Refinement of Singer-Nicolson fluid-mosaic model by microscopy imaging: Lipid rafts and actin-induced membrane compartmentalization

This year celebrates the 50th anniversary of the Singer-Nicolson fluid mosaic model for biological membranes. The next level of sophistication we have achieved for understanding plasma membrane (PM) structures, dynamics, and functions during these 50 years includes the PM interactions with cortical actin filaments and the partial demixing of membrane constituent molecules in the PM, particularly raft domains. Here, first, we summarize our current knowledge of these two structures and emphasize that they are interrelated. Second, we review the structure, molecular dynamics, and function of raft domains, with main focuses on raftophilic glycosylphosphatidylinositol-anchored proteins (GPI-APs) and their signal transduction mechanisms. We pay special attention to the results obtained by single-molecule imaging techniques and other advanced microscopy methods. We also clarify the limitations of present optical microscopy methods for visualizing raft domains, but emphasize that single-molecule imaging techniques can “detect” raft domains associated with molecules of interest in the PM.     

Nanotubes connecting B lymphocytes: High impact of differentiation-dependent lipid composition on their growth and mechanics

Nanotubes (NTs) are thin, long membranous structures forming novel, yet poorly known communication pathways between various cell types. Key mechanisms controlling their growth still remained poorly understood. Since NT-forming capacity of immature and mature B cells was found largely different, we investigated how lipid composition and molecular order of the membrane affect NT-formation. Screening B cell lines with various differentiation stages revealed that NT-growth linearly correlates with membrane ganglioside levels, while it shows maximum as a function of cholesterol level. NT-growth of B lymphocytes is promoted by raftophilic phosphatidylcholine and sphingomyelin species, various glycosphingolipids, and docosahexaenoic acid-containing inner leaflet lipids, through supporting membrane curvature, as demonstrated by comparative lipidomic analysis of mature versus immature B cell membranes. Targeted modification of membrane cholesterol and sphingolipid levels altered NT-forming capacity confirming these findings, and also highlighted that the actual lipid raft number may control NT-growth via defining the number of membrane-F-actin coupling sites. Atomic force microscopic mechano-manipulation experiments further proved that mechanical properties (elasticity or bending stiffness) of B cell NTs also depend on the actual membrane lipid composition. Data presented here highlight importance of the lipid side in controlling intercellular, nanotubular, regulatory communications in the immune system.     

Cholesterol-dependent localization of NAP-22 on a neuronal membrane microdomain (raft).

A membrane microdomain called raft has been under extensive study since the assembly of various signal-transducing molecules into this region has been envisaged. This domain is isolated as a low buoyant membrane fraction after the extraction with a nonionic detergent such as Triton X-100. The characteristic low density of this fraction is ascribed to the enrichment of several lipids including cholesterol. To clear the molecular mechanism of raft formation, several extraction methods were applied to solubilize raft components. Cholesterol extraction using methyl-beta-cyclodextrin was found to be effective to solubilize NAP-22, a neuron-enriched Ca(2+)-dependent calmodulin-binding protein as well as one of the main protein components of brain raft. Purified NAP-22 bound to the liposomes that were made from phosphatidylcholine and cholesterol. This binding was dependent on the amount of cholesterol in liposomes. Calmodulin inhibited this binding in a dose-dependent manner. These results suggest that the presence of a calcium-dependent regulatory mechanism works on the assembly of raft within the neuron.     

