The AMF-R tubule is a smooth ilimaquinone-sensitive subdomain of the endoplasmic reticulum

Autocrine motility factor receptor (AMF-R) is a marker for a distinct smooth membranous tubule. Ilimaquinone (IQ) is a sea sponge metabolite which induces the complete vesiculation of the Golgi apparatus and we show here that the addition of IQ to MDCK cells also results in the disruption of the AMF-R tubule. By immunofluorescence microscopy, the resultant punctate AMF-R label resembles the products of IQ-mediated vesiculation of the trans-Golgi network, however, the two labels can be distinguished by confocal microscopy. AMF-R tubule fragmentation occurs after nocodazole or taxol treatment of the cells demonstrating that the action of IQ on AMF-R tubules is not related to the ability of IQ to depolymerize microtubules. IQ activity is therefore not Golgi-specific. Electron microscopy of IQ-treated cells reveals that AMF-R is distributed to fenestrated networks of narrow interconnected tubules which are distinguishable from the uniform Golgi-derived vesicles and morphologically equivalent to smooth ER. Distinct fenestrations are visible in incompletely fragmented tubules which may represent intermediates in the fragmentation process. Smooth AMF-R labeled tubules exhibit continuity with rough ER cisternae and IQ selectively targets smooth and not rough ER. AMF-R tubules can be distinguished from the intermediate compartment labeled for ERGIC-53 by confocal microscopy and thus constitute a distinct IQ-sensitive subdomain of the smooth ER.

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Calcium Regulates the Association between Mitochondria and a Smooth Subdomain of the Endoplasmic Reticulum

The Journal of Cell Biology ◽

10.1083/jcb.150.6.1489 ◽

2000 ◽

Vol 150 (6) ◽

pp. 1489-1498 ◽

Cited By ~ 111

Author(s):

Hui-Jun Wang ◽

Ginette Guay ◽

Liviu Pogan ◽

Remy Sauvé ◽

Ivan R. Nabi

Keyword(s):

Confocal Microscopy ◽

Mdck Cells ◽

Close Association ◽

Cytosolic Calcium ◽

Free Calcium ◽

Autocrine Motility Factor ◽

Cytosolic Free Calcium ◽

Motility Factor ◽

Rat Liver Cytosol ◽

Close Contacts

Association between the ER and mitochondria has long been observed, and the formation of close contacts between ER and mitochondria is necessary for the ER-mediated sequestration of cytosolic calcium by mitochondria. Autocrine motility factor receptor (AMF-R) is a marker for a smooth subdomain of the ER, shown here by confocal microscopy to be distinct from, yet closely associated with the calnexin- or calreticulin-labeled ER. By EM, smooth ER AMF-R tubules exhibit direct interactions with mitochondria, identifying them as a mitochondria-associated smooth ER subdomain. In digitonin-permeabilized MDCK cells, the addition of rat liver cytosol stimulates the dissociation of smooth ER and mitochondria under conditions of low calcium. Using BAPTA chelators of various affinities and CaEGTA buffers of defined free Ca2+ concentrations and quantitative confocal microscopy, we show that free calcium concentrations <100 nM favor dissociation, whereas those >1 μM favor close association between these two organelles. Therefore, we describe a cellular mechanism that facilitates the close association of this smooth ER subdomain and mitochondria when cytosolic free calcium rises above physiological levels.

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Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle.

The Journal of Cell Biology ◽

10.1083/jcb.129.2.459 ◽

1995 ◽

Vol 129 (2) ◽

pp. 459-471 ◽

Cited By ~ 30

Author(s):

N Benlimame ◽

D Simard ◽

I R Nabi

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Immunofluorescence Microscopy ◽

Mdck Cells ◽

Electron Microscopic ◽

Autocrine Motility Factor ◽

Lysosomal Fraction ◽

Motility Factor ◽

Variable Diameter ◽

Triton X

Autocrine motility factor (AMF) is secreted by tumor cells and is capable of stimulating the motility of the secreting cells. In addition to being expressed on the cell surface, its receptor, AMF-R, is found within a Triton X-100 extractable intracellular tubular compartment. AMF-R tubules can be distinguished by double immunofluorescence microscopy from endosomes labeled with the transferrin receptor, lysosomes labeled with LAMP-2, and the Golgi apparatus labeled with beta-COP. AMF-R can also be separated from a LAMP-2 containing lysosomal fraction by differential centrifugation of MDCK cells and is found within a 100,000 g membrane pellet. By electron microscopic immunocytochemistry, AMF-R is localized predominantly to smooth vesicular and tubular membranous organelles as well as to a lesser extent to the plasma membrane and rough endoplasmic reticulum. AMF-R tubules have a variable diameter of 50-250 nm and can acquire an elaborate branched morphology. By immunofluorescence microscopy, AMF-R tubules are clearly distinguished from the calnexin labeled rough endoplasmic reticulum and AMF-R tubule expression is stable to extended cycloheximide treatment. The AMF-R tubule is therefore not a biosynthetic subcompartment of the endoplasmic reticulum. The tubular morphology of the AMF-R tubule is modulated by both the actin and microtubule cytoskeletons. In a similar fashion to that described previously for the tubular lysosome and endoplasmic reticulum, the linear extension and peripheral cellular orientation of the AMF-R tubule are dependent on the integrity of the microtubule cytoskeleton. The AMF-R tubule may thus form part of a family of microtubule-associated tubular organelles.

