Barriers to diffusion of plasma membrane proteins form early during guinea pig spermiogenesis.

The plasma membrane of the mature guinea pig sperm is segregated into at least four domains of different composition. Previous studies have shown that some proteins localized within these domains are free to diffuse laterally, suggesting that barriers to protein diffusion are responsible for maintaining the nonuniform distribution of at least some surface proteins in mature sperm. The different membrane domains appear sequentially during sperm morphogenesis in the testis and during later passage through the epididymis. To determine when diffusion barriers become functional during sperm development, we examined the diffusion of two proteins that are expressed on the cell surface of developing spermatids and become segregated to different plasma membrane domains during the course of spermiogenesis. Both proteins exhibited rapid lateral diffusion throughout spermiogenesis, even after they become localized to specific regions of the surface membrane. These results suggest that barriers to membrane diffusion form concomitantly with membrane domains during spermiogenesis.     

Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis

Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrate distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and show dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies.     

Isobaric tags for relative and absolute quantification- based comparative proteomics reveals the features of plasma membrane-associated proteomes of pollen grains and pollen tubes from Lilium davidii

Mature pollen grains (PGs) from most plant species are metabolically quiescent. However, once pollinated onto stigma, they quickly hydrate and germinate. A PG can give rise to a vegetative cell-derived polarized pollen tube (PT), which represents a specialized polar cell. The polarized PT grows by the tip and requires interaction of different signaling molecules localized in the apical plasma membrane and active membrane trafficking. The mechanisms underlying the interaction and membrane trafficking are not well understood. In this work, we purified PG and PT plasma-membrane vesicles from Lilium davidii Duch. using the aqueous two-phase partition technique, then enriched plasma membrane proteins by using Brij58 and KCl to remove loosely bound contaminants. We identified 223 integral and membrane-associated proteins in the plasma membrane of PGs and PTs by using isobaric tags for relative and absolute quantification (iTRAQ) and 2-D high-performance liquid chromatography-tandem mass spectrometry. More than 68% of the proteins have putative transmembrane domains and/or lipid-modified motifs. Proteins involved in signal transduction, membrane trafficking and transport are predominant in the plasma-membrane proteome. We revealed most components of the clathrin-dependent endocytosis pathway. Statistical analysis revealed 14 proteins differentially expressed in the two development stages: in PTs, six upregulated and eight downregulated are mainly involved in signaling, transport and membrane trafficking. These results provide novel insights into polarized PT growth.     

Ras nanoclusters: molecular structure and assembly

H-, N- and K-ras4B are lipid-anchored, peripheral membrane guanine nucleotide binding proteins. Recent work has shown that Ras proteins are laterally segregated into non-overlapping, dynamic domains of the plasma membrane called nanoclusters. This lateral segregation is important to specify Ras interactions with membrane-associated proteins, effectors and scaffolding proteins and is critical for Ras signal transduction. Here we review biological, in vitro and structural data that provide insight into the molecular basis of how palmitoylated Ras proteins are anchored to the plasma membrane. We explore possible mechanisms for how the interactions of H-ras with a lipid bilayer may drive nanocluster formation.     

Proteins and cholesterol-rich domains

Biological membranes are composed of many molecular species of lipids and proteins. These molecules do not mix ideally. In the plane of the membrane components are segregated into domains that are enriched in certain lipids and proteins. Cholesterol is a membrane lipid that is not uniformly distributed in the membrane. Proteins play an important role in determining cholesterol distribution. Certain types of protein lipidation are known to cause the lipoprotein to sequester with cholesterol and to stabilize cholesterol-rich domains. However, proteins that are excluded from such domains also contribute to the redistribution of cholesterol. One of the motifs that favor interaction with cholesterol is the CRAC motif. The role of the CRAC motif of the gp41 fusogenic protein of HIV is discussed. The distribution of the multianionic lipid, phosphatidylinositol(4,5)bis-phosphate (PtnIns(4,5)P2), is also not uniform in cell membranes. This lipid has several functions in the cell, including a morphological role in determining the sites of attachment of the actin cytoskeleton to the plasma membrane. PtnIns(4,5)P2 is sequestered by proteins having clusters of cationic residues in their sequence. Certain proteins containing cationic clusters also contain moieties such as myristoylation or a CRAC segment that would also endow them with the ability to sequester to a cholesterol-rich domain. These proteins interact with PtnIns(4,5)P2 in a cholesterol-dependent manner forming domains that are enriched in both cholesterol and in PtnIns(4,5)P2 but can also be distinct from liquid-ordered raft-like domains.     

