Figure 5. The filament forming properties of vimentin are altered in a CPAF-dependent manner at late stages of infection.

A) A subset of infected cells contain vimentin filaments that are sensitive to detergent extraction. Top panel: Immunofluorescence images of HeLa cells infected with Rif^R (WT) at 52 hpi. Infected cells were either untreated or treated with 0.5% Tx100 for 5 minutes on ice, prior to fixation and immunostaining for vimentin localization. Vimentin cage forms around the inclusion of infected cells (arrowhead), which are sensitive to Tx100 extraction (arrow). Bottom panel: Infected HeLa cells were treated with 0.5 % Tx100 in the presence of 100 μM CPAF-inhibitory peptide (Pep2). Note the presence of inclusions with both detergent resistant (arrowhead) and detergent sensitive (arrow) vimentin filaments. Blue: Hoescht; Green: vimentin. B) The proportion of inclusions with detergent extractable vimentin filaments increases with infection times in a CPAF-dependent manner. Cells were infected with Rif^R (WT) or CPAF-mutant strains and the percentage of infected cells with Tx100 extractable vimentin (Figure 5A, bottom right) was quantified at 44 hpi, 52 hpi, and 60 hpi. All extractions were done in the presence of 100 μM Pep2. Cells with extractable intermediate filaments were not apparent in HeLa cells infected CPAF-deficient mutants. ***:p<0.001, SEM, n=3.