Mark S. Bretscher

Membrane structure: some general principles.

The arrangement of lipids and some proteins in the erythrocyte membrane has been discussed. The conclusions from this are listed here as a set of general guidelines for the structure of membranes of higher organisms: some of these rules may be wrong. But at this stage it seems useful to sharpen our thoughts in this way and thereby focus attention on various specific points. 1) The basis of a membrane is a lipid bilayer with (i) choline phospholipids and glycolipids in the external half and (ii) amino (and possibly some choline) phospholipids in the cytoplasmic half. There is effectively no lipid exchange across the bilayer (unless enzymatically catalyzed) (68). 2) Some proteins extend across the bilayer. Where this is so, they will in general have carbohydrate on their surface remote from the cytoplasm. This carbohydrate may prevent the protein diffusing out of the membrane into the cytoplasm; it acts as a lock on the protein. 3) Just as lipids do not flip-flop, proteins do not rotate across the membrane. Lateral motion or rotation of lipids and proteins in the plane of the bilayer may be expected. 4) Most membrane protein is associated with the inner, cytoplasmic, urface of the membrane. Proteins are not usually associated exclusively with the outer half of the lipid bilayer. 5) Membrane proteins are a special class of cytoplasmic proteins, not of secreted proteins.

A major protein which spans the human erythrocyte membrane.

Abstract Component a is the major protein component on the surface of human erythrocytes. It has been labelled from one, or both, sides of the cell membrane by labelling intact cells or cell ghosts. Fingerprint maps of labelled peptides derived from component a , labelled in these two ways, indicate that parts of a reside on each side of the membrane. Treatment of whole cells with pronase degrades all copies of component a ; a smaller polypeptide, component c , is now found in the membrane. The results indicate that component a , a single polypeptide chain of about 105,000 daltons, extends through the cell membrane and that c is derived from a .

Phosphatidyl-ethanolamine: Differential labelling in intact cells and cell ghosts of human erythrocytes by a membrane-impermeable reagent

Abstract Much more phosphatidyl-ethanolamine is labelled when erythrocyte ghosts, rather than intact cells, are exposed to the reagent [35S]FMMP ‡ The ability to label phosphatidyl-ethanolamine from the cell exterior is not increased by prior treatment of the cells with pronase. This suggests that phosphatidyl-ethanolamine may exist in an unreactive state in the outer half of the lipid bilayer, or that most of it resides in the inner half of the bilayer of the erythrocyte membrane.

Human erythrocyte membranes: Specific labelling of surface proteins

Abstract A very reactive, highly radioactive (about 10 Ci/m-mole) reagent designed to acylate amino groups has been synthesized. This compound, the sulphone of 35 S-labelled formylmethionyl methyl phosphate, cannot pass through the red blood cell membrane. When added to intact red blood cells, it reacts with, and labels, two proteins on the outside surface of the cell membrane: these have molecular weights of about 105,000 and 90,000 daltons. All membrane proteins appear to be labelled if erythrocyte ghosts are exposed to the reagent. The two proteins exposed on the outside surface of the erythrocyte are major membrane components: they probably cover a significant portion of the surface of the cell. The smaller of these two proteins carries the bulk of the cell surface sialic acid and a large portion of cell surface carbohydrate. The other, a larger protein, carries very little, if any, carbohydrate or sialic acid.

Endocytosis and recycling of the fibronectin receptor in CHO cells

An anti‐fibronectin receptor monoclonal antibody preferentially labels the leading edges of freshly plated CHO fibroblasts, suggesting that this receptor circulates through the endocytic cycle. Using a new labelling reagent, I show that this receptor is indeed endocytosed at 37 degrees C and then returned to the cell surface. These findings imply that fibronectin receptors are recirculated to the leading edge of a motile cell by the endocytic cycle, and establish that the processes of endocytosis/exocytosis and cell locomotion are intimately linked.

