Clathrin-coated Structures Research Articles

Syncytial-type cell plates: a novel kind of cell plate involved in endosperm cellularization of Arabidopsis.

Cell wall formation in the syncytial endosperm of Arabidopsis was studied by using high-pressure-frozen/freeze-substituted developing seeds and immunocytochemical techniques. The endosperm cellularization process begins at the late globular embryo stage with the synchronous organization of small clusters of oppositely oriented microtubules ( approximately 10 microtubules in each set) into phragmoplast-like structures termed mini-phragmoplasts between both sister and nonsister nuclei. These mini-phragmoplasts produce a novel kind of cell plate, the syncytial-type cell plate, from Golgi-derived vesicles approximately 63 nm in diameter, which fuse by way of hourglass-shaped intermediates into wide ( approximately 45 nm in diameter) tubules. These wide tubules quickly become coated and surrounded by a ribosome-excluding matrix; as they grow, they branch and fuse with each other to form wide tubular networks. The mini-phragmoplasts formed between a given pair of nuclei produce aligned tubular networks that grow centrifugally until they merge into a coherent wide tubular network with the mini-phragmoplasts positioned along the network margins. The individual wide tubular networks expand laterally until they meet and eventually fuse with each other at the sites of the future cell corners. Transformation of the wide tubular networks into noncoated, thin ( approximately 27 nm in diameter) tubular networks begins at multiple sites and coincides with the appearance of clathrin-coated budding structures. After fusion with the syncytial cell wall, the thin tubular networks are converted into fenestrated sheets and cell walls. Immunolabeling experiments show that the cell plates and cell walls of the endosperm differ from those of the embryo and maternal tissue in two features: their xyloglucans lack terminal fucose residues on the side chain, and callose persists in the cell walls after the cell plates fuse with the parental plasma membrane. The lack of terminal fucose residues on xyloglucans suggests that these cell wall matrix molecules serve both structural and storage functions.

Lectin-induced retardation of subcellular organelles during preparative density gradient electrophoresis: selective purification of plasma membranes.

Plasma membranes (PM) are difficult to separate by conventional means from other cellular compartments. Using a density gradient electrophoresis (DGE) apparatus (7 cm, x 2.2 cm), mammalian subcellular organelles were separated from a total postnuclear supernatant. The sialic acid-binding lectin wheat germ agglutinin (WGA) permitted 1.5-fold electrophoretic retardation of plasma membranes lagging far behind endoplasmic reticulum, endosomes, Golgi and lysosomes (in order of increasing electrophoretic mobility). Mobilities of the latter organelles were not affected by wheat germ agglutinin. The retarded plasma membrane was monitored by surface iodination, the presence of Ca(++)- and Na+/K(+)-ATPases and by the presence of clathrin-coated pits using Western immunoblotting. In the presence of WGA two clathrin-containing compartments were detected; in the absence of WGA three clathrin populations were seen in the electropherogram: clathrin-coated vesicles, clathrin-coated pits (on the PM) and clathrin-coated structures on the trans-Golgi network (TGN). Both in the presence and absence of WGA, plasma membrane domains of different electrophoretic mobilities were detected.

Nef-mediated clathrin-coated pit formation.

The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.

Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes.

Many plasma membrane proteins destined for endocytosis are concentrated into clathrin-coated pits through the recognition of a tyrosine-based motif in their cytosolic domains by an adaptor (AP-2) complex. The mu2 subunit of isolated AP-2 complexes binds specifically, but rather weakly, to proteins bearing the tyrosine-based signal. We now demonstrate, using peptides with a photoreactive probe, that this binding is strengthened significantly when the AP-2 complex is present in clathrin coats, indicating that there is cooperativity between receptor-AP-2 interactions and coat formation. Phosphoinositides with a phosphate at the D-3 position of the inositol ring, but not other isomers, also increase the affinity of the AP-2 complex for the tyrosine-based motif. AP-2 is the first protein known (in any context) to interact with phosphatidylinositol 3-phosphate. Our findings indicate that receptor recruitment can be coupled to clathrin coat assembly and suggest a mechanism for regulation of membrane traffic by lipid products of phosphoinositide 3-kinases.

