Characterization of the Autophagy Marker Protein Atg8 Reveals Atypical Features of Autophagy in Plasmodium falciparum
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{"title"=>"Characterization of the autophagy marker protein Atg8 reveals atypical features of autophagy in Plasmodium falciparum", "type"=>"journal", "authors"=>[{"first_name"=>"Rahul", "last_name"=>"Navale", "scopus_author_id"=>"56635448900"}, {"last_name"=>"Atul", "scopus_author_id"=>"55752775600"}, {"first_name"=>"Aparna Devi", "last_name"=>"Allanki", "scopus_author_id"=>"56074725500"}, {"first_name"=>"Puran Singh", "last_name"=>"Sijwali", "scopus_author_id"=>"6603228978"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84913554658", "pmid"=>"25426852", "isbn"=>"1932-6203", "scopus"=>"2-s2.0-84913554658", "issn"=>"19326203", "pui"=>"600595855", "doi"=>"10.1371/journal.pone.0113220"}, "id"=>"d081dacb-bffb-32f8-b2e8-d7bc188703c5", "abstract"=>"Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8) employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64) and aspartic (pepstatin) protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine), indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in apicoplast biogenesis. Collectively, our results reveal several atypical features of autophagy in malaria parasites, which may be largely associated with non-degradative processes.", "link"=>"http://www.mendeley.com/research/characterization-autophagy-marker-protein-atg8-reveals-atypical-features-autophagy-plasmodium-falcip", "reader_count"=>44, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Researcher"=>10, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>10, "Student > Postgraduate"=>2, "Student > Master"=>11, "Other"=>1, "Student > Bachelor"=>4, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Researcher"=>10, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>10, "Student > Postgraduate"=>2, "Student > Master"=>11, "Other"=>1, "Student > Bachelor"=>4, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>12, "Nursing and Health Professions"=>1, "Agricultural and Biological Sciences"=>21, "Medicine and Dentistry"=>3, "Veterinary Science and Veterinary Medicine"=>1, "Physics and Astronomy"=>1, "Social Sciences"=>1, "Computer Science"=>1, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Social Sciences"=>{"Social Sciences"=>1}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>21}, "Computer Science"=>{"Computer Science"=>1}, "Nursing and Health Professions"=>{"Nursing and Health Professions"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>12}, "Unspecified"=>{"Unspecified"=>1}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>1}}, "reader_count_by_country"=>{"Canada"=>1, "Brazil"=>1, "United Kingdom"=>1, "India"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1808541"], "description"=>"<p>The indicated stages of <i>P. falciparum</i> were evaluated for the presence of Atg8 by IFA using anti-Atg8 antibodies as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section. The images of each indicated stage show the presence of Atg8 specific signal (Atg8), nucleic acid staining (DAPI), the parasite and the erythrocyte boundaries (Bright field), and the merged of all three images (Merge). The Atg8 signal is present throughout the parasite in all the stages shown, and appears to be associated with punctate structures, most likely autophagosomes. The scale bar shown is identical for this and all the figures containing IFA images.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253088, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.g002", "stats"=>{"downloads"=>1, "page_views"=>33, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_and_localization_of_Atg8_in_asexual_erythrocytic_stages_/1253088", "title"=>"Expression and localization of Atg8 in asexual erythrocytic stages.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808594"], "description"=>"a<p>Multiple homologs were predicted for the Atg proteins in italicized font, and homologs could not be identified for Atg proteins in bold font. Atg proteins predicted in previous analyses are underlined <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#pone.0113220-Rigden1\" target=\"_blank\">[38]</a>, <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#pone.0113220-Kitamura1\" target=\"_blank\">[39]</a>.</p>b<p>The <i>Plasmodium</i> genome database indicates the presence of transcript and/or peptides for the corresponding <i>P. falciparum</i> proteins in erythrocytic (E), gametocyte (G), and sporozoite (S) stages.</p><p>The components of autophagy machinery in <i>P. falciparum</i>.