Dissecting lipid raft facilitated cell signaling pathways in cancer

Cancer is one of the most devastating disorders in our lives. Higher rate of proliferation than death of cells is one of the essential factors for development of cancer. The dynamicity of cell membrane plays some vital roles in cell survival and cell death, including protection, endocytosis, signaling, and increases in mechanical stability during cell division, as well as decrease of shear forces during separation of two cells after division, and cell separation from tissues for cancer metastasis. Within the membrane, there are specialized domains, known as lipid rafts. A raft can coordinate various signaling pathways. Recent data on the proteomics of lipid rafts/caveolae have highlighted the enigmatic role of various signaling proteins in cancer development. Analysis of these data of raft proteome from various tumors, cancer tissues, and cell lines cultured without and with therapeutic agents, as well as from model rafts revealed that there may be two subsets of raft assemblage in cell membrane. One subset of raft is enriched with cholesterol-sphingomyeline-ganglioside-cav-1/Src/EGFR (hereafter, "chol-raft") that is involved in normal cell signaling, and when dysregulated promotes cell transformation and tumor progression; another subset of raft is enriched with ceramide-sphingomyeline-ganglioside-FAS/Ezrin (hereafter, "cer-raft") that generally promotes apoptosis. In view of this, and to focus insight into the cancer cell physiology caused by the lipid rafts mediated signals and their receptors, and the downstream transmitters, either proliferative (for example, EGF and EGFR) or death-inducing (for example, FASL and FAS), and the precise roles of some therapeutic drugs and endogenous acid sphingomylenase in this scenario in in situ transformation of "chol-raft" into "cer-raft" are summarized and discussed in this contribution.     

Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function

Ceramide is a membrane lipid involved in a number of crucial biological processes. Recent evidence suggests that ceramide is likely to reside and function within lipid rafts; ordered sphingolipid and cholesterol-rich lipid domains believed to exist within many eukaryotic cell membranes. Using lipid vesicles containing co-existing raft domains and disordered fluid domains, we find that natural and saturated synthetic ceramides displace sterols from rafts. Other raft lipids remain raft-associated in the presence of ceramide, showing displacement is relatively specific for sterols. Like cholesterol-containing rafts, ceramide-rich "rafts" remain in a highly ordered state. Comparison of the sterol-displacing abilities of natural ceramides with those of saturated diglycerides and an unsaturated ceramide demonstrates that tight lipid packing is critical for sterol displacement by ceramide. Based on these results, and the fact that cholesterol and ceramides both have small polar headgroups, we propose that ceramides and cholesterol compete for association with rafts because of a limited capacity of raft lipids with large headgroups to accommodate small headgroup lipids in a manner that prevents unfavorable contact between the hydrocarbon groups of the small headgroup lipids and the surrounding aqueous environment. Minimizing the exposure of cholesterol and ceramide to water may be a strong driving force for the association of other molecules with rafts. Furthermore, displacement of sterol from rafts by ceramide is very likely to have marked effects upon raft structure and function, altering liquid ordered properties as well as molecular composition. In this regard, certain previously observed physiological processes may be a result of displacement. In particular, a direct connection to the previously observed sphingomyelinase-induced displacement of cholesterol from plasma membranes in cells is proposed.     

Looking at lipid rafts?

The notion that microdomains enriched in certain specialized lipids exist in membranes has been both attractive and controversial since it was first proposed that such domains, termed rafts, might act as apical sorting devices in epithelial cells. The observation that certain lipids are not extractable in cold nonionic detergent supports the raft concept, but the nature of the in vivo correlate of such detergent-resistant membranes remains enigmatic. In principle, microscopy should be able to determine whether the postulated rafts exist. This article focuses on recent microscopy experiments addressing this question. Several, but not all, results support the raft concept, but further definition of the structure, dynamics and function of lipid domains in various biological contexts is urgently required.     

Ectopically expressed gamma-aminobutyric acid receptor B is functionally down-regulated in isolated lipid raft-enriched membranes

Lipid raft domains have attracted much recent attention as platforms for plasma membrane signalling complexes. In particular, evidence is emerging that shows them to be key regulators of G protein coupled receptor function. The G protein coupled gamma-aminobutyric acid receptor B (GABA(B) receptor) co-isolates with lipid raft domains from rat brain cerebellum. In the present study, we show that the GABA(B1a,2) receptor was also present in lipid raft domains when expressed ectopically in a Chinese hamster ovary cell line. Lipid raft-associated receptor was functionally active, displaying a concentration-dependent increase in GTPgammaS binding in response to the receptor agonist GABA. Compared with whole cell membranes, lipid raft-associated receptor displayed an increased EC(50) and a reduced magnitude of response to GABA. We conclude that lipid raft association is an intrinsic property of the GABA(B1a,2) receptor and is not cell-type specific. In addition, localisation to lipid raft domains may provide a mechanism to inhibit receptor function.     

