JC4 Slides-notes

Introduction

  • Overview of the assignment and organization within the group

  • Emphasis on preparing a 5-minute presentation

Group Assignment Instructions

  • Find your group: Locate your assigned figure for the assignment

  • Presentation Preparation:

    • Title: Should convey the essential message of the slide

    • Method: Brief explanation of the experiment

    • Interpret: Discussion of the results obtained

    • Critique: Questions and suggestions for further discussion

  • Class Assignment: ‘Tweetorial’ format encouraged, with memes allowed

  • Personal Note: Mention of snacks (candy) brought for class

  • Art Exhibits: Brief reference to visiting Dali and O’ exhibits at MFA

Scientific Content Summary

Page 2: Key Terms

  • Wnt5A: A ligand from the Wnt family that interacts with ROR2

  • ROR2: Receptor Tyrosine Kinase-like Orphan Receptor 2, involved in signaling pathways

  • JNK: Jun N-terminal Kinase

  • Cytonemes: Specialized cellular projections for protein exchange between cells

Page 3: Figure 1 Description

  • CAF Filopodia: Cancer-associated fibroblasts (CAFs) use filopodia to transfer Wnt ligand and ROR2 to AGS cells.

  • Comparison: Presentation of CAFs from different patients against normal fibroblasts.

  • Localization: Demonstrates the proximity of Wnt ligand and receptor on cytonemes using immunofluorescence techniques.

Page 4: Supplementary Material

  • Referencing Movie S1 that corresponds to Figure 1.

Page 5: Figure 2 Observations

  • Colocalization of ROR2 and Wnt5A: Through imaging using GFP and Cherry labeling.

  • Complex Behavior: Time-lapse showing molecular transfer during interaction.

  • Critique Suggestions: Need for clearer visuals on cell contact mechanisms and transfer processes, along with possible inclusion of additional time points in video.

Page 6: Figure 2 Continued

  • Co-culturing Details: Insight into CAF and AGS cells interactions with expressing Wnt and ROR2.

  • Timelapse Observations: Visualization of complexes entering recipient AGS cells through cytonemes.

  • Conclusion on Transfer Mechanics: Complex transfer directly via cytonemes, emphasizing the ability to reach long distances.

Page 7: Figure 3 A-C

  • Endocytosis of ROR2 by AGS: Confirmed by colocalization with endosomes indicating that ROR2 was internalized.

  • Fluorescence Tracking: Distinction made using membrane markers and pH antibodies to indicate internalized state.

Page 8: Further Insights on Endocytosis

  • Studies on Primary Gastric Cancer CAM Cells: Examining internalization of ROR2 into AGS cells.

  • Dynamin Role in Endocytosis: Evidence suggests that dynamin is crucial for the endocytosis mechanism of ROR2.

Page 9: Figure 4 - Activation of JNK

  • ROR2 Activation: Assay indicating kinase activity via cellular localization.

  • Quantitative Overview: Assessing co-culture impacts on kinase activation.

Page 10: Figure 5 - Actin Polymerization

  • Observations of Actin Dynamics: Effects of ROR2 on actin polymerization in AGS cells.

  • Control Comparisons: Analysis of results using dominant negative ROR in experiments.

Page 11: Figure 6 - Migration Assessments

  • Migration Experiments: Evaluation of AGS and CAF interactions regarding migration potentials.

  • Zebrafish Model: Illustrates how ROR2 transfer influences proliferation and migration.

Page 12: Key Takeaways

  • Trade of ROR2 and Wnt Complexes: Indicating potential impacts on metastatic behavior and migratory capabilities of tumor cells.

  • Role of Cytonemes: Highlight the importance of cytonemes in complex transfer processes.

  • Significance of ROR2 Internalization: Discussing how ROR2's endocytosis allows retention of kinase activity in the cytoplasm.