Tubular Invaginations Research Articles

Expression of glutamate transporters in rat cardiomyocytes and their localization in the T-tubular system.

Glutamate and aspartate play important roles in the intermediary metabolism of the myocardium and have been shown to improve cardiac recovery after hypoxia or ischemia. Limited data are available about the expression of glutamate transporters that are involved in the uptake of glutamate and aspartate in cardiomyocytes. In this study, non-radioactive in situ hybridization (ISH) using complementary RNA probes was applied to detect the glutamate transporters GLT1 variant (GLT1v) and EAAC1 mRNA in rat cardiomyocytes. The transporter proteins were demonstrated by Western blotting and immunocytochemistry using affinity-purified antibodies against transporter peptides. ISH and immunocytochemistry showed that both glutamate transporters are coexpressed in cardiomyocytes. The ISH labeling indicates the distribution of transporter mRNA throughout the cytoplasm of cardiomyocytes. GLT1v and EAAC1 proteins, which showed in Western blots a molecular mass of approximately 60 kD, are strongly enriched and colocalized in the transverse (T)-tubular system of cardiomyocytes. These results may indicate that glutamate/aspartate uptake into cardiomyocytes could be mediated by the high-affinity transporters GLT1v and EAAC1. A high efficiency of glutamate/aspartate transport into cardiomyocytes could be achieved by their localization in the T-tubular system, which consists of tubular invaginations of the sarcolemma extending deep into the cell.

A Novel Dynamin-associating Molecule, Formin-binding Protein 17, Induces Tubular Membrane Invaginations and Participates in Endocytosis

Dynamin associates with a variety of SH3 domain-containing molecules via a C-terminal proline-rich motif and takes part, with them, in endocytic processes. Here, we have investigated a new dynamin-associating molecule, formin-binding protein 17 (FBP17), involved in deforming the plasma membrane and in endocytosis. FBP17 formed tubular invaginations originating from the plasma membrane. Its N-terminal Fer/CIP4 homology domain, a coiled-coil domain, and a proline-rich motif were required for tubular invagination and self-assembly, by which tubular invagination might be induced. Using anti-FBP17 antibody, we detected positive immunoreactions in the testis that were restricted to the germ cells. We also detected FBP17 in the brain by immunoblotting and in situ hybridization. When COS cells expressing enhanced green fluorescent protein-tagged FBP17 were incubated with fluorescently labeled transferrin, epidermal growth factor, and cholera toxin, these molecules co-localized with FBP17-induced tubular invaginations, suggesting that FBP17 is involved in dynamin-mediated endocytosis in both a clathrin-dependent and -independent manner. These observations therefore indicate that FBP17 interacts with dynamin and regulates endocytosis by forming vesicotubular structures.

Ultrastructural study of spermiogenesis in the turkey, Meleagris gallopavo

1. Ultrastructural changes that take place in the spermatids of the turkey were studied. Twelve steps of spermiogenesis are described. 2. Acrosomal formation is similar to that in the domestic fowl and quail. The perforatorium develops as a dense granule in a tubular invagination of the thickened part of the nuclear membrane during the acrosomal phase of spermatid development. 3. Patchy appearances of the circular (CM) and longitudinal (LM) manchette occur concurrently for a very brief period before the complete disappearance of the former and full establishment of the latter, an indication that the CM reorganises to become the LM. 4. A chromatoid body, as found in mammals, is present in the mid-piece of the turkey spermatid, and glycogen aggregations occur in the spermatid cytoplasm, but not in the mature spermatid or spermatozoon. 5. Mitochondrial alignment around the axoneme in the mid-piece takes place only after the dissolution of the LM, unlike in the Japanese quail. 6. The merits and disadvantages of the 4-phase and step-wise systems of spermatid classification and evaluation are discussed.

Clinical experience of vasoepididymostomy using a triangulation technique.

