The Membrane Phase Transition Gives Rise to Responsive Plasma Membrane Structure and Function.

Several groups have recently reported evidence for the emergence of domains in cell plasma membranes when membrane proteins are organized by ligand binding or assembly of membrane proximal scaffolds. These domains recruit and retain components that favor the liquid-ordered phase, adding to a decades-old literature interrogating the contribution of membrane phase separation in plasma membrane organization and function. Here we propose that both past and present observations are consistent with a model in which membranes have a high compositional susceptibility, arising from their thermodynamic state in a single phase that is close to a miscibility phase transition. This rigorous framework naturally allows for both transient structure in the form of composition fluctuations and long-lived structure in the form of induced domains. In this way, the biological tuning of plasma membrane composition enables a responsive compositional landscape that facilitates and augments cellular biochemistry vital to plasma membrane functions.

TorsinA is essential for the timing and localization of neuronal nuclear pore complex biogenesis.

Nuclear pore complexes (NPCs) regulate information transfer between the nucleus and cytoplasm. NPC defects are linked to several neurological diseases, but the processes governing NPC biogenesis and spatial organization are poorly understood. Here, we identify a temporal window of strongly upregulated NPC biogenesis during neuronal maturation. We demonstrate that the AAA+ protein torsinA, whose loss of function causes the neurodevelopmental movement disorder DYT-TOR1A (DYT1) dystonia, coordinates NPC spatial organization during this period without impacting total NPC density. Using a new mouse line in which endogenous Nup107 is Halo-Tagged, we find that torsinA is essential for correct localization of NPC formation. In the absence of torsinA, the inner nuclear membrane buds excessively at sites of mislocalized, nascent NPCs, and NPC assembly completion is delayed. Our work implies that NPC spatial organization and number are independently regulated and suggests that torsinA is critical for the normal localization and assembly kinetics of NPCs.

Measuring the Co-Localization and Dynamics of Mobile Proteins in Live Cells Undergoing Signaling Responses.

Single molecule imaging in live cells enables the study of protein interactions and dynamics as they participate in signaling processes. When combined with fluorophores that stochastically transition between fluorescent and reversible dark states, as in super-resolution localization imaging, labeled molecules can be visualized in single cells over time. This improvement in sampling enables the study of extended cellular responses at the resolution of single molecule localization. This chapter provides optimized experimental and analytical methods used to quantify protein interactions and dynamics within the membranes of adhered live cells. Importantly, the use of pair-correlation functions resolved in both space and time allows researchers to probe interactions between proteins on biologically relevant distance and timescales, even though fluorescence localization methods typically require long times to assemble well-sampled reconstructed images. We describe an application of this approach to measure protein interactions in B cell receptor signaling and include sample analysis code for post-processing of imaging data. These methods are quantitative, sensitive, and broadly applicable to a range of signaling systems.

Membrane phase separation drives responsive assembly of receptor signaling domains.

Plasma membrane heterogeneity has been tied to a litany of cellular functions and is often explained by analogy to membrane phase separation; however, models based on phase separation alone fall short of describing the rich organization available within cell membranes. Here we present comprehensive experimental evidence motivating an updated model of plasma membrane heterogeneity in which membrane domains assemble in response to protein scaffolds. Quantitative super-resolution nanoscopy measurements in live B lymphocytes detect membrane domains that emerge upon clustering B cell receptors (BCRs). These domains enrich and retain membrane proteins based on their preference for the liquid-ordered phase. Unlike phase-separated membranes that consist of binary phases with defined compositions, membrane composition at BCR clusters is modulated through the protein constituents in clusters and the composition of the membrane overall. This tunable domain structure is detected through the variable sorting of membrane probes and impacts the magnitude of BCR activation.

Chemical potential measurements constrain models of cholesterol-phosphatidylcholine interactions.

