Decision letter: A lipid bound actin meshwork organizes liquid phase separation in model membranes

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The eukaryotic cell membrane is connected to a dense actin rich cortex. We present FCS and STED experiments showing that dense membrane bound actin networks have severe influence on lipid phase separation. A minimal actin cortex was bound to a supported lipid bilayer via biotinylated lipid streptavidin complexes (pinning sites). In general, actin binding to ternary membranes prevented macroscopic liquid-ordered and liquid-disordered domain formation, even at low temperature. Instead, depending on the type of pinning lipid, an actin correlated multi-domain pattern was observed. FCS measurements revealed hindered diffusion of lipids in the presence of an actin network. To explain our experimental findings, a new simulation model is proposed, in which the membrane composition, the membrane curvature, and the actin pinning sites are all coupled. Our results reveal a mechanism how cells may prevent macroscopic demixing of their membrane components, while at the same time regulate the local membrane composition. https://doi.org/10.7554/eLife.01671.001 eLife digest All cells are surrounded by a lipid membrane that protects the cell, controls the movement of molecules into and out of the cell, and passes messages about environmental conditions to the cell. This membrane is made of two layers of molecules called lipids, with various proteins embedded in it. There are many different types of lipid molecules that together help to keep the membrane flexible. Moreover, lipid molecules of particular types can also come together to form ‘rafts’ that help the membrane to carry out its various roles. Given the complexity of the cell membrane, cell biologists often use simpler model membranes and computer simulations to explore how the different types of lipid molecules are organized within the membrane. According to the ‘picket fence’ model the cell membrane is divided into small compartments as a result of its interaction with the dense network of actin fibers that acts as a skeleton inside the cell. Recent computer simulations have predicted that these interactions can influence the distribution of lipids and proteins within the membrane. In particular, they can prevent the drastic re-arrangement of lipids into regions of high and low viscosity at low temperature. This temperature dependent re-arrangement of the membrane is known as lipid phase separation. Honigmann et al. have now used computer simulations and two advanced techniques—super-resolution optical STED microscopy and fluorescence correlation spectroscopy—to explore the properties of a model membrane in the presence of a dense network of actin fibers in fine detail. The results show that, in agreement with the simulation predictions of the ‘picket fence’ model, the actin fibers bound to the membrane prevent lipid phase separation happening at low temperatures. Moreover, the actin fibers also help to organize the distribution of lipids and proteins within the membrane at physiological temperatures. Honigmann et al. also suggest that the actin fibers cause the membrane to curve in a way that can reinforce the influence of the ‘picket fence’. The results show that the ‘raft’ and ‘picket fence’ models are connected, and that a cell can control the properties of its membrane by controlling the interactions between the membrane and the actin fibers that make up the skeleton of the cell. https://doi.org/10.7554/eLife.01671.002 Introduction The lateral heterogeneity of lipids and proteins in the plasma membrane of eukaryotic cells is an important feature for regulating biological function. The most prominent concept for membrane organization, the lipid raft theory, relates lipid phase separation (driven by interactions between cholesterol, sphingolipids, and saturated phospholipids) to membrane protein partitioning and regulation (Simons and Ikonen, 1997; Simons et al., 2011). Consequently, understanding lipid phase separation in membranes is a topic of extreme interest. A convenient starting point is to envision the membrane as a two-dimensional (2D) fluid environment through which the various membrane components freely diffuse. This simple picture successfully captures ternary model membranes containing two phospholipid species and cholesterol. At low temperature, these systems macroscopically phase separate into liquid-ordered (Lo) and liquid-disordered (Ld) domains (Veatch and Keller, 2003) and the nature of the transition is consistent with that of a 2D fluid (Honerkamp-Smith et al., 2008, 2009). Similar behavior was observed in plasma membrane-derived vesicles (Baumgart et al., 2007; Sezgin et al., 2012). Despite these successes for model membranes, there is growing consensus that this simple picture needs to be refined for the plasma membrane. For example, a remaining puzzle is that the Lo/Ld domains observed in model membranes grow macroscopic in size (micrometers), whereas lipid domains in plasma membrane are postulated to be tiny (nanometers) (Lenne and Nicolas, 2009). Additionally, the temperature Tc below which Lo/Ld domains start to