Microtubule-based cup-like structures appear during microparticle uptake in U2OS cells cultured on different...

Area of Science:

• Cell Biology
• Biophysics
• Cancer Research

Background:

• Substrate stiffness influences cancer cell behavior, affecting morphology, mechanics, and cytoskeletal organization.
• Understanding these mechanosensitive responses is crucial for cancer cell research.

Purpose of the Study:

• To investigate how substrate stiffness affects U2OS osteosarcoma cell morphology, mechanical properties, and microparticle uptake.
• To explore the role of cytoskeletal remodeling, particularly microtubules and associated proteins, in substrate-dependent cellular processes.

Main Methods:

• Culturing U2OS cells on polyacrylamide (PA) hydrogels (40 kPa) and rigid glass substrates.
• Assessing cell morphology and cytoskeletal organization via fluorescence microscopy.
• Measuring cell mechanical properties using Atomic Force Microscopy (AFM).
• Quantifying microparticle internalization using fluorescent microspheres and analyzing cytoskeletal structures via 3D imaging.

Main Results:

• Cells on glass exhibited larger surface area, more actin stress fibers, and increased stiffness compared to cells on 40 kPa PA gels.
• Microparticle uptake was significantly higher on 40 kPa PA gels, with cells forming microtubule-based "cup-like" structures around particles.
• IQGAP1 was identified as a key microtubule-associated protein localized to these structures, potentially involved in endocytosis.

Conclusions:

• Substrate stiffness significantly modulates U2OS cell behavior, including morphology, mechanics, and endocytic activity.
• Novel microtubule cup-like structures and the role of IQGAP1 in microparticle uptake on softer substrates were revealed.
• These findings emphasize the mechanosensitive nature of osteosarcoma cells and offer insights into cellular uptake mechanisms.