Journal: Parasites & Vectors

Article Title: A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia

doi: 10.1186/s13071-021-04687-5

Figure Lengend Snippet: Expression and localization of G. lamblia PLK1 (GlPLK) in G. lamblia -expressing hemagglutinin (HA)-tagged GlPLK. a A schematic diagram of plasmid pGlPLK.neo. HA-tagged GlPLK was expressed from its own promoter, P glplk . Transfected trophozoites were selected by neomycin resistance conferred by the neo gene expressed by the P glggi promoter, a strong promoter of the γ-giardin gene. As a control, Giardia trophozoites were also transfected with pKS-3HA.neo, a vector control. b Western blotting to examine the expression of HA-tagged GlPLK. Extracts were prepared from G. lamblia containing empty vector (lane 1) or pGlPLK.neo (lane 2) and incubated with monoclonal mouse anti-HA antibodies. Membranes were first incubated in stripping buffer and then reacted with polyclonal rat antibodies specific to protein disulfide isomerase 1 (PDI1) of G. lamblia . c Localization of GlPLK. Giardia lamblia expressing HA-tagged GlPLK was probed with mouse anti-HA antibodies. The cells were then incubated with Alexa Fluor 488-conjugated anti-mouse IgG. Slides were mounted with ProLong™ Gold Antifade Mountant with the fluorescent stain DAPI, and then examined with a Zeiss LSM700 inverted confocal laser scanning microscope. Scale bars: 2 µm. d Co-localization of GlPLK and α-tubulin in G. lamblia . Giardia cells expressing HA-tagged GlPLK were probed with rat anti-HA antibodies and mouse anti-acetylated-α-tubulin monoclonal antibodies. e Co-localization of GlPLK and G. lamblia centrin (GlCentrin) in G. lamblia . Cells were reacted with mouse anti-HA antibodies and rat anti-GlCentrin polyclonal antibodies. Cells were then incubated with Alexa Fluor 555-conjugated anti-rat IgG and Alexa Fluor 488-conjugated anti-mouse IgG. A differential interference contrast image was acquired to show cell morphology. Scale bars: 2 μm. DIC Differential interference contrast

Article Snippet: The samples were mounted with ProLong™ Gold Antifade Mountant with DAPI (Molecular Probes, Waltham, MA, USA) and then examined inverted confocal laser scanning microscopy (model LSM700 microscrope; Carl Zeiss AG).

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Incubation, Stripping Membranes, Staining, Laser-Scanning Microscopy

Journal: Parasites & Vectors

Article Title: A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia

doi: 10.1186/s13071-021-04687-5

Figure Lengend Snippet: Expression and localization of truncated GlPLKs in G. lamblia . a A schematic diagram of plasmids pGlPLKKDL.neo and pGlPLKPBD.neo. Two truncated GlPLK proteins are expressed in an HA-tagged form from their own promoter, P glplk . Plasmid pGlPLKKDL encodes GlPLK with the KD and linker region, whereas pGlPLKPBD contains DNA coding for the polo-box domains (PBDs) of GlPLK. Plasmid pKS-3HA.neo was transfected into Giardia trophozoites as a control. b Western blotting to examine the expression of HA-tagged truncated GlPLKs. Extracts were prepared from G. lamblia containing empty vector (lane 1), pGlPLKKDL.neo (lane 2), or pGlPLKPBD.neo (lane 3), and incubated with monoclonal mouse anti-HA antibodies. Membranes were incubated in stripping buffer, and then reacted with polyclonal rat antibodies specific to GlPDI1. c Co-localization of GlPLK-KDL with α-tubulin ( i ) or GlCentrin ( ii , iii ). Giardia lamblia cells expressing HA-tagged truncated GlPLK-KDL were probed with rat anti-HA antibodies and mouse anti-acetylated-α-tubulin monoclonal antibodies. Otherwise, these cells were reacted with rat anti-GlCentrin polyclonal antibodies instead of anti-acetylated-α-tubulin monoclonal antibodies. Panel iii is an extended view of panel ii . Incorrectly positioned basal bodies are indicated with white arrows. d Co-localization of GlPLK-PBD with α-tubulin ( i ) or GlCentrin ( ii , iii ). G. lamblia cells expressing HA-tagged truncated GlPLK-PBD were probed with rat or mouse anti-HA antibodies along either with mouse anti-acetylated-α-tubulin antibodies ( i ), or rat anti-GlCentrin polyclonal antibodies ( ii , iii ), respectively. Panel iii is an extended view of panel ii . The cells were then incubated with Alexa Fluor 488-conjugated anti-rat IgG and Alexa Fluor 568-conjugated anti-mouse IgG (α-tubulin co-localization) or Alexa Fluor 555-conjugated anti-rat IgG and Alexa Fluor 488-conjugated anti-mouse IgG (for centrin co-localization). Slides were mounted with ProLong™ Gold Antifade Mountant with DAPI, and then examined with a Zeiss LSM700 inverted confocal laser scanning microscope. A differential interference contrast image was acquired to show cell morphology. Scale bars: 2 μm

Article Snippet: The samples were mounted with ProLong™ Gold Antifade Mountant with DAPI (Molecular Probes, Waltham, MA, USA) and then examined inverted confocal laser scanning microscopy (model LSM700 microscrope; Carl Zeiss AG).

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Incubation, Stripping Membranes, Laser-Scanning Microscopy

Journal: Parasites & Vectors

Article Title: A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia

doi: 10.1186/s13071-021-04687-5

Figure Lengend Snippet: Expression and localization of phosphorylated GlPLK in G. lamblia . a Giardia lamblia cells expressing HA-tagged GlPLK in the interphase (lane 1), G1/S phase (lane 2), and G2/M phase (lane 3). Western blot analysis. Extracts of these cells were probed with anti-phospho-PLK, anti-HA, anti-GlPLK or anti-GlPDI1 antibodies. Levels of phospho-GlPLK-HA, phospho-GlPLK, GlPLK-HA and GlPLK were normalized to GlPDI1, a protein loading control. Amounts of these GlPLK proteins are expressed as relative values to those observed in the interphase cells. The presented western blot is a representative of three independent experiments, and averages of three experiments are presented as a bar graph. b Localization of GlPLK and phosphorylated GlPLK in Giardia . G. lamblia expressing HA-tagged GlPLK were incubated with antibodies specific to the phosphorylated form of PLK (1:100) along with anti-HA antibodies. These cells were then reacted with anti-Alexa Fluor 568-conjugated anti-mouse IgG and Alexa Fluor 488-conjugated anti-rat IgG. Slides were mounted with ProLong™ Gold Antifade Mountant with DAPI, and then examined with a Zeiss LSM700 inverted confocal laser scanning microscope. Scale bars: 2 µm

Article Snippet: The samples were mounted with ProLong™ Gold Antifade Mountant with DAPI (Molecular Probes, Waltham, MA, USA) and then examined inverted confocal laser scanning microscopy (model LSM700 microscrope; Carl Zeiss AG).

Techniques: Expressing, Western Blot, Incubation, Laser-Scanning Microscopy