R.G.W. ANDERSON
Department of Cell Biology and Anatomy
University of Texas Southwestern Medical Center
5323 Harry Hines Boulevard, Dallas, Texas 75235, USA
Inhibitors of Coated Pit Function
Reconstitution of the Endocytic Cycle in Vitro
Future Directions
References
Cells constantly sample their environment by internalizing portions of the extracellular fluid and carrying the fluid in membrane-bound vacuoles to various intracellular targets. The efficiency of this process is increased when receptors for extracellular molecules are located at sites of internalization, a process called receptor-mediated endocytosis.¹ For nearly 24 years the clathrin-coated pit has been implicated as the major site of internalization for receptor-bound ligands.1,2
There are two components to coated pit function. First, the polygonal lattice of clathrin or the clathrin associated proteins control the shape of the membrane leading to the formation of a coated vesicle. Second, molecular elements in coated pits cause certain membrane receptors to cluster so that when a ligand binds it is rapidly internalized.2 The goal of current research efforts, therefore, is to understand at the molecular level how invagination and receptor clustering are achieved.
The evidence that coated pits mediate the internalization of receptor-bound molecules is almost entirely derived from microscopic studies. The first description of this specialized region of surface membrane provided a confusing picture of its possible function;3 some researchers even thought coated pits were an artifact of tissue preservation. The observation that the number of coated pits is increased in oocytes that are internalizing yolk proteins,4 as well as the finding that ferritin binding sites are located over coated pits of reticulocytes,5 were two findings that suggested a function for this region of membrane. Beginning with the discovery of the low-density lipoprotein (LDL) receptor in coated pits,6 there are now some 20-25 molecules or macromolecular complexes that have been found to enter cells by this route.2 The list will undoubtedly grow; however, in each case the identification of the route of entry will depend upon microscopic techniques.
While progress has been made in identifying the receptors that use this internalization mechanism, researchers have made rapid progress in isolating coated vesicles, identifying and characterizing coated vesicle proteins, and delineating the architecture of the polygonal coat.7,8 Although many of the coat proteins have been identified and it is now possible to use them to assemble coated vesicles7 as well as coated pits9 in vitro, we know nothing about how coated pits function.
Despite the widespread morphologic documentation that certain ligands enter cells by coated pits, it is possible that static electron microscopic images do not reveal the true pathway of internalization. For this reason, many investigators have searched for drugs or culture conditions that would affect the function of coated pits in some informative way. Whereas there is no drug or chemical that specifically interferes with coated pit function, there are at least three culture conditions or treatments that profoundly affect coated pit function: depletion of intracellular potassium,10 incubation of cells in a hypertonic medium11,12 and acidification of the cell cytoplasm.13 Each treatment affects coated pits in a different way, which gives clues about the role of the clathrin lattice in endocytosis.
Larkin et al., 10 were the first to describe a method for inhibiting receptor mediated endocytosis. Using 125 I-labelled low density lipoprotein (LDL) as an endocytic marker, they found that when the level of intracellular potassium was lowered in cultured human fibroblasts below 40% of normal, the internalization of LDL was inhibited. Moreover, under conditions of maximal inhibition, these cells had a reduced number of coated pits on the cell surface; greater than 80% of the coated pits were missing. Daukas and Zigmond2 reported that the receptor mediated internalization of chemotactic peptide was inhibited in cultured polymorphonuclear leukocytes that had been incubated in a hypertonic medium. Hypertonic medium affects the endocytosis of LDL11 as well as bulk phase markers. 11 As judged by immunofluorescence and electron microscopic evaluation of the inner surface of rapid-freeze, deep-etched membranes, cells incubated in a hypertonic media have a markedly reduced number of coated pits.11 Finally, Sandvig et al.,13 found that when intracellular pH was lowered below pH 6.0, there was a dramatic and specific inhibition of endocytosis through coated pits. Unlike potassium depletion and hypertonic treatment, cells that are treated in this manner have normal numbers of coated pits; however, these pits appear to be paralyzed and unable to internalize ligand.
These studies imply that when clathrin lattices are absent from the surface membrane, a cell is unable to internalize receptor-bound molecules. Moreover, they indicate that the clathrin lattice has some direct role to play in the endocytic event because in acidified cells the lattice is still present but apparently unable to function. The exact reason for the effects of these agents on coated pit structure and function are not known. Therefore, these studies have failed to identify the active components that account for receptor clustering and invagination.
More than likely, the development of additional inhibitory treatments will not have sufficient molecular resolution to elucidate how coated pits achieve their endocytic function in cells. For this reason, there has been widespread interest in the development of in vitro methods for studying endocytosis.14,15,16 Our laboratory has focused on developing a method for preparing isolated plasma membranes that are capable of reproducing part or all of the endocytic cycle in vitro.