Getting Membrane Flow and the Cytoskeleton to Cooperate in Moving Cells

I have tried here to provide a balanced view of cap formation and its extension to cell locomotion, as seen from two different perspective.The cytoskeletal model described is based on a polarized actin cycle (Bray and White 1988xCortical flow in animal cells. Bray, D and White, J.G. Science. 1988; 239: 883–888Crossref | PubMedSee all ReferencesBray and White 1988). Filament assembly occurs at the leading edge where it seems to strengthen substrate attachments. Actin may also lead to filopodial extension and help push the leading edge forward either directly by polymerization or in conjunction with other microfilament-associated proteins. Filaments adjacent to the plasma membrane flow from the leading edge towards the rear of the cell or growth cone, where they disassemble. This cycle is completed by the transfer of actin from the back to the front for reuse, presumably by actin subunits diffusing forward through the cytoplasm. The cells feet, or the rakes tines, poke through the plasma membrane and, being attached to the actin network, also migrate rearwards. This could provide the motive force for capping and cell locomotion. How the actin network is moved backward in this scheme is unclear: myosin, the most likely motor, would have to be linked to a structure to provide a buffer against which to push. Other models for effecting the actin flow can be envisaged which incorporate different microfilament-associated proteins, many of which are well-characterized molecules (Bretscher 1991xMicrofilament structure and function in the cortical cytoplasm. Bretscher, A.P. Annu. Rev. Cell Biol. 1991; 7: 337–374Crossref | PubMedSee all ReferencesBretscher 1991). This general scheme, as presented, has no obvious role for microtubules in cap formation.I favor a flow mechanism, driven by a polarized endocytic cycle in which both microtubules and microfilaments effect the intracellular transport of endocytosed membrane, because it appears to explain cap formation and particle migration naturally. In addition, the experimental evidence for the sites of endocytosis and exocytosis and the speed at which the endocytic cycle operates seem able to explain the observed phenomena, at least in fibroblasts. I therefore see related processes from that viewpoint, and offer three examples of this.First, the rearward movement of filamentous actin in migrating fibroblasts may be caused by its association with, or proximity to, a rearwardly flowing membrane. This could fit with the observation (Forscher and Smith 1988xActions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. Forscher, P and Smith, S.J. J. Cell Biol. 1988; 107: 1505–1516Crossref | PubMedSee all ReferencesForscher and Smith 1988) that actin polymerization at the front of a growth cone is not needed for the rearward movement of microfilaments. However, this possibility would require that, in the few minutes after cytochalasin B addition when these observations are made, membrane continues to be transported, and added, to the leading edge in the apparent absence of adjacent microtubules and microfilaments.Second, the shape of a migrating cell could depend on the efficiency with which its attaching feet circulate. If they are able to detach easily from the substratum and circulate quickly, feet would mainly exist near the leading edge of the cell; this is where other rapidly circulating receptors, such as the transferrin receptor, are found (19xDistribution of the transferrin receptor in normal human fibroblasts and fibrosarcoma cells. Ekblom, P, Thesleff, I, Lehto, V-P, and Virtanen, I. Int. J. Cancer. 1983; 31: 111–116Crossref | PubMedSee all References, 7xDistribution of receptors for transferrin and low density lipoprotein on the surface of giant HeLa cells. Bretscher, M.S. Proc. Natl. Acad. Sci. USA. 1983; 80: 454–458Crossref | PubMedSee all ReferencesBretscher 1996xMoving membrane up to the front of migrating cells. Bretscher, M.S. Cell. 1996; 85: 465–467Abstract | Full Text | Full Text PDF | PubMed | Scopus (122)See all ReferencesBretscher 1996; 24xThe appearance and internalization of transferrin receptors at the margins of spreading human tumor cells. Hopkins, C.R. Cell. 1985; 40: 199–208Abstract | Full Text PDF | PubMed | Scopus (39)See all References, 25xIn migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella. Hopkins, C.R, Gibson, A, Shipman, M, Strickland, D.K, and Trowbridge, I.S. J. Cell Biol. 1994; 125: 1265–1274Crossref | PubMed | Scopus (207)See all References). This might cause the cell to be snail-shaped (see alsoLawson and Maxfield 1995xCa2+- and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils. Lawson, M.A and Maxfield, F.R. Nature. 1995; 377: 75–79Crossref | PubMedSee all ReferencesLawson and Maxfield 1995). Alternatively, if they circulate rather slowly (as do α5β1 fibronectin receptors on fibroblastsBretscher 1989xEndocytosis and recycling of the fibronectin receptor in CHO cells. Bretscher, M.S. EMBO J. 1989; 8: 1341–1348PubMedSee all ReferencesBretscher 1989), the feet would be more uniformly distributed and the cells would be long and stretched out, as are fibroblasts on a fibronectin substratum.Third, the speed at which a cell migrates would depend on the rate at which adhesive membrane extends the cell forward. For a given surface area of the cell, this would depend on the rate of surface uptake and the breadth of the leading lamella: the narrower the front, the more quickly the cell could move.These views may be wrong, but at least they may help focus the picture and some of them may be testable. For example, it is possible that fish scale keratocytes move too quickly for their advance (about 30 μ/minute) to be explained by a membrane circulation scheme—but then their rates of membrane circulation have not been measured. Indeed, it is difficult to measure the absolute rate of constitutive endocytosis in any cell because part of the internalized membrane (e.g. LDL-receptors in fibroblasts) may spend so little time—a fraction of a minute—inside the cell before returning to the cell surface. A method for measuring the rate of area uptake by individual cells over a period of a few seconds is therefore required. Although no such method exists at present, that which comes closest to achieving this uses a dye, such as FM1–43, which rapidly partitions into only the external leaflet of the lipid bilayer and can therefore be used to measure surface uptake (see, for example,Smith and Betz 1996xSimultaneous independent measurement of endocytosis and exocytosis. Smith, C.B and Betz, W.J. Nature. 1996; 380: 531–534Crossref | PubMed | Scopus (175)See all ReferencesSmith and Betz 1996); it is unclear whether this method could be made sufficiently sensitive to measure very short periods of endocytosis during locomotion.It seems probable that a polarized endocytic cycle exists in migrating cells whose function is, at least in part, to transport the cells feet from the rear to its front. Whether this membrane cycle is sufficient to extend a cell forward and to propel patches of crosslinked antigens rearward, or whether the polarized actin cycle is the principle driving force for these movements, is the nub of how a cell puts its best foot forward.

