Journal: bioRxiv

Article Title: The Chlamydia trachomatis type III effector TarP coordinates a functional collaboration between the actin nucleators Formin 1 and Arp2/3 during invasion

doi: 10.1101/2021.03.18.436027

Figure Lengend Snippet: (A) Cos7 cells were transfected with GFP-Fmn1, mEmerald-mDia1, or mEmerald-mDia2 for 24hrs prior to infection with Chlamydia stained with CMTPX at MOI=20. Infection was monitored by live-cell confocal microscopy using a Leica SD6000 AF Spinning Disc microscope with a 60x (NA 1.40) objective. Images were obtained every 20 seconds for 30 minutes and compiled into videos in ImageJ to identify sites exhibiting colocalization of host formins (GFP) and Chlamydia (RFP). (B) Cos7 cells were mock-treated or pretreated with inhibitors against formin (10µM SMIFH2), Arp2/3 (100µM CK666) or both (CK+SMI) for 1hr, infected with C. trachomatis serovar L2 at MOI=50 and stained using the “in-and-out” method which distinguishes non-internalized EBs from total cell-associated EBs, as described in Materials and Methods. Results were normalized against mean invasion efficiency of mock treated cells (Untreated mean = 49% invasion efficiency) and plotted as normalized mean +/- SEM. Data was collected from 20 fields, with each field containing an average of 108 Chlamydiae. Statistical significance was determined by T- test. (C) Cos7 cells were transfected with GFP-actin or a GFP empty vector plasmid for 24hrs prior to mock-treatment or pretreatment with 10µM SMIFH2, 100µM CK666, or both for 1hr. Transfected cells were infected with Chlamydia stained with CMTPX at MOI=20 and imaged by quantitative live-cell imaging, collecting images every 20 seconds for 30 minutes. Recruitment events were isolated by selecting regions containing CMTPX Chlamydia and elevated actin-GFP fluorescence. Background fluorescence was subtracted, and fold recruitment was calculated as a function of the fold increase in mean fluorescence intensity of recruited GFP-actin compared to basal GFP-actin fluorescence. Detailed visualization of this process can be found in Fig. S1. Data is displayed as mean fold recruitment for each timepoint +/- SEM compiled from a minimum N=16 recruitment events. (D,E) All actin recruitment events used to create the averaged plot shown previously were individually divided into recruitment, fast turnover and slow turnover phases (Fig. S2). (D) Individual rates of recruitment were plotted on a violin plot with inset boxplot, reporting the median rate +/- SD for each inhibitor treatment condition. (E) Individual rates of fast turnover (Left) and slow turnover (Right) were plotted using the same method as recruitment, with fast and slow turnover plots grouped together according to condition. Violin plots contain a minimum N=16 individual rates. Statistical significance was determined by Kolmogorov-Smirnov test. All data are representative of at least 3 independent experiments, *** P ≤ 0.001.

Article Snippet: Fixed cells were labeled with a mouse monoclonal anti- Chlamydia LPS antibody (BioRad CF 6J12, Hercules, CA, USA), rinsed with 1x PBS, and fixed once more in 4% paraformaldehyde for 10 min. Next, cells were permeabilized using 0.1% (w/v) Triton X-100 for 10 minutes at room temperature, rinsed with HBSS and labeled with rabbit polyclonal anti- Chlamydia trachomatis antibody (Abcam ab252762, Cambridge, MA, USA).

Techniques: Transfection, Infection, Staining, Confocal Microscopy, Microscopy, Plasmid Preparation, Live Cell Imaging, Isolation, Fluorescence

Journal: bioRxiv

Article Title: The Chlamydia trachomatis type III effector TarP coordinates a functional collaboration between the actin nucleators Formin 1 and Arp2/3 during invasion

