scholarly journals Caveolae and Endoplasmic Reticulum: Immunofluorescence Microscopy and Time-Lapse Analysis.

1997 ◽  
Vol 30 (5/6) ◽  
pp. 593-599 ◽  
Author(s):  
Hiroshi Kogo ◽  
Mariko Shioya ◽  
Yukiko Takahashi ◽  
Toyoshi Fujimoto
Keyword(s):  
Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Time Lapse

scholarly journals Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia.

1979 ◽  
Vol 83 (2) ◽  
pp. 320-337 ◽  
Author(s):  
E Wang ◽  
R K Cross ◽  
P W Choppin
Keyword(s):  
Protein Synthesis ◽  
Cell Fusion ◽  
Structural Integrity ◽  
Immunofluorescence Microscopy ◽  
Time Lapse ◽  
Nuclear Migration ◽  
High Concentration ◽  
Nuclear Movement ◽  
Filament Bundles ◽  
10 Nm Filaments

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501-520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.

scholarly journals Reshaping of the endoplasmic reticulum limits the rate for nuclear envelope formation

2008 ◽  
Vol 182 (5) ◽  
pp. 911-924 ◽  
Author(s):  
Daniel J. Anderson ◽  
Martin W. Hetzer
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Mammalian Cells ◽  
Time Lapse ◽  
Principal Mechanism ◽  
Nuclear Assembly ◽  
Nuclear Envelope Formation ◽  
Rate Limiting

During mitosis in metazoans, segregated chromosomes become enclosed by the nuclear envelope (NE), a double membrane that is continuous with the endoplasmic reticulum (ER). Recent in vitro data suggest that NE formation occurs by chromatin-mediated reorganization of the tubular ER; however, the basic principles of such a membrane-reshaping process remain uncharacterized. Here, we present a quantitative analysis of nuclear membrane assembly in mammalian cells using time-lapse microscopy. From the initial recruitment of ER tubules to chromatin, the formation of a membrane-enclosed, transport-competent nucleus occurs within ∼12 min. Overexpression of the ER tubule-forming proteins reticulon 3, reticulon 4, and DP1 inhibits NE formation and nuclear expansion, whereas their knockdown accelerates nuclear assembly. This suggests that the transition from membrane tubules to sheets is rate-limiting for nuclear assembly. Our results provide evidence that ER-shaping proteins are directly involved in the reconstruction of the nuclear compartment and that morphological restructuring of the ER is the principal mechanism of NE formation in vivo.

The Marginal Microtubule Coil in the Resting Blood Platelet Is a Dynamic Bipolar Array.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1653-1653 ◽  
Author(s):  
Joseph E. Italiano ◽  
Jennifer L. Richardson ◽  
Harald Schulze ◽  
Ksenija Drabek ◽  
Chloe Bulinski ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Immunofluorescence Microscopy ◽  
Blood Platelet ◽  
Time Lapse ◽  
Microtubule Dynamics ◽  
Cell Periphery ◽  
Structural Studies ◽  
Time Lapse Microscopy ◽  
Marginal Band ◽  
Tyrosinated Tubulin

Abstract The discoid shape of the resting blood platelet is maintained by its marginal microtubule band. Structural studies have concluded that this band is composed of a single microtubule coiled 8-12 times around the cell periphery. To understand the dynamics of the microtubule coil, we took advantage of EB1 and EB3, proteins that highlight the ends of growing microtubules. Immunofluorescence microscopy with anti-EB1 revealed clear staining of numerous (8.7 +/− 2.0, range 4–12) comet-like dashes in the microtubule coil, suggesting the presence of several microtubule plus ends. Consistent with this observation, rhodamine-tubulin added to permeabilized platelets incorporates at multiple (7.9 +/−1.9) points throughout the microtubule coil. To visualize microtubule dynamics in platelets, we retrovirally directed megakaryocytes to express the microtubule plus-end marker EB3-GFP and isolated platelets released in these cultures. Fluorescence time-lapse microscopy of EB3-GFP-expressing resting platelets revealed multiple microtubule plus ends that grew in both clockwise and counterclockwise directions. Antibodies that recognize tyrosinated tubulin, which preferentially label newly assembled microtubules and not stable microtubules, stain the microtubule coil. These results indicate that resting platelets contain a bipolar array of microtubules that undergoes continuous assembly. When EB3-GFP-expressing platelets are activated with thrombin, the number of polymerizing microtubules increases dramatically and the microtubules grow into filopodia. Collectively, these results suggest that the marginal band of the resting blood platelet is highly dynamic, bipolar, and contains multiple microtubule plus ends. These ends are amplified in platelet activation and point towards the active edges of the cells and the tips of filopodia.