Palmitoylation and intracellular domain interactions both contribute to raft targeting of linker for activation of T cells

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc- and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly protein-protein interactions, target LAT to rafts.     

Persistence and transport of fauna on drifting kelp (Macrocystis pyrifera (L.) C. Agardh) rafts in the Southern California Bight

Drifting rafts of Macrocystis pyrifera may connect isolated kelp forests in the Southern California Bight. To determine which species might utilize this dispersal mechanism, faunal samples from natural drifting rafts and attached M. pyrifera plants were collected during five cruises between March 1995 and December 1997. These rafts, which can be considered as floating islands, were aged and the macroinvertebrate assemblage enumerated. There was no significant relationship between raft age and species richness, or between species richness and distance offshore, which contrasts with predictions based on island biogeography. Species richness, however, was related to raft weight. Patterns of species presence and density were investigated relative to raft age for the species most frequently associated with rafts. Only one species, the isopod Idotea resecata, was found on all sampled rafts. Some species increased in frequency with raft age and others decreased, but only one relationship, a decline in the frequency of the anemone Epiactis prolifera with raft age was significant. When species density was examined over all cruises, only I. resecata had a significant change in density (an increase) with raft age, but additional significant relationships were found when species density patterns were considered by cruise. The results of all the tests were combined to provide a measure of "raft success". Nine of the most frequent 19 species had a positive score, indicating a favorable response to rafting, seven were unaffected, and two species had negative responses to rafting. Extinction times were calculated using species abundance and raft age relationships. Two species (E. prolifera and Paracerceis cordata), were predicted to persist on rafts for only about 100 days, which is the maximum estimated raft lifetime. All other species were predicted to persist for longer periods if the rafts floated longer. Kelp fauna that begin rafting appear to be largely unaffected by rafting, and hence dispersal on kelp rafts is possible for many members of the kelp forest community.     

The influence of an antitumor lipid – erucylphosphocholine – on artificial lipid raft system modeled as Langmuir monolayer

Abstract

Outer layer of cellular membrane contains ordered domains enriched in cholesterol and sphingolipids, called ‘lipid rafts’, which play various biological roles, i.e., are involved in the induction of cell death by apoptosis. Recent studies have shown that these domains may constitute binding sites for selected drugs. For example alkylphosphocholines (APCs), which are new-generation antitumor agents characterized by high selectivity and broad spectrum of activity, are known to have their molecular targets located at cellular membrane and their selective accumulation in tumor cells has been hypothesized to be linked with the alternation of biophysical properties of lipid rafts. To get a deeper insight into this issue, interactions between representative APC: erucylphosphocholine, and artificial lipid raft system, modeled as Langmuir monolayer (composed of cholesterol and sphingomyelin mixed in 1:2 proportion) were investigated. The Langmuir monolayer experiments, based on recording surface pressure-area isotherms, were complemented with Brewster angle microscopy results, which enabled direct visualization of the monolayers structure. In addition, the investigated monolayers were transferred onto solid supports and studied with AFM. The interactions between model raft system and erucylphosphocholine were analyzed qualitatively (with mean molecular area values) as well as quantitatively (with ΔG^exc function). The obtained results indicate that erucylphosphocholine introduced to raft-mimicking model membrane causes fluidizing effect and weakens the interactions between cholesterol and sphingomyelin, which results in phase separation at high surface pressures. This leads to the redistribution of cholesterol molecules in model raft, which confirms the results observed in biological studies.     

