scholarly journals Rab4 Regulates Formation of Synaptic-like Microvesicles from Early Endosomes in PC12 Cells

2001 ◽  
Vol 12 (11) ◽  
pp. 3703-3715 ◽  
Author(s):  
Heidi de Wit ◽  
Yael Lichtenstein ◽  
Regis B. Kelly ◽  
Hans J. Geuze ◽  
Judith Klumperman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Pc12 Cells ◽  
Early Endosome ◽  
Small Gtpase ◽  
Immunogold Electron Microscopy ◽  
Cell Periphery ◽  
Early Endosomes ◽  
Cytoplasmic Vesicles ◽  
Important Site

Early endosomes in PC12 cells are an important site for the formation of synaptic-like microvesicles and constitutive recycling vesicles. By immunogold electron microscopy, the small GTPase rab4 was localized to early endosomes and numerous small vesicles in the cell periphery and Golgi area of PC12 cells. Overexpression of GTPase-deficient Q67Lrab4 increased the number of early endosome-associated and cytoplasmic vesicles, whereas expression of GDP-bound S22Nrab4 significantly increased the length of early endosomal tubules. In parallel, Q67Lrab4 induced a shift in rab4, VAMP2, and TfR label from early endosomes to peripheral vesicles, whereas S22Nrab4 increased early endosome labeling of all three proteins. These observations were corroborated by early endosome budding assays. Together, our data document a thus far unrecognized role for rab4 in the formation of synaptic-like microvesicles and add to our understanding of the formation of constitutive recycling vesicles from early endosomes.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals The 17β-oestradiol dehydrogenase of pig endometrial cells is localized in specialized vesicles

1993 ◽  
Vol 290 (3) ◽  
pp. 777-782 ◽  
Author(s):  
J Adamski ◽  
B Husen ◽  
F Marks ◽  
P W Jungblut
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Immunogold Electron Microscopy ◽  
The Novel ◽  
Electron Dense Material ◽  
Endometrial Cells ◽  
Functional Aspects ◽  
Cytoplasmic Vesicles ◽  
Similar Appearance ◽  
High Degree

Two monoclonal antibodies against the 17 beta-oestradiol dehydrogenase of pig endometrial cells have been used in localization studies with immunogold electron microscopy. The antibodies attach both to a fraction of dehydrogenase-rich cytoplasmic vesicles isolated from homogenates and to vesicles of similar appearance in cells. The vesicles are filled with electron-dense material. Their tagging intensity indicates a high degree of specialization. Endometrial cells from mature animals contain a host of dehydrogenase vesicles, and cells from prepubertal animals only a few. Functional aspects of the novel organelle are discussed.

scholarly journals Correlative SMLM and electron tomography reveals endosome nanoscale domains

2019 ◽  
Author(s):  
Christian Franke ◽  
Urska Repnik ◽  
Sandra Segeletz ◽  
Nicolas Brouilly ◽  
Yannis Kalaidzidis ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Single Molecule ◽  
Electron Tomography ◽  
Small Gtpase ◽  
Molecular Organization ◽  
Functional Domains ◽  
Localization Microscopy ◽  
Early Endosomes ◽  
Single Molecule Localization Microscopy

AbstractMany cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which however cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional sub-domains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-colour SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labelled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nano-domains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.SynopsisSuborganelle compartmentalization cannot be resolved by diffraction limited light microscopy and not interpreted without knowledge of the underlying ultrastructure. This work shows a novel superCLEM workflow that combines multi-colour single-molecule localization-microscopy with electron tomography to map several functional domains on early endosomes. superCLEM reveals that the small GTPase Rab5 is organized in nano-domains largely devoid from cargo molecules Transferrin and EGF and opens new possibilities to perform structure-function analysis of organelles at the nanoscale.

scholarly journals The melanoma proteoglycan: restricted expression on microspikes, a specific microdomain of the cell surface.