Bilayer membranes composed of cholesterol and phospholipids exhibit diverse forms of nonideal mixing. In particular, many previous studies document macroscopic liquid-liquid phase separation as well as nanometer-scale heterogeneity in membranes of phosphatidylcholine (PC) lipids and cholesterol. Here, we present experimental measurements of cholesterol chemical potential (µc) in binary membranes containing dioleoyl PC (DOPC), 1-palmitoyl-2-oleoyl PC (POPC), or dipalmitoyl PC (DPPC), and in ternary membranes of DOPC and DPPC, referenced to crystalline cholesterol. µc is the thermodynamic quantity that dictates the availability of cholesterol to bind other factors, and notably must be equal between coexisting phases of a phase separated mixture. It is simply related to concentration under conditions of ideal mixing, but is far from ideal for the majority of lipid mixtures investigated here. Measurements of µc can vary with phospholipid composition by 1.5 kBT at constant cholesterol mole fraction implying a more than fivefold change in its availability for binding receptors and other reactions. Experimental measurements are fit to thermodynamic models including cholesterol-DPPC complexes or pairwise interactions between lipid species to provide intuition about the magnitude of interactions. These findings reinforce that µc depends on membrane composition overall, suggesting avenues for cells to alter the availability of cholesterol without varying cholesterol concentration.

The plasma membrane as an adaptable fluid mosaic.

The fluid mosaic model proposed by Singer and Nicolson established a powerful framework to interrogate biological membranes that has stood the test of time. They proposed that the membrane is a simple fluid, meaning that proteins and lipids are randomly distributed over distances larger than those dictated by direct interactions. Here we present an update to this model that describes a spatially adaptable fluid membrane capable of tuning local composition in response to forces originating outside the membrane plane. This revision is rooted in the thermodynamics of lipid mixtures, draws from recent experimental results, and suggests new modes of membrane function.

Estimating the localization spread function of static single-molecule localization microscopy images.

Single-molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM data set is an essential aspect of the image processing and analysis pipeline. Here, we present a simple method to estimate the localization spread function (LSF) of a static SMLM data set directly from acquired localizations, exploiting the correlated dynamics of individual emitters and properties of the pair autocorrelation function evaluated in both time and space. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labeled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. We show that experimentally obtained images have LSFs that are broader than expected from the localization precision alone, due to additional uncertainty accrued when localizing molecules imaged over time.

Surface densities prewet a near-critical membrane.

Recent work has highlighted roles for thermodynamic phase behavior in diverse cellular processes. Proteins and nucleic acids can phase separate into three-dimensional liquid droplets in the cytoplasm and nucleus and the plasma membrane of animal cells appears tuned close to a two-dimensional liquid-liquid critical point. In some examples, cytoplasmic proteins aggregate at plasma membrane domains, forming structures such as the postsynaptic density and diverse signaling clusters. Here we examine the physics of these surface densities, employing minimal simulations of polymers prone to phase separation coupled to an Ising membrane surface in conjunction with a complementary Landau theory. We argue that these surface densities are a phase reminiscent of prewetting, in which a molecularly thin three-dimensional liquid forms on a usually solid surface. However, in surface densities the solid surface is replaced by a membrane with an independent propensity to phase separate. We show that proximity to criticality in the membrane dramatically increases the parameter regime in which a prewetting-like transition occurs, leading to a broad region where coexisting surface phases can form even when a bulk phase is unstable. Our simulations naturally exhibit three-surface phase coexistence even though both the membrane and the polymer bulk only display two-phase coexistence on their own. We argue that the physics of these surface densities may be shared with diverse functional structures seen in eukaryotic cells.

Comparative analysis of TCR and CAR signaling informs CAR designs with superior antigen sensitivity and in vivo function.

Chimeric antigen receptor (CAR)-modified T cell therapy is effective in treating lymphomas, leukemias, and multiple myeloma in which the tumor cells express high amounts of target antigen. However, achieving durable remission for these hematological malignancies and extending CAR T cell therapy to patients with solid tumors will require receptors that can recognize and eliminate tumor cells with a low density of target antigen. Although CARs were designed to mimic T cell receptor (TCR) signaling, TCRs are at least 100-fold more sensitive to antigen. To design a CAR with improved antigen sensitivity, we directly compared TCR and CAR signaling in primary human T cells. Global phosphoproteomic analysis revealed that key T cell signaling proteins-such as CD3δ, CD3ε, and CD3γ, which comprise a portion of the T cell co-receptor, as well as the TCR adaptor protein LAT-were either not phosphorylated or were only weakly phosphorylated by CAR stimulation. Modifying a commonplace 4-1BB/CD3ζ CAR sequence to better engage CD3ε and LAT using embedded CD3ε or GRB2 domains resulted in enhanced T cell activation in vitro in settings of a low density of antigen, and improved efficacy in in vivo models of lymphoma, leukemia, and breast cancer. These CARs represent examples of alterations in receptor design that were guided by in-depth interrogation of T cell signaling.