Regulation of desmosome assembly in MDCK epithelial cells: coordination of membrane core and cytoplasmic plaque domain assembly at the plasma membrane

Desmosomes are major components of the intercellular junctional complex in epithelia. They consist of at least eight different cytoplasmic and integral membrane proteins that are organized into two biochemically and structurally distinct domains: the cytoplasmic plaque and membrane core. We showed previously that in MDCK epithelial cells major components of the cytoplasmic plaque (desmoplakin I and II; DPI/II) and membrane core domains (desmoglein I; DGI) initially enter a pool of proteins that is soluble in buffers containing Triton X-100, and then titrate into an insoluble pool before their arrival at the plasma membrane (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685; Pasdar. M., and W. J. Nelson. 1989. J. Cell Biol. 109:163-177). We have now examined whether either the soluble or insoluble pool of these proteins represents an intracellular site for assembly and interactions between the domains before their assembly into desmosomes at the plasma membrane. Interactions between the Triton X-100-soluble pools of DPI/II and DGI were analyzed by sedimentation of extracted proteins in sucrose gradients. Results show distinct differences in the sedimentation profiles of these proteins, suggesting that they are not associated in the Triton X-100-soluble pool of proteins; this was also supported by the observation that DGI and DPI/II could not be coimmunoprecipitated in a complex with each other from sucrose gradient fractions. Immunofluorescence analysis of the insoluble pools of DPI/II and DGI, in cells in which desmosome assembly had been synchronized, showed distinct differences in the spatial distributions of these proteins. Furthermore, DPI/II and DGI were found to be associated with different elements of cytoskeleton; DPI/II were located along cytokeratin intermediate filaments, whereas DGI appeared to be associated with microtubules. The regulatory role of cytoskeletal elements in the intracellular organization and assembly of the cytoplasmic plaque and membrane core domains, and their integration into desmosomes on the plasma membrane is discussed.     

Plasma Membranes Are Subcompartmentalized into a Plethora of Coexisting and Diverse Microdomains in Arabidopsis and Nicotiana benthamiana

Eukaryotic plasma membranes are highly compartmentalized structures. So far, only a few individual proteins that function in a wide range of cellular processes have been shown to segregate into microdomains. However, the biological roles of most microdomain-associated proteins are unknown. Here, we investigated the heterogeneity of distinct microdomains and the complexity of their coexistence. This diversity was determined in living cells of intact multicellular tissues using 20 different marker proteins from Arabidopsis thaliana, mostly belonging to the Remorin protein family. These proteins associate with microdomains at the cytosolic leaflet of the plasma membrane. We characterized these membrane domains and determined their lateral dynamics by extensive quantitative image analysis. Systematic colocalization experiments with an extended subset of marker proteins tested in 45 different combinations revealed the coexistence of highly distinct membrane domains on individual cell surfaces. These data provide valuable tools to study the lateral segregation of membrane proteins and their biological functions in living plant cells. They also demonstrate that widely used biochemical approaches such as detergent-resistant membranes cannot resolve this biological complexity of membrane compartmentalization in vivo.     

Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway

Epithelial cell polarity depends on mechanisms for targeting proteins to different plasma membrane domains. Here, we dissect the pathway for apical delivery of several raft-associated, glycosyl phosphatidylinositol (GPI)-anchored proteins in polarized MDCK cells using live-cell imaging and selective inhibition of apical or basolateral exocytosis. Rather than trafficking directly from the trans-Golgi network (TGN) to the apical plasma membrane as previously thought, the GPI-anchored proteins followed an indirect, transcytotic route. They first exited the TGN in membrane-bound carriers that also contained basolateral cargo, although the two cargoes were laterally segregated. The carriers were then targeted to and fused with a zone of lateral plasma membrane adjacent to tight junctions that is known to contain the exocyst. Thereafter, the GPI-anchored proteins, but not basolateral cargo, were rapidly internalized, together with endocytic tracer, into clathrin-free transport intermediates that transcytosed to the apical plasma membrane. Thus, apical sorting of these GPI-anchored proteins occurs at the plasma membrane, rather than at the TGN.     