1986 ◽  
Vol 103 (5) ◽  
pp. 1699-1710 ◽  
Author(s):  
H J Garrigues ◽  
M W Lark ◽  
S Lara ◽  
I Hellström ◽  
K E Hellström ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Chondroitin Sulfate ◽  
Chondroitin Sulfate Proteoglycan ◽  
Immunogold Electron Microscopy ◽  
Cell Contact ◽  
Cell Periphery ◽  
Sulfate Proteoglycan ◽  
Whole Mount ◽  
Specific Localization

A cell surface chondroitin sulfate proteoglycan associated with human melanomas and defined by mAb's F24.47 and 48.7 has been characterized biochemically and localized by indirect immunogold electron microscopy. These antibodies recognize distinct epitopes on the intact proteoglycan. In addition, mAb 48.7 also recognizes an epitope on a 250,000-D glycoprotein and is therefore similar to antibody 9.2.27 (described by Bumol, T.F., and R.A. Reisfeld, 1982, Proc. Natl. Acad. Sci. USA., 79:1245-1249). Furthermore, it was shown that the glycosaminoglycan chains released by alkaline borohydride treatment of the proteoglycan recognized by mAb 48.7 had a size of approximately 60,000 D. Since the intact proteoglycan was estimated to be 420,000 D, there are probably three chondroitin sulfate chains attached to the 250,000-D core glycoprotein. Furthermore, an oligosaccharide fraction containing 42% of the 3H activity (glucosamine as precursor) was isolated. Immunolocalization studies using whole-mount electron microscopy revealed that the chondroitin sulfate proteoglycan was present almost exclusively on microspikes, a microdomain of the melanoma cell surface. These processes were present as 1-2-micron structures on the upper cell surface and as longer (up to 20 micron) structures at the cell periphery. Peripheral microspikes were involved in the initial interactions between adjacent cells and formed complex footpads that made contact with the substratum. Immunogold-labeled cells were also thin sectioned and the specific localization of the chondroitin sulfate proteoglycan antigen was quantitated. The data confirmed the results of whole-mount microscopy and demonstrated a statistically significant association of the antigen with the microspike processes as compared with other areas of the cell surface. By using two different mAb's (48.7 and F24.47) that recognize epitopes on either the core glycoprotein or the intact proteoglycan, respectively, we have demonstrated that both molecules have the same restricted distribution at the cell surface. The specific localization of the antigen to microspikes at the cell surface suggests it may play a role in cell-cell contact and cell-substratum adhesion, which could be important in the metastatic process.

scholarly journals In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome.

1990 ◽  
Vol 111 (2) ◽  
pp. 329-345 ◽  
Author(s):  
J Tooze ◽  
M Hollinshead ◽  
T Ludwig ◽  
K Howell ◽  
B Hoflack ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Lysosomal Enzymes ◽  
Early Endosome ◽  
Endocytic Pathway ◽  
Cell Periphery ◽  
Type I ◽  
Type Ii ◽  
Autophagic Vacuoles ◽  
Early Endosomes ◽  
Mannose 6 Phosphate

Intracisternal granules (ICGs) are insoluble aggregates of pancreatic digestive enzymes and proenzymes that develop within the lumen of the rough endoplasmic reticulum of exocrine pancreatic cells, especially in guinea pigs. These ICGs are eliminated by autophagy. By morphological criteria, we identified three distinct and sequential classes of autophagic compartments, which we refer to as phagophores, Type I autophagic vacuoles, and Type II autophagic vacuoles. Lobules of guinea pig pancreas were incubated in media containing HRP for periods of 5-120 min to determine the relationship between the endocytic and autophagic pathways. Incubations with HRP of 15 min or less labeled early endosomes at the cell periphery that were not involved in autophagy of ICGs, but after these short incubations none of the autophagic compartments were HRP positive. After 30-min incubation with HRP, early endosomes at the cell periphery, late endosomes in the pericentriolar region, and, in addition, Type I autophagic vacuoles containing ICGs were all labeled by the tracer. Type II autophagic vacuoles were not labeled after 30-min incubation with HRP but were labeled after incubations of 60-120 min. Phagophores did not receive HRP even after 120 min incubations. We concluded that the autophagic and endocytic pathways converge immediately after the early endosome level and that Type I autophagic vacuoles precede Type II autophagic vacuoles on the endocytic pathway. We studied the distribution of acid phosphatase, lysosomal proteases and cation-independent-mannose-6-phosphate receptor (CI-M6PR) in the three classes of autophagic compartments by histochemical and immunocytochemical methods. Phagophores, the earliest autophagic compartment, contained none of these markers. Type I autophagic vacuoles contained acid phosphatase but, at most, only very low levels of cathepsin D and CI-M6PR. Type II autophagic vacuoles, by contrast, are enriched for acid phosphatase, cathepsin D, and other lysosomal enzymes, and they are also enriched for CI-M6PR. Moreover, soluble fragments of bovine CI-M6PR conjugated to colloidal gold particles heavily labeled Type II but not Type I autophagic vacuoles, and this labeling was specifically blocked by mannose-6-phosphate. This indicates that the lysosomal enzymes present in Type II autophagic vacuoles carry mannose-6-phosphate monoester residues. Using 3-C2, 4-dinitroanilino-3'-amino-N-methyldipropylamine (DAMP), we showed that Type II autophagic vacuoles are acidic. We interpret these findings as indicating that Type II autophagic vacuoles are a prelysosomal compartment in which the already combined endocytic and autophagic pathways meet the delivery pathway of lysosomal enzymes.