The endocytic cycle in intact cells has several components:2 (a) receptor clustering over coated pits; (b) invagination of the coated pit to form a coated vesicle; (c) removal of the coat from the vesicle to form an endosome; (d) the sorting of receptor from ligand in an early endosome compartment; (e) the return of the receptor in a transport vesicle to the cell surface; (f) the movement of the ligand to a specific intracellular target such as the lysosome; and (g) the formation of a new coated pit at the cell surface. Our goal has been to develop the methodology needed for studying each step in vitro.
To accomplish this goal, we reasoned that we needed large numbers of isolated plasma membranes attached to a solid substratum by the extracellular surface.9 In these membranes, the numerous coated pits would be available for experimental manipulation. The method we chose relied upon the ability of cells to attach to a poly-L-lysine coated surface. The bulk of the cell can easily be removed by gentle sonication, leaving behind the plasma membrane. Since the membranes can be prepared on a variety of different surfaces, it is possible to analyse coated pits using carbon platinum replicas, indirect immunofluorescence or radioimmunoassay. With appropriate monoclonal antibodies, clathrin as well as important receptor molecules such as the LDL receptor can be easily detected by both visual and quantitative techniques.
Thus far our main focus has been on using these membranes to study coated pit assembly. The endogenous coated pits can be removed by brief treatment with a high pH buffer. This leaves behind sites on the membrane that are capable of initiating the formation of coated pits when suitable coat proteins are available. Cytoplasm prepared from tissues or cells can serve as a source of clathrin and clathrin associated proteins.9 Some of the important conclusions from these studies include: (1) clathrin and clathrin associated proteins are recruited from the cytoplasm to the surface of the cells and form normal appearing coated pits; (2) the membranes contain a limited number of assembly sites; (3) assembly occurs equally well at 4 °C and 3 °C with a half time of assembly of approximately 5 mini (4) assembly seems not to require a source of ATP; (5) when assembly is carried out at 37 °C, after initial assembly there is a rapid disappearance of the clathrin from the membrane, and this disappearance is inhibited by the ATP-destroying enzyme apyrase.
We have used this system to determine whether it is possible to assemble a coated pit from coat proteins that have been extracted from isolated coated vesicles.17 These coat proteins will form normal coated pits and the assembly reaction shares many of the properties described above.
This membrane preparation has also afforded us the opportunity to investigate whether the assembly sites have specific ultrastructural features as viewed in carbon platinum replicas of rapid-freeze, deep-etched membranes. The stripping procedure always leaves behind 20-30% of the clathrin that was originally on the membrane.9 The replicas reveal that this clathrin is in the form of incomplete polygons that are occasionally seen on the membrane. More importantly, these incomplete polygons are associated with well-defined particles that are arranged into clusters the size of a coated pit. These particles account for the fact that in stereo images, the flat lattices on unstripped membranes appear to be raised above the surface of the membrane; in other words, there's a definite molecular linkage between the clathrin lattice and the membrane. At least one component of the particles making up the assembly site most likely is the 100-50 Kd assembly complex identified by several laboratories as involved in coated vesicle assembly.18 19
More recently, we have fumed our attention to investigating whether the coated pits that are on these membranes are capable of rounding up and pinching off to form a vesicle. Although we have yet to prove definitively that endocytic vesicles can form from these membranes, we have found that the clathrin lattices will spontaneously round up when the temperature is shifted from 4 to 37 °C and that attendant with the rounding up process is the loss of clathrin from the membrane. The loss of clathrin is inhibited at pH below pH 6.0 and by treatment of the membranes with apyrase. Therefore, the behaviour of the clathrin coated pits in these isolated membranes is what one would predict based on the extensive morphologic studies carried out on a variety of cell systems.
To understand the molecular basis of coated pit function, it will be necessary to exploit an in vitro system such as the one we have developed. There is much to learn about the assembly phase. For example, we would like to know the nature of the assembly sites and whether or not their activity depends upon the assembly protein complex. More importantly, we would like to identify the membrane determinants that specify where and when a coated pit will form.
We are also optimistic that we will be able to use this system to study the invagination phase of endocytosis. If we can recreate this event and find conditions that lead to endocytosis, then there are innumerable studies that will be possible, ranging from the use of recombinant clathrin to the study of mutations that effect endocytosis. Ultimately, we would like to understand coated pits as dynamic cellular structures.
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18. KEEN, J.H. 1987. J. Cell Biol. 105: 1989-1998.
19. BAR-ZVI, D. and D. BRANTON. 1986. J. Biol. Chem. 262: 9614-9621.