The dynamics of spatio-temporal Rho GTPase signaling: formation of signaling patterns

Doss

et al. 2020

Preprint

SummaryAt the basis of cell shape and behavior, actomyosin organization and force-generating property are widely studied, however very little is known about the regulation of the contractile network in space and time. Here we study the role of the epithelial-specific protein EpCAM, a contractility modulator, in cell shape and motility, and we show that it is required for the maturation of stress fibers and frontrear polarity acquisition at the single cell level. There, EpCAM ensures the remodeling of a transient active RhoA zone in the cortex of spreading epithelial cells. GTP-RhoA follows the endosomal pathway mediated by Rab35 and EHD1, where it co-evolves together with EpCAM. In fact, EpCAM balances GTP-RhoA turnover in order to tune actomyosin remodeling for cell shape, polarity and mechanical property acquisition. Impairment of GTP-RhoA endosomal trafficking either by EpCAM silencing or Rab35 / EHD1 mutant expression prevents correct myosin-II activity, stress fiber formation, and ultimately cell polarization. Collectively, this work shows that the coupling of EpCAM/RhoA co-trafficking to actomyosin rearrangement is critical for spreading, and advances our understanding of how biochemical and mechanical properties can be coupled for cell plasticity.

Section: Discussionmentioning

confidence: 99%

Endosomal spatio-temporal modulation of the cortical RhoA zone conditions epithelial cell organization

Gaston

Doss

et al. 2020

Preprint

“…Yet, activity patterns governed by excitable systems have a propensity to propagate through the whole cell, and inhibitory mechanisms are required to limit their expansion. Rho GTPases are inefficient enzymes, with a slow GTP hydrolysis rate in vitro 33 such that additional processes are required to prevent a propagation of active Rho GTPases species by diffusion or actin-driven advection [34][35][36] , where the term advection refers to the oriented (ballistic) transport of material. Three classes of mechanisms could confine Rho GTPases activities.…”

Section: Introductionmentioning

confidence: 99%

“…First, GAPs hydrolyze active Rho GTPases and can shorten their lifetimes in the GTP-bound state over orders of magnitude. Rho GTPase cycles can then be locally regulated by GEF and GAP concentrations, whose distributions along the cell would shape Rho GTPase intracellular gradients [37][38][39]36 . Second, anchoring or trapping in the cortical acto-myosin network can decrease diffusion considerably.…”

Section: Introductionmentioning

confidence: 99%

Optogenetic dissection of Rac1 and Cdc42 gradient shaping

Vaidžiulytė

Manzi

et al. 2018

Preprint

During migration, cells present a polarized activity that is aligned with the direction of motion. This cell polarity is established by an internal molecular circuitry, without the requirement of extracellular cues. At the heart of this circuitry, Rho GTPases spontaneously form spatial gradients that define the front and back of migrating cells. At the front of the cell, active Cdc42 forms a steep gradient whereas active Rac1 forms a more extended pattern peaking a few microns away from the cell tip. What are the mechanisms shaping these gradients, and what is the functional role of the shape of these gradients? Combining optogenetics and cell micopatterning, we show that Cdc42 and Rac1 gradients are set by spatial patterns of activators and deactivators and not directly by advection or diffusion mechanisms. Cdc42 simply follows the distribution of GEFs thanks to a uniform GAP activity, whereas Rac1 shaping requires the activity of an additional GAP, β2-chimaerin, which is sharply localized at the tip of the cell. We find that β2-chimaerin recruitment depends on feedbacks from Cdc42 and Rac1. Functionally, the extent -neither the slope nor the amplitude-of RhoGTPases gradients governs cell migration. A Cdc42 gradient with a short spatial extent is required to maximize directionality during cell migration while an extended Rac1 gradient controls the speed of the cell.

“…Rac1 shuttles to and from the plasma membrane through its interaction with Rho GDP-dissociation inhibitors (GDIs), which mask its prenyl group. Rac1 presents spatiotemporal patterns of activity (6) (7) which extend over few micrometers and last for few minutes during cell migration (8). Localized shuttling of Rac1 by GDIs and localized activation by GEFs are two mechanisms capable of producing and maintaining activation profiles.…”

Section: Introductionmentioning

confidence: 99%

“…Yet, we do not know whether the spatial extent of Rac1 activity gradients in the cell, generated by a specific distribution of activators and deactivators, are maintained due to low mobility or short lifetimes. Some of the mechanisms that localize GEFs and GAPs have been identified and described (reviewed in (8)). Lipidinteraction domains with varying lipid specificity, BAR domains, tyrosine kinases, scaffold proteins, adhesion complexes and the cytoskeleton have been shown to selectively direct GEFs and GAPs to different plasma membrane (PM) subdomains.…”

Section: Introductionmentioning

confidence: 99%

Gradients of Rac1 nanoclusters support spatial patterns of Rac1 signaling

Remorino

Cayrac

et al. 2017

Preprint

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. Significance 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.