Eisosome proteins assemble into a membrane scaffold

Spatial organization of membranes into domains of distinct protein and lipid composition is a fundamental feature of biological systems. The plasma membrane is organized in such domains to efficiently orchestrate the many reactions occurring there simultaneously. Despite the almost universal presence of membrane domains, mechanisms of their formation are often unclear. Yeast cells feature prominent plasma membrane domain organization, which is at least partially mediated by eisosomes. Eisosomes are large protein complexes that are primarily composed of many subunits of two Bin-Amphiphysin-Rvs domain-containing proteins, Pil1 and Lsp1. In this paper, we show that these proteins self-assemble into higher-order structures and bind preferentially to phosphoinositide-containing membranes. Using a combination of electron microscopy approaches, we generate structural models of Pil1 and Lsp1 assemblies, which resemble eisosomes in cells. Our data suggest that the mechanism of membrane organization by eisosomes is mediated by self-assembly of its core components into a membrane-bound protein scaffold with lipid-binding specificity.     

Probing for membrane domains in the endoplasmic reticulum: retention and degradation of unassembled MHC class I molecules

Quality control of protein biosynthesis requires ER-retention and ER-associated degradation (ERAD) of unassembled/misfolded molecules. Although some evidence exists for the organization of the ER into functionally distinct membrane domains, it is unknown if such domains are involved in the retention and ERAD of unassembled proteins. Here, it is shown that unassembled MHC class I molecules are retained in the ER without accumulating at ER-exit sites or in the ERGIC of beta2m-/- cells. Furthermore, these molecules did not cluster in the ER membrane and appeared to be highly mobile even when ERAD or their association with calnexin were inhibited. However, upon ATP depletion, they were reversibly segregated into an ER membrane domain, distinct from ER exit sites, which included calnexin and COPII, but not the ERGIC marker protein p58. This quality control domain was also observed upon prolonged inhibition of proteasomes. Microtubules were required for its appearance. Segregation of unfolded proteins, ER-resident chaperones, and COPII may be a temporal adaptation to cell stress.     

Recycling endosomes of polarized epithelial cells actively sort apical and basolateral cargos into separate subdomains

The plasma membranes of epithelial cells plasma membranes contain distinct apical and basolateral domains that are critical for their polarized functions. However, both domains are continuously internalized, with proteins and lipids from each intermixing in supranuclear recycling endosomes (REs). To maintain polarity, REs must faithfully recycle membrane proteins back to the correct plasma membrane domains. We examined sorting within REs and found that apical and basolateral proteins were laterally segregated into subdomains of individual REs. Subdomains were absent in unpolarized cells and developed along with polarization. Subdomains were formed by an active sorting process within REs, which precedes the formation of AP-1B-dependent basolateral transport vesicles. Both the formation of subdomains and the fidelity of basolateral trafficking were dependent on PI3 kinase activity. This suggests that subdomain and transport vesicle formation occur as separate sorting steps and that both processes may contribute to sorting fidelity.     

Tracing pathways of transport protein evolution

We have conducted bioinformatic analyses of integral membrane transport proteins belonging to dozens of families. These families rarely include proteins that function in a capacity other than transport. Many transporters have arisen by intragenic duplication, triplication and quadruplication events, in which the numbers of transmembrane alpha-helical hydrophobic segments (TMSs) have increased. The elements multiplied may encode two, three, four, five, six, 10 or 12 TMSs and gave rise to proteins with four, six, seven, eight, nine, 10, 12, 20, 24 and 30 TMSs. Gene fusion, splicing, deletion and insertion events have also contributed to protein topological diversity. Amino acid substitutions have allowed membrane-embedded domains to become hydrophilic domains and vice versa. Some evidence suggests that amino acid substitutions occurring over evolutionary time may in some cases have drastically altered protein topology. The results summarized in this microreview establish the independent origins of many transporter families and allow postulation of the specific pathways taken for their appearance.     