Non-plasmalemmal localisation of the major ganglioside in sea urchin eggs

Zygote ◽  
1993 ◽  
Vol 1 (3) ◽  
pp. 215-223 ◽  
Author(s):  
Hidehiko Shogomori ◽  
Kazuyoshi Chiba ◽  
Hideo Kubo ◽  
Motonori Hoshi
Keyword(s):  
Endoplasmic Reticulum ◽  
Sea Urchin ◽  
Indirect Immunofluorescence ◽  
Immunofluorescence Microscopy ◽  
Mitotic Apparatus ◽  
Sea Urchin Eggs ◽  
Indirect Immunofluorescence Microscopy ◽  
The One ◽  
Major Ganglioside ◽  
Hemicentrotus Pulcherrimus

SummaryM5 ganglioside (NeuGcα2–6Glcβl-' Cer) is the predominant glycosphingolipid in sea urchin eggs. Distribution of M5 ganglioside was studied in unfertilised and fertilised eggs of the sea urchin Hemicentrotus pulcherrimus by indirect immunofluorescence microscopy. In the cortices of unfertilised eggs, anti-M5 antibody strongly stained the submembranous, polygonal and tubular network of endoplasmic reticulum that was revealed by a membrane-staining dye, DiIC18(3). In addition to the cortical network of endoplasmic reticulum, at least two morphologically distinct vesicles were positive to the antibody. In the cortices isolated from fertilised eggs 30 min after insemination, the antibody stained only a similar network of endoplasmic reticulum, presumably the one reconstructed 5–10 min after fertilisation. During mitosis the endoplasmic reticulum is known to aggregate within the asters of the mitotic apparatus. Indeed, the antibody stained the asters and (more strongly) the vesicular components attaching to the periphery of the mitotic apparatus.

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

1995 ◽  
Vol 129 (2) ◽  
pp. 459-471 ◽  
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.

scholarly journals Granule exocytosis is required for platelet spreading: differential sorting of α-granules expressing VAMP-7

Blood ◽  
2012 ◽  
Vol 120 (1) ◽  
pp. 199-206 ◽  
Author(s):  
Christian G. Peters ◽  
Alan D. Michelson ◽  
Robert Flaumenhaft
Keyword(s):  
Immunofluorescence Microscopy ◽  
Time Lapse ◽  
Granule Exocytosis ◽  
Time Lapse Microscopy ◽  
Gray Platelet Syndrome ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Discrete Functions ◽  
Single Granule

Abstract There has been recent controversy as to whether platelet α-granules represent a single granule population or are composed of different subpopulations that serve discrete functions. To address this question, we evaluated the localization of vesicle-associated membrane proteins (VAMPs) in spread platelets to determine whether platelets actively sort a specific subpopulation of α-granules to the periphery during spreading. Immunofluorescence microscopy demonstrated that granules expressing VAMP-3 and VAMP-8 localized to the central granulomere of spread platelets along with the granule cargos von Willebrand factor and serotonin. In contrast, α-granules expressing VAMP-7 translocated to the periphery of spread platelets along with the granule cargos TIMP2 and VEFG. Time-lapse microscopy demonstrated that α-granules expressing VAMP-7 actively moved from the granulomere to the periphery during spreading. Platelets from a patient with gray platelet syndrome lacked α-granules and demonstrated only minimal spreading. Similarly, spreading was impaired in platelets obtained from Unc13dJinx mice, which are deficient in Munc13-4 and have an exocytosis defect. These studies identify a new α-granule subtype expressing VAMP-7 that moves to the periphery during spreading, supporting the premise that α-granules are heterogeneous and demonstrating that granule exocytosis is required for platelet spreading.

scholarly journals Dynamics of the Endoplasmic Reticulum and Golgi Apparatus during Early Sea Urchin Development

2000 ◽  
Vol 11 (3) ◽  
pp. 897-914 ◽  
Author(s):  
Mark Terasaki
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Permeability Barrier ◽  
Cell Cycles ◽  
Nuclear Envelope Breakdown ◽  
Quantitative Measurements ◽  
Green Fluorescent

The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2–3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is ∼30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-μm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.

scholarly journals EB1 binding provides a diffusion trap mechanism regulating STIM1 localization and Ca2+ signaling

2017 ◽  
Author(s):  
Chi-Lun Chang ◽  
Yu-Ju Chen ◽  
Jen Liou
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Cellular Functions ◽  
Binding Ability ◽  
Spatiotemporal Regulation ◽  
Time Lapse Imaging

AbstractThe endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion. STIM1 also directly interacts with end binding protein 1 (EB1) at microtubule (MT) plus-ends and resembles comet-like structures during time-lapse imaging. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with pharmacological perturbation and a reconstitution approach, we revealed that EB1 binding constitutes a diffusion trap mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. EB1 binding delayed the translocation of STIM1 oligomers to ER-PM junctions and recaptured STIM1 to prevent excess SOCE and ER Ca2+ overload. Thus, the counterbalance of EB1 binding and PM targeting of STIM1 shapes the kinetics and amplitude of local SOCE in regions with growing MTs, and contributes to precise spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.SummarySTIM1 activates store-operated Ca2+ entry (SOCE) by translocating to endoplasmic reticulum-plasma membrane junctions. Chang et al. revealed that STIM1 localization and SOCE are regulated by a diffusion trap mechanism mediated by STIM1 binding to EB1 at growing microtubule ends.