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253133, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.t001", "stats"=>{"downloads"=>8, "page_views"=>35, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_components_of_autophagy_machinery_in_P_falciparum_/1253133", "title"=>"The components of autophagy machinery in <i>P. falciparum</i>.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808593"], "description"=>"<p>Early/mid trophozoite stage parasites were exposed to a variety of stresses by culturing in complete medium (Control) or in HBSS containing DMSO (Starved) or the indicated inhibitors (22 µM E64 (Starved+E64), 220 µM pepstatin (Starved+pepstatin), or both (Starved+E64+Pep)) at 37°C for 8 hours. For oxidative stress, parasites were grown in complete medium containing the indicated oxidative stress-causing agents (90 nM artemisinin (Artemisinin), 100 µM H<sub>2</sub>O<sub>2</sub> (H<sub>2</sub>O<sub>2</sub>)) at 37°C for 8 hours. For heat shock (T43), parasites were cultured in complete medium at 43°C for 8 hours. After 8 hours of the indicated treatments, equal amounts of parasite samples were evaluated for expression levels of Atg8 and Hsp70 by western blotting as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section. Signal intensities of Atg8 in stressed parasite samples were compared with that in the control sample as mentioned in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section. <b>A</b>. The blot clearly shows significantly lower Atg8 levels in starved and heat shock-stressed parasites than the control parasites; whereas artemisinin and H<sub>2</sub>O<sub>2</sub>-treated parasites have Atg8 expression levels similar to that of the control. Hsp70 expression, as expected, is significantly upregulated in starved and heat shock-stressed parasites. Notably, no effect of E64 and pepstatin alone or together on expression level of Atg8 in starved parasites suggests that downregulation of Atg8 levels is not due to degradation of Ag8 in the food vacuole. The coomassie-stained SDS-PAGE (B) below the western blot has the same sample amounts used for the western blot, which shows that sample amounts were similar across the lanes. <b>C</b>. The bar graph shows fold reduction in Atg8 expression levels in stressed parasites compared to the control parasites, and it clearly indicates almost 3-fold reduction in Atg8 levels upon starvation or heat shock. The results shown in B are means with standard deviation error bars of three western blots, which were carried out with parasite samples from two independent experiments. The experiment was repeated twice, and the results were reproducible.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253132, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.g010", "stats"=>{"downloads"=>0, "page_views"=>37, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_stresses_on_autophagy_/1253132", "title"=>"Effects of stresses on autophagy.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808549"], "description"=>"<p>The trophozoite and schizont stages of wild type <i>P. berghei</i> were evaluated for expression and localization of native PbAtg8 using anti-Atg8 antibodies (A). Similarly, recombinant <i>P. berghei</i> parasites were assessed for localization of episomally expressed GFP-PfAtg8 (B) using anti-GFP antibodies and for colocalization of the signals (C) with anti-PfAtg8 and anti-GFP antibodies as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section. The images show signal for native PbAtg8 (A; Atg8) or episomally expressed GFP-PfAtg8 (B; GFP) or colocalization of anti-Atg8 and anti-GFP antibody signals (C; Atg8+GFP), staining of the nucleus (DAPI), the parasite and the erythrocyte boundaries (Bright field), and the merged of all three images (Merge). The punctate signal for both native and episomally expressed Atg8 throughout the parasite is similar to the localization pattern of native PfAtg8 in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#pone-0113220-g002\" target=\"_blank\">Figure 2</a>. The yellow spots (Atg8+GFP) in C indicate that both the antibodies label the same structures and that anti-PfAtg8 antibodies are specific to PfAtg8. For representation purpose, the GFP signal was false-coloured to green and the Atg8 signal was false-coloured to red.