AFM of the Ultrastructural and Mechanical Properties of Lipid-Raft-Disrupted and/or Cold-Treated Endothelial Cells

The nonionic detergent extraction at 4 °C and the cholesterol-depletion-induced lipid raft disruption are the two widely used experimental strategies for lipid raft research. However, the effects of raft disruption and/or cold treatment on the ultrastructural and mechanical properties of cells are still unclear. Here, we evaluated the effects of raft disruption and/or cold (4 °C) treatment on these properties of living human umbilical vein endothelial cells (HUVECs). At first, the cholesterol-depletion-induced raft disruption was visualized by confocal microscopy and atomic force microscopy (AFM) in combination with fluorescent quantum dots. Next, the cold-induced cell contraction and the formation of end-branched filopodia were observed by confocal microscopy and AFM. Then, the cell-surface ultrastructures were imaged by AFM, and the data showed that raft disruption and cold treatment induced opposite effects on cell-surface roughness (a significant decrease and a significant increase, respectively). Moreover, the cell-surface mechanical properties (stiffness and adhesion force) of raft-disrupted- and/or cold-treated HUVECs were measured by the force measurement function of AFM. We found that raft disruption and cold treatment induced parallel effects on cell stiffness (increase) or adhesion force (decrease) and that the combination of the two treatments caused dramatically strengthened effects. Finally, raft disruption was found to significantly impair cell migration as previously reported, whereas temporary cold treatment only caused a slight but nonsignificant decrease in cell migration performed at physiological temperature. Although the mechanisms for causing these results might be complicated and more in-depth studies will be needed, our data may provide important information for better understanding the effects of raft disruption or cold treatment on cells and the two strategies for lipid raft research.     

Polysialic acid chains exhibit enhanced affinity for ordered regions of membranes

Polysialic acid (polySia) forms linear chains which are usually attached to the external surface of the plasma membrane mainly through the Neural Cell Adhesion Molecule (NCAM) protein. It is exposed on neural cells, several types of cancer cells, dendritic cells, and egg and sperm cells. There are several lipid raft-related phenomena in which polySia is involved; however the mechanisms of polySia action as well as determinants of its localization in lipid raft microdomains are still unknown, although the majority of NCAM molecules in the liquid-ordered raft membrane fractions of neural cells appear to be polysialylated. Here we investigate the affinity of polySia (both soluble and NCAM-dependent plasma membrane-bound) for liquid-ordered- and liquid-disordered regions of lipid vesicle and neuroblastoma cell membranes. Our studies indicate that polySia chains have a higher affinity for ordered regions of membranes as determined by the dissociation constant values for polySia-lipid bilayer complex, the fluorescence intensity of polySia bound to giant vesicles, the polySia-to-membrane FRET signal at the plasma membrane of live cells, and the decrease of the FRET signals after Endo-N treatment of the cells. These results suggest that polysialylation may be one of the determinants of protein association with liquid-ordered membrane lipid raft domains.     

Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine

Wnt signaling pathways regulate many developmental responses; however, little is known about how Wnt ligands function on a biochemical level. Recent studies have shown that Wnt-3a is palmitoylated before secretion. Here we report that Drosophila Wnt-1 (Wingless) also undergoes a lipid modification. Lipidation occurs in the endoplasmic reticulum and is dependent on Porcupine, a putative O-acyltransferase. After modification, DWnt-1 partitions as a membrane-anchored protein and is sorted into lipid raft detergent-insoluble microdomains. Lipidation, raft targeting, and secretion can be blocked by the addition of 2-bromopalmitate, a competitive inhibitor of O-acyltransferase activity. Based on these results we propose a model whereby lipidation targets Wnt-1 to secretory vesicles that deliver the ligand to specialized microdomains at the cell surface where it can be packaged for secretion.     

Regulation of expression and function of Lck tyrosine kinase by high cell density

For many types of cells, an increase in cell density leads to characteristic changes in intracellular signalling and cell function. It is unknown, however, whether cell density affects the function of T lymphocytes. It is presented here that aggregation of Jurkat T cells, murine thymocytes or human peripheral blood T cells, results in gradual modification of the Lck tyrosine kinase. Within one hour of aggregation, Lck in the detergent-insoluble lipid raft fraction is dephosphorylated mainly at the carboxy-terminal tyrosine. Further aggregation leads to gradual loss of Lck protein from both lipid raft and non-raft fractions which is accompanied by increased protein ubiquitination, a process that is more evident in the detergent-soluble fraction. In contrast, the expression of LAT, which like Lck distributes to raft and non-raft membrane, or Csk, a kinase with a structure similar to Lck, is not affected by cell aggregation. Dephosphorylation of lipid raft-associated Lck, albeit with reduced kinetics, is observed in aggregated Jurkat CD45-deficient cells as well, suggesting involvement of additional tyrosine phosphatases. Changes in Lck structure and expression correlate with reduced ability of aggregated cells to fully activate protein tyrosine phosphorylation after stimulation of the TCR, and with changes in the activation of down-stream signalling cascades.     