Rapid endocytosis of interleukin 2 receptors when clathrin-coated pit endocytosis is inhibited

The cytokine interleukin 2 (IL2) is produced by activated helper T lymphocytes and modulates the growth and activity of cells expressing high-affinity surface IL2 receptors that transduce its signaling. After ligand binding to receptors on the plasma membrane, receptor-ligand complexes are rapidly endocytosed and IL2 is degraded in acidic compartments. The best known receptor-mediated endocytosis pathway involves clathrin-coated pits. Receptors that carry an internalization signal recognized by adaptors on the cytosolic side of the plasma membrane are clustered into the coated pits and enter cells very efficiently. Many receptors use this pathway, but other endocytic pathways have also been reported, for ricin, EGF and insulin, for instance, which seem to be less efficient than the coated one. We compared the endocytosis of IL2 and its receptors to that of transferrin, a marker of the coated pit pathway. Under normal conditions, the kinetics of entry of IL2 was two times slower than that of transferrin. When internalization via coated pits was inhibited by two different methods, potassium depletion and cytosol acidification, endocytosis of IL2 and its receptors was only partly inhibited, while transferrin entry was strongly affected. Treatment with the cationic amphiphilic drug chlorpromazine, which induces a redistribution of a clathrin-coated pit component, AP-2, to endosomes, reduced transferrin, but not IL2 internalization. Thus, unexpectedly, this cytokine and its receptors can still be rapidly endocytosed in the absence of functional clathrin-coated structures. We propose a model for receptor-mediated endocytosis that may account for these results and published data on other receptors.

Coated pits and coated vesicles — sorting it all out

Clathrin-coated structures are involved in the first steps of membrane vesiculation which lead to receptor sorting and directed traffic. This review focuses on our current understanding of clathrin and its associated proteins, the major components of the coat. Recent experiments provide new insights into the interactions between these proteins.

Presence of an immunologically-defined class of peri-Golgi vesicles in cultured mammalian cells

Immunsera of mice injected with clathrin-depleted coated vesicle membranes, purified from rat liver, revealed a preferential labeling of some perinuclear structures by immunofluorescence microscopy on NRK cells. Subsequent production of 4 monoclonal antibodies was achieved. The antigen was strictly located in the Golgi area of the cell but was not an intrinsic element of the Golgi complex. The restricted location of the structures excluded these were lysosomes which appeared more dispersed in these cells. After nocodazole treatment the material was found dispersed in the cytoplasm. This provided a means of distinguishing the antigen from clathrin-coated structures and Golgi intrinsic elements. Immunolocalization at the electron microscope level confirmed the data obtained at the light level. Some peroxidase reaction product was rarely associated with Golgi elements, but predominantly stained small neighboring Golgi vesicles (50 nm diameter), as well as tubulo-elongated structures and some large (500 nm) irregular-shaped vesicles. A 32 kDa molecular weight antigen was characterized by immunopurification from NRK cells metabolically labeled with 35S-Met. This 32 kDa antigen appeared as part of a higher multimolecular membrane component of 300 kDa. A 170 kDa and a 70 kDa components were immunodetected in a semi-purified membrane fraction from rat liver, demonstrating that the antigen was a minor but very antigenic contaminant of the coated vesicle preparation used as immunogen. In conclusion, the labeled peri-Golgi structures may be part of the newly characterized trans-Golgi network and/or of the reticular/vesicular endosomal, prelysosomal structure recently described.

