Lysosomal acid phosphatase is internalized via clathrin-coated pits.

The presence of lysosomal acid phosphatase (LAP) in coated pits at the plasma membrane was investigated by immunocytochemistry in thymidine kinase negative mouse L-cells (Ltk-) and baby hamster kidney (BHK) cells overexpressing human LAP (Ltk-LAP and BHK-LAP cells). Double immunogold labeling showed that at various stages of invaginating coated pits LAP colocalized with clathrin and plasma membrane adaptors (HA-2 adaptors). Quantitation of the immunogold label showed similar density of wild-type LAP in coated over non-coated areas of the plasma membrane, whereas an internalization-deficient, truncated mutant of LAP which lacks the cytoplasmic tail was less efficiently included into coated pits. Internalization of anti-LAP antibodies into endosomal vesicles was accompanied by rapid dissociation of the coat proteins as shown by an immunofluorescence assay. The role of clathrin-coated vesicles in internalization of LAP was further corroborated by microinjecting monoclonal antibodies against clathrin or HA-2 adaptors into BHK-LAP cells. Internalization of LAP as detected by an immunofluorescence assay was transiently blocked by microinjected antibodies against clathrin or HA-2 adaptors, whereas unrelated antibodies did not affect internalization. These data suggest that LAP is included into clathrin-coated pits of the plasma membrane for rapid internalization.     

Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification

The effects of methods known to perturb endocytosis from clathrin- coated pits on the localization of clathrin and HA2 adaptors in HEp-2 carcinoma cells have been studied by immunofluorescence and ultrastructural immunogold microscopy, using internalization of transferrin as a functional assay. Potassium depletion, as well as incubation in hypertonic medium, remove membrane-associated clathrin lattices: flat clathrin lattices and coated pits from the plasma membrane, and clathrin-coated vesicles from the cytoplasm, as well as those budding from the TGN. In contrast, immunofluorescence microscopy using antibodies specific for the alpha- and beta-adaptins, respectively, and immunogold labeling of cryosections with anti-alpha- adaptin antibodies shows that under these conditions HA2 adaptors are aggregated at the plasma membrane to the same extent as in control cells. After reconstitution with isotonic K(+)-containing medium, adaptor aggregates and clathrin lattices colocalize at the plasma membrane as normally and internalization of transferrin resumes. Acidification of the cytosol affects neither clathrin nor HA2 adaptors as studied by immunofluorescence microscopy. However, quantitative ultrastructural observations reveal that acidification of the cytosol results in formation of heterogeneously sized and in average smaller clathrin-coated pits at the plasma membrane and buds on the TGN. Collectively, our observations indicate that the methods to perturb formation of clathrin-coated vesicles act by different mechanisms: acidification of the cytosol by affecting clathrin-coated membrane domains in a way that interferes with budding of clathrin-coated vesicles from the plasma membrane as well as from the TGN; potassium depletion and incubation in hypertonic medium by preventing clathrin and adaptors from interacting. Furthermore our observations show that adaptor aggregates can exist at the plasma membrane independent of clathrin lattices and raise the possibility that adaptor aggregates can form nucleation sites for clathrin lattices.     

An internalization-competent influenza hemagglutinin mutant causes the redistribution of AP-2 to existing coated pits and is colocalized with AP-2 in clathrin free clusters

Image correlation spectroscopy and cross correlation spectroscopy were used to demonstrate that approximately 25% of the internalization-competent influenza virus hemagglutinin mutant, HA+8, is colocalized with clathrin and AP-2 at the plasma membrane of intact cells, while wild-type HA (which is excluded from coated pits) does not colocalize with either protein. Clathrin and AP-2 clusters were saturated when HA+8 was overexpressed, and this was accompanied by a redistribution of AP-2 into existing coated pits. However, de novo coated pit formation was not observed. In nontreated cells, the number of clusters of clathrin or AP-2 colocalized with HA+8 was always comparable. Hypertonic treatment which disperses the clathrin lattices resulted in more clusters containing AP-2 and HA+8 than clathrin and HA+8. Less colocalization of HA+8 with clathrin was also observed after cytosol acidification, which causes the formation of deeply invaginated pits, where the HA+8 may be inaccessible to extracellular labeling by antibodies, and blocks coated vesicle budding. However, cytosol acidification elevated the number of clusters containing both HA+8 and AP-2, suggesting an increase in their level of association outside of the deep invaginations. Our results imply that AP-2 and HA+8 can colocalize in clusters devoid of clathrin, at least in cells treated to alter the clathrin lattice structure. Although we cannot ascertain whether this also occurs in untreated cells, we propose that AP-2 binding to membrane proteins carrying internalization signals can occur prior to the binding of AP-2 to clathrin. While such complexes can in principle serve to recruit clathrin for the formation of new coated pits, the higher affinity of the internalization signals for clathrin-associated AP-2 [Rapoport, I., et al. (1997) EMBO J. 16, 2240-2250] makes it more likely that once the AP-2-membrane protein complexes form, they are quickly recruited into existing coated pits.     