Direct evidence for cholesterol crystalline domains in biological membranes: role in human pathobiology

This review will discuss the use of small-angle X-ray diffraction approaches to study the organization of lipids in plasma membranes derived from two distinct mammalian cell types: arterial smooth muscle cells and ocular lens fiber cells. These studies indicate that cholesterol at an elevated concentration can self-associate and form immiscible domains in the plasma membrane, a phenomenon that contributes to both physiologic and pathologic cellular processes, depending on tissue source. In plasma membrane samples isolated from atherosclerotic smooth muscle cells, the formation of sterol-rich domains is associated with loss of normal cell function, including ion transport activity and control of cell replication. Analysis of meridional diffraction patterns from intact and reconstituted plasma membrane samples indicates the presence of an immiscible cholesterol domain with a unit cell periodicity of 34 Å, consistent with a cholesterol monohydrate tail-to-tail bilayer, under disease conditions. These cholesterol domains were observed in smooth muscle cells enriched with cholesterol in vitro as well as from cells obtained ex vivo from an animal model of atherosclerosis. By contrast, well-defined cholesterol domains appear to be essential to the normal physiology of fiber cell plasma membranes of the human ocular lens. The organization of cholesterol into separate domains underlies the role of lens fiber cell plasma membranes in maintaining lens transparency. These domains may also interfere with cataractogenic aggregation of soluble lens proteins at the membrane surface. Taken together, these analyses provide examples of both physiologic and pathologic roles that sterol-rich domains may have in mammalian plasma membranes. These findings support a model of the membrane in which cholesterol aggregates into structurally distinct regions that regulate the function of the cell membrane.     

Direct evidence for cholesterol crystalline domains in biological membranes: role in human pathobiology

This review will discuss the use of small-angle X-ray diffraction approaches to study the organization of lipids in plasma membranes derived from two distinct mammalian cell types: arterial smooth muscle cells and ocular lens fiber cells. These studies indicate that cholesterol at an elevated concentration can self-associate and form immiscible domains in the plasma membrane, a phenomenon that contributes to both physiologic and pathologic cellular processes, depending on tissue source. In plasma membrane samples isolated from atherosclerotic smooth muscle cells, the formation of sterol-rich domains is associated with loss of normal cell function, including ion transport activity and control of cell replication. Analysis of meridional diffraction patterns from intact and reconstituted plasma membrane samples indicates the presence of an immiscible cholesterol domain with a unit cell periodicity of 34 A, consistent with a cholesterol monohydrate tail-to-tail bilayer, under disease conditions. These cholesterol domains were observed in smooth muscle cells enriched with cholesterol in vitro as well as from cells obtained ex vivo from an animal model of atherosclerosis. By contrast, well-defined cholesterol domains appear to be essential to the normal physiology of fiber cell plasma membranes of the human ocular lens. The organization of cholesterol into separate domains underlies the role of lens fiber cell plasma membranes in maintaining lens transparency. These domains may also interfere with cataractogenic aggregation of soluble lens proteins at the membrane surface. Taken together, these analyses provide examples of both physiologic and pathologic roles that sterol-rich domains may have in mammalian plasma membranes. These findings support a model of the membrane in which cholesterol aggregates into structurally distinct regions that regulate the function of the cell membrane.     

Taking the Scenic Route: Biosynthetic Traffic to the Plasma Membrane in Polarized Epithelial Cells

The maintenance of epithelial cell function requires the establishment and continuous renewal of differentiated apical and basolateral plasma membrane domains with distinct lipid and protein compositions. Newly synthesized proteins destined for either surface domain are processed along the biosynthetic pathway and segregated into distinct subsets of transport carriers emanating from the trans -Golgi network. Recent studies have illuminated additional complexities in the subsequent delivery of these proteins to the cell surface. In particular, multiple routes to the apical and basolateral cell surfaces have been uncovered, and many of these involve indirect passage through endocytic compartments. This review summarizes our current understanding of these routes and discusses open issues that remain to be clarified.     

Highly purified bile-canalicular vesicles and lateral plasma membranes isolated from rat liver on Nycodenz gradients. Biochemical and immunolocalization studies.