Recovery of structurally intact and infectious poliovirus type 1 from HeLa cells during Receptor-mediated endocytosis

Poliovirus type 1 enters HeLa cells by receptor-mediated endocytosis as an intact virus. Up to 30 min after adsorption complete virus particles still containing VP4 and sedimenting with 156 S could be recovered from the cells. These virus particles were N-antigenic and infectious. Thirty minutes after adsorption the recovery of intact and infectious virus decreased. This decrease presumably reflects viral uncoating in the acidic endosomes and/or lysosomes because virus particles could be localized in endosomes at this time. The direct involvement of clathrin-coated structures in the endocytosis of poliovirus has been deduced from the enclosure of poliovirus in coated vesicles at 10 min after adsorption. At this time intact and infectious virus could be recovered only after the coated vesicles were disrupted by treatment with 0.5 M Tris at pH 7.0.

Endocytic pathway of recombinant murine tumor necrosis factor in L-929 cells.

The fate of TNF after binding to the surface of L-929 cells was followed by using murine rTNF coupled to colloidal gold as a probe. A time-course study using electron microscopy was performed. Our results confirm previous indications obtained from biochemical studies suggesting that TNF is internalized by this cell type. They further directly show that internalization proceeds through the classical receptor-mediated endocytosis pathway, i.e., via clathrin-coated structures and endosomes before accumulation in secondary lysosomes.

Selective cleavage of human ACTH, beta-lipotropin, and the N-terminal glycopeptide at pairs of basic residues by IRCM-serine protease 1. Subcellular localization in small and large vesicles.

We present a study of the cleavage specificity of IRCM-serine protease 1 from frozen porcine pituitary neurointermediate lobes using polypeptide substrates representing different segments of human pro-opiomelanocortin. Using 125I-labeled ACTH(11-24) and a 125I-labeled model beta-lipotropin (beta-LPH) peptide, the preference of this protease for cleavage C-terminal to the pairs of basic residues Lys-Arg and Lys-Lys was clearly seen. This study was extended to larger unlabeled natural human polypeptides including ACTH(1-39), beta-LPH(1-89), and the N-terminal glycopeptide (1-76), which are known to serve as substrates for further cleavage in vivo. In these substrates IRCM-serine protease 1 cleaved C-terminal to all pairs of basic residues known to be cleaved in vivo. In addition, the enzyme cleaved between two pairs of basic amino acids found in NT(1-76) which are also known to be cleaved in vivo. Many potential "tryptic-like" cleavage sites were not cleaved by the enzyme. However, IRCM-serine protease 1 cleaved C-terminal to Phe-Arg in the three melanocyte-stimulating hormone sequences of pro-opiomelanocortin. In order to better understand the physiological role of IRCM-serine protease 1, differential centrifugation was used to study the subcellular distribution of the enzyme from porcine pituitary anterior lobe homogenates. We present evidence that the active enzyme form, isolated from the subcellular fractions, possesses a similar molecular architecture as the enzyme isolated from frozen tissue (Mr 38,000 catalytic domain linked via disulfide bridge(s) to another polypeptide chain(s) to form an Mr 88,000 monomeric structure). The majority of IRCM-serine protease activity is found to be associated with small vesicles (150,000 X g for 5 h) of as yet undetermined nature. In addition, a latent activity was found to be associated with a 27,000 X g (15 min) pellet containing the majority of mature secretory granules. If IRCM-serine protease 1 participates in prohormone maturation in vivo, we propose a model in which this protease is present in an enzymatically active form in small vesicles, possibly within clathrin-coated structures (prosecretory granules) which are then transformed to mature secretory granules by a process which would also inactivate most of the enzyme.

