DOI: 10.5061/dryad.brv15dvh5
title: Dynamin-dependent entry of Chlamydia trachomatis is sequentially regulated by the effectors TarP and TmeA

description: Chlamydia invasion of epithelial cells is a pathogen-driven process
 involving two functionally distinct effectors – TarP and TmeA. They
 collaborate to promote robust actin dynamics at sites of entry. Here, we
 extend studies on the molecular mechanism of invasion by implicating the
 host GTPase dynamin 2 (Dyn2) in the completion of pathogen uptake.
 Importantly, Dyn2 function is modulated by TarP and TmeA at the levels of
 recruitment and activation through oligomerization, respectively.
 TarP-dependent recruitment requires phosphatidylinositol 3-kinase and the
 small GTPase Rac1, while TmeA has a post-recruitment role related to Dyn2
 oligomerization. This is based on the rescue of invasion duration and
 efficiency in the absence of TmeA by the Dyn2 oligomer-stabilizing small
 molecule activator Ryngo 1-23. Notably, Dyn2 also regulated the turnover
 of TarP- and TmeA-associated actin networks, with disrupted Dyn2 function
 resulting in aberrant turnover dynamics, thus establishing the
 interdependent functional relationship between Dyn2 and the effectors TarP
 and TmeA.
description: This repository contains spreadsheets compiling and analyzing the
 recruitment of proteins during <em>Chlamydia</em> invasion,
 quantification of <em>Chlamydia</em> invasion efficiency under
 various conditions, quantification of fixed-cell immunofluorescence, and
 dextran uptake data. Broadly speaking, data were obtained through
 fluorescence microscopy analysis of invading <em>C.
 trachomatis</em> and subsequent quantitative analysis of
 invasion-associated parameters.
description: # Dynamin-dependent entry of *Chlamydia trachomatis* is sequentially
 regulated by the effectors TarP and TmeA Author Information: Matthew D.
 Romero, Rey A. Carabeo Department of Pathology and Microbiology, College
 of Medicine, University of Nebraska Medical Center, Omaha, NE
 Corresponding Author Email:
 [rey.carabeo@unmc.edu](mailto:rey.carabeo@unmc.edu) Date of Collection:
 September 2022 - February 2024 Collected at: University of Nebraska
 Medical Center (2022-Current) Project Funding: This study was supported by
 funding from the U.S. National Institutes of Health, and the National
 Institutes of Allergy and Infectious Disease grants R01 AI065545 (R.A.C.).
 M.D.R. was further supported by a fellowship from the Seattle Chapter of
 Achievement Rewards for College Scientists (ARCS). --- ## Data and File
 Overview: This repository contains spreadsheets compiling and analyzing
 the recruitment of proteins during *Chlamydia* invasion, quantification of
 *Chlamydia* invasion efficiency under various conditions, quantification
 of fixed-cell immunofluorescence, and dextran uptake data. Within these
 data, there are five main subfolders, Actin Excel Summary Sheets, Dextran
 Uptake Assay Summary Sheets, Dyn2 Excel Summary Sheets, HCEC
 Immunofluorescence Sheets, and Invasion Assay Summary Sheets. Furthermore,
 this repository contains raw data containing .nd2 files that may be opened
 using ImageJ or Nikon NIS Elements ("Raw Data"), in addition to
 the finalized figures utilized in this manuscript ("Nature
 Communications Submission Files"). ### Actin Excel Summary Sheets *
 Actin Excel Summary Sheets contains four Excel spreadsheets containing
 summarized data regarding actin recruitment across various
 inhibitor/bacterial strain combinations. The first tab of the spreadsheet
 is titled Summary Sheet and is labeled to denote whether data corresponds
 to Recruitment Curves, Kinetics Data, Internalization Duration, or Violin
 Plot Inputs. ##### Summary Sheet Contents * Recruitment Curves data is
 comprised of the percent maximal mean fluorescence intensity for the
 indicated strain/inhibitor combination across all time points. Data is
 averaged across a minimum of 3 experiments, reporting the average of these
 experiments. * Kinetics Data reports the recruitment (Rec) or turnover
