1. Volume 125, Issue 1, Pages 216-228 (July 2003)
Cryptosporidium parvum invasion of biliary epithelia requires host cell tyrosine phosphorylation of cortactin via c-Src 
Xian-Ming Chen, Bing Q Huang, Patrick L Splinter, Hong Cao, Guan Zhu, Mark A Mcniven, Nicholas F Larusso 
Gastroenterology 
Volume 125, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Figure 1
Host cell cytoskeleton accumulation at the parasite-host cell interface as assessed by confocal microscopy and TEM. H69 cells were exposed to C. parvum sporozoites for 2 hours followed by confocal microscopy and TEM. (A1–B3) Confocal images of costaining of C. parvum with actin or tubulin. Labels indicate staining of C. parvum or proteins or the mergers of the corresponding red and green panels. Inset shows a higher magnification of the boxed region in each image. (A1–A3) Actin recruited to the parasite-host cell interface. (B1–B3) No tubulin accumulation was found at the interface. (C1) A representative TEM image of the parasite-host cell interface. A direct parasite-host cell interaction was observed at the attachment site (arrow). (C2) A high magnification of the boxed region in C1. Filaments with the typical characteristics of actin in nature were accumulated at the parasite-host cell interface around the dense band (arrows). Bar = 1 μm.
Gastroenterology  , DOI: ( /S (03) )

3. Figure 2
Accumulation of c-Src and cortactin at the parasite-host cell interface as assessed by confocal microscopy. H69 cells were exposed to C. parvum sporozoites for 2 hours followed by confocal microscopy. Labels indicate staining of C. parvum or proteins or the mergers of the corresponding red and green panels. Inset shows a higher magnification of the boxed region in each image. (A1–B3) c-Src and cortactin accumulated at the parasite-host cell interface. (C ) Quantitative analysis of accumulation of c-Src and cortactin at the parasite-host cell interface. Bar = 5 μm.
Gastroenterology  , DOI: ( /S (03) )

4. Figure 3
c-Src and cortactin accumulation at the parasite-host cell interface as assessed by immunoelectron microscopy. H69 cells were exposed to C. parvum sporozoites for 2 hours followed by immunoelectron microscopy. (A1) Immunogold labeling of c-Src at the parasite-host cell interface. (A2) A higher magnification of the boxed region in A1. Many gold particles were found along the dense band or in the adjacent cytoplasm area. No obvious accumulation of gold particles was found along the membrane not directly associated with the parasite-host cell interface. (B1) Immunogold labeling of cortactin at the parasite-host cell interface. (B2) A higher magnification of the boxed region in B1 shows gold particle labeling of cortactin around the dense band. (C and D) Representative images of gold particle labeling of c-Src and cortactin in uninfected control cells. (E ) Quantitative analysis of immunogold particles for c-Src and cortactin around the dense band (in C. parvum-infected cells) or along the membrane (in uninfected control cells). ∗P < 0.05 compared with uninfected control cells. Bar = 0.1 μm.
Gastroenterology  , DOI: ( /S (03) )

5. Figure 4
Host cell protein phosphotyrosine at the parasite-biliary epithelial cell interface. H69 cells were exposed to C. parvum sporozoites for 2 hours followed by confocal microscopy and immunoprecipitation. (A1–A3) Accumulation of protein tyrosine phosphorylation at the parasite-biliary epithelial cell interface. Labels indicate staining of C. parvum or protein phosphotyrosine or the merger of the corresponding red and green panels. Inset shows a higher magnification of the boxed region in each image. (B) Tyrosine phosphorylation of cortactin, but not c-Src, was increased in infected biliary epithelial cells as assessed by immunoblot analysis of immunoprecipitates using an antibody to phosphotyrosine. P-Tyr, phosphotyrosine. Bar = 5 μm.
Gastroenterology  , DOI: ( /S (03) )

6. Figure 5
Inhibition of c-Src blocks C. parvum-induced actin and cortactin accumulation and tyrosine phosphorylation of cortactin at the parasite-host cell interface. H69 cells, or cells transfected with c-Src (K297M) or empty vector, were exposed to C. parvum sporozoites for 2 hours in the absence or presence of PP2 followed by confocal microscopy and immunoprecipitation. Images show the mergers of the corresponding red (proteins) and green (C. parvum) panels. Inset shows a higher magnification of the boxed region of the protein panel in each image. (A1) A representative immunofluorescence microscopy image shows no obvious actin accumulation in c-Src (K297M) transfected cells. (A2) Quantitative analysis shows only about 50% of the organisms infecting c-Src (K297M) transfected cells with actin accumulation at the parasite-host cell interface, whereas more than 95% of parasite attachment sites were found with actin accumulation in both the normal or empty vector controls. No significant accumulation of (B1) phosphotyrosine or (C1) cortactin was found in c-Src (K297M) transfected cells at the parasite attachment sites. (B2 and C2) Quantitative analysis shows a significant inhibition (up to 80%) of both (B2) phosphotyrosine and (C2) cortactin accumulation in c-Src transfected cells compared with the normal or empty vector controls. (D1) Inhibition of C. parvum-induced tyrosine phosphorylation of cortactin in c-Src (K297M) transfected cells or cells treated with a selective c-Src inhibitor, PP2, as assessed by immunoblot analysis of immunoprecipitates. Cortactin was also immunoblotted from the same nitrocellulose membrane to ensure that the same amount of total cortactin protein was immunoprecipitated for the analysis. (D2) Densitometric analysis of cortactin tyrosine phosphorylation from 3 separate experiments. P-Tyr, phosphotyrosine. #P < 0.05 compared with sham-infected cells. ∗P < 0.05 compared with normal control or empty vector control cells. Bar = 5 μm.
Gastroenterology  , DOI: ( /S (03) )

