https://doi.org/10.5061/dryad.j9kd51cnf

Description of the data and file structure

The experimental efforts involved investigating the polarization of the microtubule-organizing center (MTOC) and multivesicular bodies (MVB) in Jurkat C3 cells. The study aimed to analyze the effects of different constructs related to the interference and expression of various versions of FMNL1 on the organization and signaling in the immune synapse. Data were collected to generate linear regression models, assess correlations between the polarization of the MTOC and MVB, and conduct statistical analyses to evaluate the significance of the observed effects. As can be seen in the different data sheets and analyses, the sample size for each of the conditions being compared is not identical. Therefore, there are empty cells in some of the conditions in the data tables. Nothing has been written to avoid potential interference with analysis tools. In general, in the attached data tables, the different study conditions are organized in columns, while each row represents a single analyzed Jurkat cell. This comprehensive approach helps to elucidate the role of FMNL1 in the formation and signaling of the immune synapse.

Files and variables

Files:

• Figure_2-figure_supplement_1_plot.xlsx
• Figure_4_plot.xlsx
• Figure_4-figure_supplement_1_plot.xlsx
• Figure_4-figure_supplement_2_plot.xlsx
• Figure_4-figure_supplement_5_regression_line.xlsx
• Figure_5_plot.xlsx
• Figure_7_plot.xlsx

Description: Include the different Excel files that are attached (each corresponding to a figure in the article).

• Figure_2-figure_supplement_1_plot.xlsx: The levels of mean fluorescence intensity were measured in Jurkat C3 cells transfected with various constructs of interest expressing YFP, which were subsequently labeled by immunofluorescence using an anti-FMNL1 antibody. In the first sheet, the different levels measured for each condition are presented in columns, including both YFP and anti-FMNL1. Each row represents a distinct cell. In all cases, "CELL-BACKGROUND" is indicated, as the non-specific background contribution was removed from each image. At the bottom, the means and sample sizes for each condition are displayed. The second sheet presents only the levels of anti-FMNL1.
• Figure_4_plot.xlsx: Experiments were conducted on the polarization of the MTOC and MVB in Jurkat C3 clones transfected with the constructs of interest, forming synapses with SEE-pulsed Raji cells. The first sheet of the Excel file includes the data for the MTOC. The second one includes the data for the MVB. The values shown in the table represent the polarization index (PI=A/B), calculated as explained in Figure 4-figure supplement 1 of the original article. To the right of the polarization values table, a table is shown containing the different p-values resulting from the analysis using an ANOVA test. The values were obtained using NIS-Elements software.
• Figure 4-figure_supplement_1_plot.xlsx: Experiments were conducted on the polarization of the MTOC in Jurkat C3 and P5 clones transfected with the constructs of interest, forming synapses with Raji cells pulsed or not with SEE. The values shown in the table represent the polarization index (PI=A/B), calculated as explained in Figure 4-figure supplement 1 of the original article. The values were obtained using NIS-Elements software.
• Figure_4-figure_supplement_2_plot.xlsx: Experiments were conducted on the polarization of the MTOC and MVB in Jurkat C3 clones transfected with the constructs of interest, forming synapses with SEE-pulsed Raji cells. The first sheet of the Excel file includes the data for the MTOC. The second one includes the data for the MVB. The values were obtained using NIS-Elements software.
• Figure_4-figure_supplement5_regression_line.xlsx: Data on the polarization of the MTOC and MVB are collected, from which a linear regression line is generated. The first sheet corresponds to the analysis of control YFP- Jurkat C3 cells. The second sheet pertains to the analysis of Jurkat C3 cells transfected with the shFMNL1-YFP construct, which interferes with the endogenous levels of FMNL1.
• Figure_5_plot.xlsx: Experiments were conducted on the polarization of the MTOC in Jurkat C3 and P5 clones transfected with the constructs of interest, forming synapses with SEE-pulsed Raji cells. The values shown in the table represent the polarization index (PI=A/B), calculated as explained in Figure 4-figure supplement 1 of the original article. The values were obtained using NIS-Elements software.
• Figure_7_plot.xlsx: Experiments were conducted to measure the reorganization of cortical F-actin in clones of Jurkat C3 cells forming synapses with Raji cells. In the Excel file, there is a table summarizing the values of the hypodense cortical F-actin region for each condition, with the mean highlighted in bold. The values were calculated by determining the ratio between the area of the F-actin hypodense region and the area of the total synaptic region. The measurements of these areas were obtained using ImageJ, based on three-dimensional projections generated from the XZ plane of the synapse oriented from the Jurkat cell, as explained in the original article.