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253097, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.g004", "stats"=>{"downloads"=>2, "page_views"=>35, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_and_localization_of_native_and_episomally_expressed_Atg8_in_P_berghei_/1253097", "title"=>"Expression and localization of native and episomally expressed Atg8 in <i>P. berghei</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808583"], "description"=>"<p>Early/mid trophozoite stage parasites were cultured in HBSS (Starved) or in complete medium containing DMSO (Control) or the indicated autophagy inhibitors (5 mM 3MA, 30 nM CQ, 22 µM E64, and 220 µM pepstatin (Pep); all except 3MA are at concentrations 3× IC<sub>50</sub>) for 8 hours, and then parasites were evaluated for expression of Atg8 by western blotting (A) or for localization of Atg8 by IFA (B) using anti-Atg8 antibodies as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section. The same parasite samples were assessed for expression of the control proteins β-actin (β-Actin) and Hsp70 by western blotting as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section. <b>A</b>. The immunoblot shows drastically reduced Atg8 levels in starved parasites and almost similar Atg8 levels in other parasite samples compared to control parasites. Both Hsp70 and β-actin levels are similar in all except the starved parasites, which may be due to a starvation-induced stress response. <b>B</b>. The parasite images are labelled as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#pone-0113220-g002\" target=\"_blank\">Figure 2</a>, and show similar Atg8 signal regardless of the treatment. The experiment was repeated three times, and the results were reproducible.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253126, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.g008", "stats"=>{"downloads"=>0, "page_views"=>26, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_typically_used_autophagy_inducer_inhibitors_on_Atg8_levels_and_localization_/1253126", "title"=>"Effects of typically used autophagy inducer/inhibitors on Atg8 levels and localization.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808542"], "description"=>"<p><i>P. falciparum</i> trophozoite/schizont stage parasites were first lysed by freeze-thaw method, and equal aliquots of the lysate were processed for total freeze-thaw lysate (TL), soluble (S) and pellet (P) fractions of the freeze-thaw lysate, extraction of peripheral membrane proteins (PM) with Na<sub>2</sub>CO<sub>3</sub>, and extraction of integral membrane proteins (IM) with Triton X-100 as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section. Equivalent amounts of all the fractions, including the pellets remaining after Na<sub>2</sub>CO<sub>3</sub> (PN) and Triton X-100 (PT) extractions, were analyzed for the presence of Atg8, the proteasome α2 subunit (α2), and β-actin (β-actin) by western blotting as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section. The blot showed predominant Atg8 signal in the total freeze-thaw lysate, the pellet fraction of the freeze-thaw lysate, and the integral membrane protein fraction. Soluble fraction of the freeze-thaw lysate did not have any Atg8 signal and the peripheral membrane fraction had very low signal, indicating that Atg8 is exclusively present in the freeze-thaw pellet fraction, mostly as an integral membrane protein. The α2 was used as a marker for soluble protein, and it is exclusively present in the soluble fraction of the freeze-thaw lysate. β-actin was used as a general control and it appears to be predominantly exist as a peripheral membrane protein. The experiment was repeated three times and the results were reproducible.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253089, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.g003", "stats"=>{"downloads"=>0, "page_views"=>30, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Distribution_of_Atg8_in_different_subcellular_fractions_/1253089", "title"=>"Distribution of Atg8 in different subcellular fractions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808557"], "description"=>"<p>The indicated stages of <i>P. falciparum</i> D10 parasites expressing the apicoplast marker ACP-GFP were evaluated for colocalization of PfAtg8 with the apicoplast as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section. The images show PfAtg8 (Atg8) and apicoplast (ACP-GFP), merged Atg8 and apicoplast signals (Atg8+GFP), nuclear stain (DAPI), parasite and erythrocyte boundaries (Bright field), and the merged of all the four images (Merge). Note that PfAtg8 signal is distributed throughout the parasite as puncta, which partially overlaps with the apicoplast, particularly in the late trophozoite stage.