Background: Although the number of patients receiving vasoepididymostomies is gradually increasing, these individuals are limited in the recent advanced assisted reproductive technology (ART) era. A novel technique involving vasoepididymostomy with epididymal tubular invagination has been reported. We attempted to define the results of this method and to compare them with the conventional end-to-side technique in patients with suspected epididymal obstruction and no previous history of vasectomy. Methods and Results: Eight eligible triangulation end-to-side vasoepididymostomy procedures performed on five azoospermic patients exhibiting either unilateral or bilateral epididymal obstruction are described. The overall patency rate following operation was 100% (five of five). Two pregnancies were achieved by natural intercourse and one was accomplished via artificial insemination. A single pregnancy was obtained with an intracytoplasmic sperm injection using frozen-thawed sperm collected during the operation. Conclusion: Vasoepididymostomy, using the triangulation technique for epididymal obstruction, resulted in an earlier patency in all patients. This method may afford advantages when compared with the conventional end-to-side approach; however, larger subject populations are required in order to assess further the efficacy of this procedure. In addition, long-term follow up is necessary. (Reprod Med Biol 2003; 2: 101-104).

The nucleus of HeLa cells contains tubular structures for Ca 2+ signaling with the involvement of mitochondria

Calcium is an important messenger that controls many nuclear functions such as gene expression in mammalian cells but the regulation of nuclear Ca 2+ remains unclear. It has long been thought that Ca 2+ is translocated from the cytosol by a long distance to the nucleus through the nuclear pore complexes to activate or suppress gene transcription. However, this model is at best an incomplete one. With an aid of confocal and transmission electron microscopy, we demonstrated here that tubules, in a vertical or horizontal orientation, extended deep inside the nucleus of HeLa cells. These nuclear tubules (NTs) are double-membraned invaginations of the nuclear envelope and are usually associated with nucleolus. Also, membrane bound vesicles are found inside and inositol 1,4,5 trisphosphate (IP 3) receptors are enriched in some but not all of these tubular structures. Interestingly, shuttling of mitochondria was observed in the NT and cytoplasm of the HeLa cells loaded with dihydro-rhod-2/AM. After stimulation with histamine that increases cytosolic [Ca 2+] through IP 3 production, a slow rise of dihydro-rhod-2 fluorescence for the measurement of intra-mitochondrial Ca 2+ was observed in the area of NT indicating that Ca 2+ was sequestered by mitochondria inside the tubular invagination. Our work therefore suggests that the NTs and mitochondrial activities represent a specialized compartment and dynamic process involved in the regulation of Ca 2+ inside the cell nucleus.

Defining epithelial cell progenitors in the human oxyntic mucosa.

In the human stomach, the oxyntic epithelium includes numerous tubular invaginations consisting of short pits opening into long glands. The pit is lined by pit cells, whereas the gland is composed of three regions: the base, containing zymogenic cells; the neck, containing neck cells; and the isthmus, composed of little known immature cells and of parietal cells, which are also scattered through the neck and base. The aim of this study was to examine the ultrastructure of the immature cells and to determine their relation to mature cells. To do so, normal oxyntic mucosal biopsies from subjects ranging from 20-43 years old were fixed in aldehydes and postfixed in reduced osmium for electron microscopy and morphometric analysis. The immature cells were sorted out into four classes, whose roles were clarified by comparison with the thoroughly investigated mouse oxyntic epithelium. The first class was composed of the least differentiated immature cells, which were rare and characterized by minute, dense, or cored secretory granules and were accordingly named mini-granule cells. Their function was not clarified. The second class consisted of pre-pit cells, which were characterized by few dense mucous granules and give rise to pit cells that ascend the pit wall and, after reaching the luminal surface, die or are extruded. Both pre-pit and pit cells underwent continuous renewal and, therefore, together constituted a renewal system referred to as pit cell lineage. The third class, or pre-neck cells, characterized by cored secretory granules, give rise to neck cells that descend toward the base region and differentiate further into pre-zymogenic cells, which finally become zymogenic cells. The latter eventually degenerate and die. Thus pre-neck cells and their progeny constitute a renewing system, designated zymogenic cell lineage. The fourth class, or pre-parietal cells, characterized by long microvilli and few tubulovesicles, differentiate into parietal cells that descend along the neck and base regions and eventually degenerate and die. Pre-parietal and parietal cells represent a renewing system referred to as parietal cell lineage. While the origin of the last three classes of progenitor cells has not been elucidated, it is likely that they arise either from an unidentified multipotential stem cell, possibly the mini-granule cell itself, or from the mitotic activity of pre-pit and pre-neck cells. In conclusion, the human oxyntic epithelium is composed of continually renewing cells organized in distinct cell lineages.

