Swider, Z. | bioRxiv Science

bioRxiv2021

Rho and F-actin self-organize within an artificial cell cortex

The cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic reorganizations during a variety of essential biological processes including cell adhesion, cell migration, and cell division1,2. During cell division and cell locomotion, for example, waves of filamentous-actin (F-actin) assembly and disassembly develop in the cell cortex in a process termed "cortical excitability"3-7. In developing frog and starfish embryos, cortical excitability is generated through coupled positive and negative feedback, with rapid activation of Rho-mediated F-actin assembly followed in space and time by F-actin-dependent inhibition of Rho8,9. These feedback loops are proposed to serve as a mechanism for amplification of active Rho signaling at the cell equator to support furrowing during cytokinesis, while also maintaining flexibility for rapid error correction in response to movement of the mitotic spindle during chromosome segregation10. In this paper, we develop an artificial cortex based on Xenopus egg extract and supported lipid bilayers (SLBs), to investigate cortical Rho and F-actin dynamics11. This reconstituted system spontaneously develops two distinct dynamic patterns: singular excitable Rho and F-actin waves and non-traveling oscillatory Rho and F-actin patches. Both types of dynamic patterns have properties and dependencies similar to the cortical excitability previously characterized in vivo9. These findings directly support the longstanding speculation that the cell cortex is a self-organizing structure and present a novel approach for investigating mechanisms of Rho-GTPase-mediated cortical dynamics. HighlightsO_LIAn artificial cell cortex comprising Xenopus egg extract on a supported lipid bilayer self-organizes into complex, dynamic patterns of active Rho and F-actin C_LIO_LIWe identified two types of reconstituted cortical dynamics - excitable waves and coherent oscillations C_LIO_LIReconstituted waves and oscillations require Rho activity and F-actin polymerization C_LI

cell biology↗

bioRxiv2018

Calcium nanodomains in spindles

The role of calcium signaling in specific events of animal cell meiosis or mitosis (M-phase) is a subject of enduring controversy. Early efforts suggested that increases in intracellular free calcium ([Ca2+]i) promote spindle disassembly 1, 2 while subsequent work suggested that global [Ca2+]i increases trigger nuclear envelope breakdown, spindle assembly, the metaphase-anaphase transition, and cytokinesis 3-6. However, further studies led to the conclusion that elevation of [Ca2+]i either has no role in these events, plays a permissive role in these events, or functions as an auxiliary signaling pathway that supplements other mechanisms 7. One potential explanation of the controversy is that specific M-phase events might depend on highly localized increases in [Ca2+]i, variously referred to as microdomains 8 or nanodomains 9, as proposed recently 10. Such domains are hypothesized to arise from rapid shuttling of calcium between closely positioned sources and sinks, rendering them potentially difficult to detect with traditional dyes and largely insensitive to slow chelators such as EGTA 9. Here a novel microtubule-binding calcium sensor--TubeCamp--was used to test the hypothesis 10 that spindles are associated with calcium nanodomains. TubeCamp imaging revealed that spindles in Xenopus eggs, Xenopus embryos, and HeLa cells were all associated with calcium nanodomains at the spindle poles. Calcium nanodomains also formed in spindles assembled in cell extracts and at the center of monopolar spindles, suggesting that they are a basic feature of spindle self-assembly. Disruption of calcium nanodomains via perturbation of inositol-1,4,5-trisphosphate signaling or rapid chelation of [Ca2+]i resulted in spindle disassembly in vivo and vitro. The results demonstrate the existence of spindle-associated calcium nanodomains and indicate that such domains are an essential and common feature of spindles in vertebrates.

cell biology↗