The obligate intracellular pathogen Chlamydia trachomatis manipulates the host actin cytoskeleton to assemble actin-rich structures that drive pathogen entry. The recent discovery of TmeA, which, like TarP, is an invasion-associated type III effector implicated in actin remodeling, raised questions regarding the nature of their functional interaction. Quantitative live-cell imaging of actin remodeling at invasion sites revealed differences in recruitment and turnover kinetics associated with the TarP and TmeA pathways, with the former accounting for most of the robust actin dynamics at invasion sites. TarP-mediated recruitment of actin nucleators, i.e. formins and the Arp2/3 complex, was crucial for rapid actin kinetics, generating a collaborative positive feedback loop that enhanced their respective actin-nucleating activities within invasion sites. In contrast, the formin Fmn1 was not recruited to invasion sites and did not collaborate with Arp2/3 within the context of Tme-aassociated actin recruitment. Although the TarP-Fmn1-Arp2/3 signaling axis is responsible for the majority of actin dynamics, its inhibition had similar effects as the deletion of TmeA on invasion efficiency, consistent with the proposed model that TarP and TmeA act on different stages of the same invasion pathway.

The mean fluorescence intensity (MFI) of individual invasion event was measured for each timepoint alongside the local background MFI of a concentric region immediately outside the recruitment event with NIH ImageJ. Background MFI was subtracted from recruitment MFI for each timepoint and converted into fold recruitment by dividing the MFI of each timepoint against the baseline MFI, defined as the average MFI of five timepoints (100 sec) prior to the start of recruitment.

Datasets include representative time-lapse imaging videos from which data were acquired. In addition, results from statistical analyses, graphical outputs, etc are included.