Unexpected role of curvature sensing protein IRSp53 in mediating mechanotransduction during migration

Introduction:: The cell migration cycle proceeds with the formation of protrusions at the cell membrane followed by the establishment of actin stress fiber driven contractile machinery. While the process of cell migration is well documented on flat 2D substrates, how nascent protrusions aid 3D migration in fibrous extracellular matrix (ECM) of various diameters need further development. In particular, not much is known about the role of curvature sensing proteins in formation of protrusions and their role in cell migration. Here, using previously reported synthetic nanoscale suspended 1D fiber networks that recapitulate 3D migratory behavior, we investigated the role of membrane curvature sensing protein IRSp53 (Insulin receptor substrate protein of 53 kDa) in mediating the migration cycle. We found that IRSp53 deletion limited actin polymerization at the cell membrane resulting in both impaired protrusive activity and a lack of cell-length spanning actin stress fibers. These effects resulted in a reduction in contractile forces exerted by the cell, reduced compression of the nucleus, and a decoupling of the nucleus from the cell body during migration causing a shift from traditional stick-slip to gliding migratory mode. Overall, our work unravels a previously unknown role of IRSp53 protein spatially positioned at the extremities of the cell in controlling membrane dynamics, cytoskeletal contractility, and nucleus shapes deep inside the cell to mediate the migratory cycle.


Materials and Methods:: he non-electrospinning Spinneret Based Tunable Engineered Parameters (STEP) method was used to fabricate suspended two-layer fiber networks (large ‘base fibers’ and small diameter orthogonal). Briefly, polystyrene (PS, Scientific Polymer Products, Ontario, NY) of ≈2 million g mol⁻¹ molecular weight was dissolved in xylene (Thermo Fischer Scientific, Pittsburgh, PA) at 6% (w/w) concentration and 10% (w/w) concentration to prepare the solutions for spinning the ≈135 nm and ≈500 nm diameter fibers, respectively. To spin the ≈2 μm base fibers, a 5% (w/w) concentration solution of polystyrene of ≈15 × 10⁶ g mol⁻¹ molecular weight was used.

U-251 glioma cells used for this study were obtained from the Japanese Collection of Research Bioresources Cell Bank. The IRSp53 knock-out (KO) cells were generated by the CRISPR/Cas9 system. The cells were imaged using the AxioObserver Z.1 (with mRm camera) microscope (Carl Zeiss, Germany) at 20× for the protrusion, force, and migration studies and at 63× (water immersion objective) magnification for the protrusion coiling studies. The imaging intervals used were 5 min for the migration study, 3 minutes for the force studies, 2 min for the protrusion study, and 1 s for the coiling study. Cell forces were estimated by Nanonet Force Microscopy (NFM). Briefly, force vectors were directed from adhesion sites (found to occur at poles of cells attached to fibers) along the actin stress fibers inside the cell.


Results, Conclusions, and Discussions:: In this study we wanted to investigate the role of IRSp53 in regulating the migration cycle on 1D suspended fiber networks (Figure 1A). First, we investigated the effect of IRSp53 deletion on protrusive activity and coiling at the protrusion tip. We seeded IRSp53 KO and WT cells on networks comprising of orthogonally deposited large diameter “base fiber” (~2 µm) and smaller diameter “protrusive fiber” (either ~135 or ~500 nm). This configuration allowed us to quantify protrusive activity decoupled from bulk cell migration which was constrained along the base fiber. We found that although both WT and KO cells exhibited similar protrusion lengths, the KO cells extended the protrusions slower and displayed a smaller coil width at the protrusion tip on both protrusive fiber diameters tested (Figure 1B.ii). We then wondered if impaired protrusive activity at cell membrane tip correlated with an impairment in the actin-mediated contractile machinery. Using nanonet force microscopy we found that KO cells displayed a significant reduction in cell-length spanning actin stress fibers resulting in a reduction in contractile forces exerted and nuclear compression (Figure 1B.iii-v). Given the impairment in both protrusive activity and actin mediated contractile force generation we enquired if IRSp53 KO hindered migration on 1D fibers. We found IRSp53 deletion resulted in a decrease in migration speed and persistence and unexpectedly, led to a decoupling of the nucleus from the cell body (Figure 1B.vi). To understand our results further we developed a theoretical migration model comprising of two key parameters: the cell-fiber adhesiveness (r) and the maximal actin polymerization speed (β). Based on our protrusion and force results we proposed that the KO cells have a lower β placing them in a slower, smooth migration regime, while WT cells have a higher level of 𝛽, placing them in the stick-slip migration regime. We experimentally confirmed these different migration regimes on our suspended fiber networks and the differential actin polymerization rate using fluorescence speckled microscopy (Figure 1B.i). Overall, our study highlights the crucial role played by IRSp53, previously thought to primarily drive membrane curvature, in mediating contractility and the migration cycle on 1D fibrous environments.


Acknowledgements (Optional): : The authors thank Prof. Masahito Ikawa (Osaka University) and Prof. Taro Kawai (Nara Institute of Science and Technology) for the CRISPR/Cas9 system, Prof Naoyuki Inagaki, and Dr. Takunori Minegishi for the Halo-tagged actin, and all the members of the laboratories for technical assistance and helpful discussions. This work was supported by grants from the JSPS (KAKENHI JP20H03252, JP20KK0341, and JP21H05047) and JST CREST (JPMJCR1863) to S.S, and JST CREST (JPMJCR1863) to Y.M.K. N.S.G. is the incumbent of the Lee and William Abramowitz Professorial Chair of Biophysics and acknowledges support by the Ben May Center for Theory and Computation and the Israel Science Foundation (Grant No. 207/22). This research was made possible in part by the historic generosity of the Harold Perlman Family. A.S.N. acknowledges partial funding support from National Science Foundation (NSF, Grant No. 1762468 and 2119949). A.S.N. and B.B. acknowledge the Institute of Critical Technologies and Science (ICTAS) and Macromolecules Innovative Institute (MII) at Virginia Tech for their support in conducting this study

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