form in plasma membrane derived vesicles is distinctly below T = 37°C (Sezgin et al., 2012), and so its relevance at physiological temperature requires further justification. In the case of a free-standing membrane (i.e., in the absence of an actin cortex), experiments have shown that lipid domains at temperatures above Tc can be induced by crosslinking low abundant membrane constituents (Hammond et al., 2005; Lingwood et al., 2008). Furthermore, there are numerous theoretical proposals of how a finite domain size above Tc might be accounted for: vicinity of a critical point (Honerkamp-Smith et al., 2009), hybrid lipids (Palmieri and Safran, 2013), coupling between composition and membrane curvature (Schick, 2012), electrostatic forces (Liu et al., 2005). In addition to this, the cortical cytoskeleton has also been identified as a key player affecting membrane domain formation (Kusumi et al., 2005). The latter is a dense fiber network of actin and spectrin on the cytoplasmic side of the eukaryotic plasma membrane. This network is connected to the membrane via pinning sites, such as lipid-binding proteins, transmembrane proteins, or membrane-attached proteins (Janmey, 1998; Mangeat et al., 1999; Janmey and Lindberg, 2004; Saarikangas et al., 2010 ). This has led to the hypothesis of the membrane being laterally compartmentalized: the pinning sites structure the membrane into small compartments whose perimeters are defined by the underlying actin network (the so-called ‘picket-fence’ model). This picket-fence network then acts as a barrier to diffusion, which elegantly accounts for confined diffusion of lipids and proteins observed in a single molecule tracking experiments (Kusumi et al., 2005). In a recent series of simulations, it was subsequently shown that a picket-fence network also acts as a barrier to macroscopic phase separation of lipids (Ehrig et al., 2011; Fischer and Vink, 2011; Machta et al., 2011). Instead, a stable mosaic of Lo and Ld domains is predicted, with a domain structure that strongly correlates to the actin fibers. Moreover, this mosaic structure already appears at physiological temperatures. These simulation findings are promising in view of the lipid raft hypothesis, since rafts are postulated to be small, as opposed to macroscopic. In this paper, we present the first experimental confirmation of these simulation results. To this end, we use an in vitro model system consisting of a supported lipid bilayer bound to an actin network. Complementing previous studies (Liu and Fletcher, 2006; Subramaniam et al., 2012; Heinemann et al., 2013; Vogel et al., 2013), our system enables direct observation of Lo/Ld domain formation in the presence of a lipid bound actin network using superresolution STED microscopy (Hell and Wichmann, 1994; Hell, 2007) and fluorescence correlation spectroscopy (FCS) (Magde et al., 1972; Kim et al., 2007). Based on our results, we propose an extension of the picket-fence model by including a coupling of the local membrane curvature to the membrane composition. Computer simulations of this extended model show that the pinning effect of the actin network is dramatically enhanced by such a coupling. These results imply that even a low density of pinning sites can induce significant structuring of lipids and proteins in the plasma membrane. Results Experimental results Domain formation in membranes without actin Our model system is a lipid mixture of unsaturated DOPC, saturated DPPC, and cholesterol. Such ternary mixtures are routinely used to approximate the complex lipid composition of the eukaryotic plasma membrane (Dimova et al., 2006). The phase diagram for this system in the absence of actin is well known (Veatch and Keller, 2003). For a large range of compositions this system reveals macroscopic Lo/Ld phase coexistence below the transition temperature Tc, while above Tc they are homogenously mixed. The size, shape, and the exact phase transition temperature of Lo/Ld domains depend on the specific model membrane system used. Domains grow largest in unsupported membranes (Honigmann et al., 2010), they are smaller in Mica supported membranes (Jensen et al., 2007) and are below the diffraction limit on glass-supported membranes (Honigmann et al., 2013). Depending on the composition, the transition to the coexistence region can be first-order, or continuous passing through a critical point. In the present work, we choose a composition ratio of (DOPC:DPPC:cholesterol) = (35:35:30) mol%, including 1 mol% of a biotinylated lipid (DOPE-biotin) to eventually connect the membrane to an actin network. To facilitate high-resolution microscopy flat, single membranes of that mixture were prepared on a Mica support. After membrane preparation the sample was heated to T = 37°C for 15 min to equilibrate the bilayer in the mixed phase. At this stage, the connector protein streptavidin was bound to the biotinylated lipid (but not yet to actin). After this preparation the membrane was slowly cooled and the distribution of the red fluorescent Ld-marker (DPPE-KK114) was determined by superresolution STED imaging. Visual inspection of images revealed that, upon cooling, there is a well-defined temperature at