doi: 10.1101/2021.03.18.436027

Figure Lengend Snippet: (A,B) Cos7 cells were transfected with (A) GFP-formin 1 isoform 1B (GFP-Fmn1) or (B) GFP-Arp3 for 24 hours prior to mock-treatment or pretreatment with 10µM SMIFH2 or 100µM CK666 for 1 hour. Quantitative live-cell imaging of invading CMTPX- labeled Chlamydia (MOI=20) was performed as described in . Briefly, images were acquired once every 20 seconds for 30 minutes and assembled into videos. MFI of recruitment events were obtained, and background fluorescence subtracted, before quantifying and plotting the fold recruitment values +/- SEM of Fmn1 or Arp3 for each timepoint. Data was compiled from a minimum N=16 recruitment events. (C) Cos7 cells were co-transfected with GFP- Fmn1 and mCherry-Arp3 for 24 hours prior to infection with unlabeled C. trachomatis (MOI=20) followed by quantitative live-cell imaging of invasion. Image acquisition and processing was performed as described above with a few modifications. Since CMTPX and mCherry-Arp3 fluorescence overlap, unlabeled Chlamydia was used, thus recruitment events are defined simply as regions containing elevated GFP-Fmn1 fluorescence compared to local background. MFI of both GFP-Fmn1 and mCherry-Arp3 were obtained from the recruitment ROI, and background was subtracted from both GFP and RFP channels independently. Maximal MFI was derived from both channels and used to normalize fluorescence as percent maximal MFI for each protein. Normalized MFI values for each timepoint were compiled from 33 events, plotting mean %Max MFI +/- SEM. (D) Recruitment of mEmerald-mDia1 and mEmerald-mDia2 was monitored in the presence and absence of 100µM CK666 using the method described above for monitoring the recruitment of GFP-Fmn1 and GFP-Arp3. Data is displayed as mean fold recruitment for each timepoint +/- SEM compiled from a minimum N=22 recruitment events. (E,F) Kinetics of Fmn1 and Arp3 recruitment and turnover were analyzed in the presence and absence of 100µM CK666 or 10µM SMIFH2, respectively, using the same methodology described in . Violin plots contain a minimum N=16 individual rates, reporting the median rate +/- SD. Statistical significance for violin plots was determined by Kolmogorov-Smirnov test. All data are representative of at least 3 independent experiments, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Fixed cells were labeled with a mouse monoclonal anti- Chlamydia LPS antibody (BioRad CF 6J12, Hercules, CA, USA), rinsed with 1x PBS, and fixed once more in 4% paraformaldehyde for 10 min. Next, cells were permeabilized using 0.1% (w/v) Triton X-100 for 10 minutes at room temperature, rinsed with HBSS and labeled with rabbit polyclonal anti- Chlamydia trachomatis antibody (Abcam ab252762, Cambridge, MA, USA).

Techniques: Transfection, Live Cell Imaging, Labeling, Fluorescence, Infection, Derivative Assay

Journal: bioRxiv

Article Title: The Chlamydia trachomatis type III effector TarP coordinates a functional collaboration between the actin nucleators Formin 1 and Arp2/3 during invasion

doi: 10.1101/2021.03.18.436027

Figure Lengend Snippet: (A) Cos7 cells were transfected with mRuby-LifeAct for 24 hours prior to infection with either a TarP deletion mutant of C. trachomatis (ΔTarP) or a strain in which TarP was restored by cis -complementation ( cis -TarP) at MOI=20. Infection was monitored by live-cell confocal microscopy, obtaining images every 20 seconds for 30 minutes to identify sites exhibiting actin recruitment. (B) Cos7 cells were mock-treated or pretreated with inhibitors against formin (10µM SMIFH2), Arp2/3 (100µM CK666) or both (CK+SMI) for 1hr. Cells were infected with either ΔTarP or cis -TarP at MOI=50 and stained using the “in-and-out” method described in . Results were normalized against mean invasion efficiency of cis -TarP in mock treated cells ( cis -TarP untreated mean = 52% invasion efficiency) and plotted as normalized mean +/- SEM. Data was collected from 12 fields, with each field containing an average of 127 Chlamydiae. Statistical significance was determined by T- test. (C) Cos7 cells were transfected with mRuby-LifeAct for 24 hours prior to infection with either cis -TarP or ΔTarP at MOI=20. Quantitative live-cell imaging of invading Chlamydia was performed as described in . Briefly, images were acquired once every 20 seconds for 30 minutes and assembled into videos. MFI of recruitment events were obtained, and background fluorescence subtracted, before quantifying and plotting the fold recruitment values +/- SEM of mRuby- LifeAct for each timepoint compiled from a minimum N=22 recruitment events. (D,E) Kinetics of mRuby-LifeAct recruitment and turnover were analyzed for cis -TarP and ΔTarP using the same methodology described in . Violin plots contain a minimum N=22 individual rates, reporting the median rate +/- SD. Statistical significance was determined by Kolmogorov-Smirnov test. All data are representative of at least 3 independent experiments, * P ≤ 0.05, *** P ≤ 0.001.

Article Snippet: Fixed cells were labeled with a mouse monoclonal anti- Chlamydia LPS antibody (BioRad CF 6J12, Hercules, CA, USA), rinsed with 1x PBS, and fixed once more in 4% paraformaldehyde for 10 min. Next, cells were permeabilized using 0.1% (w/v) Triton X-100 for 10 minutes at room temperature, rinsed with HBSS and labeled with rabbit polyclonal anti- Chlamydia trachomatis antibody (Abcam ab252762, Cambridge, MA, USA).

