scholarly journals Partitioning of the Golgi Apparatus during Mitosis in Living HeLa Cells

1997 ◽  
Vol 137 (6) ◽  
pp. 1211-1228 ◽  
Author(s):  
David T. Shima ◽  
Kasturi Haldar ◽  
Rainer Pepperkok ◽  
Rose Watson ◽  
Graham Warren
Keyword(s):  
Golgi Apparatus ◽  
Hela Cells ◽  
Direct Evidence ◽  
Fluorescent Protein ◽  
Immunofluorescence Microscopy ◽  
Critical Feature ◽  
Directed Movement ◽  
Late Telophase ◽  
Green Fluorescent ◽  
Retention Signal

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I–GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.

Important role for the V-type H+-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor

2004 ◽  
Vol 286 (5) ◽  
pp. E704-E710 ◽  
Author(s):  
Hesham A. W. Tawfeek ◽  
Abdul B. Abou-Samra
Keyword(s):  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Receptor Recycling ◽  
Bafilomycin A1 ◽  
Coated Pits ◽  
Concanamycin A ◽  
Hydrogen Atpase ◽  
Green Fluorescent ◽  
Pthrp Receptor

Our previous studies demonstrated that a green fluorescent protein-tagged parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor stably expressed in LLCPK-1 cells undergoes agonist-dependent internalization into clathrin-coated pits. The subcellular localization of the internalized PTH/PTHrP receptor is not known. In the present study, we explored the intracellular pathways of the internalized PTH/PTHrP receptor. Using immunofluorescence and confocal microscopy, we show that the internalized receptors localize at a juxtanuclear compartment identified as the Golgi apparatus. The receptors do not colocalize with lysosomes. Furthermore, whereas the internalized receptors exhibit rapid recycling, treatment with proton pump inhibitors (bafilomycin-A1 and concanamycin A) or brefeldin A, Golgi disrupting agents, reduces PTH/PTHrP receptor recycling. Together, these data indicate an important role for the vacuolar-type hydrogen-ATPase and the Golgi apparatus in postendocytic PTH/PTHrP receptor recovery.

scholarly journals Internalization of Monomeric Lipopolysaccharide Occurs after Transfer Out of Cell Surface Cd14

1999 ◽  
Vol 190 (4) ◽  
pp. 509-522 ◽  
Author(s):  
Thierry Vasselon ◽  
Eric Hailman ◽  
Rolf Thieringer ◽  
Patricia A. Detmers
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Soluble Cd14 ◽  
Stable Transfectants ◽  
Intracellular Vesicles ◽  
Green Fluorescent

Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14–EGFP) was used to follow trafficking of mCD14 and BODIPY–LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex–like structure. mCD14–EGFP was functional in mediating binding of and responses to LPS. BODIPY–LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14–EGFP on the cell surface. However, within 5–10 min, the BODIPY–LPS distributed to intracellular vesicles that did not contain mCD14–EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

scholarly journals A C-Terminal Domain Targets the Pseudomonas aeruginosa Cytotoxin ExoU to the Plasma Membrane of Host Cells

2006 ◽  
Vol 74 (5) ◽  
pp. 2552-2561 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Jeffrey L. Veesenmeyer ◽  
Kathryn T. Bieging ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Type Iii ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.

scholarly journals Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells

2002 ◽  
Vol 158 (5) ◽  
pp. 841-847 ◽  
Author(s):  
Marko J. Kallio ◽  
Victoria A. Beardmore ◽  
Jasminder Weinstein ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Anaphase Promoting Complex ◽  
Late Prophase ◽  
Checkpoint Signaling ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Late Telophase ◽  
Green Fluorescent

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20–GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.

scholarly journals Golgi-bound Rab34 Is a Novel Member of the Secretory Pathway

2007 ◽  
Vol 18 (12) ◽  
pp. 4762-4771 ◽  
Author(s):  
Neil M. Goldenberg ◽  
Sergio Grinstein ◽  
Mel Silverman
Keyword(s):  
Hela Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Golgi Stack ◽  
Green Fluorescent ◽  
Intracellular Vesicle Transport

Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.