Additional data dictionary / codebook

This section provides additional information to make the Excel files interpretable as an independent dataset. The data are organized by figure-related Excel files. In most spreadsheets, experimental conditions are arranged in columns and individual analyzed cells or cell conjugates are arranged in rows. The last rows usually contain summary information, including the arithmetic mean and the sample size (n) for each condition.

General notes on empty cells and rows

Some spreadsheets contain empty cells, empty rows, or empty columns. These empty cells are intentional and reflect the structure of the original data tables. They are mainly due to one or more of the following reasons:

1. Different experimental conditions contain different sample sizes.
2. Empty cells are used to keep columns aligned when one condition has fewer analyzed cells than another.
3. Empty rows are used as visual separators between individual measurements and summary rows.
4. Empty columns are used as visual separators between primary data tables, explanatory notes, and statistical summary tables.

Unless explicitly indicated, empty cells should not be interpreted as zero values. They should also not necessarily be interpreted as missing experimental measurements. In most cases, they simply indicate that no additional cell was analyzed for that specific condition because the corresponding sample size was smaller than in other conditions.

General abbreviations and terms

Jurkat C3:
Jurkat C3 T lymphocyte clone used as a control cellular model in the study.

Jurkat P5:
PKCδ-interfered Jurkat clone used in the study.

Raji:
Raji B cells used as antigen-presenting cells.

SEE:
Staphylococcal enterotoxin E. In the synapse assays, SEE-pulsed Raji cells were used to induce immune synapse formation with Jurkat T cells.

sin SEE:
Spanish abbreviation meaning “without SEE”. These conditions correspond to Jurkat/Raji conjugates in which Raji cells were not pulsed with SEE.

FMNL1:
Formin-like 1, the actin cytoskeleton-regulatory protein analyzed in the study.

FMNL1β:
Beta isoform of FMNL1.

shFMNL1:
Short hairpin RNA-mediated interference of FMNL1. In the corresponding transfection conditions, endogenous FMNL1 isoforms are depleted or strongly reduced.

FMNL1-WT:
Wild-type FMNL1β construct.

S1086A / FMNL1β-S1086A:
Non-phosphorylatable FMNL1β mutant in which serine 1086 is replaced by alanine.

S1086D / FMNL1β-S1086D:
Phosphomimetic FMNL1β mutant in which serine 1086 is replaced by aspartic acid.

YFP:
Yellow fluorescent protein. In these experiments, YFP fluorescence identifies cells expressing the corresponding YFP-tagged construct.

Control / Controles:**
Control Jurkat cells or control transfection condition, depending on the specific spreadsheet.

MTOC:
Microtubule-organizing center.

MVB / MVBs:
Multivesicular body / multivesicular bodies.

IS:
Immune synapse, defined as the contact interface between a Jurkat T cell and a Raji antigen-presenting cell.

MFI:
Mean fluorescence intensity.

ROI:
Region of interest used for image analysis.

CELL-BACKGROUND:
Background-corrected cell fluorescence intensity. The fluorescence value corresponds to the cell signal after subtracting the non-specific background contribution measured in the corresponding image.

PI:
Polarization index. This is a dimensionless value used to quantify the relative polarization of the MTOC or MVBs with respect to the immune synapse. Positive values indicate polarization toward the immune synapse, values close to zero indicate a more central or non-polarized distribution, and negative values indicate orientation away from the immune synapse.

A, B, C:
Distances used to calculate polarization indexes in the MTOC/MVB polarization analyses. These distances were measured in the image-analysis workflow from the cell geometry and the position of the MTOC or MVBs relative to the immune synapse. The variable C is the normalization distance used to scale the measurement to the size and geometry of each analyzed cell. For MVB polarization, the index corresponds to A/C. For MTOC polarization, the index corresponds to B/C. The resulting polarization index is dimensionless and normalized between negative and positive values according to the orientation of the analyzed structure relative to the immune synapse.

A/B:
Some spreadsheet headers refer to “A/B” because the original analysis table used this notation for the polarization measurement workflow. In these files, the numerical values under this label correspond to the polarization index values used for the corresponding figure. These are dimensionless normalized values and should be interpreted as polarization index measurements, not as raw fluorescence intensity values.

p-value:
Probability value obtained from the statistical comparison indicated in the corresponding row of the statistical summary table.

