Actin Filaments Couple the Protrusive Tips to the Nucleus through the I‐BAR Domain Protein IRSp53 during the Migration of Cells on 1D Fibers

Abstract

The cell migration cycle, well-established in 2D, proceeds with forming new protrusive structures at the cell membrane and subsequent redistribution of contractile machinery. Three-dimensional (3D) environments are complex and composed of 1D fibers, and 1D fibers are shown to recapitulate essential features of 3D migration. However, the establishment of protrusive activity at the cell membrane and contractility in 1D fibrous environments remains partially understood. Here the role of membrane curvature regulator IRSp53 is examined as a coupler between actin filaments and plasma membrane during cell migration on single, suspended 1D fibers. IRSp53 depletion reduced cell-length spanning actin stress fibers that originate from the cell periphery, protrusive activity, and contractility, leading to uncoupling of the nucleus from cellular movements. A theoretical model capable of predicting the observed transition of IRSp53-depleted cells from rapid stick-slip migration to smooth and slower migration due to reduced actin polymerization at the cell edges is developed, which is verified by direct measurements of retrograde actin flow using speckle microscopy. Overall, it is found that IRSp53 mediates actin recruitment at the cellular tips leading to the establishment of cell-length spanning fibers, thus demonstrating a unique role of IRSp53 in controlling cell migration in 3D.

Experimental and theoretical model for the origin of coiling of cellular protrusions around fibers

“…This prediction is verified in our experiments, comparing fibers with circular cross-section and flattened fibers. Furthermore, the essential role of the curved membrane proteins in our model can explain the reduced coiling observed in cells with reduced amount of IRSp53 proteins 49 , which have convex curvature, and are involved with the recruitment of Arp2/3 actin nucleation to the membrane.…”

“…Such bundled actin induces filopodia growth, which are observed in cellular protrusions (such as neuronal growth cones 50 ) and may inhibit the ability of the lamellipodia-like leading edge to induce coiling. Finally, it has to be remembered that our simulations describe a simplified membrane protrusion, while cells spreading over fibers have to accommodate internals organelles, such as the nucleus, and contain cytoskeletal structures, such as stress-fibers 49 , which can further modify the coiling behavior.…”

Theoretical model of membrane protrusions driven by curved active proteins

“…Note that the concentration of the reconstituted GFP-IRSp53 in the cells is 2–3 times higher than in the normal cells, but it is still within the range if the endogenous level ( Mukherjee et al, 2023 ).…”

Confinement in fibrous environments positions and orients mitotic spindles

“…Deflections of nanofibers during interphase (contractile cell state, inward fiber deflections) and cell rounding (expansive rounded state, outward fiber deflections) were converted to force (nN) values using our previously reported Nanonet Force Microscopy 29,30,58 . Briefly, fibers are modeled as Euler Bernoulli beams with fixed boundary conditions and subjected to point loads at the cell-fiber contact regions (see Appendix I in Supplementary Materials ).…”

“…The bending of fibers allowed us to estimate the forces during interphase, mitosis, and postcell division (Fig. 1b (i, ii), Supplementary Movie S3) using Nanonet Force Microscopy (NFM, Appendix I in Supplementary Materials) [29][30][31] . During interphase, cells are in their natural contractile state and thus deflect the fibers inward (pink arrows, Fig.…”