Serial section analysis of clathrin-coated pits in rat liver sinusoidal endothelial cells

Electron microscopy and serial sections were used to examine the shape of clathrincoated pits in sinusoidal endothelial cells of rat livers. Livers were perfused at 4 °C with either concanavalin A-horseradish peroxidase (conA-HRP), or HRP alone, followed by warm-up to 37 °C and fixation with glutaraldehyde. Alternatively, the livers were perfused with HRP at 37 °C, followed by fixation. All tissue was preserved using a membrane contrast enhancement technique (R-OTO) consisting of sequential osmium-ferrocyanide, thiocarbohydrazide, and osmium-ferrocyanide treatment. Peroxidase reaction product was used to identify structures participating in endocytosis. One hundred and ninety-three clathrin-coated structures were examined. Sixty-six were from livers perfused with conA-HRP at 4 °C, 63 were from livers perfused with only HRP at 4 °C, and 64 were from livers perfused with HRP at 37 °C. These coated structures were morphologically classified into three categories: ( a) flat pits; ( b) cup-shaped pits; ( c) pits with a narrow neck. No isolated coated vesicles were found. In cells perfused at 4 °C followed by wanning to 37 °C, the percentage of coated pits found connected to the cell surface by narrow necks was 31 %, using conA-HRP, and 27% using HRP alone. In cells perfused continuously at 37 °C, the percentage of coated pits with narrow neck connections was 21 % using HRP alone. These results suggest that the formation of coated pits connected to the surface by narrow necks is not an artifact of cell type, of experimental protocol or of incubation with a lectin.

Initial steps in receptor-mediated endocytosis: The influence of temperature on the shape and distribution of plasma membrane clathrin-coated pits in cultured mammalian cells

We have examined the shape and distribution of clathrin-coated pits in Swiss 3T3 cells at 4 or 37 °C using electron microscopy with serial sections and immunofluorescence light microscopy. Both groups were fixed in glutaraldehyde and preserved further using a membrane contrast enhancement technique consisting of sequential osmium-ferrocyanide, thiocarbohydrazide ande osmium-ferrocyanide treatment in situ. Concanavalin A-horseradish peroxidase (conA-HRP) was used to identify these structures participating in endocytosis. Two hundred twenty-two clathrin-coated structures were analysed; 126 from cells fixed at 4 °C, and 96 from cells fixed after a 3 min warm-up to 37 °C. All coated structures labeled with conA-HRP had demonstrable connections to the plasma membrane. These coated structures were morphologically classified into three categories: (a) flat pits; (b) curved pits; and (c) pits with narrow-neck connections to the plasma membrane. At 37 °C, 27% of coated pits had narrow neck connections to the plasma membrane whereas at 4 °C only 1% had such connections. Receptosomes (endosomes) labeled with conA-HRP were found only after incubation at 37 °C, indicating that active endocytosis was occurring in cells at 37 °C, but not at 4 °C. Immunofluorescence with anti-clathrin antibody was used to quantitate the number of clathrin-coated pits in Swiss 3T3 cells incubated at 4 and 37 °C prior to fixation. No difference was detected. There were 426 ± 122 pits per cell at 37 °C and 441 ± 106 at 4 °C. These results support the hypothesis that formation of a narrow neck connected a coated pit to the cell surface is an early step in the mechanism of receptor-mediated endocytosis.

Morphologic demonstration of clathrin-coated pits in frog and turkey erythrocytes

We have examined nucleated erythrocytes of frog and turkey for the presence of clathrin-coated structures using electron microscopy and immunocytochemistry. By electron microscopy, coated pits were found on the plasma membrane of peripheral blood erythrocytes of both species. These structures had an appearance similar to coated pits seen in non-erythroid mammalian cells. Using immunofluorescence with anti-(bovine) clathrin antibody, erythrocytes of both species showed punctate membrane fluorescence similar to the pattern of coated pits seen in other cells. By both methods, frog erythrocytes showed considerable heterogeneity, such that only about 50% of the cells showed significant numbers of coated pits, usually fewer than 20–50 per cell. In contrast, the vast majority of turkey erythrocytes showed no detectable coated pits, but occasional cells (less than 10%) showed large numbers of coated structures. These results suggest that a functional endocytic system may be present in a subpopulation of these nucleated erythrocytes. These findings may be of significance in understanding the ligand-induced loss of some receptors from the surface of these cells, and may serve as an indication of morphologic differentiation.