Significance:
Qualitative interpretation of the p-value. “sí” or “si” means that the comparison was considered statistically significant. “no” means that the comparison was not considered statistically significant.

mean:
Arithmetic mean of the values listed above in the same column.

sample size (n):
Number of individual cells, synapses, or cell conjugates analyzed for the corresponding condition.

standard desviation:
Standard deviation of the values listed in the corresponding column. The spelling “standard desviation” is preserved as it appears in the original spreadsheet.

File-specific data dictionary

Figure 2-figure supplement 1 plot.xlsx

This file contains fluorescence intensity measurements from Jurkat C3 cells transfected with different YFP-tagged FMNL1-related constructs and stained with an anti-FMNL1 antibody. The purpose of the file is to document the expression levels of the YFP-tagged constructs and the corresponding anti-FMNL1 immunofluorescence signal.

Sheet: YFP-FMNL1

This sheet contains paired fluorescence measurements for each condition. Each condition is represented by two adjacent columns:

• YFP: mean fluorescence intensity of the YFP-tagged construct expressed in the analyzed cell.
• FMNL1: mean fluorescence intensity of the anti-FMNL1 immunofluorescence signal in the same analyzed cell.

The experimental conditions are:

• Controles: control Jurkat C3 cells.
• shFMNL1: Jurkat C3 cells transfected with the FMNL1-interfering construct.
• FMNL1-WT: Jurkat C3 cells expressing wild-type YFP-FMNL1β.
• S1086A: Jurkat C3 cells expressing the non-phosphorylatable YFP-FMNL1β-S1086A mutant.
• S1086D: Jurkat C3 cells expressing the phosphomimetic YFP-FMNL1β-S1086D mutant.

The row labeled CELL-BACKGROUND indicates that values are background-corrected. Individual rows correspond to individual analyzed cells. The final rows provide the mean and sample size for each column.

Sheet: FMNL1

This sheet contains only the anti-FMNL1 immunofluorescence signal extracted from the previous paired measurements. Each column corresponds to one experimental condition:

• Controles
• shFMNL1
• FMNL1-WT
• S1086A
• S1086D

Values correspond to background-corrected anti-FMNL1 mean fluorescence intensity. Individual rows correspond to individual analyzed cells. The final rows provide the mean and sample size for each condition.

Figure 4 plot.xlsx

This file contains MTOC and MVB polarization index measurements in Jurkat C3 cells forming immune synapses with SEE-pulsed Raji cells. The values were obtained using NIS-Elements image-analysis software. The file also contains statistical comparison tables with p-values.

Sheet: MTOC datos

This sheet contains MTOC polarization index values. Each column corresponds to one experimental condition:

• Control
• shFMNL1
• FMNL1-WT
• S1086A
• S1086D

Each numerical value corresponds to one analyzed Jurkat/Raji synaptic conjugate. Values are dimensionless polarization index measurements. Positive values indicate MTOC polarization toward the immune synapse, values around zero indicate weak or absent polarization, and negative values indicate positioning away from the immune synapse.

The right-hand table contains pairwise statistical comparisons. The first column of that table indicates the comparison, the second column contains the p-value, and the third column indicates whether the comparison was considered significant.

Sheet: MVB datos

This sheet contains MVB polarization index values for the same type of experiment and the same experimental conditions:

• Control
• shFMNL1
• FMNL1-WT
• S1086A
• S1086D

Each numerical value corresponds to one analyzed Jurkat/Raji synaptic conjugate. Values are dimensionless MVB polarization index measurements. The right-hand table contains pairwise statistical comparisons, p-values, and significance calls.

Figure 4-figure supplement 1 plot.xlsx

This file contains MTOC polarization index measurements comparing Jurkat C3 and Jurkat P5 clones, with or without SEE stimulation.

Sheet: C3 y P5 graphs

Each column corresponds to one experimental condition:

• Control (C3): control Jurkat C3 cells forming synapses with SEE-pulsed Raji cells.
• Control (C3) sin SEE: control Jurkat C3 cells in the absence of SEE stimulation.
• shFMNL1 (C3): FMNL1-interfered Jurkat C3 cells.
• Control (P5): control condition in the Jurkat P5 clone.
• Control (P5) sin SEE: Jurkat P5 condition in the absence of SEE stimulation.
• shFMNL1 (P5): FMNL1-interfered Jurkat P5 cells.

Each numerical value corresponds to the MTOC polarization index of one analyzed cell conjugate. The final rows provide the mean and sample size for each condition.

Figure 4-figure supplement 2 plot.xlsx

This file contains MTOC and MVB polarization index measurements in Jurkat C3 cells transfected with the constructs of interest and forming synapses with SEE-pulsed Raji cells. The file also includes statistical comparison tables.

