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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session C

Poster F8 - IQGAP1 Regulates HeLa Cell Mechanosensing, shape and Motility

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

AS

Alberto Salgado, n/a

Undergraduate
Worcester Polytechnic Institute, United States

Co-Author(s)

PW

Pengbo Wang, n/a

Undergraduate
Worcester Polytechnic Institute, United States

Last Author(s)

QW

Qi Wen

Professor
Worcester Polytechnic Institute
Worcester, Massachusetts, United States

Introduction: The IQ Motif Containing GTPase Activating Protein 1 (IQGAP1) is a scaffold protein involved in cellular activities such as invasion, migration, and morphology. It is ubiquitously expressed in multiple cell lines across the human body and is a necessity for normal cell function. In this study, we used HeLa cells, an immortal human epithelial cancer cell line derived from cervical cancer, which on average, shows a high expression of IQGAP1 protein compared to other cells in the cervix. Cancer cells can sense the stiffness adjustments of their environment, in this case the PAA gel, and can adapt their behavior accordingly. By knocking down IQGAP1 in HeLa cells, we compared their migration speed and traction force with non-knockdown cells to understand cancer spread and aid in metastasis prevention research. Previous data showed a significant decrease in traction force with IQGAP1 knockdown, leading us to hypothesize a similar reduction in migration speed.

Materials and

Methods: HeLa cells were cultured in a controlled environment until it reached a confluency of 80-90%. Fluorescent beads of 0.1 um in diameter were placed on a coverslip. The cells were plated on polyacrylamide gels (PAA) of different stiffness to mimic human cervix tissue. The gel stiffness prepared were: 2, 7.5, 13, 20, and 40 kPa. The gel substrate was then placed on the bead coated slip before being placed on a glutaraldehyde slide inside a 60mm petri dish. After the gels polymerized, they were coated in Sulfo-SANPAH, a chemical used for cell-surface protein crosslinking that will allow the IQGAP1 protein to link together. The gel is also coated in collagen (9.37mg/mL) to support cell adhesion and proliferation. Before plating HeLa cells, they were subjected to IQGAP1 suppression by using Lipofectamine 3000. Phase contrast images were taken of single cells to compare cell area, aspect ratio, and circularity using free hand tool in ImageJ. Traction force microscopy was done by taking fluorescent images of beads underneath the HeLa cell attached to the gel. To detach the cells from the gel surface, trypsin is added. Fluorescent images were taken again in the same location to check the displacement of the beads. A MATLAB script was used to measure the displacement of the fluorescent beads before and after cells were released using trypsin.

Results, Conclusions, and Discussions: Cell imaging shows that IQGAP1 knockdown has steady cell area across the 5 gel stiffness with low fluctuations. Compared to non-knocked IQGAP1 HeLa cells, their area increased until 13kPa when it reached a plateau and maintained a similar area for 20 and 40 kPa. For the aspect ratio, the opposite is shown. IQGAP1 knockdown starts low and increases as the stiffness of the gel increases. Non-knockdown HeLa cells maintain their average aspect ratio of ~2 with a slight decrease in the 13kPa gel. Circularity of IQGAP1 knockdown starts high at 2kPa and gradually decreases as gel stiffness increases. HeLa cells maintained a similar circularity of ~0.4. The maximum traction stress of HaLa cells increased as the gel stiffness increased. However, it was the opposite for IQGAP1 knockdown. IQGAP1 knockdown started with slightly greater traction stress than HeLa cells in the 2kpa gel but decreased as the stiffness increased.
The HeLa cells shows that it can sense the stiffness of the gel and gradually increase their area while IQGAP1 knockdown stays at a similar area. This indicates that suppressing IQGAP1 expression decreases the cells mechanosensing abilities. The maximum traction stress of IQGAP1 knockdown is relatively low compared to that of HeLa cells with a normal expression of IQGAP1. Future research on IQGAP1 will be done on visualizing F-Acting staining Furthermore, IQGAP1 knockdown reduces the cells' mechanosensing ability, significantly decreasing traction force. This highlights IQGAP1's crucial role in cells' response to substrate stiffness, providing insights for future cancer metastasis research.

Acknowledgements (Optional):