "full-text"=>"18", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"18", "full-text"=>"15", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"10", "supp-data"=>"9", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"20", "full-text"=>"20", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"11", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"13", "full-text"=>"14", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}
  • {"unique-ip"=>"23", "full-text"=>"22", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"8"}

Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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