A novel yellowish-green fluorescent protein from the marine copepod, Chiridius poppei, and its use as a reporter protein in HeLa cells

Gene ◽  
2006 ◽  
Vol 372 ◽  
pp. 18-25 ◽  
Author(s):  
Hiromi Masuda ◽  
Yasuhiro Takenaka ◽  
Atsushi Yamaguchi ◽  
Satoshi Nishikawa ◽  
Hiroshi Mizuno
Keyword(s):  
Green Fluorescent Protein ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
Marine Copepod ◽  
Yellowish Green ◽  
Green Fluorescent

scholarly journals Endoplasmic Reticulum Dynamics, Inheritance, and Cytoskeletal Interactions in Budding Yeast

2002 ◽  
Vol 13 (3) ◽  
pp. 854-865 ◽  
Author(s):  
K. L. Fehrenbacher ◽  
D. Davis ◽  
M. Wu ◽  
I. Boldogh ◽  
Liza A. Pon
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Filamentous Actin ◽  
Protein Fusion ◽  
Directed Movement ◽  
Apical Bud ◽  
M Phase ◽  
Green Fluorescent

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae consists of a reticulum underlying the plasma membrane (cortical ER) and ER associated with the nuclear envelope (nuclear ER). We used a Sec63p-green fluorescent protein fusion protein to study motility events associated with inheritance of cortical ER and nuclear ER in living yeast cells. During M phase before nuclear migration, we observed thick, apparently rigid tubular extensions emanating from the nuclear ER that elongate, undergo sweeping motions along the cell cortex, and shorten. Two findings support a role for microtubules in this process. First, extension of tubular structures from the nuclear ER is inhibited by destabilization of microtubules. Second, astral microtubules, structures that undergo similar patterns of extension, cortical surveillance and retraction, colocalize with nuclear ER extensions. During S and G2 phases of the cell cycle, we observed anchorage of the cortical ER at the site of bud emergence and apical bud growth. Thin tubules of the ER that extend from the anchored cortical ER display undulating, apparently random movement and move into the bud as it grows. Finally, we found that cortical ER morphology is sensitive to a filamentous actin–destabilizing drug, latrunculin-A, and to mutations in the actin-encoding ACT1 gene. Our observations support 1) different mechanisms and cytoskeletal mediators for the inheritance of nuclear and cortical ER elements and 2) a mechanism for cortical ER inheritance that is cytoskeleton dependent but relies on anchorage, not directed movement.

scholarly journals Monitoring Nuclear Transport in HeLa Cells Using the Green Fluorescent Protein

BioTechniques ◽  
1996 ◽  
Vol 21 (1) ◽  
pp. 62-63 ◽  
Author(s):  
Shivani Chatterjee ◽  
Ursula Stochaj
Keyword(s):  
Green Fluorescent Protein ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Nuclear Transport ◽  
Green Fluorescent

scholarly journals The Rab6 Effector Bicaudal D1 Associates with Chlamydia trachomatis Inclusions in a Biovar-Specific Manner

2006 ◽  
Vol 75 (2) ◽  
pp. 781-791 ◽  
Author(s):  
A. R. Moorhead ◽  
K. A. Rzomp ◽  
M. A. Scidmore
Keyword(s):  
Gene Expression ◽  
Chlamydia Trachomatis ◽  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Rab Gtpases ◽  
Specific Manner ◽  
Guanine Nucleotide Binding ◽  
Obligate Intracellular Bacteria ◽  
Green Fluorescent

ABSTRACT Chlamydia species are obligate intracellular bacteria that replicate within a membrane-bound vacuole, the inclusion, which is trafficked to the peri-Golgi region by processes that are dependent on early chlamydial gene expression. Although neither the host nor the chlamydial proteins that regulate the intracellular trafficking have been clearly defined, several enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, including Rab6, are recruited to Chlamydia trachomatis inclusions. To further characterize the association of Rab6 with C. trachomatis inclusions, we examined the intracellular localization of guanine nucleotide-binding mutants of Rab6 and demonstrated that only active GTP-bound and not inactive GDP-bound EGFP-Rab6 mutants were recruited to the inclusion, suggesting that EGFP-Rab6 interacts with the inclusion via a host Rab6 effector or a chlamydial protein that mimics a Rab6 effector. Using EGFP-tagged fusion proteins, we also demonstrated that the Rab6 effector Bicaudal D1 (BICD1) localized to C. trachomatis inclusions in a biovar-specific manner. In addition, we demonstrated that EGFP-Rab6 and its effector EGFP-BICD1 are recruited to the inclusion in a microtubule- and Golgi apparatus-independent but chlamydial gene expression-dependent mechanism. Finally, in contrast to the Rab6-dependent Golgi apparatus localization of endogenous BICD1, EGFP-BICD1 was recruited to the inclusion by a Rab6-independent mechanism. Collectively, these data demonstrate that neither Rab6 nor BICD1 is trafficked to the inclusion via a Golgi apparatus-localized intermediate, suggesting that each protein is trafficked to the C. trachomatis serovar L2 inclusion by a unique, but as-yet-undefined, mechanism.

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