Di Federico, F. | bioRxiv Science

bioRxiv2022

Cell adhesion and spreading on fluid membranes through microtubules-dependent mechanotransduction

During cell adhesion, integrins form clusters that transmit mechanical forces to the substrate (mechanotransduction) and regulate biochemical signaling depending on substrate stiffness. Studies on mechanotransduction significantly advanced our understanding of cell adhesion and were mostly performed on rigid substrates. In contrast to rigid substrates, integrins ligands on fluid supported lipid bilayers (SLBs) are mobile and adhesive complexes cannot serve as anchoring points promoting cell spreading. Here, we demonstrate that cells spread on SLBs coated with Invasin, a high-affinity integrin ligand. We show that in contrast to SLBs functionalized with RGD peptides, integrin clusters grow in size and complexity on Invasin-SLBs to a similar extent as on glass. While actomyosin contraction dominates adhesion maturation on stiff substrates, we find that integrin mechanotransduction and cell spreading on fluid SLBs rely on dynein pulling forces along microtubules perpendicular to membranes and microtubules pushing on adhesive complexes, respectively. These forces that may also occur on non-deformable surfaces are revealed in fluid substrate set ups. Our findings, supported by a theoretical model, demonstrate a new mechanical role for microtubules in integrin clustering.

cell biology↗

bioRxiv2017

Gradients Of Rac1 Nanoclusters Support Spatial Patterns Of Rac1 Signaling

The dynamics of the cytoskeleton and cell shape relies on the coordinated activation of RhoGTPase molecular switches. Among them, Rac1 participates to the orchestration in space and time of actin branching and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single molecule imaging and super-resolution microscopy, we reveal an additional supramolecular organization of Rac1. We find that, similarly to H-Ras, Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble due to the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs, GAPs, downstream effectors, and possibly other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Using optogenetics and micropatterning tools, we find that activation of Rac1 leads to its immobilization in nanoclusters and that the local level of Rac1 activity matches the local density of nanoclusters. Altogether, our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms. This finding implies that graded distributions of nanoclusters might encode spatial information.\n\nSignificance statementThe plasma membrane of eukaryotic cells is a highly organized surface where hundreds of incoming signals are transduced to the intracellular space. How cells encode faithfully this myriad of signals is a fundamental question. Here we show that Rac1, a critical membrane-bound protein involved in the regulation of cytoskeletal dynamics, forms small aggregates together with other regulating proteins. These supramolecular assemblies, called nanoclusters, are the \"quantal\" units of signaling. By increasing the local concentration, nanoclusters set thresholds for downstream signaling and ensure the fidelity of information transduction. We found that Rac1 nanoclusters are distributed as spatial gradients matching the patterns of Rac1 activity. We propose that cells can encode positional information through distributed signaling quanta, hereby ensuring spatial fidelity.

biophysics↗