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253104, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.g005", "stats"=>{"downloads"=>2, "page_views"=>50, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Colocalization_of_Atg8_and_the_apicoplast_/1253104", "title"=>"Colocalization of Atg8 and the apicoplast.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808574"], "description"=>"<p>The <i>P. falciparum</i> early/mid trophozoite stage parasites were cultured in complete medium (Control) or HBSS (Starved), samples were collected after four (4-hrs) and eight (8-hrs) hours of culture, and processed for localization of Atg8 by IFA (A) or expression level of Atg8 by western blotting (B) using anti-Atg8 antibodies. As a loading control, β-actin expression was also assessed in the same samples. <b>A</b>. The labels of images are as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#pone-0113220-g002\" target=\"_blank\">Figure 2</a>, and parasite images show almost similar localization patterns of Atg8 in control and starved parasites at both the time points. <b>B</b>. The western blot shows significantly reduced level of Atg8 in parasites starved for 8 hours compared to the 8 hour control parasites, suggesting downregulation of Atg8. Similar β-actin levels in control and starved parasites indicate that similar sample amounts were loaded.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253117, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.g007", "stats"=>{"downloads"=>5, "page_views"=>64, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_starvation_on_Atg8_levels_and_localization_/1253117", "title"=>"Effect of starvation on Atg8 levels and localization.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808591"], "description"=>"<p>Synchronized early/mid trophozoite stage parasites were cultured in the presence of DMSO (Control) or inhibitors (22 µM E64, 220 µM pepstatin) for 15 hours, and then analyzed by IFA using anti-Atg8 antibodies. <b>A</b>. The parasite images are labelled as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#pone-0113220-g002\" target=\"_blank\">Figure 2</a>, and show punctate localization pattern of Atg8, which is distributed throughout the control and pepstatin-treated parasites. The E64-treated parasite show accumulation of Atg8 signal in a narrow region around the swollen food vacuole, most likely because cytoplasm has been pushed to the periphery in these parasites due to the enlarged food vacuole. <b>B</b>. Z-sections for control and E64-treated parasites were captured as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section, and the composite image shows punctate signal for Atg8 (Atg8), the stained-nucleus (DAPI), erythrocyte and parasite boundaries (Bright field), and a merge of all three images (Merge). Note that both control and E-64 treated parasites appear to have a few Atg8 puncta in the food vacuole, but not any significant accumulation of the puncta in the food vacuole of E64-treated parasites.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253130, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.g009", "stats"=>{"downloads"=>7, "page_views"=>213, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_E64_and_pepstatin_treatments_on_autophagy_/1253130", "title"=>"Effect of E64 and pepstatin treatments on autophagy.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808531"], "description"=>"<p><b>A</b>. Western blot of uninfected erythrocyte (RBC, approximately 1×10<sup>8</sup>/lane) and mixed stage parasite (Parasite, approximately 1×10<sup>8</sup>/lane) lysates with anti-Atg8 antibodies showed a single band around the predicted size of native PfAtg8 (∼14 kD) exclusively in the parasite, confirming specificity of the antibodies. Antibodies against the α2 subunit of the proteasome were used as a loading control, which showed similar intensity signal in both the samples. <b>B</b>. Western blot of ring (R), early trophozoite (ET), late trophozoite (LT), and schizont (S) stage lysates of <i>P. falciparum</i> (approximately 1×10<sup>8</sup> parasites/lane) with anti-Atg8 (Atg8) and anti-β-actin (β-Actin) antibodies. The blot showed expression of PfAtg8 and the control protein (β-actin) in all stages. M, molecular weight in kD.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253078, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.g001", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Specificity_of_anti_PfAtg8_antibody_and_expression_of_native_PfAtg8_/1253078", "title"=>"Specificity of anti-PfAtg8 antibody and expression of native PfAtg8.