 slopes (Turn). Rec slopes correspond to the time point in which actin
 recruitment is initiated (start) and terminate at the time point in which
 actin recruitment is at its maximal intensity (max). Using the same actin
 recruitment curve, a corresponding turnover Turn slope is generated,
 derived from the timepoint of maximal actin intensity (max), terminating
 at the timepoint when actin recruitment returns to basal intensity end). *
 Internalization Duration data are derived from calculating the elapsed
 time between the start of actin recruitment, Start of Invasion,
 terminating when *Chlamydia* signal disappears, and End of Invasion,
 indicating that *Chlamydia* elementary bodies have achieved
 engulfment/internalization. Internalization duration calculations were
 performed for each recruitment event monitored and are reported according
 to the strain/inhibitor combination being observed. * Violin Plot Inputs
 contain kinetics data for recruitment and turnover, in addition to
 internalization duration data. These inputs compile all data from all
 strain/inhibitor combinations associated with the dataset and are
 formatted for further analysis in rStudio to generate violin/boxplot/dot
 plot figures for publication. ##### Other Tabs: * Excel documents also
 contain other tabs explicating the data found in the Summary Sheet tab in
 greater detail. These tabs are labeled corresponding to the
 strain/inhibitor combination being investigated in addition to the type of
 investigation being conducted (e.g., AcGFP Mock WT [ AcGFP=Protein,
 Mock=Inhibitor, WT=Strain]). Generally speaking, tabs either report
 recruitment intensity information (e.g., AcGFP Mock WT) or internalization
 duration information (e.g., AcGFP Mock WT IntDur). * The former (AcGFP
 Mock WT) contains a labeled header denoting the replicate number (e.g.,
 Rep 1) and date of data acquisition (e.g., "052723 [May 27, 2023]).
 The mean value of all recruitment events is reported in the column with
 the header Averaged Data, and the standard deviation for statistical
 analysis is in the column with the header Standard Deviation. These
 recruitment curves were further analysed as described in the Kinetics Data
 section of the Summary sheet; briefly, these data calculate the slopes
 corresponding to the rate of protein recruitment and turnover for each
 recruitment curve, labeled according to the replicate being measured and
 its date of acquisition. * The latter AcGFP Mock WT IntDur contains a
 labeled header denoting the replicate number (e.g., Rep 1) and date of
 data acquisition (e.g., "052723 [May 27, 2023]). The row labeled
 Internalization Duration is the quantified duration between the start of
 protein recruitment and pathogen engulfment. The row labeled "Start
 of Invasion denotes the time point in which protein recruitment starts,
 and the row labeled End of Invasion denotes the time point in which
 *Chlamydia* is engulfed. ### Dextran Uptake Assay Summary Sheet * Dextran
 Uptake Assay Summary Sheet contains one Excel spreadsheet that summarizes
 dextran colocalization data. This sheet contains color coding representing
 the replicate being analyzed and the date of replicate collection. These
 data can be used to cross-reference the raw data found in the Raw Data
 folder in the archive. Each section is divided according to the inhibitor
 being used (Mock, Ryngo, Dyni) or strain being applied (WT, dTmeA, dTarP).
 Tables report the number of dextran positive events versus the total
 number of events analyzed, quantifying the %dextran positive events. These
 values are summarized and color-coded to ease identification of the
 replicate from which the data is derived. All values from all replicates
 are collated into a two-column series (Column AI, AJ) that serves as input
 data for further analysis in rStudio. Starting in column AO, statistical
 tests are performed via pairwise T-tests, reporting the p-values that
 correspond to degrees of significance after Bonferroni post-testing.
