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First published online 5 August 2003
doi: 10.1242/jcs.00695

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Chlamydia trachomatis uses host cell dynein to traffic to the microtubule-organizing center in a p50 dynamitin-independent process

Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, NIAID, NIH, Rocky Mountain Laboratories, Hamilton, MT 59840, USA

View larger version (91K): [in a new window]   Fig. 1. Nascent inclusions of LGV and non-LGV serovars of C. trachomatis localize in the vicinity of the MTOC. Cos-7 cells were infected with C. trachomatis serovar L2 (LGV) or serovar D (non-LGV) chlamydial EBs at an MOI of 50. Cells were fixed 5 hours postinfection and stained via indirect immunofluorescence simultaneously with anti-L2 EB (red) and anti-ß-tubulin antibodies (green). The fluorescent staining revealed that nascent inclusions of either serovar aggregated at a single site corresponding to the MTOC (arrows). In dividing cells in which the MTOC has been duplicated, nascent inclusions were observed to localize at both spindle poles (arrowheads). Bar, 10 µM.

View larger version (98K): [in a new window]   Fig. 2. Aggregation is dependent on host cell microtubules but does not require the actin cytoskeleton or an intact Golgi apparatus. Cos-7 cells were infected with C. trachomatis L2 for 1 hour before treatment with the indicated drug. Infected cells were incubated with nocodazole for 4 hours before fixing and staining for C. trachomatis and tubulin. In these cells the chlamydial early inclusions did not aggregate at a single perinuclear site but remained widely dispersed throughout the cytoplasm (A). The tubulin staining shows that the microtubule network (green) was disrupted (B). Cells transfected with GFP-Golgi were infected and treated with brefeldin A for 4 hours. The cells were fixed and stained for chlamydiae (C) and observed simultaneously with GFP signal. The nascent inclusions aggregated normally even thought the Golgi was dispersed, as can be seen by the dispersed GFP-Golgi signal (D). Disruption of the actin cytoskeleton with cytochalasin D also does not inhibit chlamydial aggregation. FITC-phalloidin staining of the F-actin cytoskeleton shows that the actin cytoskeleton has been disrupted (F) but the nascent inclusions are still aggregated at a single site within the cell (E). Bar, 10 µM.

View larger version (93K): [in a new window]   Fig. 3. Chlamydial protein synthesis is required for aggregation at the MTOC. Cos-7 cells were infected for 5 hours with C. trachomatis in the absence (control) or presence of chloramphenicol or rifampicin and stained simultaneously with anti-L2 and anti-ß-tubulin antibodies. In the control cells the majority of nascent inclusions migrate to a single site (arrowheads) corresponding to the MTOC as can be seen in the merged image. However, the chlamydial inclusions do not migrate to the MTOC and remain scattered throughout the cytoplasm when the infection is carried out in presence of chloramphenicol or rifampicin. Bar, 10 µM.

View larger version (104K): [in a new window]   Fig. 4. Chlamydiae are capable of rapid migration in host cells. Chlamydial migration was inhibited after entry by treating infected cells with nocodazole for 4 hours. This allowed time for chlamydial protein synthesis and modification of the inclusion membrane. Nocodazole was replaced with complete media and cultures were further incubated for 15 minutes or 1 hour. Simultaneous visualization of cells stained with antibodies to tubulin and L2 EBs revealed that after 15 minutes of nocodazole washout a few microtubules had reformed and there was an obvious MTOC (arrowheads). The merged image shows many of the nascent inclusions are aggregated at this site. After one hour the microtubule network is nearly restored. The tubulin staining shows two MTOC in this cell (arrowheads). In the merged image the majority of the chlamydial staining localizes to one or the other MTOC. Bar, 10 µM.

View larger version (43K): [in a new window]   Fig. 5. Simultaneous visualization of host cell vesicle trafficking and chlamydial migration. Host vesicles were visualized by labeling cells with C6-NBD-ceramide and chlamydiae were visualized by intrinsically labeling EBs with CMTMR. Both the host vesicles and chlamydiae were observed simultaneously via time-lapse confocal microscopy. Time-lapse images shown were taken at 30-second intervals and show a vesicle (green; arrowheads) and a chlamydial nascent inclusion (red; arrowheads) traveling parallel to one another and migrating at approximately the same speed. Bar, 2.5 µM.