scholarly journals Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

2011 ◽  
Vol 22 (20) ◽  
pp. 3853-3860 ◽  
Author(s):  
Liang Wang ◽  
Zhimin Liang ◽  
Guangpu Li
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Small Gtpase ◽  
Rab Gtpase ◽  
Early Endosomes ◽  
Ngf Signaling

Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22–Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22–Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation.

Transmission Electron Microscopy Studies of Pore Cells in Limax Maximus

1977 ◽  
Vol 35 ◽  
pp. 656-657
Author(s):  
B. S. Beltz
Keyword(s):  
Electron Microscopy ◽  
Cell Periphery ◽  
Salivary Duct ◽  
Terrestrial Mollusc ◽  
Buccal Ganglia ◽  
Transmission Electron ◽  
Pore Cells ◽  
Gland Tissue ◽  
Storage Area ◽  
Limax Maximus

The cells which are described in this study surround the salivary nerve of the terrestrial mollusc, Limax maximus. The salivary system of Limax consists of bilateral glands, ducts, and nerves. The salivary nerves originate at the buccal ganglia, which are situated on the posterior face of the buccal mass, and run along the salivary duct to the gland. The salivary nerve branches several times near the gland, and eventually sends processes into the gland.The pore cells begin to appear at the first large branch point of the salivary nerve, near the gland (Figure 1). They follow the nerve distally and eventually accompany the nerve branches into the gland tissue. The cells are 20-50 microns in diameter and contain very small nuclei (1-5 microns) (Figure 2).The cytoplasm of the pore cells is segregated into a storage area of glycogen and an organelle region located in a band around the cell periphery (Figure 3).

Immunogold electron microscopy of surface antigens of oral bacteria

1984 ◽  
Vol 30 (8) ◽  
pp. 1008-1013 ◽  
Author(s):  
C. Mouton ◽  
L. Lamonde
Keyword(s):  
Electron Microscopy ◽  
Surface Antigens ◽  
Oral Bacteria ◽  
Immunogold Electron Microscopy ◽  
Bacterial Surface ◽  
Outer Aspect ◽  
Bacteroides Gingivalis ◽  
Cell Envelopes ◽  
Inexpensive Procedure ◽  
Monospecific Antibodies

Colloidal gold particles 3–6 nm in diameter were prepared and stabilized with the IgG fraction of polyspecific rabbit antisera produced against four different oral bacteria. The immunogold markers were used in homologous reactions to label the bacteria in a preembedding procedure for electron microscopy. An indirect immunofluorescence procedure was concurrently used to optimize the labelling conditions before observation with the electron microscope. The immunogold markers labelled fibrillar structures extending outward 50–275 nm from the Gram-positive cell envelopes and a fuzzy 5–10 nm thick capsulelike layer on the outer aspect of Bacteroides gingivalis. The immunogold method appears to be a simple, rapid, and inexpensive procedure suitable for the study of bacterial surface antigens and can be upgraded with the use of monospecific antibodies.

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