Early in infection, CHIR99021 multiple inclusions cluster tightly at the MTOC and remain associated as these inclusions begin to fuse. After fusion is complete, the single inclusion retains its close association with the MTOC as it continues to expand. The MTOC contains the cells centrosomes and acts as an organizing foci for the cell. Additionally, the MTOC acts as the nucleation point for cellular microtubules.

Host microtubules are polymerized in a polar fashion; the plus ends undergo rapid polymerization while the minus ends are anchored at the MTOC which allows for directional transport along the microtubules. We previously demonstrated that the the nascent chlamydial inclusion trafficks along microtubules using the microtubule motor protein dynein [5]. This study demonstrates that inclusion migration is a critical component for efficient fusion as both the dynein motor protein and intact microtubules are important for inclusion fusion. The requirement for both an intact microtubule network and the dynein motor protein along with the observation

that fusion takes place between closely adjacent inclusions suggests that migration to a central location in the cell is a mechanism to physically drive the inclusions together. This increases the likelihood that the fusogenic protein IncA on neighboring inclusions will interact, thereby enhancing a timely fusion. This hypothesis is further AZD8055 mw supported by the observation that when the minus ends of the microtubules are not anchored (EB1.84 Cytidine deaminase expressing cells) or not anchored at a single site in the cell (neuroblastomas), fusion was severely delayed. Interestingly, in neuroblastoma cells, the non fused inclusions appear to be in close proximity to each other however the resolution of fluorescence microscopy cannot resolve molecular level interactions. This suggests that for the chlamydial fusion protein IncA to interact with an IncA protein on a second

inclusion, the distance between them would likely need to be very small. Interestingly, fusion is only delayed under these circumstances suggesting that eventually multiple inclusions in the cell come in close enough contact for the IncA driven fusion system to mediate fusion. Overall our data support a model where nascent chlamydia-containing inclusions traffic along microtubules using the dynein motor protein to directionally traffic to the minus ends of microtubules. If the minus ends of the microtubules are anchored at the MTOC, then the multiple inclusions make close contact and are spatially arranged to encourage fusion. Interestingly, this trafficking takes place prior to IncA expression. Inclusion migration is rapid and occurs within the first few hours of infection however IncA is only expressed during the mid cycle of chlamydial infection, about 8 hours after infection [22].