Sheet: MTOC plot

This sheet contains MTOC polarization index values. The experimental conditions are:

• Control
• shFMNL1
• FMNL1-WT
• S1086A
• S1086D

The row labeled A/B indicates that the values below correspond to the polarization index calculated from the distance-based image-analysis workflow. Each numerical value corresponds to one analyzed cell conjugate. The final rows provide the mean and sample size.

The right-hand table contains pairwise statistical comparisons. The comparison column indicates the two conditions being compared. The p-value column contains the corresponding p-value. The significance column indicates whether the comparison was significant.

Sheet: MVB plot

This sheet contains MVB polarization index values for the same experimental conditions:

• Control
• shFMNL1
• FMNL1-WT
• S1086A
• S1086D

The row labeled A/B indicates that the values below correspond to the polarization index calculated from the distance-based image-analysis workflow. Each numerical value corresponds to one analyzed cell conjugate. The final rows provide the mean and sample size.

The right-hand table contains pairwise statistical comparisons, p-values, and significance calls.

Figure 4-figure supplement 5 regression line.xlsx

This file contains paired MVB and MTOC polarization index measurements used to generate linear regression analyses. Each row contains paired measurements from the same analyzed cell conjugate.

Sheet: Regression Control MTOC vs MVB

This sheet contains paired MVB and MTOC polarization index values from control YFP-negative Jurkat C3 cells forming synapses.

Columns:

• MVBs polarization index: MVB polarization index for one analyzed cell conjugate.
• MTOC polarization index: MTOC polarization index for the same analyzed cell conjugate.

Each row corresponds to one cell conjugate for which both MVB and MTOC polarization could be quantified. The final rows provide the mean and standard deviation for each variable.

Sheet: Regression shFMNL1 MTOC vs MVB

This sheet contains paired MVB and MTOC polarization index values from shFMNL1-YFP Jurkat C3 cells forming synapses.

Columns:

• MVBs polarization index: MVB polarization index for one analyzed cell conjugate.
• MTOC polarization index: MTOC polarization index for the same analyzed cell conjugate.

Each row corresponds to one cell conjugate for which both MVB and MTOC polarization could be quantified. The final rows provide the mean and standard deviation for each variable.

Figure 5 plot.xlsx

This file contains MTOC polarization index measurements comparing Jurkat P5 and Jurkat C3 conditions after transfection with the indicated constructs.

Sheet: MTOC plot

Each column corresponds to one experimental condition:

• Control P5: control Jurkat P5 cells.
• shFMNL1 P5: FMNL1-interfered Jurkat P5 cells.
• FMNL1-WT P5: Jurkat P5 cells expressing wild-type FMNL1β.
• S1086A P5: Jurkat P5 cells expressing the non-phosphorylatable FMNL1β-S1086A mutant.
• S1086D P5: Jurkat P5 cells expressing the phosphomimetic FMNL1β-S1086D mutant.
• Controles C3: control Jurkat C3 cells.
• shFMNL1 C3: FMNL1-interfered Jurkat C3 cells.

Each numerical value corresponds to the MTOC polarization index of one analyzed cell conjugate. The final rows provide the mean and sample size for each condition.

Figure 7 plot.xlsx

This file contains measurements of cortical F-actin reorganization at the immune synapse. The values represent the relative size of the hypodense cortical F-actin region at the synaptic interface.

Sheet: Fig. 7 cortical F-actin region

Each column corresponds to one experimental condition:

• Control
• shFMNL1
• FMNL1-WT
• FMNL1β-S1086A
• FMNL1β-S1086D

Each numerical value corresponds to one analyzed synapse. The value represents the ratio between the area of the hypodense cortical F-actin region and the total synaptic region area. Therefore, higher values indicate a larger relative hypodense cortical F-actin region at the synaptic interface, whereas lower values indicate a smaller relative hypodense region. Values of 0 indicate that no measurable hypodense cortical F-actin region was detected for that analyzed synapse under the applied segmentation/measurement criteria.

The final rows provide the mean and sample size for each condition.

Code/software

To view the data, Microsoft Excel (version 2016 or later) is required. This software allows users to open and edit .xlsx files, perform preliminary analyses, and create visualizations. The workflow involved organizing the data into separate sheets within Excel, using built-in functions to calculate averages and conduct basic statistical analyses, as well as generating scatter plots and correlation graphs for clear result presentation. If any formulas or specific instructions were included, they are documented to assist users in navigating the data effectively.

Access information

Other publicly accessible locations of the data:

• None

Data was derived from the following sources:

• https://doi.org/10.7554/eLife.96942.2