Clathrin-coated membrane: a distinct membrane domain in acetylcholine receptor clusters of rat myotubes

We have used antibodies to clathrin light chains in immunocytochemical studies of acetylcholine receptor (AChR) clusters of cultured rat myotubes. Immunofluorescence and ultrastructural experiments show that clathrin is present in coated pits and in large plaques of coated membrane. Coated membrane plaques are spatially and structurally distinct from AChR-rich membrane domains and the bundles of microfilaments that are also present in AChR clusters. Clusters contain a relatively constant amount of clathrin light chain protein, which is not dependent on the amount of AChR. Clathrin plaques remain after AChR domains are disrupted by azide, or after microfilament bundles are destabilized by cytochalasin D. Extraction of myotubes with saponin removes clathrin without disrupting AChR domains. Thus, clathrin plaques, microfilament bundles, and AChR-rich domains are independently stabilized.     

Role of coated vesicles, microfilaments, and calmodulin in receptor- mediated endocytosis by cultured B lymphoblastoid cells

Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) binding of the multivalent ligand in a diffuse cell surface distribution, (b) clustering of the ligand-receptor complexes, (c) recruitment of clathrin coats to the cytoplasmic surface of the cell membrane opposite ligand-receptor clusters, (d) assembly and (e) internalization of coated vesicles, and (f) delivery of label into a large vesicular compartment, presumably partly lysosomal. Most of the labeled ligand enters this pathway. The recruitment of clathrin coats to the membrane opposite ligand-receptor clusters is sensitive to the calmodulin-directed drug Stelazine (trifluoperazine dihydrochloride). In addition, Stelazine inhibits an alternate pathway of endocytosis that does not involve coated vesicle formation. The actin-directed drug dihydrocytochalasin B has no effect on the recruitment of clathrin to the ligand-receptor clusters and the formation of coated pits and little effect on the alternate pathway, but this drug does interfere with subsequent coated vesicle formation and it inhibits capping. Cortical microfilaments that decorate with heavy meromyosin with constant polarity are observed in association with the coated regions of the plasma membrane and with coated vesicles. SDS-polyacrylamide gel electrophoresis analysis of a coated vesicle preparation isolated from WiL2 cells demonstrates that the major polypeptides in the fraction are a 175-kdalton component that comigrates with calf brain clathrin, a 42- kdalton component that comigrates with rabbit muscle actin and a 18.5- kdalton minor component that comigrates with calmodulin as well as 110- , 70-, 55-, 36-, 30-, and 17-kdalton components. These results clarify the pathways of endocytosis in this cell and suggest functional roles for calmodulin, especially in the formation of clathrin-coated pits, and for actin microfilaments in coated vesicle formation and in capping.     

Ultrastructural immunocytochemical localization of clathrin in cultured fibroblasts

Using an affinity-purified antibody prepared against the major coat protein of brain-coated vesicles, clathrin, we have localized this protein by ultrastructural immunocytochemistry in Swiss 3T3 cultured fibroblasts. Fixation, permeabilization, and labeling were performed using the EGS and ferritin bridge labeling techniques. Localization of clathrin was detected on the coated regions of both the plasma membrane and the GERL apparatus. Almost no clathrin was found in the cytosol or in association with any other organelles. A very low concentration of labeling was occasionally seen randomly distributed on the inner surface of the plasma membrane and reticular membranous structures near the plasma membrane. The significance of these results in the role of the clathrin-coated regions in receptor-mediated endocytosis and Golgi function are discussed.     

Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices

Previous studies have shown that when human fibroblasts are depleted of intracellular K+, coated pits disappear from the cell surface and the receptor-mediated endocytosis of low density lipoprotein (LDL) is inhibited. We have now used the K+ depletion protocol to study several aspects of coated pit function. First, since coated pits rapidly form when K+-depleted fibroblasts are incubated in the presence of 10 mM KCl, we studied the sequence of assembly of coated pits as visualized in carbon-platinum replicas of inner membrane surfaces from cells that had been incubated in the presence of K+ for various times. New coated pits initially appeared as planar clathrin lattices that increased in size by the formation of polygons at the margin of the lattice. Once the lattice reached a critical size it invaginated to form coated vesicles. Second, we determined that LDL-ferritin can induce clustering of LDL receptors over noncoated membrane on the surface of K+-depleted fibroblasts; however, when these cells are subsequently incubated in the presence of K+, these clusters become associated with newly formed coated pits and are internalized. Finally, we determined that K+ depletion inhibits the assembly of coated pits, but that existing coated pits in K+-depleted cells are able to internalize LDL. These results suggest that the clathrin lattice of coated pits is actively involved in membrane shape change during endocytosis and that the structural proteins of the lattice are cyclically assembled and disassembled in the process.     

In vitro, insulin receptor catalyses phosphorylation of clathrin heavy chain and a plasma membrane 180,000 molecular weight protein

Insulin receptor mutation studies that the receptor tyrosine kinase activity is necessary for receptor endocytosis, and several insulin receptor-containing tissues have a plasma membrane-associated protein (M_r 180,000, p180) whose tyrosine phosphorylation is receptor catalysed. Since clathrin heavy chain (M_r 180,000 in dodecyl sulphate gel electrophoresis) is a major component of coated vesicles, the latter functioning in receptor endocytosis, we investigated whether insulin receptors can catalyse clathrin phosphorylation and whether p180 is clathrin. Bovine brain triskelion or coated vesicles and ³²P-ATP were added to prephosphorylated insulin receptor preparations (wheat ferm agglutinin-purified human placenta membrane proteins). Antiphosphotyrosine immunoprecipitated a phosphorylated 180,000 molecular weight protein. Insulin (10⁻⁷M) increased the rate of phosphorylation. Monoclonal anti-clathrin antibody immunoprecipitated the phosphorylated 180,000 molecular weight protein, whereas monoclonal anti-insulin receptor antibodies (-IR1, MA10) immunoprecipitated both insulin receptors and the phosphorylated 180,000 molecular weight protein. In the absence of added clathrin, anticlathrin immunoprecipitated no proteins, and -IR1 imunoprecipitated only the insulin receptor. Density gradient (glycerol 7.5–30%, w/v) centrifugation separated human placenta microsomal membrane proteins into endosomal, plasma membrane, cytoplasmic and coated vesicle fractions. Antiphosphotyrosine immunoprecipitated phosphorylated-microsomal proteins that centrifugated into endosomal and plasma membrane fractions. Addition of glycerol gradient fractions to a prephosphorylated insulin receptor preparation, however, gave a tyrosine-phosphorylated 180,000 molecular weight protein when cytoplasmic and coated vesicle fractions were added. Taken together these results suggest: (1) that, in vitro, human placenta insulin receptors can phosphorylate bovine brain and human placenta clathrin heavy chain; (2) that both assembled and unassembled clathrin can be phosphorylated; and (3) that p180, the plasma membrane-associated insulin receptor substrate, is not clathrin heavy chain.     

Serial-section analysis of coated pits and vesicles involved in adsorptive pinocytosis in cultured fibroblasts

We have examined, by analyzing thin (15-20 nm) serial sections, whether coated pits involved in adsorptive pinocytosis in cultured fibroblasts give rise to free coated vesicles or represent permanently surface-associated structures from the neck of which uncoated receptosomes pinch off and carry ligand into the cell. Human skin fibroblasts and mouse L-929 fibroblasts were incubated with cationized ferritin (CF), a ligand known to bind to coated pit regions, at 37 degrees C before fixation. In thin sections, CF was found in coated vesicular profiles within the cytoplasm. Serial sections revealed that whereas many of these coated profiles communicated with the cell surface, thus representing pits, about 10% in L-cells and 36% in skin fibroblasts were actually free coated vesicles. Moreover, evidence for uncoated vesicular structures (receptosomes) budding off from the coated pits was not obtained. We therefore conclude that coated pits do pinch off from the plasma membrane to form free, coated vesicles (pinosomes).     

Annexin VI-mediated Loss of Spectrin during Coated Pit Budding Is Coupled to Delivery of LDL to Lysosomes

Previously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH₂-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI–dependent budding is accompanied by the loss of ∼50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI–dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.     

Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors

Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-dependent stimulation of Tfn sequestration requires cytosolic clathrin, but is unaffected by clathrin purified from coated vesicles suggesting that soluble and assembled clathrin pools are functionally distinct. In addition to adaptors and cytosolic clathrin other, as yet unidentified, cytosolic factors are also required for efficient coated pit invagination. These results provide new insight into the mechanisms and regulation of coated pit assembly and invagination.     

Immunocytochemical visualization of coated pits and vesicles in human fibroblasts: relation to low density lipoprotein receptor distribution.

The coated pit-coated vesicle system has a key role in the uptake of plasma low density lipoprotein (LDL) and other receptor-bound proteins in human fibroblasts. To study the distribution of coated pits and coated vesicles in fibroblasts by immunochemical techniques at both the light and electron microscopic levels, we immunized rabbits with coat protein extracted from bovine brain-coated vesicles. The resulting anti-coat protein antibody was directed predominantly against clathrin, the 180,ooo dalton protein that constitutes the major component of coat protein. By indirect immunoperoxidase electron microscopy, the anti-coat protein antibody was observed to bind specifically to coated pits on the surface of human fibroblasts and to coated vesicles within the cell. Indirect immunofluorescence and immunoperoxidase staining techniques at the light microscopic level revealed that the coat protein was distributed in fibroblasts in two distinctive patterns: as discrete foci on or near the cell surface that were linearly aligned in association with phase-dense cellular fibers (first pattern), and as intracellular foci that were randomly arranged around the cell nucleus (second pattern). The distribution of coat protein in fibroblasts was compared with the distribution of ferritin-labeled LDL, which was studied with the use of similar electron microscopic and immunofluorescence techniques. As previously reported, electron microscopic studies revealed that the LDL-ferritin binding sites at 4 degrees C were clustered in coated pits. By immunofluorescence microscopy, the LDL-ferritin that was bound to receptors within coated pits was shown to be arranged linearly over the cell surface in a pattern that was similar to the linear arrangement of coat protein (first pattern). Considered together, the current data indicate that coated pits in human fibroblasts contain a protein analogous to clathrin, and that those coated pits which contain receptors for LDL are located over intracellular fibers most likely corresponding to stress fibers. These observationa may have relevance to the mechanisms by which the coated pit-coated vesicle system efficiently delivers recptor-bound ligands to lysosomes.     

Coat proteins isolated from clathrin coated vesicles can assemble into coated pits

Isolated human fibroblast plasma membranes that were attached by their extracellular surface to a solid substratum contained numerous clathrin coated pits that could be removed with a high pH buffer (Moore, M.S., D.T. Mahaffey, F.M. Brodsky, and R.G.W. Anderson. 1987. Science [Wash. DC]. 236:558-563). When these membranes were incubated with coat proteins extracted from purified bovine coated vesicles, new coated pits formed that were indistinguishable from native coated pits. Assembly was dependent on the concentration of coat protein with half maximal assembly occurring at 7 micrograms/ml. Assembly was only slightly affected by the presence of divalent cations. Whereas normal appearing lattices formed in a low ionic strength buffer, when assembly was carried out in a low pH buffer, few coated pits were evident but numerous small clathrin cages decorated the membrane. Coated pits did not form randomly on the surface; instead, they assembled at differentiated regions of membrane that could be distinguished in carbon/platinum replicas of frozen and etched membranes by the presence of numerous particles clustered into patches the size and shape of a coated pit.     

Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.     

Biochemical characterization of algal coated vesicles

Thin sections of tissue preparations from a green alga, Ulva lactuca (Ulvophyceae), and brown alga, Laminaria digitata (Pheophyceae) showed the presence of coated pits and coated vesicles in these 2 species. A discontinuous sucrose gradient after subcellular fractionation of the tissue homogenate resulted in an enriched coated vesicle fraction. Electron microscopy of negatively stained samples revealed the presence of coated vesicles of diameter ranging from 40-125 nm, together with large sheets of polygonal nets of clathrin. Electrophoresis of the CV purified fraction revealed various polypeptide components. Two of them, a 175 kDa and a 70 kDa, exhibited a positive response to bovine brain anticlathrin antibodies raised in goat or in rabbit. A third component of 30-40 kDa also gave a faint positive response. These 3 components corresponded to the clathrin heavy and light chains already described in higher plants. Clathrin was released from the CV algal preparations by treatment with 2M urea in Tris buffer, pH 8.5. Interestingly, in Ulva lactuca, the proportion of clathrin relative to the other proteins from the CV decreased with plant growth. Biochemical analysis of the purified CV revealed the presence of all the major phospholipids characterized in mammalian CV. The ratio of protein over lipid was also in the same range as that calculated for mammalian CV. Carbohydrate analysis demonstrated a high proportion of N-acetylgalactosamine and N-acetylglucosamine in both algal CV whereas these sugars were not detectable in the crude homogenate. These results demonstrate the presence of clathrin and coated vesicles in 2 species of algae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibronectin Receptor Internalization and AP-2 Complex Reorganization in Potassium-Depleted Fibroblasts

