Localization of Procollagen I in the Lysosome/Endosome System of Human Fibroblasts

Ripley, Catherine R.; Bienkowski, Robert S.

doi:10.1006/excr.1997.3707

Cited by 24 publications

(14 citation statements)

References 45 publications

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“…In fibroblasts, upward of 15% of newly synthesized collagen is degraded intracellularly rather than secreted (55). Studies of procollagen in fibroblasts showed a partial co-distribution or procollagen with lysosome markers (56), suggesting that lysosomes play an active role in the basal degradation of collagen. Collagen secretion requires MTs (57), and dispersed lysosomes in osteogenic cells may situate the lysosomes along the same highway for rapid interactions with vesicles containing structurally abnormal procollagen.…”

Section: Discussionmentioning

confidence: 99%

Lysosome Dispersion in Osteoblasts Accommodates Enhanced Collagen Production during Differentiation

Nabavi

Urukova

Cardelli

et al. 2008

Journal of Biological Chemistry

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Lysosomes are essential organelles for intracellular degradation and are generally sequestered near the cell center to receive vesicles with contents targeted for destruction. During ascorbic acid (AA)-induced differentiation of osteogenic cells (Beck, G. R., Jr., Zerler, B., and Moran, E. (2001) Cell Growth Differ. 12, 61-83), we saw a marked increase in total lysosome organelles in osteoblastic cells, in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating osteoblasts. We determined that lysosome dispersion in differentiated osteoblasts required intact microtubules for long range transport and was dependent on kinesin motors but did not involve cytosolic acidification. Impairment of lysosome dispersion markedly reduced AA-induced osteoblast differentiation. Lysosomes were not secreted in differentiated osteoblasts, implicating them instead in intracellular degradation. We assayed the degradative capacity and saw a significant increase in DQ-ovalbumin fluorescence in differentiated osteogenic cells compared with undifferentiated control cells. Osteogenic cells are specialized for type I collagen production, and we noted enhanced secreted and intracellular collagen in AA-differentiated osteoblasts versus control cells. Importantly, osteoblasts displayed procollagen-containing vesicles that were distributed throughout the cytoplasm, a portion of which colocalized with lysosomes. Treatment of cells with 2,2-dipyridyl to inhibit procollagen trimerization enhanced colocalization of lysosomes with procollagen-containing organelles, implicating dispersed lysosomes in collagen processing in osteogenic cells.Proper organelle positioning is essential in all eukaryotic cells for inter-organelle trafficking, protein targeting, cellular homeostasis, and response to extracellular stimuli (2). The intracellular organellar architecture is dictated by the protein scaffold of the cell, namely the cytoskeleton. The major cytoskeletal component that dictates the final location of organelles is the microtubule (MT) 3 cytoskeleton (3), with intermediate filaments also playing a role for certain organelles (4). MTs are long polarized polymers that interact indirectly with organelles through motor and adaptor proteins (5). The relative abundance and/or activity of motor proteins on the organelles is believed to determine whether the organelle will be positioned in the cell center (toward the MT minus ends) or toward the plasma membrane (at MT plus ends). Organelles, such as mitochondria and melanosomes, are held toward the cell periphery through the actions of the kinesin motor (6, 7), whereas the minus end-directed motor, dynein, is responsible for holding the nucleus, Golgi complex, lysosomes, and recycling endosomes within the cell interior (8).Although most eukaryotic cells have a similar spatial organization of organelles, there are cell-specific differences in organelle quantities and positioning. The relative abundance of organelles can vary from cell to ce...

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Section: Discussionmentioning

confidence: 99%

Lysosome Dispersion in Osteoblasts Accommodates Enhanced Collagen Production during Differentiation

Nabavi

Urukova

Cardelli

et al. 2008

Journal of Biological Chemistry

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“…A major advantage of the new model system is that the beadbound biotinylated collagen permits simultaneous study of fusion processes and intracellular degradation of exogenous collagen in fibroblasts. This system enables clear cut discrimination of the exogenous collagen from nascent collagen (15) that also undergoes intracellular degradation (16).…”

Section: Discussionmentioning

confidence: 99%

“…Electron microscopic studies on the digestion of phagocytosed exogenous collagen have shown cross-banded collagen profiles in the phagolysosomes of fibroblasts (12,13), and there is an apparent requirement for lysosomal serine proteinases for collagen degradation (13,14). A confounding factor in fibroblasts is that not all nascent collagen is secreted (15)(16)(17); a significant fraction is degraded intracellularly in the endoplasmic reticulum, in the Golgi network, and in the endosome/lysosome system, thereby complicating the distinction between exogenous (i.e. phagocytosed) and newly synthesized endogenous collagen.…”

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confidence: 99%

A Novel Model System for Characterization of Phagosomal Maturation, Acidification, and Intracellular Collagen Degradation in Fibroblasts