The mannose 6‐phosphate receptor: function, biosynthesis and translocation

This report summarizes studies concerning the role of the lysosomal protein: Man-6-P receptor and describes some recent data on its biosynthesis and cellular translocation. The receptor functions both in the Golgi apparatus (or GERL) and on the cell surface where it binds lysosomal proteins and mediates their transport to lysosomes. Consistent with its dual role, the receptor in several cell types has been localized to the plasma membrane and Golgi cisternae, to clathrin-coated structures at both locations, and to vesicles characteristic of endosomes or CURL. Biosynthetic studies have shown that the receptor undergoes several post-translational modifications including the processing of its asparagine-linked oligosaccharides, phosphorylation of serine residues, and unknown modifications required for acquisition of immunoreactivity and functional activity. Cellular pools of mature receptor readily mix as evidenced by rapid labeling of intracellular receptor by exogenously added receptor antibodies. Degradation of the receptor occurs non-lysosomally and is perhaps mediated by extracellular Man-6-P-containing hydrolases. A working hypothesis for the mechanism of Man-6-P receptor function that is consistent with these observations is presented.

Formation of receptosomes from plasma membrane coated pits during endocytosis: analysis by serial sections with improved membrane labeling and preservation techniques.

The early events in the entry of ligands into cultured fibroblasts through plasma membrane clathrin-coated pits were examined by using serial sections and electron microscopy. Swiss 3T3 cells were labeled at 4 degrees C with concanavalin A-horseradish peroxidase and warmed to 37 degrees C for 1 or 2 min to label organelles participating in endocytosis. Cells were fixed in glutaraldehyde and further preserved with a membrane contrast-enhancement technique that utilized sequential osmium/ferrocyanide, thiocarbohydrazide, and osmium/ferrocyanide treatment in situ. This technique provides exceptional preservation and enhanced contrast of membranous structures. Serial sections taken both parallel to and perpendicular to the substratum were examined by electron microscopy, and 263 clathrin-coated structures containing horseradish peroxidase were evaluated for their connection to the plasma membrane. All of these coated structures were found to be connected to the plasma membrane either directly or via necks approximately equal to 170 A in width and up to 7,000 A in length. These necks were often surrounded by a dense filamentous network. These results strongly suggest that isolated coated vesicles do not form during the endocytosis process in Swiss 3T3 cells.

Ultrastructural immunocytochemical localization of calmodulin in cultured cells.

Using an antibody prepared against performic acid-treated calmodulin, we have localized calmodulin in cultured fibroblastic cells by immunofluorescence and immunoelectron microscopy. In interphase cells, calmodulin was found to be diffusely distributed throughout the cytosol. An increased amount of calmodulin was found in the pericentriolar region of interphase cells. No significant aggregation of calmodulin was found in association with microfilaments, peripheral cytoplasmic microtubules or clathrin-coated structures. Calmodulin was present in moderate amounts in microvilli, ruffles, and zeiotic blebs of the cell surface. In motitic cells, calmodulin was found concentrated in the pericentriolar region, and appeared to concentrate along radiating spindle microtubules proximal to the centrioles. Redistribution of calmodulin was seen between early and late telophase, in which the pericentriolar pattern of calmodulin in early telophase shifted to an aggregation on the intercellular bridge, with a large part of the midbody portion of the bridge being devoid of calmodulin. These results show that calmodulin is distributed throughout the cytosol, but is markedly concentrated in the region of the microtubule organizing center in interphase cells, as well as in elements of the mitotic spindle apparatus. This distribution suggests that calmodulin has a regulatory role in the organization and function of microtubules during interphase, as well as during mitosis.

Microinjection of anticlathrin antibodies into cultured fibroblasts: clathrin-coated structures in receptor-mediated endocytosis and in exocytosis.

Microinjection of anticlathrin antibodies into cultured fibroblasts: clathrin-coated structures in receptor-mediated endocytosis and in exocytosis.