Techniques: Transfection, Infection, Mutagenesis, Confocal Microscopy, Staining, Live Cell Imaging, Fluorescence

Journal: bioRxiv

Article Title: The Chlamydia trachomatis type III effector TarP coordinates a functional collaboration between the actin nucleators Formin 1 and Arp2/3 during invasion

doi: 10.1101/2021.03.18.436027

Figure Lengend Snippet: (A) Cos7 cells were transfected with Fmn1-mCherry or Arp3-mCherry for 24 hours prior to infection with either ΔTarP or cis -TarP at MOI=20. Infection was monitored by live-cell confocal microscopy, obtaining images every 20 seconds for 30 minutes to identify sites exhibiting Fmn1 or Arp3 recruitment. (B,C) Cos7 cells were transfected with (B) Fmn1-mCherry or (C) Arp3-mCherry for 24 hours prior to infection with either cis -TarP or ΔTarP at MOI=20. Quantitative live-cell imaging of invading Chlamydia was performed as described in . Briefly, images were acquired once every 20 seconds for 30 minutes and assembled into videos. MFI of recruitment events were obtained, and background fluorescence subtracted, before quantifying and plotting the fold recruitment values +/- SEM of Fmn1 or Arp3 for each timepoint compiled from a minimum N=18 recruitment events. (D,E) Kinetics of Fmn1-mCherry or Arp3-mCherry recruitment and turnover were analyzed for cis -TarP and ΔTarP using the same methodology described in . Violin plots contain a minimum N=18 individual, reporting the median rate +/- SD. Statistical significance was determined by Kolmogorov-Smirnov test. All data are representative of at least 3 independent experiments, *** P ≤ 0.001.

Article Snippet: Fixed cells were labeled with a mouse monoclonal anti- Chlamydia LPS antibody (BioRad CF 6J12, Hercules, CA, USA), rinsed with 1x PBS, and fixed once more in 4% paraformaldehyde for 10 min. Next, cells were permeabilized using 0.1% (w/v) Triton X-100 for 10 minutes at room temperature, rinsed with HBSS and labeled with rabbit polyclonal anti- Chlamydia trachomatis antibody (Abcam ab252762, Cambridge, MA, USA).

Techniques: Transfection, Infection, Confocal Microscopy, Live Cell Imaging, Fluorescence

Journal: bioRxiv

Article Title: The Chlamydia trachomatis type III effector TarP coordinates a functional collaboration between the actin nucleators Formin 1 and Arp2/3 during invasion

doi: 10.1101/2021.03.18.436027

Figure Lengend Snippet: (A) C. trachomatis engages and activates a multitude of host receptors whose activation is linked to the recruitment of Arp2/3 and actin in non-invasion contexts. It is currently unknown whether receptor activation contributes to actin remodeling during invasion. Concurrent with receptor engagement, Chlamydia secretes the effectors TmeA and TarP via Type III secretion system; each effector activates Arp2/3 through N-WASP or WAVE2, respectively. (B) In addition to recruiting Arp2/3, TarP signaling through Rac1 may also function to recruit mDia1 and/or mDia2. TarP also serves as a platform for actin nucleation by direct interaction with filamentous actin, and thus may be a scaffold for the variety of recruited actin nucleators (Arp2/3, Fmn1, mDia1/2) to act upon. (C) Collaboration between Arp2/3 and formin via their respective actin branching and elongating activities increases the rate of actin recruitment. Inhibition of Arp2/3 by CK666 or formin by SMIFH2 prevents this collaboration and constrains the actin network into either filamentous-only or branching-only networks. (D) The actin network generated by Chlamydia is rapidly turned over alongside the actin nucleators found within it. Although the precise mechanism by which this process occurs remains unknown, four host actin depolymerization factors (ADF-Cofilin, cyclase associated protein (CAP), Coronin, and β-thymosin) have been implicated by an siRNA screen to participate in invasion.

Article Snippet: Fixed cells were labeled with a mouse monoclonal anti- Chlamydia LPS antibody (BioRad CF 6J12, Hercules, CA, USA), rinsed with 1x PBS, and fixed once more in 4% paraformaldehyde for 10 min. Next, cells were permeabilized using 0.1% (w/v) Triton X-100 for 10 minutes at room temperature, rinsed with HBSS and labeled with rabbit polyclonal anti- Chlamydia trachomatis antibody (Abcam ab252762, Cambridge, MA, USA).

Techniques: Activation Assay, Inhibition, Generated