View larger version (105K): [in a new window]   Fig. 6. Dynein colocalizes with nascent inclusions by 5 hours postinfection and remains associated with the inclusion as late as 24 hours postinfection. Cos-7 cells were infected with C. trachomatis for 5 or 24 hours in the presence or absence of nocodazole. Cells were stained for simultaneous visualization with mAb DIC 70.1 to visualize the dynein intermediate chain and a polyclonal antibody to L2 EBs to visualize the nascent inclusions. By 5 hours postinfection, the dynein antibody revealed staining consistent with it being localized along microtubules, as well as associated with the aggregated chlamydiae. In cells treated with nocodazole, dynein staining of the microtubules is absent and much of the staining is evident around the nuclear envelope. The nascent inclusions show dramatic dynein recruitment even in the presence of nocodazole. Dynein remains associated with the mature chlamydial inclusion even after infection for 24 hours. Bars, 10 µM.

View larger version (117K): [in a new window]   Fig. 7. Chlamydial migration is not inhibited by disruption of the dynactin complex due to overexpression of p50 dynamitin. Cos-7 cells were transiently transfected with GFP-dynamitin before infection with C. trachomatis or labeling with Alexa-Tf or Alexa-CTX. Cells expressing GFP-dynamitin (green in merged image) show no defects in chlamydial migration. The nascent inclusions stained with an antibody to L2 EBs (red in merged image) aggregate at a single perinuclear site in GFP-positive cells (arrowhead). Transiently transfected cells incubated with Alexa-Tf (red in merged image) show that Tf accumulated normally at a peri-Golgi region (arrowheads) in untransfected cells (no green signal in merged image) but Tf trafficking was inhibited in a neighboring cell expressing GFP-dynamitin (green cell in merged image). Similar results were observed for Alexa-CTX trafficking. CTX (red in merged image) was delivered to the Golgi (arrowhead) in the untransfected cell (no green signal in merged image) but delivery was abolished in GFP-dynamitin-expressing cells (green in merged image). Bars, 10 µM.

View larger version (77K): [in a new window]   Fig. 8. Microinjection of antibodies to the dynein intermediate chain abolishes chlamydial migration and aggregation. Monoclonal antibodies to the dynein intermediate chain were injected into the cytoplasm of Cos-7 cells and subsequently infected with C. trachomatis. The injected antibodies were detected with AlexaFluor-488-conjugated secondary antibodies (green) and chlamydial nascent inclusions were stained with antibodies to L2 EBs and detected with AlexaFluor-594 conjugated secondary antibodies (red). Chlamydial migration and aggregation was abolished in cells injected with a monoclonal antibody to dynein (green cells). Uninjected cells (no green signal in merged image) on the same coverslip show normal chlamydial migration (arrowheads). Injection of an irrelevant antibody, R. rickettsii, or an antibody to the positive directed microtubule motor protein, kinesin, had no effect on chlamydial movement as chlamydiae aggregated normally (arrowheads) at the MTOC of both injected (green cells) and noninjected cells (no green signal in merged image). Bars, 10 µM.

View larger version (127K): [in a new window]   Fig. 9. The nascent inclusion recruits p150(Glued) and this recruitment is not inhibited by nocodazole treatment or over expression of p50 dynamitin. Simultaneous visualization of p150(Glued) and the chlamydial nascent inclusions by indirect immunofluorescent microscopy show colocalization of p150(Glued) with the aggregated chlamydiae after infection for 5 hours. Inhibition of chlamydial migration with nocodazole does not block association with p150(Glued). Furthermore, colocalization of p150(Glued) and Chlamydia is not disrupted by overexpression of GFP-dynamitin (blue in the merged images). Bar, 10 µM.

View larger version (36K): [in a new window]   Fig. 10. Model of molecular components in chlamydial migration. Model adapted from Hirokawa (Hirokawa, 1998) shows the differences between classical dynein-dynactin-dependent microtubule movement and that which we propose to be used by C. trachomatis.

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