Amphiphysin 2 (Bin1) and T-Tubule Biogenesis in Muscle

In striated muscle, the plasma membrane forms tubular invaginations (transverse tubules or T-tubules) that function in depolarization-contraction coupling. Caveolin-3 and amphiphysin were implicated in their biogenesis. Amphiphysin isoforms have a putative role in membrane deformation at endocytic sites. An isoform of amphiphysin 2 concentrated at T-tubules induced tubular plasma membrane invaginations when expressed in nonmuscle cells. This property required exon 10, a phosphoinositide-binding module. In developing myotubes, amphiphysin 2 and caveolin-3 segregated in tubular and vesicular portions of the T-tubule system, respectively. These findings support a role of the bilayer-deforming properties of amphiphysin at T-tubules and, more generally, a physiological role of amphiphysin in membrane deformation.

Morphology and evolution of the respiratory apparatus in the family Eubelidae (Crustacea, Isopoda, Oniscidea).

The morphology of the respiratory apparatus in the pleopodal lungs of the family Eubelidae was investigated. The family is a monophyletic group including more than 240 species in 53 genera (three of which are nomina dubia), mostly distributed in the Afrotropical Region (tropical Africa and Arabian Peninsula). In all the Eubelidae, except for the monospecific genus Parelumoides and two species of the genus Elumoides, the exopods of pleopods have lungs. All the pulmonary morphologies present in the entire suborder Oniscidea are found: 1) uncovered lungs, composed of a pleated respiratory surface, directly exposed to the air (Atracheodillo-type) or partially enclosed within the appendage (Synarmadilloides-type); 2) covered lungs with several spiracles and respiratory trees, housed within the appendages, with spiracles surrounded by a specialized, nonrespiratory, structure (perispiracular area) (Eubelum- and Somaloniscus-types); 3) covered lungs with only one spiracle, with or without perispiracular area, and one respiratory tree (Aethiopopactes- and Periscyphis-types), which in taxa with Periscyphis-type lung crosses the insertion of the appendage and penetrates into the pleon with bundles of respiratory tubules. The evolution of the various types of lungs is discussed. It is concluded that the two main evolutionary lines, i.e., uncovered lungs and covered lungs, originated independently from an ancestral respiratory structure-the semilunar area. A first mechanism of development of the semilunar area by folding of its surface produced the Atracheodillo-type (all folds coplanar with the surface of the exopod) and Synarmadilloides-type (folds partly coplanar and partly intraflexed inside the exopod) uncovered lungs. A second mechanism of development by tubular invagination of the cuticle of the semilunar area produced the polyspiracular Eubelum-type lungs (numerous arborescent invaginations) and the monospiracular Aethiopopactes-type lungs (only one arborescent invagination), probably passing through a common intermediate pattern. From the common pattern, both the poly- and monospiracular types would have inherited the characteristic concave cell arrangement of the perispiracular area. The Somaloniscus-type and Periscyphis-type lungs are forms specialized for arid environments, directly derived from the Eubelum-type and Aethiopopactes-type, respectively.

Pinguiochrysis pyriformis gen. et sp. nov. (Pinguiophyceae), a new picoplanktonic alga isolated from the Pacific Ocean

SUMMARY Pinguiochrysis pyriformis gen. et sp. nov. is a brown, naked, non-motile, marine picoplankton. A culture was established from a surface sample collected in 1991 from the tropical Western Pacific Ocean. Typical cells of P. pyriformis are distinctively pear-shaped and have one ovoid chloroplast; these two features distinguish this species from the other picophytoplankton species. However, the pyriform morphology is not consistent and cells frequently change to a subspherical shape. The chloroplast and mitochondrion ultrastructure confirm that this species belongs to the photosynthetic stramenopiles (chromophytes). Additional distinctive ultrastructural characteristics of P. pyriformis include (i) a chloroplast envelope forming a tubular invagination that penetrates into the pyrenoid; (ii) thylakoid lamellae consisting of more than three layers in some cells; (iii) the lack of basal bodies and centrioles; and (iv) the lack of scales or other extracellular structures. Based on the morphological features, this picoplanktonic species was described as a new species and placed in the Pinguiophyceae on the basis of the molecular phylogenetic analysis and biochemical data published elsewhere.