which Lo/Ld domains became visible (Figure 1A). This temperature is around Tc ≈ 28°C and thus marks the transition between the mixed one-phase state at high temperature, and the low-temperature two-phase coexistence state. This phase transition temperature is close but slightly decreased compared to the reported value for lipid vesicles made of the same mixture (Tc ≈ 30°C [Veatch et al., 2004]). At Tc, domains were constantly forming and re-forming, implying that the line tension is small, and so the transition is close to critical. The transition from a one-phase to a Lo/Ld separated two-phase membrane can be analyzed more quantitatively via the cumulant U1 of the images (see ‘Materials and methods’ for definition). In the one-phase region, U1 = π/2, whereas for a two-phase system U1 = 1 (Binder, 1981; Fischer and Vink, 2011). As shown in Figure 1C, U1 markedly dropped toward unity below Tc. Once the membrane enters the two-phase region, domains were seen to coarsen within a couple of minutes. The typical domain size R was obtained from the radial distribution function g(r) of the images. As shown in Figure 1D, R increased rapidly at Tc, from <75 nm to >200 nm, and continued to grow into the near micrometer range as the temperature was lowered further. These findings are consistent with atomic force microscopy experiments (Connell et al., 2013), where the transition was shown to be continuous as well. The observation that the domains do not fully coalesce at low temperature (as opposed to free-standing membranes) can be attributed to the interaction of the membrane with the mica support (Jensen et al., 2007). Our data do not allow the nature of the transition to be unambiguously identified. We emphasize, however, that this does not affect the overall conclusions of our work. All that we require is that the system without actin reveals two-phase coexistence at low temperatures. Figure 1 with 4 supplements see all Download asset Open asset Binding of an actin network to ternary membranes dramatically affects lipid domain structure. (A) In the absence of actin, our Mica supported model membrane revealed a phase transition at Tc ≈ 28°C, below which macroscopic Lo/Ld phase separation was observed. The membrane was stained with the Ld-marker DPPE-KK114 (magenta), and imaged by STED-microscopy with a lateral resolution of 70 nm. (B) The same membrane as in (A) but now in the presence of an actin network (green) bound to the membrane via a streptavidin linker. The Ld domains were strongly correlated to the actin network, as can be seen in the overlay, resulting in a meshwork like structure of Ld channels. This structure was stable even at temperatures above Tc. (C) Variation of the cumulant U1 of the images with temperature T in the absence of actin (red) and presence (blue). In the absence of actin, there is a transition toward a two-phase coexistence region at low temperature, as manifested by a pronounced drop of U1 toward unity. The transition occurs at Tc ≈ 28°C. In the presence of actin, no such transition is detected. (D) Typical domain size R as a function of T. In the absence of actin, there was pronounced domain coarsening below Tc. In the presence of actin, R was essentially temperature independent, and restricted to at most 90 nm. Note that R could not be measured for temperatures above T = 32°C as the contrast of the domains became too low. (E) Pearson correlation coefficient (PCC) between Ld domains and actin vs temperature. This coefficient measures the degree of correlation between Ld domains and the actin fibers. The correlation is largest at low temperature, but it persists at high temperature also, including the physiological temperature T = 37°C. Inset shows the graphical representation of the correlation between high intensities in the actin channel with high intensities in the lipid channel (ld phase). For another example of phase reorganization by actin binding see Figure 1—figure supplement 1. For actin meshwork binding to single component DOPC membranes see Figure 1—figure supplement 2. https://doi.org/10.7554/eLife.01671.003 Domain formation in membranes with actin Having characterized the phase behavior of the membrane without actin, we heated the same membrane back to the mixed state at T = 37°C to bind actin fibers via phalloidin–biotin to the streptavidin–biotin–lipid complexes (a molecular sketch is provided in Figure 1—figure supplement 3). The resulting binding sites strongly attract the Ld phase with a partitioning value of Lo% = 10 for DOPE-biotin (Figure 3—figure supplement 1). The actin fibers were stained with a green fluorescent phalloidin. After the actin meshwork was bound to the membrane, the temperature was again decreased, and the distributions of the Ld marker DPPE-KK114 and the actin were imaged simultaneously using (superresolution) STED and (diffraction limited) confocal microscopy, respectively. Visual inspection of images (Figure 1B) revealed that, with actin, domains already appeared at T ≈ 37°C. The spatial domain structure, however, was very different compared to the actin-free case. We observed a partitioning of the membrane into compartments of Lo-enriched domains, separated by ‘channels’ of Ld-enriched domains. These channels were clearly correlated to the actin fibers (middle row), as shown in the overlay (lower row). This correlation was analyzed quantitatively via the Pearson measure (PCC), which is an estimate of the colocalization between the Ld domains and the actin fibers (see ‘Materials and methods’ for definition). In general, a large value PCC>0 indicates a pronounced overlay of structures, PCC = 0 indicates randomly distributed objects, whereas PCC<0 represents inverted features (i.e., anti-correlations). We observed that PCC is largest at low temperature, implying that the correlation between Ld domains and actin is strongest there (Figure 1E). However, PCC remains finite at high temperatures also, with a significant correlation up to our highest accessible temperatures of T = 37°C, which is a stunning 9°C above Tc of the actin-free membrane. For T > 28°C, the decrease of PCC is approximately linear. This decrease was caused by an increasingly even partitioning of the Ld-marker, and not by a redistribution of Ld domains away from the actin fibers (as follows from Figure 1B, where correlated domains remain clearly visible at T = 37°C). In contrast to the actin-free case, our data suggest that there is no phase transition associated with the formation of the domain structure: Neither the cumulant nor the domain size revealed any pronounced temperature dependence (Figure 1C,D). Also the variation of PCC with temperature is entirely smooth, and does not indicate a transition either. Furthermore, in the presence of actin, the average domain size was significantly smaller, R ≈ 90 nm at most, and no coarsening was observed. Our conclusion is that the phase transition of the actin-free membrane is effectively eliminated by the actin network, in line with theoretical expectations (Fischer and Vink, 2011; Machta et al., 2011). As control experiment, we also considered a single component bilayer (DOPC) bound to actin. In this case, no domains were induced by the actin (Figure 1—figure supplement 2). Additionally, we excluded the possibility that the actin-correlated phases were induced by the green-fluorescent phalloidin. To this end, we stained the membrane with the Ld-marker (red) and the Lo marker (green) but not the actin itself. The result was a comparable structure as observed in Figure 1B, see Figure 1—figure supplement 1. The lateral diffusion of lipids is restricted by actin-organized domains To determine the lateral movement of lipids in the membrane in relation to the actin fibers, we applied scanning FCS (Digman et al., 2005; Ries and Schwille, 2006; Digman and Gratton, 2009). We used the same system as described above, but now with a lower density of actin such that single fibers could be resolved by confocal microscopy. The temperature was set to T = 32°C, that is slightly above Tc of the actin-free membrane. As a control, we started with actin fibers on a single component DOPC membrane including 1 mol% DOPE-biotin (compare Figure 1—figure supplement 2B). A small circle (diameter d = 500 nm) was scanned over a single actin fiber, as depicted in Figure 2E. For each position on the circle, the diffusion of the Ld-marker was determined by standard FCS analysis (Kim et al., 2007). The upper panel in Figure 2A shows the intensity of the Ld-marker (red) and of the actin marker (green) along the scan circle for the single component membrane (A). The intensity maximum of the green channel indicates the position of the actin fiber (corresponding to 0 and 180°), the intensity of the red channel shows the corresponding distribution of the Ld marker. In the single component membrane, the Ld marker was homogenously distributed. The lower panels of Figure 2A shows the decay of the autocorrelation function of the Ld marker measured along the scan circle, with the color representing the amplitude of the correlation. For the single component membrane, the autocorrelation analysis revealed no significant differences in mobility between areas with actin and without. Next, we used the same data to determine the diffusion of lipids across the scanning circle by calculating the pair-correlation function of ‘opposing’ pixels on the scanning circle, that is pixel pairs that are separated by a rotation of 180° (Cardarelli et al., 2012). This correlation measures how long the probe on average needs to diffuse across the circle and is therefore more sensitive to detect diffusion barriers than the autocorrelation analysis of a single excitation spot. The resulting pair-correlation is shown in Figure 2B. For the single component membrane, the amplitude and correlation time revealed no clear The average correlation time across the circle was ≈ that the pinning of the actin no significant diffusion barrier for These results are in agreement with point FCS measurements on single component membranes in the presence of actin which are reported in Figure 1—figure supplement 2E. Also the lateral diffusion was to the presence of actin. Figure Download asset Open asset diffusion was restricted by actin-organized domains. (A) analysis of the Ld marker DPPE-KK114 in a single component DOPC membrane in the presence of a low density actin