Mechanosensitive mTORC2 independently coordinates leading and trailing edge polarity programs during neutrophil migration.

By acting both upstream of and downstream from biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanosensitive mTOR complex 2 (mTORC2) programs in neutrophil polarity and motility. We find that the tension-based inhibition of leading-edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the regulation of RhoA and myosin II-based contractility at the trailing edge depend on mTORC2 kinase activity. mTORC2 is essential for spatial and temporal coordination of the front and back polarity programs for persistent migration under confinement. This mechanosensory pathway integrates multiple upstream signals, and we find that membrane stretch synergizes with biochemical co-input phosphatidylinositol (3,4,5)-trisphosphate to robustly amplify mTORC2 activation. Our results suggest that different signaling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs for efficient coordination of neutrophil shape and movement.

Active emulsions in living cell membranes driven by contractile stresses and transbilayer coupling.

The spatiotemporal organization of proteins and lipids on the cell surface has direct functional consequences for signaling, sorting, and endocytosis. Earlier studies have shown that multiple types of membrane proteins, including transmembrane proteins that have cytoplasmic actin binding capacity and lipid-tethered glycosylphosphatidylinositol-anchored proteins (GPI-APs), form nanoscale clusters driven by active contractile flows generated by the actin cortex. To gain insight into the role of lipids in organizing membrane domains in living cells, we study the molecular interactions that promote the actively generated nanoclusters of GPI-APs and transmembrane proteins. This motivates a theoretical description, wherein a combination of active contractile stresses and transbilayer coupling drives the creation of active emulsions, mesoscale liquid order (lo) domains of the GPI-APs and lipids, at temperatures greater than equilibrium lipid phase segregation. To test these ideas, we use spatial imaging of molecular clustering combined with local membrane order, and we demonstrate that mesoscopic domains enriched in nanoclusters of GPI-APs are maintained by cortical actin activity and transbilayer interactions and exhibit significant lipid order, consistent with predictions of the active composite model.

Joining forces: crosstalk between biochemical signalling and physical forces orchestrates cellular polarity and dynamics.

Dynamic processes like cell migration and morphogenesis emerge from the self-organized interaction between signalling and cytoskeletal rearrangements. How are these molecular to sub-cellular scale processes integrated to enable cell-wide responses? A growing body of recent studies suggest that forces generated by cytoskeletal dynamics and motor activity at the cellular or tissue scale can organize processes ranging from cell movement, polarity and division to the coordination of responses across fields of cells. To do so, forces not only act mechanically but also engage with biochemical signalling. Here, we review recent advances in our understanding of this dynamic crosstalk between biochemical signalling, self-organized cortical actomyosin dynamics and physical forces with a special focus on the role of membrane tension in integrating cellular motility.This article is part of the theme issue 'Self-organization in cell biology'.

GPI-anchored protein organization and dynamics at the cell surface.

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite.

Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin.

Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24-37 °C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an "active actin-membrane composite" cell surface.

Live cell plasma membranes do not exhibit a miscibility phase transition over a wide range of temperatures.

Lipid/cholesterol mixtures derived from cell membranes as well as their synthetic reconstitutions exhibit well-defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid/cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid-phase separation is not generally observed in cell membranes, and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown. A fundamental property of phase transitions is that the variation of tagged particle diffusion with temperature exhibits an abrupt change as the system passes through the transition, even when the two phases are distributed in a nanometer-scale emulsion. We support this using a variety of Monte Carlo and atomistic simulations on model lipid membrane systems. However, temperature-dependent fluorescence correlation spectroscopy of labeled lipids and membrane-anchored proteins in live cell membranes shows a consistently smooth increase in the diffusion coefficient as a function of temperature. We find no evidence of a discrete miscibility phase transition throughout a wide range of temperatures: 14-37 °C. This contrasts the behavior of giant plasma membrane vesicles (GPMVs) blebbed from the same cells, which do exhibit phase transitions and macroscopic phase separation. Fluorescence lifetime analysis of a DiI probe in both cases reveals a significant environmental difference between the live cell and the GPMV. Taken together, these data suggest the live cell membrane may avoid the miscibility phase transition inherent to its lipid constituents by actively regulating physical parameters, such as tension, in the membrane.

Homo-FRET imaging highlights the nanoscale organization of cell surface molecules.