Ultrastructural investigations of testes and spermiogenesis in two species of halacarid mites (Halacaridae, Actinedida, Actinotrichida): Thalassarachna basteri from the Baltic Sea and Halacarellus thomasi from McMurdo Sound (Antarctica)

Testes and sperm cells of two species of halacarid mites, Thalassarachna basteri from the Baltic Sea and Halacarellus thomasi from McMurdo Sound (Antarctica), were investigated. Testes are paired structures, composed of a glandular and a germinal part. The testicular lumen is filled with a very complex secretion that contributes to sperm cell aggregates. Early spermatids of T. basteri display unusual chromatin condensation within the nucleus, but the formation of an acrosomal complex with a small acrosomal vesicle and a long acrosomal filament can be regarded as typical for the group. Tubular invaginations of the plasmalemma occur at the cell periphery. A deep, ring-like infold divides the cell into one part containing the chromatin body and another containing mainly the invaginations and the acrosomal complex. The mature sperm cell is ovoid, aflagellate and surrounded by a distinct secretion sheath. In H. thomasi only a limited number of spermiogenesis stages were observable. Chromatin condensation was rather similar and peripheral invaginations also occurred. However, no acrosomal complex was observed in the early stages. The division of the mature sperm cells into two halves was even more pronounced in H. thomasi, since one half of the cell contained masses of convoluted structures. The same half also contained a structure that remotely resembled an acrosomal complex. The observed differences between T. basteri and H. thomasi sperms support the placing of the two halacarids in separate genera.

Cryo-Electron Tomography of Isolated Triad Junctions from Skeletal Muscle

In skeletal muscle, depolarization of the plasma membrane, which is initiated at the neuromuscular junction, is transduced to a rise in cytoplasmic calcium at specialized structures known as triad junctions (TJs). TJs occur in the myofiber’s interior at regions near the z-lines, where transversely oriented tubular invaginations of the plasma membrane (T-tubules) form junctions with two elements of the sarcoplasmic reticulum (SR). Isolation of membrane fractions that are enriched in junctional complexes and which retain function has been reported.Figure 1 shows a region of an electron micrograph containing an isolated TJ in the frozen-hydrated state. in the orientation shown, two SR-derived vesicles sandwich a flattened vesicle derived from the T-tubule. The junctional regions contain a complex distribution of density, presumably due to proteins that are known to be present in TJs. Electron tomography offers the means to determine the three-dimensional mass density from such micrographs, which would greatly aid in their interpretation. Only recently has the automated data collection technology for determining tomograms of non-stained, frozen-hydrated specimens become available. Here we describe the first tomographic reconstruction of a frozen-hydrated triad junction by automated electron tomography.

Imaging of the Human Vomeronasal Duct

The human vomeronasal duct (VND) is described as a tubular or pouch-like mucosal invagination of the anterior nasal septum. This study investigated shape, size and orientation of the VND using magnetic resonance imaging (MRI). Fifteen subjects participated (eight women, seven men; mean age 39 years, age range 18-66 years); they had been pre-selected with regard to the presence of a VND opening of 1 mm. MRI was performed before and after application of diluted gadolinium-diethylene-triamino-penta-acetic actetate (Gd-DTPA) into the left or right VND. A tubular structure was found in 12 subjects with a median length of 7 mm (range 3-22 mm; one VND with a length 47 mm). In three subjects a nearly circular, pouch-like structure was observed. Seven of the tubular VNDs were slightly bent upwards, the other five VNDs ran parallel to the floor of the nasal cavity. There was no significant gender-related difference in the length of VNDs. These data indicate considerable variability of shape, size and orientation of the human VND.