scholarly journals Wolbachia endosymbionts subvert the endoplasmic reticulum to acquire host membranes without triggering ER stress

2018 ◽  
Author(s):  
Nour Fattouh ◽  
Chantal Cazevieille ◽  
Frédéric Landmann
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Host Cell ◽  
Pathogenic Bacteria ◽  
Secretory Pathway ◽  
Immune Surveillance ◽  
Lipid Synthesis ◽  
Time Lapse ◽  
Intracellular Bacteria ◽  
Arthropod Species

AbstractThe reproductive parasite Wolbachia are the most common endosymbionts on earth, present in a plethora of arthropod species. They have been introduced into mosquitos to successfully prevent the spread of vector-borne diseases, yet the strategies of host cell subversion underlying their obligate intracellular lifestyle remain to be explored in depth in order to gain insights into the mechanisms of pathogen-blocking. Like some other intracellular bacteria, Wolbachia reside in a host-derived vacuole in order to replicate and escape the immune surveillance. Using here the pathogen-blocking Wolbachia strain from Drosophila melanogaster, introduced into two different Drosophila cell lines, we show that Wolbachia subvert the endoplasmic reticulum to acquire their vacuolar membrane and colonize the host cell at high density. Wolbachia redistribute the endoplasmic reticulum to increase contact sites, and time lapse experiments reveal tight coupled dynamics suggesting important signalling events or nutrient uptake. They however do not affect the tubular or cisternal morphologies. A fraction of endoplasmic reticulum becomes clustered, allowing the endosymbionts to reside in between the endoplasmic reticulum and the Golgi apparatus, possibly modulating the traffic between these two organelles. Gene expression analyses and immunostaining studies suggest that Wolbachia achieve persistent infections at very high titers without triggering endoplasmic reticulum stress or enhanced ERAD-driven proteolysis, suggesting that amino acid salvage is achieved through modulation of other signalling pathways.Author summaryWolbachia are a genus of intracellular bacteria living in symbiosis with millions of arthropod species. They have the ability to block the transmission of arboviruses when introduced into mosquito vectors, by interfering with the cellular resources exploited by these viruses. Despite the biomedical interest of this symbiosis, little is known about the mechanisms by which Wolbachia survive and replicate in the host cell. We show here that the membrane composing the Wolbachia vacuole is acquired from the endoplasmic reticulum, a central organelle required for protein and lipid synthesis, and from which originates a vesicular trafficking toward the Golgi apparatus and the secretory pathway. Wolbachia modify the distribution of this organelle to increase their interactions with this source of membrane and likely of nutrients as well. In contrast to some intracellular pathogenic bacteria, the effect of Wolbachia on the cell homeostasis does not induce a stress on the endoplasmic reticulum. One of the consequences of such a stress would be an increased proteolysis used to relieve the cell from an excess of misfolded proteins. Incidentally, this shows that Wolbachia do not acquire amino acids from the host cell through this strategy.

Recycling of the yeast v-SNARE Sec22p involves COPI-proteins and the ER transmembrane proteins Ufe1p and Sec20p

1998 ◽  
Vol 111 (11) ◽  
pp. 1507-1520 ◽  
Author(s):  
W. Ballensiefen ◽  
D. Ossipov ◽  
H.D. Schmitt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Proteins ◽  
Retrograde Transport ◽  
Subcellular Fractionation ◽  
Hybrid Protein ◽  
Wild Type ◽  
Alpha Factor ◽  
Membrane Compartments

Vesicle-specific SNAP receptors (v-SNAREs) are believed to cycle between consecutive membrane compartments. The v-SNARE Sec22(Sly2)p mediates the targeting of vesicles between endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors involved in targeting of Sec22(Sly2)p, an alpha-factor-tagged Sec22 protein (Sec22-alpha) was employed. Only on reaching the late Golgi, can alpha-factor be cleaved from this hybrid protein by Kex2p, a protease localized in this compartment. In wild-type cells Kex2p-cleavage is observed only when Sec22-alpha is greatly overproduced. Immunofluorescence microscopy and subcellular fractionation studies showed that Sec22-alpha is returned to the ER from the late Golgi (Kex2p) compartment. When Sec22-alpha is expressed in wild-type cells at levels comparable to the quantities of endogenous Sec22p, very little of this protein is cleaved by Kex2p. Efficient cleavage, however, occurs in mutants defective in the retrograde transport of different ER-resident proteins indicating that Sec22-alpha rapidly reaches the late Golgi of these cells. These mutants (sec20-1, sec21-1, sec27-1 and ufe1-1) reveal Golgi structures when stained for Sec22-alpha and do not show the ER-immunofluorescence observed in wild-type cells. These results show consistently that Sec22p recycles from the Golgi back to the ER and that this recycling involves retrograde COPI vesicles.

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