Several models have been proposed to understand the structure and organization of the plasma membrane in living cells. Predicated on equilibrium thermodynamic principles, the fluid-mosaic model of Singer and Nicholson and the model of lipid domains (or membrane rafts) are dominant models, which account for a fluid bilayer and functional lateral heterogeneity of membrane components, respectively. However, the constituents of the membrane and its composition are not maintained by equilibrium mechanisms. Indeed, the living cell membrane is a steady state of a number of active processes, namely, exocytosis, lipid synthesis and transbilayer flip-flop, and endocytosis. In this active milieu, many lipid constituents of the cell membrane exhibit a nanoscale organization that is also at odds with passive models based on chemical equilibrium. Here we provide a detailed description of microscopy and cell biological methods that have served to provide valuable information regarding the nature of nanoscale organization of lipid components in a living cell.

PSF decomposition of nanoscopy images via Bayesian analysis unravels distinct molecular organization of the cell membrane.

The spatial organization of membrane receptors at the nanoscale has major implications in cellular function and signaling. The advent of super-resolution techniques has greatly contributed to our understanding of the cellular membrane. Yet, despite the increased resolution, unbiased quantification of highly dense features, such as molecular aggregates, remains challenging. Here we describe an algorithm based on Bayesian inference of the marker intensity distribution that improves the determination of molecular positions inside dense nanometer-scale molecular aggregates. We tested the performance of the method on synthetic images representing a broad range of experimental conditions, demonstrating its wide applicability. We further applied this approach to STED images of GPI-anchored and model transmembrane proteins expressed in mammalian cells. The analysis revealed subtle differences in the organization of these receptors, emphasizing the role of cortical actin in the compartmentalization of the cell membrane.

Light driven ultrafast electron transfer in oxidative redding of Green Fluorescent Proteins.

Fluorescent proteins undergoing green to red (G/R) photoconversion have proved to be potential tools for investigating dynamic processes in living cells and for photo-localization nanoscopy. However, the photochemical reaction during light induced G/R photoconversion of fluorescent proteins remains unclear. Here we report the direct observation of ultrafast time-resolved electron transfer (ET) during the photoexcitation of the fluorescent proteins EGFP and mEos2 in presence of electron acceptor, p-benzoquinone (BQ). Our results show that in the excited state, the neutral EGFP chromophore accepts electrons from an anionic electron donor, Glu222, and G/R photoconversion is facilitated by ET to nearby electron acceptors. By contrast, mEos2 fails to produce the red emitting state in the presence of BQ; ET depletes the excited state configuration en route to the red-emitting fluorophore. These results show that ultrafast ET plays a pivotal role in multiple photoconversion mechanisms and provide a method to modulate the G/R photoconversion process.

Active remodeling of cortical actin regulates spatiotemporal organization of cell surface molecules.

Many lipid-tethered proteins and glycolipids exist as monomers and nanoclusters on the surface of living cells. The spatial distribution and dynamics of formation and breakup of nanoclusters does not reflect thermal and chemical equilibrium and is controlled by active remodeling of the underlying cortical actin. We propose a model for nanoclustering based on active hydrodynamics, wherein cell surface molecules bound to dynamic actin are actively driven to form transient clusters. This consistently explains all of our experimental observations. Using FCS and TIRF microscopy, we provide evidence for the existence of short, dynamic, polymerizing actin filaments at the cortex, a key assumption of the theoretical framework. Our theory predicts that lipid-anchored proteins that interact with dynamic actin must exhibit anomalous concentration fluctuations, and a cell membrane protein capable of binding directly to actin can form nanoclusters. These we confirm experimentally, providing an active mechanism for molecular organization and its spatiotemporal regulation on the plasma membrane.

Dynamic imaging of homo-FRET in live cells by fluorescence anisotropy microscopy.

Multiple lipid and protein components of the plasma membrane of a living cell are organized, both compositionally and functionally, at different spatial and temporal scales. For instance, Rab protein domains in membranes the clathrin-coated pit, or the immunological synapse are exquisite examples of functional compartmentalization in cell membranes. These assemblies consist in part of nanoscale complexes of lipids and proteins and are necessary to facilitate some specific sorting and signaling functions. It is evident that cellular functions require a regulated spatiotemporal organization of components at the nanoscale, often comprising of countable number of molecular species. Here, we describe multiple homo-FRET-based imaging methods that provide information about nanoscale interactions between fluorescently tagged molecules in live cells, at optically resolved spatial resolution.