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Septins arrange F-actin-containing fibers on the Chlamydia trachomatis inclusion and are required for normal release of the inclusion by extrusion.

Volceanov L, Herbst K, Biniossek M, Schilling O, Haller D, Nölke T, Subbarayal P, Rudel T, Zieger B, Häcker G - MBio (2014)

Bottom Line: RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells.Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol.Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology and Hygiene, University Medical Center Freiburg, Freiburg, Germany.

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Related in: MedlinePlus

(A) Fibers containing SEPT2, -9, and -11 encase the chlamydial inclusion. HeLa cells infected for 48 h were fixed and stained for SEPT2 or SEPT9 or infected for 30 h, fixed, and stained for SEPT11. Chlamydial inclusions are indicated by arrows. Cell nuclei are marked with asterisks. Scale bar, 10 µm. Images are representative of at least 3 independent experiments. Proteomic analysis found the four septins SEPT2, -7, -9, and -11 on purified inclusions (see Materials and Methods for details; the following numbers of peptides were identified by mass spectrometry in infected/uninfected cells: SEPT2, 64/29; SEPT7, 6/11; SEPT9, 13/5; SEPT11, 7/11). (B) Infection increases the amount of SEPT9 in SEPT2-containing complexes. Uninfected HeLa cells or HeLa cells infected with C. trachomatis for 30 h were lysed, and proteins in the lysate supernatants (Input) were immunoprecipitated with anti-SEPT2 antibodies. Input, unbound, and IP fractions from the immunoprecipitations (IPs) were analyzed by SDS-PAGE, followed by immunoblotting with anti-SEPT2, -7, -9, or -11 or anti-GAPDH antibody. The smaller band for SEPT2 is consistent with cleavage by CPAF during sample preparation. A number of isoforms exist for SEPT9; one isoform especially appears to be recruited to septin fibers during infection.
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fig2: (A) Fibers containing SEPT2, -9, and -11 encase the chlamydial inclusion. HeLa cells infected for 48 h were fixed and stained for SEPT2 or SEPT9 or infected for 30 h, fixed, and stained for SEPT11. Chlamydial inclusions are indicated by arrows. Cell nuclei are marked with asterisks. Scale bar, 10 µm. Images are representative of at least 3 independent experiments. Proteomic analysis found the four septins SEPT2, -7, -9, and -11 on purified inclusions (see Materials and Methods for details; the following numbers of peptides were identified by mass spectrometry in infected/uninfected cells: SEPT2, 64/29; SEPT7, 6/11; SEPT9, 13/5; SEPT11, 7/11). (B) Infection increases the amount of SEPT9 in SEPT2-containing complexes. Uninfected HeLa cells or HeLa cells infected with C. trachomatis for 30 h were lysed, and proteins in the lysate supernatants (Input) were immunoprecipitated with anti-SEPT2 antibodies. Input, unbound, and IP fractions from the immunoprecipitations (IPs) were analyzed by SDS-PAGE, followed by immunoblotting with anti-SEPT2, -7, -9, or -11 or anti-GAPDH antibody. The smaller band for SEPT2 is consistent with cleavage by CPAF during sample preparation. A number of isoforms exist for SEPT9; one isoform especially appears to be recruited to septin fibers during infection.

Mentions: Large septin structures typically are formed by heteromeric complexes that contain three or four different septins (22), suggesting that the observed structures around the inclusion contained other septins in addition to SEPT2. Our proteomic study of isolated C. trachomatis inclusions by mass spectrometry (MS) had identified SEPT2, -7, -9, and -11 on the inclusion, with substantial enrichment of SEPT2 and -9 (see the legend to Fig. 2). We therefore also stained for these septins in infected cells. We found staining very similar to that for SEPT2 with antibodies against SEPT9 and also with antibodies against SEPT11. Staining for SEPT7 was inconclusive, most likely because the antibodies available to us did not give a very clear signal in immunofluorescence (data not shown). The data, however, clearly identify fibers containing SEPT2, -9, and -11 on the C. trachomatis inclusion. Since SEPT7 appears to be a core component of most septin structures (22, 23) and it was identified by MS on the inclusion, its involvement here appears very likely.