Localization of neuronal growth-associated, microtubule-destabilizing factor SCG10 in brain-derived raft membrane microdomains

Raft is a mobile membrane subdomain enriched in sphingolipid and cholesterol and also various signaling molecules. Previous observation suggested that brain-derived rafts contain tubulin but that rafts of non-neural origin do not. We hypothesized that SCG10, one of the neuronal growth-associated proteins (nGAPs), might be a neuron-specific molecule that anchors tubulin to neuronal rafts, and we explored biochemically its subcellular localization, interaction with tubulin, and effects on microtubule dynamics. In postnatal rat brain extracts, SCG10 was recovered mostly in membrane-associated fractions, and at least half was included in the raft fraction that was also enriched in GAP-43 and NAP-22. SCG10-enriched brain rafts also contained tubulin, and chemical cross-linking experiments revealed that SCG10 was closely associated with tubulin. In addition, SCG10 was able to inhibit polymerization of tubulin. These results indicate that SCG10 is a component of neuronal rafts as are other nGAPs, and suggest that SCG10 may be involved in signaling events in membranes for cytoskeletal reorganization around neuronal rafts.     

Regulation of expression and function of Lck tyrosine kinase by high cell density

For many types of cells, an increase in cell density leads to characteristic changes in intracellular signalling and cell function. It is unknown, however, whether cell density affects the function of T lymphocytes. It is presented here that aggregation of Jurkat T cells, murine thymocytes or human peripheral blood T cells, results in gradual modification of the Lck tyrosine kinase. Within one hour of aggregation, Lck in the detergent-insoluble lipid raft fraction is dephosphorylated mainly at the carboxy-terminal tyrosine. Further aggregation leads to gradual loss of Lck protein from both lipid raft and non-raft fractions which is accompanied by increased protein ubiquitination, a process that is more evident in the detergent-soluble fraction. In contrast, the expression of LAT, which like Lck distributes to raft and non-raft membrane, or Csk, a kinase with a structure similar to Lck, is not affected by cell aggregation. Dephosphorylation of lipid raft-associated Lck, albeit with reduced kinetics, is observed in aggregated Jurkat CD45-deficient cells as well, suggesting involvement of additional tyrosine phosphatases. Changes in Lck structure and expression correlate with reduced ability of aggregated cells to fully activate protein tyrosine phosphorylation after stimulation of the TCR, and with changes in the activation of down-stream signalling cascades.     

Selective localization of recognition complexes for leukotriene B4 and formyl-Met-Leu-Phe within lipid raft microdomains of human polymorphonuclear neutrophils

Neutrophilic polymorphonuclear leukocytes contain glycosphingolipid- and cholesterol-enriched lipid raft microdomains within the plasma membrane. Although there is evidence that lipid rafts function as signaling platforms for CXCR chemokine receptors, their role in recognition systems for other chemotaxins such as leukotriene B4 (LTB4) and fMLP is unknown. To address this question, human neutrophils were extracted with 1% Brij-58 and fractionated on sucrose gradients. B leukotriene receptor-1 (BLT-1), the primary LTB4 receptor, partitioned to low density fractions, co-isolating with the lipid raft marker, flotillin-1. By contrast, formyl peptide receptor (FPR), the primary fMLP receptor, partitioned to high density fractions, co-isolating with a non-raft marker, Cdc42. This pattern was preserved after the cells were stimulated with LTB4 or fMLP. Fluorescence resonance energy transfer (FRET) was performed to confirm the proximity of BLT-1 and FPR with these markers. FRET was detected between BLT1 and flotillin-1 but not Cdc42, whereas FRET was detected between FPR and Cdc42, but not flotillin-1. Pretreating neutrophils with methyl-beta-cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca(2+) mobilization and ERK1/2 phosphorylation in response to LTB4 but had no effect on either of these responses to fMLP. We conclude that BLT-1 is physically located within lipid raft microdomains of human neutrophils and that disrupting lipid raft integrity suppresses LTB4-induced activation. By contrast, FPR is not associated with lipid rafts, and fMLP-induced signaling does not require lipid raft integrity. These findings highlight the complexity of chemotaxin signaling pathways and offer one mechanism by which neutrophils may spatially organize chemotaxin signaling within the plasma membrane.     