Critical Phenomena in Plasma Membrane Organization and Function.

Lateral organization in the plane of the plasma membrane is an important driver of biological processes. The past dozen years have seen increasing experimental support for the notion that lipid organization plays an important role in modulating this heterogeneity. Various biophysical mechanisms rooted in the concept of liquid-liquid phase separation have been proposed to explain diverse experimental observations of heterogeneity in model and cell membranes with distinct but overlapping applicability. In this review, we focus on the evidence for and the consequences of the hypothesis that the plasma membrane is poised near an equilibrium miscibility critical point. Critical phenomena explain certain features of the heterogeneity observed in cells and model systems but also go beyond heterogeneity to predict other interesting phenomena, including responses to perturbations in membrane composition.

SMAUG: Analyzing single-molecule tracks with nonparametric Bayesian statistics.

Single-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we develop a method for the case of multiple interconverting species undergoing free diffusion and introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of single-particle trajectory datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells.

A mean shift algorithm for drift correction in localization microscopy.

Single-molecule localization microscopy techniques transcend the diffraction limit of visible light by localizing isolated emitters sampled stochastically. This time-lapse imaging necessitates long acquisition times, over which sample drift can become large relative to the localization precision. Here, we present an efficient and robust method for estimating drift, using a simple peak-finding algorithm based on mean shifts that is effective for single-molecule localization microscopy in two or three dimensions.

Criticality of plasma membrane lipids reflects activation state of macrophage cells.

Signalling is of particular importance in immune cells, and upstream in the signalling pathway many membrane receptors are functional only as complexes, co-locating with particular lipid species. Work over the last 15 years has shown that plasma membrane lipid composition is close to a critical point of phase separation, with evidence that cells adapt their composition in ways that alter the proximity to this thermodynamic point. Macrophage cells are a key component of the innate immune system, are responsive to infections and regulate the local state of inflammation. We investigate changes in the plasma membrane's proximity to the critical point as a response to stimulation by various pro- and anti-inflammatory agents. Pro-inflammatory (interferon γ, Kdo 2-Lipid A, lipopolysaccharide) perturbations induce an increase in the transition temperature of giant plasma membrane vesicles; anti-inflammatory interleukin 4 has the opposite effect. These changes recapitulate complex plasma membrane composition changes, and are consistent with lipid criticality playing a master regulatory role: being closer to critical conditions increases membrane protein activity.

Synergistic factors control kinase-phosphatase organization in B-cells engaged with supported bilayers.

B-cells become activated by ligands with varying valency and mode of presentation to the B-cell receptor (BCR). We previously demonstrated that clustering the immunoglobulin M (IgM) isotype of BCR with an artificial soluble cross-linker stabilized an ordered phase-like domain that enriched kinases and depleted phosphatases to promote receptor tyrosine phosphorylation. BCR is also activated by ligands presented at surfaces, and here we activate B-cells via supported bilayers of phosphatidylcholine lipids, a natural ligand for the IgM BCR expressed in the CH27 cells used. Using superresolution fluorescence localization microscopy, along with a quantitative cross-correlation analysis, we find that BRCs engaged with bilayers sort minimal peptide markers of liquid-ordered and liquid-disordered phases, indicating that ordered-domain stabilization is a general feature of BCR clustering. The phosphatase CD45 is more strongly excluded from bilayer-engaged BRCs than a transmembrane peptide, indicating that mechanisms other than domain partitioning contribute to its organization. Experimental observations are assembled into a minimal model of receptor activation that incorporates both ordered domains and direct phosphatase exclusion mechanisms to produce a more sensitive response.

Ion channels can be allosterically regulated by membrane domains near a de-mixing critical point.

