PMC

Als3 and Ssa1 mediate binding to the N-cadherin–SEPT7 complex. Endothelial cells were infected for 90 min with the indicated strains of C. albicans or with S. cerevisiae containing the backbone vector (pADH1 or pYES2.1) or expressing C. albicans ALS3 (pALS3) or SSA1 (pSSA1). Next, the endothelial cell proteins that bound to these strains were isolated and then identified by immunoblotting.

SEPT7 knockdown inhibits endocytosis of C. albicans. (A) Endothelial cells transfected with either control or N-cadherin siRNA were infected with C. albicans for 90 min, after which the number of endocytosed organisms was determined by a differential fluorescence assay. Results are the means ± SD from three experiments, each performed in triplicate. *, P < 0.001 compared to endothelial cells transfected with the control siRNA. (B) Verification by immunoblotting of SEPT7 knockdown, which did not affect total N-cadherin content.

SEPT7 and actin colocalize during the endocytosis of C. albicans. Endothelial cells were infected with C. albicans for the indicated times and then fixed and stained for SEPT7 and actin. (A) The numbers of organisms in 15 HPF that were surrounded by SEPT7 alone, actin alone, or SEPT7 plus actin were counted at the indicated times after infection. Results are the means ± SD from three experiments, each performed in triplicate. (B) Confocal microscopic images of SEPT7 (a, e, i) and actin (b, f, j) accumulation. The merged images are shown in panels c, g, and k, and images of the corresponding microscopic fields viewed by differential interference contrast are shown in panels d, h, and l. Arrows indicate the SEPT7 and actin that accumulated around the hyphae. Scale bar = 5 µm.

C. albicans binds to a complex of N-cadherin and SEPT7. Endothelial cells were infected with C. albicans for the indicated time points, and the endothelial cell proteins that bound to the organisms, either directly or indirectly (bound), were isolated. The nonbinding proteins (total) were also collected. The bound and total N-cadherin and SEPT7 were identified by immunoblotting. (A) Representative immunoblot. (B) Densitometric analysis of 3 independent immunoblots comparing the relative amounts of bound to total protein. Results are means ± SD. (C) Endothelial cells were infected with C. albicans for 90 min and lysed, after which the lysates were subjected to immunoprecipitation (IP) with either control IgG or an anti-N-cadherin antibody. The presence of SEPT7 and N-cadherin in immunoprecipitated proteins was detected by immunoblotting.

Colocalization of N-cadherin and SEPT7 during the endocytosis of C. albicans. Endothelial cells were infected with wild-type C. albicans for the indicated times and then fixed and stained for N-cadherin and SEPT7. (A) The number of organisms in 15 high-power fields (HPF) that were surrounded by N-cadherin alone, SEPT7 alone, or N-cadherin plus SEPT7 were counted at the indicated times after infection. Results are the means ± standard deviations (SD) from three experiments, each performed in triplicate. (B) Confocal microscopic images of N-cadherin (a, e, i) and SEPT7 (b, f, j) accumulating around the same organism. The merged images are shown in panels c, g, and k, and images of the corresponding microscopic fields viewed by differential interference contrast are shown in panels d, h, and l. Arrows indicate the N-cadherin and SEPT7 that accumulated around the hyphae. Scale bar = 5 µm.

Effects of N-cadherin siRNA on C. albicans recruitment of endothelial cell N-cadherin and SEPT7. Endothelial cells were transfected with either control or N-cadherin siRNA and then infected with wild-type C. albicans for 90 min. (A, B) The cells were fixed and then stained for N-cadherin and SEPT7. (A) The number of organisms in 15 high-power fields that were surrounded by N-cadherin alone, SEPT7 alone, or N-cadherin plus SEPT7. Results are the means ± SD from three experiments, each performed in triplicate. (B) Confocal microscopic images of N-cadherin (a, e) and SEPT7 (b, f) accumulating around the same organism. The merged images are shown in panels c and g, and images of the corresponding microscopic fields viewed by differential interference contrast are shown in panels d and h. Arrows indicate the N-cadherin and SEPT7 that accumulated around the hyphae. Scale bar = 5 µm. (C) Representative immunoblot showing the effects of N-cadherin knockdown on the amount of endothelial cell N-cadherin and SEPT7 that bound to C. albicans hyphae. (D) Densitometric analysis of 3 independent SEPT7 immunoblots. Results are means ± SD. *, P ≤ 0.05 compared to cells transfected with control siRNA.

SEPT7 knockdown reduces the accumulation of N-cadherin around C. albicans. Endothelial cells were transfected with either control or SEPT7 siRNA and then infected with C. albicans for 90 min. (A, B) The cells were fixed and then stained for N-cadherin and SEPT7. (A) The number of organisms in 15 high-power fields that were surrounded by N-cadherin alone, SEPT7 alone, or N-cadherin plus SEPT7. Results are the means ± SD from three experiments, each performed in triplicate. (B) Confocal microscopic images of N-cadherin (a, e) and SEPT7 (b, f) accumulating around the same organism. The merged images are shown in panels c and g, and images of the corresponding microscopic fields viewed by differential interference contrast are shown in panels d and h. Arrows indicate the N-cadherin and SEPT7 that accumulated around the hyphae. Scale bar = 5 µm. (C) Representative immunoblot showing the effects of SEPT7 knockdown on the amount of endothelial cell N-cadherin and SEPT7 that bound to C. albicans hyphae. (D) Densitometric analysis of 4 independent N-cadherin immunoblots. Results are means ± SD. *, P < 0.05 compared to cells transfected with control siRNA.

Cytochalasin D inhibits the interaction of N-cadherin and SEPT7 with C. albicans. Endothelial cells were incubated with diluent alone (control) or cytochalasin D (cyto. D) and then infected with C. albicans for 90 min. (A, B) The cells were fixed and then stained for N-cadherin and SEPT7. (A) The number of organisms in 15 high-power fields that were surrounded by N-cadherin alone, SEPT7 alone, or N-cadherin plus SEPT7. Results are the means ± SD from five experiments, each performed in triplicate. (B) Confocal microscopic images of N-cadherin (a, e) and SEPT7 (b, f) accumulating around the same organism. The merged images are shown in panels c and g, and images of the corresponding microscopic fields viewed by differential interference contrast are shown in panels d and h. Arrows indicate the N-cadherin and SEPT7 that accumulated around the hyphae. Scale bar = 5 µm. (C) Representative immunoblot showing the effects of cytochalasin D on the amount of endothelial cell N-cadherin and SEPT7 that bound to C. albicans hyphae. (D) Densitometric analysis of 5 independent N-cadherin and SEPT7 immunoblots. Results are means ± SD. *, P < 0.05 compared to control cells; ^†, P < 0.05 compared to N-cadherin. (E, F) Coimmunoprecipitation results showing the effects of cytochalasin D on the association between SEPT7 and N-cadherin. (E) Representative immunoblot of cell lysates that were immunoprecipitated with either an anti-N-cadherin antibody or control IgG, separated by SDS-PAGE and then probed for the presence of SEPT7 by immunoblotting. (F) Densitometric analysis of 3 independent immunoblots. Results are means ± SD. *, P = 0.03 compared to control cells that had been infected with C. albicans in the absence of cytochalasin D.