Introduction: Mechanotransduction is an essential cellular process, important for cell motility and proliferation. Cells must integrate multiple signals to both generate and interpret traction forces through intracellular signaling cascades. Many experimental paradigms have been designed to investigate different aspects of mechanotransduction, such as the micropillar array device (mPAD), which utilizes the bending of soft polymer pillars to derive forces. However, these devices are limited in modelling the complexity of the extracellular matrix, especially by the restriction of single protein patterning. We have developed a novel technique, humidified microcontact printing (HµCP), which allows for the generation of multi protein patterns[3]. Combining these two techniques, we are able to make a multi patterned mPAD (MmPAD), allowing for the quantification of traction forces in response to juxtaposed stripes of proteins. In parallel, experiments were performed on glass surfaces to elucidate the receptor recruitment to various surface-bound proteins.
Materials and Methods: HµCP on mPAD devices and glass allowed for the patterning of alternating cues of various proteins and reference surfaces. Myoblast cells were transfected with fluorescent-tagged receptors, including a new DCC-mCherry construct. These cells were then seeded on the MmPADs, allowing for measurement of traction forces in a multi cue environment. Additionally, functional blocking experiments were performed on both glass and MmPADs, to elucidate the roles of specific receptor recruitment in mechanotransduction.
Results and Discussion:
First, we patterned mPADs with proteins of varying surface affinities, and established a correlation between cell surface affinity and paxillin recruitment. Thus, linking focal adhesion density with surface affinity.
Using HµCP, we created the MmPAD, the first high resolution, multi patterned mechanotransduction sensor. This novel integration of techniques allows for real time force measurements of single cells reacting to different proteins simultaneously. We find that traction forces on proteins changes when the cell is presented with multiple cues.
In parallel, on glass surfaces a novel receptor for surface-bound netrin-1 was discovered, by visualizing fluorescent-tagged receptor recruitment via TIRF microscopy. Finally, utilizing both the MmPAD and HµCP on glass surfaces, the roles of specific receptor recruitment in mechanotransduction can be investigated. Functional blocking experiments reveal a disconnect between traction force and cell preference; the protein with the highest traction forces is not necessarily the protein which cells prefer.
Conclusion: The presented sensor, for the first time, allows us to quantify both the magnitude and distribution of cellular traction forces on different proteins with a single cell resolution. The utilization of this novel sensor reveals that cellular response varies in multi cue environments. Additionally, the experiments on glass surfaces reveal novel receptor recruitment patterns. The presented data paves the way for future works in understanding the complexity of mechanotransduction, providing further insight into cellular navigation.
NSERC-CREATE NeuroEngineering; NSERC-CREATE Integrated Sensor Systems; McGill Nanotools Facility; Dr. Christopher Chen; Greta Thompson-Steckel; NSERC; Canadian Institute of Health Research
References:
[1] Yang, M.T., et al., Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity. Nat Protoc, 2011. 6(2): p. 187-213.
[2] Ricoult, S.G., et al., Tuning cell-surface affinity to direct cell specific responses to patterned proteins. Biomaterials, 2014. 35(2): p. 727-36.
[3] Ricoult, S.G., et al., Humidified Microcontact Printing of Proteins: Universal Patterning of Proteins no Both Low and High Energy Surfaces. Langmuir, 2014