1. A liver canalicular plasma-membrane fraction enriched 115-155-fold in five marker enzymes relative to the tissue homogenate was obtained by sonication of liver plasma membranes followed by fractionation in iso-osmotic Nycodenz gradients. 2. Two lateral-plasma membrane fractions were also collected by this procedure; the lighter-density fraction was still associated with canalicular membranes, as assessed by enzymic and polypeptide analysis. 3. The polypeptide composition of the domain-defined plasma-membrane fractions was evaluated. It was demonstrated by immunoblotting that the 41 kDa alpha-subunit of the inhibitory G-protein, associated in high relative amounts with canalicular plasma-membrane fractions, was partially lost in the last stage of purification; however, this subunit was retained by lateral plasma membranes. 4. Antibodies to the proteins of bile-canalicular vesicles were shown to localize to the hepatocyte surface in thin liver sections examined by immunofluorescent and immuno-gold electron microscopy. Two subsets of antigens were identified, one present on both sinusoidal and canalicular plasma-membrane domains and another, by using antisera pre-absorbed with sinusoidal plasma membranes, that was confined to the bile-canalicular domain.     

Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells

To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.     

Plasma membrane stress induces relocalization of Slm proteins and activation of TORC2 to promote sphingolipid synthesis

The plasma membrane delimits the cell, and its integrity is essential for cell survival. Lipids and proteins form domains of distinct composition within the plasma membrane. How changes in plasma membrane composition are perceived, and how the abundance of lipids in the plasma membrane is regulated to balance changing needs remains largely unknown. Here, we show that the Slm1/2 paralogues and the target of rapamycin kinase complex 2 (TORC2) play a central role in this regulation. Membrane stress, induced by either inhibition of sphingolipid metabolism or by mechanically stretching the plasma membrane, redistributes Slm proteins between distinct plasma membrane domains. This increases Slm protein association with and activation of TORC2, which is restricted to the domain known as the membrane compartment containing TORC2 (MCT; ref.?). As TORC2 regulates sphingolipid metabolism, our discoveries reveal a homeostasis mechanism in which TORC2 responds to plasma membrane stress to mediate compensatory changes in cellular lipid synthesis and hence modulates the composition of the plasma membrane. The components of this pathway and their involvement in signalling after membrane stretch are evolutionarily conserved.     

Temporal expression and localization of protein farnesyltransferase during spermiogenesis and posttesticular sperm maturation in the hamster

Spermiogenesis and posttesticular sperm maturation in the epididymis are distinct developmental processes that result in a polarized spermatozoon possessing a plasma membrane partitioned into segment-specific domains of distinct composition and function. The mechanisms that specify the distribution of intracellular organelles and target proteins to restricted membrane domains are not well understood. In this study we examined the expression pattern and distribution of protein farnesyltransferase (FTase) in hamster spermatids and epididymal spermatozoa to determine if protein lipidation may represent a potential mechanism to regulate protein association with specific organelles or the plasma membrane. Round spermatids exhibited only weak immunostaining with antibody against the β-subunit of FTase, whereas elongating spermatids exhibited a high level of FTase expression that was segregated to the cytoplasmic lobe surrounding the anterior flagellum. Although FTase was released with the residual body, mature spermatids retained FTase within the midpiece and cytoplasmic droplet. In epididymal spermatozoa, FTase remained associated with the cytoplasmic droplet during its migration to the midpiece-principal piece junction; following release of the cytoplasmic droplet, no immunodetectable FTase was noted in the midpiece segment. Immunoblotting demonstrated the presence of both the α and β subunits of FTase in sperm lysates. The temporal expression pattern and restricted distribution of FTase in spermatids and epididymal spermatozoa suggest a potential role in regulating protein association with specific organelles and/or membrane domains of the mature spermatozoon. Mol. Reprod. Dev. 48:71–76, 1997. © 1997 Wiley-Liss, Inc.     

ERM proteins and merlin: integrators at the cell cortex

A fundamental property of many plasma-membrane proteins is their association with the underlying cytoskeleton to determine cell shape, and to participate in adhesion, motility and other plasma-membrane processes, including endocytosis and exocytosis. The ezrin-radixin-moesin (ERM) proteins are crucial components that provide a regulated linkage between membrane proteins and the cortical cytoskeleton, and also participate in signal-transduction pathways. The closely related tumour suppressor merlin shares many properties with ERM proteins, yet also provides a distinct and essential function.