Journal: Infection and Immunity

Article Title: Cholangiocyte Myosin IIB Is Required for Localized Aggregation of Sodium Glucose Cotransporter 1 to Sites of Cryptosporidium parvum Cellular Invasion and Facilitates Parasite Internalization

doi: 10.1128/iai.00077-10

Figure Lengend Snippet: FIG. 1. Immunofluorescence C. parvum invasion assay. (A) Cells pretreated with DMSO vehicle (control) are readily infected with C. parvum sporozoites, as detected with immunofluorescence. Pretreat- ment of cholangiocytes with the myosin II inhibitor blebbistatin (B) or with the myosin light chain kinase inhibitor ML-7 (C) diminishes the number of C. parvum invasion sites detected with immunofluores- cence. (D) Quantitation of attachment and attachment/invasion shows that pretreatment of cells with blebbistatin does not affect parasite attachment to H69 cells but that ML-7 reduces attachment approxi- mately 40%. Conversely, blebbistatin pretreatment results in a 10-fold decrease in cellular invasion while ML-7 results in a 2-fold decrease in cellular invasion. Bars 20 m. Data are presented as means SE. , P 0.01 for comparison to control, vehicle-treated cells by ANOVA.

Article Snippet: For studies addressing accumulation of myosin isoforms and serine19-phosphorylated myosin light chain (MLC2) to C. parvum invasion sites, infected cells were fixed as described above and incubated with monoclonal antibodies against myosin IIB (CMII-23), pMLC2 (sc-19849-R; Santa Cruz Biotechnology), and the polyclonal anti-C. parvum antibody Cp2, followed by incubation with fluorophore-conjugated secondary anti-mouse and anti-rabbit antibodies.

Techniques: Invasion Assay, Control, Infection, Quantitation Assay, Comparison

Journal: Infection and Immunity

Article Title: Cholangiocyte Myosin IIB Is Required for Localized Aggregation of Sodium Glucose Cotransporter 1 to Sites of Cryptosporidium parvum Cellular Invasion and Facilitates Parasite Internalization

doi: 10.1128/iai.00077-10

Figure Lengend Snippet: FIG. 4. Myosin IIB localizes to sites of C. parvum invasion in con- fluent cells. (A) Confocal microscopy with dual labeling of nuclei (DAPI; blue) and myosin IIA (green) demonstrates that this isoform localizes primarily to stress fibers throughout C. parvum-infected cells. Arrowheads indicate C. parvum invasion sites identified by DAPI nu- clear stain. (B) The blue channel (DAPI staining) was digitally re- moved to reveal myosin IIA immunofluorescence. No myosin IIA was detected at any infection sites. (C) Confocal microscopy of C. parvum (C.p.; red)-infected confluent H69 cells reveals localized accumulation of myosin IIB (green) to sites of invasion. (D) Both blue (DAPI) and red (C. parvum) channels were removed to reveal localized accumu- lation of myosin IIB. Bars 10 m.

Article Snippet: For studies addressing accumulation of myosin isoforms and serine19-phosphorylated myosin light chain (MLC2) to C. parvum invasion sites, infected cells were fixed as described above and incubated with monoclonal antibodies against myosin IIB (CMII-23), pMLC2 (sc-19849-R; Santa Cruz Biotechnology), and the polyclonal anti-C. parvum antibody Cp2, followed by incubation with fluorophore-conjugated secondary anti-mouse and anti-rabbit antibodies.

Techniques: Confocal Microscopy, Labeling, Infection, Staining

Journal: Infection and Immunity

Article Title: Cholangiocyte Myosin IIB Is Required for Localized Aggregation of Sodium Glucose Cotransporter 1 to Sites of Cryptosporidium parvum Cellular Invasion and Facilitates Parasite Internalization

doi: 10.1128/iai.00077-10

Figure Lengend Snippet: FIG. 5. Phospho-MLC accumulates at C. parvum invasion sites. (A) Confocal immunofluorescence was utilized to assess the distribu- tion of phosphorylated MLC in C. parvum (red)-infected cells. (B) Nearly every infection site showed a strong colocalization of phos- pho-MLC (green). (C) Merged image of panels A and B demonstrates colocalization of C. parvum and phosphorylated myosin light chain. Phosphorylated myosin light chain aggregates at a region directly ad- jacent to the invading parasite as seen in the Z section. (D) An immunoblot using a phospho-MLC specific antibody detects increased phoshorylated myosin light chain in cultured cells following C. parvum infection. Actin was blotted as a loading control. Bars 20 m.

