Triggering signaling pathways using F-actin self-organization

Abstract

The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity.

Untitled

“…The local activity of protein network involved in cell motility discovers the mechanism of the directional persistency of the cell migration through responding to local mechanical (through cell matrix adhesions) and chemical signals, as well as branching new appendages of the older ones. This branching proposes a positive feedback mechanism by which migration occurs persistently in the absence of any external signal (48)(49)(50). As assumed in the persistent random walk (PRW; also called correlated random walk), the cell persists in its direction of motion in some timescales, whereas displays a type of random movement along its trajectory in longer time scales (Figure 1d) [51].…”

Nanoparticle-based local translation reveals mRNA as translation-coupled scaffold with anchoring function

“…GFP-ABD was then cloned into pCFE between the NdeI and XhoI sites to construct the pCFE-GFP-ABD. mCherry gene was amplified by PCR from pET28-hUtrophin-mCherry (48) with BamHI_mCherry fwd and mCherry_XhoI rev primers. The gene was then replaced with ABD on pCFP-GFP-ABD between the BamHI and XhoI sites to construct the pCFP-GFP-mCherry.…”

“…5 µL of HeLa Cell Extract, 1 µL of Accessory protein and 2 µL of reaction mix (from 1-Step Human Coupled IVT Kit-DNA, Thermo Fisher Scientific) were mixed with 2 µL of mRNA-NPs and immediately encapsulated in droplets by water-in-oil emulsion process. The process has been previously described and allows the formation of active actin meshworks in droplets (48). PHS-PEO-PHS block copolymer (Arlacel P135) was first dissolved in mineral oil (0.4 mg/mL).…”

Probing the biology of cell boundary conditions through confinement of Xenopus cell‐free cytoplasmic extracts

“…Subsequent experiments exploited Ran-mediated microtubule aster formation inside similar droplets (Hoffmann et al, 2013). In a separate study, the authors quantified actin flow inside emulsion droplets (Pinot et al, 2012), and demonstrated that a polymerized actin network can promote subcompartmentalization (Colin, Bonnemay, Gayrard, Gautier, & Gueroui, 2016). Other researchers have confined Xenopus egg extracts in micropatterned wells or channels to characterize contraction of microtubule networks (Foster, Furthauer, Shelley, & Needleman, 2015).…”