Potassium-depleted fibroblasts are unable to develop polarized morphology and lack coated pits. Experiments were carried out to measure internalization of fibronectin receptors (FNR) in potassium-depleted cells and possible association of FNR with AP-2 complexes after adding potassium back to the cells, which restores cell polarization. AP-2 complexes are the cell surface component of coated pits that contain both clathrin and membrane receptor binding domains. Potassium-depleted fibroblasts endocytosed antibody-tagged FNR and also internalized fluorescent fibronectin that previously had been adsorbed to the substratum. During cell polarization, antibody-tagged FNR reorganized into fibrillar structures along stress fibers beginning from nucleation sites at cell margins. Plasma membrane AP-2 complexes, which were undetectable in potassium-depleted cells, reappeared at the cell surface above the nucleus and then spread toward the cell margins. The results show that endocytosis of FNR can occur at least partially by a coated pit-independent mechanism.     

Clathrin exchange during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein-clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate.     

Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques

Clathrin is the scaffold of a conserved molecular machinery that has evolved to capture membrane patches, which then pinch off to become traffic carriers. These carriers are the principal vehicles of receptor-mediated endocytosis and are the major route of traffic from plasma membrane to endosomes. We report here the use of in vivo imaging data, obtained from spinning disk confocal and total internal reflection fluorescence microscopy, to distinguish between two modes of endocytic clathrin coat formation, which we designate as “coated pits” and “coated plaques.” Coated pits are small, rapidly forming structures that deform the underlying membrane by progressive recruitment of clathrin, adaptors, and other regulatory proteins. They ultimately close off and bud inward to form coated vesicles. Coated plaques are longer-lived structures with larger and less sharply curved coats; their clathrin lattices do not close off, but instead move inward from the cell surface shortly before membrane fission. Local remodeling of actin filaments is essential for the formation, inward movement, and dissolution of plaques, but it is not required for normal formation and budding of coated pits in the cells we have studied. We conclude that there are at least two distinct modes of clathrin coat formation at the plasma membrane—classical coated pits and coated plaques—and that these two assemblies interact quite differently with other intracellular structures.     

Variability of the topography of low-density lipoprotein (LDL) receptors in the plasma membrane of cultured human skin fibroblasts as revealed by gold-LDL conjugates in conjunction with the surface replication technique

In this investigation the membrane-perturbing effect of filipin, a polyene antibiotic which reacts specifically with cholesterol or cholesterol-like compounds in cell membranes, has been exploited to study the distribution of coated pits in cultured human skin fibroblasts. The coated pits, showing no filipin-cholesterol complexes, occurred singly or in clusters without apparent localization of either type to a particular region of the fibroblast membrane. Colloidal gold, conjugated to low-density lipoprotein, has proven to be an excellent marker, allowing the localization of low-density lipoprotein receptors on the surface of cultured cells. A pattern similar to that for the coated pits in the plasma membrane fracture faces was observed in the distribution of gold-low-density lipoprotein conjugates in surface replicas, indicating that the low-density lipoprotein receptors are associated with these coated pits. It was shown that there is an apparent heterogeneity in the distribution of low-density lipoprotein receptors, from cell to cell and even among different areas of the same cell membrane. The binding capacity for gold-low-density lipoprotein complexes, as represented by the extent of surface labeling, was directly related to the cell variety within the culture or to the particular experimental procedure. The observation of differences in the distribution of gold-low-density lipoprotein conjugates, even among adjacent coated pits, provides evidence for various stages of activity of the low-density lipoprotein receptors corresponding to incorporation, mobility, and internalization.     

Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading

To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor- mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.