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

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).