 P-values are color-coded to identify statistically significant comparisons
 and the degree of significance indicated. ### Dyn2 Excel Summary Sheets *
 Dyn2 Excel Summary Sheets contains six Excel spreadsheets containing
 summarized data regarding Dynamin-2 recruitment across various
 inhibitor/bacterial strain combinations. Each spreadsheet is organized
 comparably to the layout described in the Actin Excel Summary Sheets
 section described earlier ### HCEC Immunofluorescence Sheets * HCEC
 Immunofluorescence Sheets contain nine Excel spreadsheets containing
 analysis of HCEC Z-stack data. Each spreadsheet is titled according to the
 strain and inhibitor combination being analyzed (e.g.,
 HCEC_FixedCellParticleTracking_dTarP_Dyni; HCEC=cell lineage,
 dTarP=Bacteria Strain, Dyni=Inhibitor). For each spreadsheet, the first
 tab Input Data contains data obtained from automated particle analysis via
 the ImageJ TrackMate plugin, which is available in the base FIJI package
 of ImageJ. Row 1 of this tab describes each measurement being depicted in
 the representative column. * Columns pertaining to spot ID, Track ID,
 Frame, and MFIs for Channels 1, 2, and 3 were each selected and copied
 onto the next tab titled Sorted Data. Spot IDs are unique ID labels
 attributed to a datapoint, and can be used to identify the exact section
 of the image being measured in the TrackMate-analyzed image. Track IDs are
 assigned for each particle, and are conserved for a given particle across
 all Z-slices; Track IDs enable researchers to quantify all relevant data
 associated with a given particle. A frame corresponds to the Z-slice from
 which the data is derived. Mean Ch1, Ch2, and Ch3 is the mean fluorescence
 intensity of Actin (Ch3), *Chlamydia* (Ch1), or Dyn2 (Ch2). Data are
 further refined to organize in a format suitable for input in rStudio,
 which is used to break down data according to Track ID (isolate data for
 each given particle) using the melt operator in rStudio. * Once data is
 broken down according to track ID, data are copied onto the Results tab.
 Here, data are represented as a line graph depicting the fluorescence
 intensity of actin, Dyn2, and *Chlamydia* across all Z-stacks. Events that
 are positive for recruitment of actin, Dyn2, or both are selected based on
 the fluorescence intensity and whether the trendlines (recruitment curves)
 trend together (run parallel). Numbers of positive events for actin, Dyn2,
 or both are compared to the total number of events to determine the
 %positive for each group. ### Invasion Assay Summary Sheets * Invasion
 Assay Summary Sheets contain eight Excel spreadsheets containing
 summarized data regarding invasion efficiency measurements across various
 inhibitor/bacterial strain combinations. Each spreadsheet is titled
 according to the treatment condition being analyzed, e.g.,
 Dyn2esiRNA_InvAssay_3RepsCompiled.xlsx reports invasion efficiency data
 from cells treated with Dyn2 siRNA or scramble control,
 Mock-Dynasore_InvAssay_3RepsCompiled reports invasion efficiency data from
 cells treated with Dynasore or DMSO mock control, etc. * Each spreadsheet
 is organized such that the *Chlamydia* strain used (CTL2WT, dTmeA, dTarP,
 DKO) and the condition (e.g., ScrRNA, Dyn2 siRNA) is clearly reported
 above the data series being analyzed. Each group has column headings
 describing the values depicted, e.g., In/Out refers to the number of
 bacteria analyzed (all bacteria in and out of the cell), Out refers to the
 number of bacteria on the external surface of the cell (all bacteria
 outside the cell), %InvEff refers to the number of bacteria that achieved
 internalization (all bacteria inside the cell). This value was calculated
 as follows: ((#In/Out - #Out)/ #In/Out) * 100 = %InvEff. Data are also
 color-coded according to the replicate being observed. Data from all
 replicates are reported at the bottom of each data frame, calculating the
 sum total of #In/Out events, sum #Out events, and the average %InvEff
 value, alongside the standard deviation of all %InvEff values. P-values
 are also calculated in a pairwise fashion for each condition to determine
 the significance between groups. ### Raw Data * Raw Data contains nine
 .tar.gz archives for raw images used in analysis throughout the study.
 Dyn2 esiRNA Data contains recruitment assays in cells treated with Dyn2
 esiRNA or scramble RNA. Dyn2-Ac CoRec Data contains recruitment assay data
 from cells co-transfected with fluorescent Dyn2 (GFP-Dyn2, RFP-Dyn2)
 alongside miRFP-670-LifeAct. Dyn2WT-DN contains recruitment assay data
 from cells singly transfected with either GFP-Dyn2 or RFP-Dyn2. Dynasore
 Data contains recruitment assays in cells treated with the Dynamin
 inhibitor Dynasore or mock DMSO control. EHop Rac Inhibitor Data contains
 recruitment assays in cells treated with the Rac1 inhibitor EHop or mock
 DMSO control. Invasion Assays Raw Data contains Z-stacks of cells treated
 with various inhibitors before infection with various *Chlamydia* strains.