Arora

Manolson²,

Downey³

et al. 2000

Journal of Biological Chemistry

104

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Intracellular collagen degradation by fibroblasts is an important but poorly understood pathway for the physiological remodeling of mature connective tissues. The objective of this study was to determine whether gingival fibroblasts that express endogenous ␣ 2 ␤ 1 integrin, the collagen receptor, would exhibit the cellular machinery required for phagosomal maturation and collagen degradation. There was a time-dependent increase of collagen bead internalization and a time-dependent decrease of bead-associated ␣ 2 ␤ 1 integrin after initial bead binding. ␤-Actin and gelsolin associated transiently with beads (0 -30 min) followed by LAMP-2 (60 -240 min) and cathepsin B (30 -240 min). Cytochalasin D prevented phagosome formation and also prevented the sequential fusion of early endosomes with lysosomes. Collagen bead-associated pH was progressively reduced from 7.25 to 5.4, which was contemporaneous with progressive increases in degradation of bead-associated collagen (30 -120 min). Concanamycin blocked acidification of phagolysosomes and collagen degradation but not phagosome maturation. Phagosomal acidification was partly dependent on elevated intracellular calcium. These studies demonstrate that the cellular machinery required for intracellular collagen degradation in fibroblasts closely resembles the vacuolar system in macrophages.Phagocytosis is central to the uptake and degradation of microorganisms as well as damaged or senescent cells and is therefore an essential process in host defense, tissue remodeling, and inflammation. Although "professional" phagocytic cells such as macrophages and neutrophils have been studied in considerable depth (1), several types of nonphagocytic cells such as epithelial cells and fibroblasts can internalize particles and matrix proteins in vivo and in culture. Collagen fibril phagocytosis is thought to be an important pathway for physiological degradation of extracellular matrix in mature connective tissues (2). Uterine, wounded dermal, and periodontal connective tissues exhibit rapid physiological turnover of matrix proteins, processes that are mediated by the intracellular degradation pathway (3). While in inflamed periodontal sites, extracellular matrix metalloproteinases are believed to be responsible for the bulk degradation of connective tissues (4), in normal turnover, fibroblasts are thought to use solely the intracellular vacuolar system for focal proteolysis of collagen (3,5). However, at present, the vacuolar system that mediates intracellular collagen degradation is incompletely characterized.In professional phagocytes, the phagocytic process is initiated by binding of particles to receptors on the plasma membrane, an event that subsequently generates a phagocytic signal (6). In fibroblasts, the initial internalization of intracellular collagen degradation is a specific process that is mediated by the adhesive interactions between ligand and collagen receptors (i.e. ␣ 2 ␤ 1 integrins; Ref. 7). However, little is known about the regulation of the downstream events and...

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“…Cells were fixed with 3% paraformaldehyde/0.1% glutaraldehyde using the pH-shift method with some modifications. 23 Cells were permeabilized with 0.1% Triton X-100 in PBS. All antibodies were diluted in 0.2% fish skin gelatin in PBS.…”

Section: Methodsmentioning

confidence: 99%

Deficient assembly and function of gap junctions in Trf1, a trafficking mutant of the human liver-derived cell line HuH-7

et al. 1999

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Gap junctions are complexes between adjacent cells that are responsible for direct intercellular communication via passive diffusion of low-molecular-weight hydrophilic molecules. The proteins present in gap junctions are termed connexins, of which at least 16 distinct gene products are known to occur in vertebrate species. 1,2 Connexin proteins differ notably from other membrane proteins in lacking glycosylation and being localized primarily at appositional regions between coupled cells. While the pathways of biogenesis, trafficking, assembly, and turnover have not yet been completely elucidated, key features appear to be more similar than not to ''typical'' plasma membrane proteins. [3][4][5][6][7][8][9][10][11] In previous studies, biochemical analysis of secretory or trafficking mutants 12-14 has elucidated many features of these pathways. This approach for analysis of gap junction biology has not been pursued in vertebrate cell lines harboring similar mutations. Characterization of connexin trafficking in the membrane protein trafficking mutant, Trf1, was undertaken as a first step in combining biochemical and mutational approaches to analysis of connexin trafficking and assembly.The Trf1 cell line was isolated as a result of a dual selection protocol. 15 Endocytic activity of 2 different carbohydratespecific receptors of the human hepatoma cell line HuH-7 were simultaneously selected against using gelonin, an inhibitor of protein synthesis, conjugated to galactose-and mannoseterminating glycoproteins. Because there was no significant reciprocal inhibition between the mannose-and galactoseterminating glycoproteins and their respective gelonin conjugates, the mutation harbored by Trf1 cells appeared to affect a common step in their endocytic pathway. This pleiotropic phenotype was extended to other cell-surface membrane proteins, including the transferrin receptor and the phenylalanine transporter.The Trf1 mutation provided the first genetic evidence for the existence of different subpopulations (States 1 and 2) of the asialoglycoprotein receptor (ASGPR) as described by Weigel and Oka. 16 Originally based on the biphasic kinetics of ligand-receptor complex dissociation, numerous characteristics have since been described that differentiate the 2 ASGPR subpopulations and the proposed endocytic pathways that they mediate. 17 While no biochemical basis for this receptor dichotomy has been established, only State 2 receptors were shown to be susceptible to modulation by a wide variety of cell perturbants and altered metabolic states of the liver. 17 The proposed existence of 2 subpopulations of receptors is not limited to ASGPR and has been suggested for a wide variety of cell-surface receptors on different cell types. The low-density lipoprotein and mannose-6-P receptors on fibroblasts, the mannose receptor and ␣ 2 macroglobulin receptor on alveolar macrophages, and the transferrin receptor on K562 and HuH-7 cells all respond to cell perturbants by a partial reduction in their surface expression, suggesting Abb...

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Localization of Procollagen I in the Lysosome/Endosome System of Human Fibroblasts

Cited by 24 publications

References 45 publications

Lysosome Dispersion in Osteoblasts Accommodates Enhanced Collagen Production during Differentiation

Lysosome Dispersion in Osteoblasts Accommodates Enhanced Collagen Production during Differentiation

A Novel Model System for Characterization of Phagosomal Maturation, Acidification, and Intracellular Collagen Degradation in Fibroblasts

Deficient assembly and function of gap junctions in Trf1, a trafficking mutant of the human liver-derived cell line HuH-7

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