Septins arrange F-actin-containing fibers on the Chlamydia trachomatis inclusion and are required for normal release of the inclusion by extrusion.

Volceanov L, Herbst K, Biniossek M, Schilling O, Haller D, Nölke T, Subbarayal P, Rudel T, Zieger B, Häcker G - MBio (2014)

(A) Fibers containing SEPT2, -9, and -11 encase the chlamydial inclusion. HeLa cells infected for 48 h were fixed and stained for SEPT2 or SEPT9 or infected for 30 h, fixed, and stained for SEPT11. Chlamydial inclusions are indicated by arrows. Cell nuclei are marked with asterisks. Scale bar, 10 µm. Images are representative of at least 3 independent experiments. Proteomic analysis found the four septins SEPT2, -7, -9, and -11 on purified inclusions (see Materials and Methods for details; the following numbers of peptides were identified by mass spectrometry in infected/uninfected cells: SEPT2, 64/29; SEPT7, 6/11; SEPT9, 13/5; SEPT11, 7/11). (B) Infection increases the amount of SEPT9 in SEPT2-containing complexes. Uninfected HeLa cells or HeLa cells infected with C. trachomatis for 30 h were lysed, and proteins in the lysate supernatants (Input) were immunoprecipitated with anti-SEPT2 antibodies. Input, unbound, and IP fractions from the immunoprecipitations (IPs) were analyzed by SDS-PAGE, followed by immunoblotting with anti-SEPT2, -7, -9, or -11 or anti-GAPDH antibody. The smaller band for SEPT2 is consistent with cleavage by CPAF during sample preparation. A number of isoforms exist for SEPT9; one isoform especially appears to be recruited to septin fibers during infection.
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Related In: Results  -  Collection

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fig2: (A) Fibers containing SEPT2, -9, and -11 encase the chlamydial inclusion. HeLa cells infected for 48 h were fixed and stained for SEPT2 or SEPT9 or infected for 30 h, fixed, and stained for SEPT11. Chlamydial inclusions are indicated by arrows. Cell nuclei are marked with asterisks. Scale bar, 10 µm. Images are representative of at least 3 independent experiments. Proteomic analysis found the four septins SEPT2, -7, -9, and -11 on purified inclusions (see Materials and Methods for details; the following numbers of peptides were identified by mass spectrometry in infected/uninfected cells: SEPT2, 64/29; SEPT7, 6/11; SEPT9, 13/5; SEPT11, 7/11). (B) Infection increases the amount of SEPT9 in SEPT2-containing complexes. Uninfected HeLa cells or HeLa cells infected with C. trachomatis for 30 h were lysed, and proteins in the lysate supernatants (Input) were immunoprecipitated with anti-SEPT2 antibodies. Input, unbound, and IP fractions from the immunoprecipitations (IPs) were analyzed by SDS-PAGE, followed by immunoblotting with anti-SEPT2, -7, -9, or -11 or anti-GAPDH antibody. The smaller band for SEPT2 is consistent with cleavage by CPAF during sample preparation. A number of isoforms exist for SEPT9; one isoform especially appears to be recruited to septin fibers during infection.
Mentions: Large septin structures typically are formed by heteromeric complexes that contain three or four different septins (22), suggesting that the observed structures around the inclusion contained other septins in addition to SEPT2. Our proteomic study of isolated C. trachomatis inclusions by mass spectrometry (MS) had identified SEPT2, -7, -9, and -11 on the inclusion, with substantial enrichment of SEPT2 and -9 (see the legend to Fig. 2). We therefore also stained for these septins in infected cells. We found staining very similar to that for SEPT2 with antibodies against SEPT9 and also with antibodies against SEPT11. Staining for SEPT7 was inconclusive, most likely because the antibodies available to us did not give a very clear signal in immunofluorescence (data not shown). The data, however, clearly identify fibers containing SEPT2, -9, and -11 on the C. trachomatis inclusion. Since SEPT7 appears to be a core component of most septin structures (22, 23) and it was identified by MS on the inclusion, its involvement here appears very likely.

Bottom Line: RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells.Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol.Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology and Hygiene, University Medical Center Freiburg, Freiburg, Germany.

Show MeSH
Related in: MedlinePlus