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AMF-R Tubules Concentrate in a Pericentriolar Microtubule Domain After MSV Transformation of Epithelial MDCK Cells

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549704501004 ◽

1997 ◽

Vol 45 (10) ◽

pp. 1351-1363 ◽

Cited By ~ 3

Author(s):

Ivan R. Nabi ◽

Ginette Guay ◽

Danièle Simard

Keyword(s):

Mdck Cells ◽

Cell Periphery ◽

Perinuclear Region ◽

Microtubule Organizing Center ◽

Autocrine Motility Factor ◽

Microtubule Cytoskeleton ◽

Motility Factor ◽

Organizing Center ◽

Sarcoma Virus ◽

Moloney Sarcoma Virus

Autocrine motility factor receptor (AMF-R) is localized to an intracellular microtubule-associated membranous organelle, the AMF-R tubule. In well-spread untrans-formed MDCK epithelial cells, the microtubules originate from a broad perinuclear region and AMF-R tubules extend throughout the cytoplasm of the cells. In Moloney sarcoma virus (mos)-transformed MDCK (MSV-MDCK) cells, microtubules accumulate around the centrosome, forming a microtubule domain rich in stabilized detyrosinated microtubules. AMF-R tubules are quantitatively associated with this pericentriolar microtubule domain and the rough endoplasmic reticulum and lysosomes also co-distribute with the pericentriolar mass of microtubules. The Golgi apparatus is closely associated with the microtubule organizing center (MTOC) within the juxtanuclear mass of AMF-R tubules, and no co-localization of AMF-R tubules with the Golgi marker β-COP could be detected by confocal microscopy. After nocodazole treatment and washout, microtubule nucleation occurs exclusively at the centrosome of MSV-MDCK cells, and only after microtubule extension to the cell periphery does the microtubule cytoskeleton reorganize to generate the pericentriolar microtubule domain after 30–60 min. AMF-R tubules dispersed by nocodazole treatment concentrate in the pericentriolar region in parallel with the reorganization of the microtubule cytoskeleton. MSV transformation of epithelial MDCK cells results in the stabilization of a pericentriolar microtubule domain responsible for the concentration and polarized distribution of AMF-R tubules.

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Localization of Autocrine Motility Factor Receptor to Caveolae and Clathrin-independent Internalization of Its Ligand to Smooth Endoplasmic Reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.9.7.1773 ◽

1998 ◽

Vol 9 (7) ◽

pp. 1773-1786 ◽

Cited By ~ 80

Author(s):

Naciba Benlimame ◽

Phuong U. Le ◽

Ivan R. Nabi

Keyword(s):

Endoplasmic Reticulum ◽

Confocal Microscopy ◽

Cell Surface ◽

Smooth Endoplasmic Reticulum ◽

Fluid Phase ◽

Cell Surface Receptor ◽

Tubular Structures ◽

Autocrine Motility Factor ◽

Motility Factor ◽

3T3 Fibroblasts

Autocrine motility factor receptor (AMF-R) is a cell surface receptor that is also localized to a smooth subdomain of the endoplasmic reticulum, the AMF-R tubule. By postembedding immunoelectron microscopy, AMF-R concentrates within smooth plasmalemmal vesicles or caveolae in both NIH-3T3 fibroblasts and HeLa cells. By confocal microscopy, cell surface AMF-R labeled by the addition of anti-AMF-R antibody to viable cells at 4°C exhibits partial colocalization with caveolin, confirming the localization of cell surface AMF-R to caveolae. Labeling of cell surface AMF-R by either anti-AMF-R antibody or biotinylated AMF (bAMF) exhibits extensive colocalization and after a pulse of 1–2 h at 37°C, bAMF accumulates in densely labeled perinuclear structures as well as fainter tubular structures that colocalize with AMF-R tubules. After a subsequent 2- to 4-h chase, bAMF is localized predominantly to AMF-R tubules. Cytoplasmic acidification, blocking clathrin-mediated endocytosis, results in the essentially exclusive distribution of internalized bAMF to AMF-R tubules. By confocal microscopy, the tubular structures labeled by internalized bAMF show complete colocalization with AMF-R tubules. bAMF internalized in the presence of a 10-fold excess of unlabeled AMF labels perinuclear punctate structures, which are therefore the product of fluid phase endocytosis, but does not label AMF-R tubules, demonstrating that bAMF targeting to AMF-R tubules occurs via a receptor-mediated pathway. By electron microscopy, bAMF internalized for 10 min is located to cell surface caveolae and after 30 min is present within smooth and rough endoplasmic reticulum tubules. AMF-R is therefore internalized via a receptor-mediated clathrin-independent pathway to smooth ER. The steady state localization of AMF-R to caveolae implicates these cell surface invaginations in AMF-R endocytosis.

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The application of video-enhanced constrast/differential interference constrast microscopy in conjunction with whole mount electron microscopy to the study of organelle transport

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102419 ◽

1988 ◽

Vol 46 ◽

pp. 66-67

Author(s):

J. H. Hayden

Keyword(s):

Electron Microscopy ◽

Rana Pipiens ◽

Silver Film ◽

Immunofluorescence Microscopy ◽

Organelle Transport ◽

Linear Element ◽

Whole Mount ◽

Carbon Coated ◽

Linear Elements ◽

Dic Microscopy

In a previous study, Allen video-enhanced constrast/differential interference constrast (AVEC-DIC) microscopy was used in conjunction with immunofluorescence microscopy to demonstrate that organelles and vesicle move in either direction along linear elements composed of microtubules. However, this study was limited in that the number of microtubules making up a linear element could not be determined. To overcome this limitation, we have used AVEC-DIC microscopy in conjunction with whole mount electron microscopy.Keratocytes from Rana pipiens were grown on glass coverslips as described elsewhere. Gold London Finder grids were Formvar- and carbon coated, and sterilized by exposure to ultraviolet light. It is important to select a Formvar film that gives a grey reflection when it is floated on water. A silver film is too thick and will detract from the image in the light microscope.

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