Glycosylphosphatidylinositol-anchored proteins as regulators of cortical cytoskeleton

Glycosylphosphatidylinositol-anchored proteins (GPI-AP) are important players in reception and signal transduction, cell adhesion, guidance, formation of immune synapses, and endocytosis. At that, a particular GPI-AP can have different activities depending on a ligand. It is known that GPI-AP oligomer creates a lipid raft in its base on plasma membrane, which serves as a signaling platform for binding and activation of src-family kinases. Yet, this does not explain different activities of GPI-APs. Meanwhile, it has been shown that short-lived actomyosin complexes are bound to GPI-APs through lipid rafts. Here, we hypothesize that cell cortical cytoskeleton is the main target of GPI-AP signaling. Our hypothesis is based on the fact that the GPI-AP-induced lipid raft bound to actin filaments and anionic lipids of this raft is known to interact with and activate various actin-nucleating factors, such as formins and N-WASP. It is also known that these and other actin-regulating proteins are activated by src-family kinases directly or through their effectors, such as cortactin and abl-kinases. Regulation of cytoskeleton by GPI-APs may have impact on morphogenesis, cell guidance, and endocytosis, as well as on signaling of other receptors. To evaluate our hypothesis, we have comprehensively considered physiological activities of two GPI-APs–urokinase receptor and T-cadherin.     

Targeting membrane proteins to liquid-ordered phases: molecular self-organization explored by fluorescence correlation spectroscopy

The complex and dynamic architecture of biological membranes comprises of various heterogeneities, some of which may include lipid-based and/or protein-based microdomains called "rafts". Due to interactions among membrane components, several types of domains can form with different characteristics and mechanisms of formation. Model membranes, such as giant unilamellar vesicles (GUVs), provide a key system to study lipid-lipid and lipid-protein interactions, which are potentially relevant to raft formation, by (single-molecule) optical microscopy. Here, we review studies of combined confocal imaging and fluorescence correlation spectroscopy (FCS) on lipid dynamics and organization in domains assembled in GUVs, prepared from various lipid mixtures, which are relevant to the problem of raft formation. Finally, we summarize the results on lipid-protein interactions, which govern the targeting of several putative raft- and non-raft-associated membrane proteins to domain-exhibiting GUVs.     

Order,disorder, and perturbations in actin-aldolase rafts

Actin-aldolase rafts provide insights into the use of rafts as models for three-dimensional actin bundles. Although aldolase has three twofold axes, filaments in actin-aldolase rafts were not strictly related by a twofold axis. Interfilament angles were on average +15 degrees off the expected 180 degrees, and most rafts appeared handed; that is, rows of cross-bridges were tilted in a clockwise direction off the perpendicular. We can account for both the deviation of the angle from 180 degrees and the handedness of the rafts by a steric constraint due to the lipid layer. We further found that the axial spacings of cross-bridges varied significantly from raft to raft. We suggest that this difference arises from variations in the twist of the filaments that nucleate raft formation; that is, filaments added to a raft adopt the symmetry of those in the raft. We conclude that the organization of filaments in rafts can be modulated by outside factors such as the lipid layer and that the variable twist of filaments in the nucleating core of the raft are imposed on all the filaments in the raft. These results provide a measure of the potential for polymorphism in actin assemblies.