 * Resubmission Data contains all new data added since initial submission
 to Nature Communications and was formatted such that previous archival
 efforts would not be disrupted with the addition of new data. This archive
 contains a variety of data, including Dextran uptake raw data, recruitment
 assays, invasion assays, and fixed-cell immunofluorescence data. Ryngo
 Data contains recruitment assays in cells treated with the Dynamin
 activator Ryngo 1-23 or mock DMSO control. Wortmannin Data contains
 recruitment assays in cells treated with the PI3K inhibitor Wortmannin or
 mock DMSO control. * Generally, files within these archives are in the
 .nd2 image format, which can be opened in ImageJ or NIS elements. Invasion
 efficiency, Dextran Uptake, and Fixed Cell immunofluorescence data are
 fixed-cell Z-stacks of cell monolayers infected with *Chlamydia* in the
 presence or absence of an inhibitor. Recruitment assays are 30-minute
 timecourses of live Cos7 cells infected with the indicated strain of
 *Chlamydia* in the presence or absence of an inhibitor. Empty cells do not
 signify missing or hidden data. All data are visible. --- ## Data-specific
 information: Recruitment Curves are calculated according to %max mean
 fluorescence intensity for each condition to enable normalization and
 conduct direct comparison across the multitude of conditions tested in
 this study. Kinetics Data report the rate as % change in intensity per
 second, using the %Max data found in Recruitment Curves as input data.
 Likewise, this methodology allows for rates of protein recruitment and
 turnover to be directly compared between conditions. Internalization
 Duration is as described in the corresponding section above. Raw data
 archives also contain representative kymographs visually depicting the
 parameters used to determine internalization duration. Violin Plot Inputs
 contain individual rates of recruitment ("Rec") and turnover
 (Turn) for input into rStudio for further analysis. Slope refers to the
 rate of protein recruitment or turnover (Fold change per second), and was
 calculated independently for each recruitment event, such that each entry
 within the "Violin Plot Inputs" tab represents a unique
 recruitment event. For each figure in the manuscript, a corresponding
 spreadsheet can be found in the Figures folder. Each spreadsheet
 containing the term Figure (e.g., Actin-AllIhnibitors_Figure_010721) has
 data from the All Replicates Summary tab from each of the spreadsheets
 found above and placed in a tab descriptively named to identify the
 protein/inhibitor/ strain combination from which the data is derived. Each
 Summary tab (e.g., Actin Untreated Summary) is further summarized into a
 single tab with all Raw fold recruitment values, along with their
 corresponding Standard Error of the Mean for each time point. These values
 were compiled into a scatterplot which shows the raw Fold Recruitment
 value at each timepoint +/- SEM. This process was repeated for each
 spreadsheet, containing all the recruitment plots (scatterplots) found
 throughout the manuscript. Each "Invasion Assay" spreadsheet
 contains a summary tab in which all replicates of the invasion assay are
 reported, including the raw numbers of cell-associated
 ("Inside/Outside") and invasion-incompetent
 ("Outside") *Chlamydia* particles, the raw invasion efficiency
 ("%Invasion efficiency", "%Inv Efficiency",
 "%InvEff", or "%Eff" each refer to this value), and
 the normalized invasion efficiency values ("Normalized %
 Invasion", "Normalized Data", or "Normalized"
 each refer to this value) for each experiment are reported in this summary
 tab. Also found within the summary tab is a statistical analysis using
 pairwise student's T-tests, via the Excel function "=TTEST"
 using a two-tailed paired T-test. --- ### Notes: For further details
 regarding the contents of this repository, methods used in their analysis,
 or other queries related to the material found herein, please contact Rey
 Carabeo ([rey.carabeo@unmc.edu](mailto:matthew.romero@unmc.edu)).