Ion channels are embedded in the plasma membrane, a compositionally diverse two-dimensional liquid that has the potential to exert profound influence on their function. Recent experiments suggest that this membrane is poised close to an Ising critical point, below which cell-derived plasma membrane vesicles phase separate into coexisting liquid phases. Related critical points have long been the focus of study in simplified physical systems, but their potential roles in biological function have been underexplored. Here we apply both exact and stochastic techniques to the lattice Ising model to study several ramifications of proximity to criticality for idealized lattice channels, whose function is coupled through boundary interactions to critical fluctuations of membrane composition. Because of diverging susceptibilities of system properties to thermodynamic parameters near a critical point, such a lattice channel's activity becomes strongly influenced by perturbations that affect the critical temperature of the underlying Ising model. In addition, its kinetics acquire a range of time scales from its surrounding membrane, naturally leading to non-Markovian dynamics. Our model may help to unify existing experimental results relating the effects of small-molecule perturbations on membrane properties and ion channel function. We also suggest ways in which the role of this mechanism in regulating real ion channels and other membrane-bound proteins could be tested in the future.

Giant Plasma Membrane Vesicles: An Experimental Tool for Probing the Effects of Drugs and Other Conditions on Membrane Domain Stability.

Giant plasma membrane vesicles (GPMVs) are isolated directly from living cells and provide an alternative to vesicles constructed of synthetic or purified lipids as an experimental model system for use in a wide range of assays. GPMVs capture much of the compositional protein and lipid complexity of intact cell plasma membranes, are filled with cytoplasm, and are free from contamination with membranes from internal organelles. GPMVs often exhibit a miscibility transition below the growth temperature of their parent cells. GPMVs labeled with a fluorescent protein or lipid analog appear uniform on the micron-scale when imaged above the miscibility transition temperature, and separate into coexisting liquid domains with differing membrane compositions and physical properties below this temperature. The presence of this miscibility transition in isolated GPMVs suggests that a similar phase-like heterogeneity occurs in intact plasma membranes under growth conditions, albeit on smaller length scales. In this context, GPMVs provide a simple and controlled experimental system to explore how drugs and other environmental conditions alter the composition and stability of phase-like domains in intact cell membranes. This chapter describes methods to generate and isolate GPMVs from adherent mammalian cells and to interrogate their miscibility transition temperatures using fluorescence microscopy.

Loss of PTEN promotes formation of signaling-capable clathrin-coated pits.

Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs) display highly heterogeneous dynamics on the plasma membrane where they can take from 20 s to over 1 min to form cytosolic coated vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], which is dephosphorylated by phosphatase and tensin homolog (PTEN), is a potent tumorigenic signaling lipid. By using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we found that EGF-bound EGFR and PTEN are enriched in a distinct subset of short-lived CCPs that correspond with clathrin-dependent EGF-induced signaling. We demonstrated that PTEN plays a role in the regulation of CCP dynamics. Furthermore, increased PI(3,4,5)P3 resulted in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Altogether, our findings provide evidence for the existence of short-lived 'signaling-capable' CCPs.

Chemoselective ratiometric imaging of protein S-sulfenylation.

Here we report a ratiometric fluorescent probe for chemoselective conjugation to sulfenic acids in living cells. Our approach couples an α-fluoro-substituted dimedone to an aminonaphthalene fluorophore (F-DiNap), which upon sulfenic acid conjugation is locked as the 1,3-diketone, changing the fluorophore excitation. F-DiNap reacts with S-sulfenylated proteins at equivalent rates to current probes, but the α-fluorine substitution blocks side-reactions with biological aldehydes.

Miscibility Transition Temperature Scales with Growth Temperature in a Zebrafish Cell Line.

Cells can alter the lipid content of their plasma membranes upon changes in their environment to maintain and adjust membrane function. Recent work suggests that some membrane functions arise because cellular plasma membranes are poised close to a miscibility transition under growth conditions. Here we report experiments utilizing giant plasma membrane vesicles (GPMVs) to explore how membrane transition temperature varies with growth temperature in a zebrafish cell line (ZF4) that can be adapted for growth between 20 and 32°C. We find that GPMV transition temperatures adjust to be 16.7 ± 1.2°C below growth temperature for four growth temperatures investigated and that adjustment occurs over roughly 2 days when temperature is abruptly lowered from 28 to 20°C. We also find that GPMVs have slightly different lipidomes when isolated from cells adapted for growth at 28 and 20°C. Similar to past work in vesicles derived from mammalian cells, fluctuating domains are observed in ZF4-derived GPMVs, consistent with their having critical membrane compositions. Taken together, these experimental results suggest that cells in culture biologically tune their membrane composition in a way that maintains specific proximity to a critical miscibility transition.