The upper panel shows the intensity of the green channel and red channel the position of the actin fiber on the scan The lower panel the decay for each pixel along the scan with a of d = 500 nm. The amplitude is by the The time through the excitation can be by the transition from to As the mobility along the scan was (B) analysis of the same data as in A for pixels on the scan The in the pair-correlation the average time the to across the scan significant dependence of the mobility was observed in case of simple component (C) as in (A) but for ternary membranes composition as in Figure 1). (D) the analysis to be a dependence of diffusion was revealed by the In the along the actin fiber, a correlation is visible with a maximum at ≈ In the the correlation amplitude is which indicates a diffusion barrier along this (E) of the scanning over a single actin fiber bound to the membrane. The the of the representation the results of the pair-correlation analysis with pronounced diffusion along and restricted diffusion to the actin fiber In contrast to the single component membrane, a pronounced dependence of lipid diffusion was observed in the ternary The intensity distribution of the Ld-marker a at the position of the actin fiber panel Figure a Ld phase along the actin the autocorrelation analysis along the scan no clear (lower panel Figure the pair-correlation function revealed at 0 and 180° (i.e., along the actin at ≈ (Figure In the to the actin fiber, a amplitude was with the to > This indicates that the actin Ld domains the diffusion of the Ld marker within the domain thus along the actin while diffusion across domain These findings the simulation of Machta et al. where lipid domains by actin pinning were also to lipid of the type of on domain structure We how domain formation is different pinning sites are used to bind the membrane to the actin network. To this end, we compared biotinylated lipids with different partitioning DOPE-biotin (i.e., the same as used in the of Figure that strongly into the Ld phase = that in the Lo phase = and that in phases (but with a small toward the Lo Lo% = The experimental to determine the Lo% is in Figure 3—figure supplement 1. In Figure we show confocal images of the domain structure for the pinning lipid The the previous of Figure We again the formation of Ld domains along the actin fibers, as by a Pearson coefficient PCC = of the lipid induced an domain structure, a PCC = (Figure The binding of actin to not result in a of lipid domains along the fibers, as manifested by a small PCC = (Figure The result of Figure is since slightly the Lo and so a PCC was that could be for this streptavidin binding to biotinylated lipids may induce lipid by or electrostatic interactions with the it is that a coupling between membrane composition and curvature also about this To this we to computer simulations, where important such as pinning density and phase partitioning of pinning sites can be Figure with 1 supplement see all Download asset Open asset The type of pinning used to bind actin to the membrane strongly affects the domain structure. The Ld phase were stained with while actin (middle was stained with The shows the overlay of images. The membrane was imaged by confocal microscopy at T = using the same lipid composition as in Figure 1. (A) Binding of actin to the Ld lipid DOPE-biotin in Ld domains along the actin fibers (as in Figure 1B, PCC = (B) actin was bound to the correlation of domains with the actin fibers was significantly but with a slightly Pearson coefficient = (C) Binding of actin to the Lo lipid in correlated Lo domains along the actin fibers, that is the structure of (A). In this case, the Pearson coefficient was = For the lipid phase partitioning of the biotinylated lipids used in this see Figure 3—figure supplement 1. results We a phase membrane to a picket-fence network actin using a model to Machta et al. but extended to membrane The and The first the membrane properties using the form with and tension the the phase separation using a the the phases to the local membrane The of the curvature coupling is to the of and the curvature between Lo and Ld domains and there is a range in the reported of and a large of in the of the curvature coupling is (Schick, 2012). For this we allow the curvature coupling to be by a in our The latter is defined such that, for > regions of curvature unsaturated lipids, that is Ld domains. For = 0 our model to the of Machta et al. In where curvature coupling is known to to finite The influence of the actin network is via pinning sites, which are distributed randomly along the actin fibers pinning density At the pinning sites, there is a to of the lipid species by the The pinning sites also the membrane which we model by = 0 at these the actin network to in a the from which the membrane is Additionally, there is a between the membrane and the the actin fibers, the membrane is restricted to We first pinning sites that slightly Lo domains = the corresponding (Figure revealed a implying a of Ld domains along the actin fibers This can be however, the membrane In Figure we show how the PCC with the curvature coupling using pinning density = corresponding to of the actin