Autophagic tubes: vacuolar invaginations involved in lateral membrane sorting and inverse vesicle budding.

Many intracellular compartments of eukaryotic cells do not adopt a spherical shape, which would be expected in the absence of mechanisms organizing their structure. However, little is known about the principles determining the shape of organelles. We have observed very defined structural changes of vacuoles, the lysosome equivalents of yeast. The vacuolar membrane can form a large tubular invagination from which vesicles bud off into the lumen of the organelle. Formation of the tube is regulated via the Apg/Aut pathway. Its lumen is continuous with the cytosol, making this inverse budding reaction equivalent to microautophagocytosis. The tube is highly dynamic, often branched, and defined by a sharp kink of the vacuolar membrane at the site of invagination. The tube is formed by vacuoles in an autonomous fashion. It persists after vacuole isolation and, therefore, is independent of surrounding cytoskeleton. There is a striking lateral heterogeneity along the tube, with a high density of transmembrane particles at the base and a smooth zone devoid of transmembrane particles at the tip where budding occurs. We postulate a lateral sorting mechanism along the tube that mediates a depletion of large transmembrane proteins at the tip and results in the inverse budding of lipid-rich vesicles into the lumen of the organelle.

A functional link between dynamin and the actin cytoskeleton at podosomes.

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2-binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aa(G273D) abolished podosomes while GFP-dynamin(K44A) was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.

MODIFIED VASOEPIDIDYMOSTOMY WITH SIMULTANEOUS DOUBLE NEEDLE PLACEMENT, TUBULOTOMY AND TUBULAR INVAGINATION

Recently a new technique for vasoepididymostomy was described which included tubular invagination by the triangulation method with a patency rate of 92%. Although these early data were encouraging, some technical problems were noted with the technique. After placing the first suture there was often tubular leakage and collapse. The tubulotomy was difficult and in some cases a suture was inadvertently cut. A modified technique is presented for vasoepididymostomy with tubular invagination, including some methods described in rat models. Only 2 sutures are used with simultaneous double needle placement. The 2-suture technique was used on 19 men who had undergone at least 1 vasoepididymostomy during vasectomy reversal procedures. Patency was demonstrated in 7 of 9 men (77.7%) who underwent modified bilateral vasoepididymostomy and 6 of 7 (85.7%) who underwent unilateral vasoepididymostomy and unilateral vasovasostomy. Pregnancies were reported in both groups. The overall operating time for the modified technique was about 35 to 45 minutes per side. Modified vasoepididymostomy with simultaneous needle placement, tubulotomy and invagination resulted in early patency in a high percentage of patients. Tubular invagination may have advantages compared to other conventional methods of vasoepididymostomy but more followup is needed.

Formation of intranuclear crystalloids and proliferation of the smooth endoplasmic reticulum in schwann cells induced by tellurium treatment: association with overexpression of HMG CoA reductase and HMG CoA synthase mRNA.

Administration of tellurium (Te) in weaning rats causes a well-established demyelinating neuropathy induced by the inhibition in myelinating Schwann cells (SC) of the synthesis of cholesterol, a major component of the myelin sheath, at the level of squalene epoxidase. We have used this experimental model of Te neuropathy to study the biogenesis and reorganization of the endomembranes of the nuclear envelope and endoplasmic reticulum (ER) in response to Te treatment by ultrastructural analysis and in situ hybridization for the detection of HMG CoA reductase and synthase mRNA, which encode key enzymes in cholesterol synthesis. The adaptive response of myelinating SC to cholesterol depletion includes cell hypertrophy, the formation of tubular invaginations of proliferating nuclear membranes giving rise to peculiar nuclear inclusions termed crystalloids, and, at the cytoplasmic level, the formation of lamellar bodies of rough ER, proliferation of the smooth ER, and overexpression of HMG CoA reductase and synthase mRNAs. The changes revert after withdrawal of Te treatment. Our results show that the biogenesis and structural organization of both endomembrane systems change dynamically upon Te-induced cholesterol depletion, indicating that this constituent plays a critical role in the organization of nuclear envelope and ER compartments in SC. The results also suggest that the HMG CoA reductase, an integral membrane protein of ER, provides the signal for the extensive membrane assembly. While the physiological meaning of crystalloid remains to be clarified, the hypertrophy of the smooth ER may represent a cytoprotective mechanism involved in detoxification of the neurotoxic agent or its metabolic derivates.