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808603", "https://ndownloader.figshare.com/files/1808604", "https://ndownloader.figshare.com/files/1808605", "https://ndownloader.figshare.com/files/1808606", "https://ndownloader.figshare.com/files/1808607", "https://ndownloader.figshare.com/files/1808608", "https://ndownloader.figshare.com/files/1808609", "https://ndownloader.figshare.com/files/1808610", "https://ndownloader.figshare.com/files/1808611", "https://ndownloader.figshare.com/files/1808612", "https://ndownloader.figshare.com/files/1808613", "https://ndownloader.figshare.com/files/1808614", "https://ndownloader.figshare.com/files/1808615", "https://ndownloader.figshare.com/files/1808616", "https://ndownloader.figshare.com/files/1808617"], "description"=>"<div><p>Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in <i>Plasmodium</i>, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated <i>Plasmodium falciparum</i> Atg8 (PfAtg8) employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64) and aspartic (pepstatin) protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine), indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in apicoplast biogenesis. Collectively, our results reveal several atypical features of autophagy in malaria parasites, which may be largely associated with non-degradative processes.</p></div>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253139, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0113220.s001", "https://dx.doi.org/10.1371/journal.pone.0113220.s002", "https://dx.doi.org/10.1371/journal.pone.0113220.s003", "https://dx.doi.org/10.1371/journal.pone.0113220.s004", "https://dx.doi.org/10.1371/journal.pone.0113220.s005", "https://dx.doi.org/10.1371/journal.pone.0113220.s006", "https://dx.doi.org/10.1371/journal.pone.0113220.s007", "https://dx.doi.org/10.1371/journal.pone.0113220.s008", "https://dx.doi.org/10.1371/journal.pone.0113220.s009", "https://dx.doi.org/10.1371/journal.pone.0113220.s010", "https://dx.doi.org/10.1371/journal.pone.0113220.s011", "https://dx.doi.org/10.1371/journal.pone.0113220.s012", "https://dx.doi.org/10.1371/journal.pone.0113220.s013", "https://dx.doi.org/10.1371/journal.pone.0113220.s014", "https://dx.doi.org/10.1371/journal.pone.0113220.s015"], "stats"=>{"downloads"=>31, "page_views"=>80, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Characterization_of_the_Autophagy_Marker_Protein_Atg8_Reveals_Atypical_Features_of_Autophagy_in_Plasmodium_falciparum_/1253139", "title"=>"Characterization of the Autophagy Marker Protein Atg8 Reveals Atypical Features of Autophagy in <i>Plasmodium falciparum</i>", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-11-26 02:58:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/1808567"], "description"=>"<p>Synchronized ring stage <i>P. falciparum</i> D10 parasites expressing the apicoplast marker ACP-GFP were cultured in the presence of DMSO (Control) or doxycycline (2.5 µM Dox) for two full cycles, samples were collected at 30-hours (30-hrs) and 78-hours (70-hrs) time points, and processed for IFA using anti-Atg8 antibodies as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#s4\" target=\"_blank\">Materials and Methods</a> section. The labels are as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#pone-0113220-g005\" target=\"_blank\">Figure 5</a>. The parasite images indicate that both control and treated parasites have strong signal over the elongated apicoplast at the 30-hours time point, indicating a healthy dividing apicoplast. The 78-hours control parasite have multiple intensely-stained apicoplasts, indicating that it has matured to multinucleate stage and the apicoplast has divided, whereas the treated parasites have weak fragmented signal, suggesting disruption of the apicoplast, which has been observed previously <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113220#pone.0113220-Dahl1\" target=\"_blank\">[66]</a>. Note that Atg8 signal pattern in the control and treated parasites mostly remains unchanged.</p>", "links"=>[], "tags"=>["malaria parasites", "Atg 8 expression level", "autophagy marker Atg 8.", "PfAtg 8 level", "food vacuole protease activity", "integral membrane protein", "Atg 8", "Plasmodium falciparum Atg 8", "Atg 8 localization", "Autophagy Marker Protein Atg 8", "33 yeast autophagy proteins"], "article_id"=>1253110, "categories"=>["Biological Sciences"], "users"=>["Rahul Navale", "Atul", "Aparna Devi Allanki", "Puran Singh Sijwali"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0113220.g006", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Atg8_in_doxycycline_treated_parasites_/1253110", "title"=>"Atg8 in doxycycline-treated parasites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-11-26 02:58:11"}

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