7. Figure 6
Inhibition of c-Src and cortactin diminishes C. parvum invasion of biliary epithelial cells. H69 cells were transfected with Cort (M3T → F)-RFP, c-Src (K297M), or empty vector, and some cells were fixed with paraformaldehyde. Both fixed and nonfixed cells were exposed to C. parvum sporozoites for 2 hours in the absence or presence of PP2 followed by confocal microscopy. Electron microscopy analysis was performed in cells stably transfected with c-Src (K297M) after a 2-hour incubation with C. parvum. (A ) Attachment assay in prefixed cells shows no significant difference of C. parvum attachment in all of the treated cells. (B) Attachment/invasion assay in nonfixed cells. A significant decrease of infection rate was found in cells treated with PP2, a relative c-Src inhibitor, or transfected with c-Src (K297M) or Cort (M3T → F) compared with the normal or empty vector controls. (C ) An immunofluorescence microscopy image shows that C. parvum (stained green) was rarely found in cells transfected with Cort (M3T → F) (in red), whereas many C. parvum were found in nontransfected cells (as outlined). (D and E ) C. parvum can only attach to the membrane surface of c-Src (K297M) transfected cells as shown by TEM and SEM. There was no obvious host cell membrane protrusion and dense-band formation at the parasite-host cell interface. (F and G) Electron micrographs show C. parvum attachment and invasion of empty vector control cells, with (F ) host cell membrane protrusion around the organism and (G) the dense band underlying the parasitophorous vacuole. (H ) Quantitative analysis of dense-band formation at the parasite-host cell interface in c-Src (K297M) transfected and control cells. ∗P < 0.05 compared with normal control or empty vector controls. Bar = 1 μm.
Gastroenterology  , DOI: ( /S (03) )

8. Figure 7
Accumulation of parasite-originated molecules at the parasite-host cell interface and inhibition of c-Src and cortactin accumulation as well as C. parvum attachment and invasion of biliary epithelial cells by antiserum against C. parvum sporozoite surface proteins. (A1 and A2) By immunoelectron microscopy using antiserum against C. parvum membrane proteins, specific gold articles were observed not only in the organism but also within the host cell around the dense-band area and at the parasitophorous vacuole membrane. (A2) A higher magnification of the boxed region of A1. (B1) By immunoblotting with the antiserum, multiple bands were recognized in C. parvum sporozoite lysates, whereas no band was detected in cell lysates of uninfected biliary epithelial cells. No significant accumulation of (C1) c-Src or (D1) cortactin was found at the parasite attachment sites in the presence of the antiserum in cells exposed to C. parvum for 2 hours. Images show the mergers of the corresponding red (proteins) and green (C. parvum) panels. Inset shows a higher magnification of the boxed region of the protein panel in each image. Quantitative analysis shows a significant inhibition (up to 75%) of both (C2) c-Src and (D2) cortactin accumulation in cells exposed to antiserum compared with the normal or serum controls. (E1 and E2) Attachment/invasion assay in fixed or nonfixed cells after a 2-hour exposure to C. parvum sporozoites. A dose-dependent decrease of both attachment and invasion rates was found in the presence of the antiserum compared with the normal or serum controls. ∗P < 0.05 compared with normal and serum control cells. Ctrl, control. Bar = 5 μm.
Gastroenterology  , DOI: ( /S (03) )

9. Figure 8
Endocytosis-related proteins, dynamin 2 and clathrin, and C. parvum invasion of biliary epithelial cells. Transfected or nontransfected H69 cells were exposed to C. parvum sporozoites for 2 hours followed by confocal microscopy. Labels indicate staining of C. parvum or proteins or the mergers of the corresponding red and green panels. (A1–B3) Immunofluorescence microscopy images show that no accumulation of dynamin 2 and clathrin occurs at the parasite-host cell interface. (C ) C. parvum (stained red) was found in both cells transfected with Dyn2 (K44A)-GFP (in green) and nontransfected cells. Both transfected and nontransfected cells were outlined. (D) Quantitative analysis shows a similar attachment/invasion of C. parvum to biliary epithelial cells transfected with dynamin 2 mutants and nontransfected cells. Bar = 5 μm.
Gastroenterology  , DOI: ( /S (03) )

10. Figure 9
Schematic model of C. parvum-induced host cell actin-based membrane alteration and dense-band formation. C. parvum attachment to host cell membrane activates c-Src and induces tyrosine phosphorylation of cortactin, thus causing actin rearrangement at the parasite attachment site. Host cell actin reorganization triggers membrane protrusion, induces dense-band formation, and consequently facilitates microbial invasion of host cells.
Gastroenterology  , DOI: ( /S (03) )