Calycophoran siphonophore muscle fibres without any sarcoplasmic reticulum but with tubular invaginations morphologically analogous to a T-system

Different marine invertebrates have different tubular or vesicular systems within their locomotor muscle fibres. The siphonophores Chelophyes, Abylopsis and Muggeia have invaginated tubules which are the morphological equivalent of the vertebrate invaginated tubular system, but lack a sarcoplasmic reticulum. In Chelophyes the previous suggestion that Ca2+ channels in the extensive invaginated tubule system allow ingress of Ca2+ is shown to be incorrect. Contraction of the swimming muscles in Chelophyes is not blocked by 20 μM ryanodine, nor is it induced by 10 mM caffeine, hence intracellular Ca2+ stores appear absent. Contraction is, however, maintained by replacement of the greater part of the usual external Na+ by Li+ or by or N-methyl-D-glucamine, although action potentials can still be evoked. Hence we conclude that following contraction, internal Ca2+ is reduced by a Na/Ca2+ exchange mechanism.

Tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen.

The fine structure of plasmalemmal tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen has been demonstrated by scanning and transmission electron microscopy. In addition, the three-dimensional structure of the tubular invagination has been revealed by computer-aided reconstruction. The tubular invaginations of the plasma membrane plunged into the cytoplasm everywhere from the apical, lateral, and basal surfaces of the plasma membrane. The invaginations were tubular and branched away, and their plasma membranes were reinvaginated to form numerous caveolae and occasional coated pits. Numerous caveolae were found in clusters that looked similar to a bunch of grapes and the coated pits were present at the base of the clusters. The caveolae and coated pits derived from the tubular invaginations were almost ultrastructurally identical to those derived from the surface plasma membrane. From examination of the fractured surfaces of the endothelial cells treated with the aldehyde prefix osmium-dimethyl sulfoxide-osmium method and of ultrathin sections of those infiltrated by lanthanum nitrate, the tubular invaginations were found to not penetrate any endothelial cells. A computer-aided reconstruction revealed that the caveolae derived from the tubular invaginations were in close apposition to the surface-connected canaliculi. The reaction product of Concanavalin A conjugated to horseradish peroxidase was present on the outer leaflet of the membranes of the coated pits and coated vesicles and also in the contents of the endosomes, but it was absent from any caveolae. Based on our observations, the functional significance of the tubular invaginations in sinus endothelial cells is discussed.

Immunocytochemical study of endocytotic structures accumulated in HeLa cells transformed with a temperature-sensitive mutant of dynamin.

Dynamin is a 100-kD GTPase, which is required for clathrin-mediated endocytosis. Recent studies have revealed that dynamin is closely involved in clathrin-coated vesicle formation. In this study we investigated the ultrastructure of endocytotic structures accumulated in HeLa cells that were transformed with a temperature-sensitive (ts) mutant of dynamin to clarify which step was blocked in dynts cells. Endocytosis of transferrin receptors was restricted at the level of surface-connected membrane structures. Tubular and vesicular membrane invaginations were accumulated in the cells' peripheral regions, suggesting that the endocytosis was blocked just before the pinching-off steps in coated vesicle formation. The "collared" tubes, which were reported to be localized in nerve terminals in shibirets1 flies and GTPgammaS-treated synaptosomes, were not observed in the dynts cells even at nonpermissive temperature. The distribution pattern of dynamin in deeply invaginated coated pits in dynts cells was similar to that in dynwt cells but not to that in dynK44A cells, which are other endocytosis-defective mutant cells. These morphological data suggest that dynts blocked the pinching-off steps in clathrin-coated vesicle formation, which may be caused by a different mechanism from that of dynK44A cells.

Bacterial Lipopolysaccharide Can Enter Monocytes Via Two CD14-Dependent Pathways

Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.