Article Snippet: For studies addressing accumulation of myosin isoforms and serine19-phosphorylated myosin light chain (MLC2) to C. parvum invasion sites, infected cells were fixed as described above and incubated with monoclonal antibodies against myosin IIB (CMII-23), pMLC2 (sc-19849-R; Santa Cruz Biotechnology), and the polyclonal anti-C. parvum antibody Cp2, followed by incubation with fluorophore-conjugated secondary anti-mouse and anti-rabbit antibodies.

Techniques: Infection, Western Blot, Cell Culture, Control

Journal: Infection and Immunity

Article Title: Cholangiocyte Myosin IIB Is Required for Localized Aggregation of Sodium Glucose Cotransporter 1 to Sites of Cryptosporidium parvum Cellular Invasion and Facilitates Parasite Internalization

doi: 10.1128/iai.00077-10

Figure Lengend Snippet: FIG. 7. Blebbistatin inhibits SGLT1 accumulation at C. parvum invasion sites. (A) Representative confocal micrographs demonstrate that SGLT1 accumulates to regions of C. parvum invasion in control, vehicle-treated cells (left column), while pretreatment of H69 cells with blebbistatin (center column) or ML-7 (right column) inhibits C. parvum-induced aggregation of SGLT1. The top row shows representative images of C. parvum-specific immunofluorescence invasion sites (green), the middle row shows SGLT1-specific immunofluorescence (red), and the bottom row shows the merged images of C. parvum and SGLT1 immunofluorescence. The inset shows a representative confocal x-z plane analysis of the respective boxed area. The x-z plane analysis demonstrates the localized accumulation of SGLT1 in control cells. Bars 20 m. (B) Quantitation of SGLT1 aggregation to infection sites from control, blebbistatin-treated, and ML-7-treated cells. Data are presented as means SE. , P 0.01 for comparison to control, vehicle-treated cells by ANOVA.

Article Snippet: For studies addressing accumulation of myosin isoforms and serine19-phosphorylated myosin light chain (MLC2) to C. parvum invasion sites, infected cells were fixed as described above and incubated with monoclonal antibodies against myosin IIB (CMII-23), pMLC2 (sc-19849-R; Santa Cruz Biotechnology), and the polyclonal anti-C. parvum antibody Cp2, followed by incubation with fluorophore-conjugated secondary anti-mouse and anti-rabbit antibodies.

Techniques: Control, Quantitation Assay, Infection, Comparison

Journal: Infection and Immunity

Article Title: Cholangiocyte Myosin IIB Is Required for Localized Aggregation of Sodium Glucose Cotransporter 1 to Sites of Cryptosporidium parvum Cellular Invasion and Facilitates Parasite Internalization

doi: 10.1128/iai.00077-10

Figure Lengend Snippet: FIG. 8. Electron microscopic analysis of invasion sites. (A) Scan- ning electron micrograph (SEM) from a representative invasion site in control, vehicle-treated H69 cells, showing a fully internalized parasite on the surface of the cell. The parasitophorous vacuole membrane consists of a typical double membrane structure (inset). (B) Scanning electron micrograph (SEM) from representative invasion site in bleb- bistatin-pretreated cells. The typical parasitophorous vacuole mem- brane is lacking; rather, a single, discontinuous membrane is present (inset). Over 25 electron micrographs were analyzed for both condi- tions. Seventy-six percent of the invasion sites from control cells were fully internalized and exhibited the bimembrane parasitophorous vac- uole membrane, while fewer than 10 percent of the blebbistatin- treated cells were fully internalized in a bimembrane parasitophorous vacuole membrane. The dense band formed in approximately 50% of invasion sites in both control and blebbistatin-treated cells. (C, D) Representative images of the plasma membrane of control (C) and blebbistatin-treated, uninfected (D) cells. No obvious ultrastructural morphological differences were noted in this region. (E) Representa- tive Immunogold electron micrograph (EM) detecting SGLT1 at C. parvum invasion sites in control, vehicle-treated cells. Gold particles localized to the host cell parasite interface (arrows), while few gold particles were detected at C. parvum invasion sites in blebbistatin- pretreated cells (F). (G) Quantitation of nanogold particles localized to invasion sites in control cells and blebbistatin-treated cells. Bars 0.5 m. Data are presented as means SE. *, P 0.02 for comparison to control, vehicle-treated cells by Student’s t test.

Article Snippet: For studies addressing accumulation of myosin isoforms and serine19-phosphorylated myosin light chain (MLC2) to C. parvum invasion sites, infected cells were fixed as described above and incubated with monoclonal antibodies against myosin IIB (CMII-23), pMLC2 (sc-19849-R; Santa Cruz Biotechnology), and the polyclonal anti-C. parvum antibody Cp2, followed by incubation with fluorophore-conjugated secondary anti-mouse and anti-rabbit antibodies.

Techniques: Control, Membrane, Clinical Proteomics, Quantitation Assay, Comparison