Membrane traffic during cell locomotion.

The key role played by a polarised endocytic cycle in extending the front of a eukaryotic cell is now becoming established. Highlights that have occurred during the past year include the visualisation of vesicles fusing with the advancing edge of Physarum, directly leading to extensions of the cell; furthermore, the fusion of vesicles at the leading edge in plant pollen tubes appears to be controlled by a small GTPase, Rop 1, which is a plant homologue of the mammalian Rac. In animal cells, Rac appears to help determine where exocytosis occurs on the surface of a polarised cell. These and other observations illuminate how the endocytic cycle assists the locomotory process in animal cells.

Transferrin receptor and its recycling in HeLa cells.

The transferrin receptor is a 180 000‐dalton protein which can be dissociated to two 90 000‐dalton polypeptides under reducing conditions. It can be labelled by lactoperoxidase‐catalysed iodination on the cell surface at 0 degree C. Trypsin digestion of labelled cells at 0 degree C can be used to degrade those receptors on the cell surface; they release a 70 000‐dalton soluble fragment which binds to transferrin. When cells are labelled at 0 degree C, then warmed to 37 degrees C, the labelled receptors enter the cells and become trypsin resistant. These receptors enter the cells, probably via coated pits, with a half‐life of approximately 5 min. Since there is about three times as much receptor inside cells as on the surface, this means that transit through the cell to the cell surface takes approximately 21 min, if all receptors are on the same cycling pathway.

EGF induces recycling membrane to form ruffles

KB cells are know to respond to epidermal growth factor (EGF) by producing prodigious ruffles in the plasma membrane within minutes. The signal transduction pathway underlying this effect in fibroblasts is mediated through Rac, a member of the Ras-like family of GTPases. As ruffles are rich in components of the cytoskeleton--particularly in actin and ezrin--it has been suggested that ruffles arise when activated Rac modulates the actin cytoskeleton to push out a membrane protrusion. We set out to see whether the surface of new ruffles arises from neighbouring membrane, or whether it comes from an intracellular pool of endocytosed membrane. If it arose by exocytosis from endosomes, it would be expected to be enriched in those recycling receptors that are concentrated in coated pits in the endocytic side of the cycle. On the other hand, if it arose passively from the adjacent plasma membrane, a uniform distribution of these receptors would be expected. Here, we show that as soon as ruffles appear on KB cells in response to EGF, their membrane surfaces are enriched in both transferrin and low density lipoprotein (LDL) receptors. Both these proteins are known to be selectively concentrated into endosomal membranes by clathrin-mediated endocytosis. Our results reveal that the surfaces of ruffles arise by exocytosis of internal membrane from the endocytic cycle and, therefore, that a primary action of Rac is to redirect the exocytosis of recycling membrane into just those specific sites where ruffles form.

A GTP requirement for binding initiator tRNA to ribosomes.

Formylmethionyl-tRNA (F-Met-tRNAF) is bound by particular codons to ribosomes in the initiation of protein synthesis. When a certain small concentration of magnesium ions is present, initiation factors and GTP are also required for this binding to take place. An analogue can be substituted for GTP, which suggests that this role of the nucleotide is uncoupled from its later hydrolysis.