scholarly journals Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex.

1992 ◽  
Vol 118 (6) ◽  
pp. 1333-1345 ◽  
Author(s):  
I Corthésy-Theulaz ◽  
A Pauloin ◽  
S R Pfeffer
Keyword(s):  
Golgi Complex ◽  
Atp Hydrolysis ◽  
Intact Cell ◽  
Cytoplasmic Dynein ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Intact Cells ◽  
Organizing Center ◽  
Cell Cytosol ◽  
Stable Association

The localization of the Golgi complex depends upon the integrity of the microtubule apparatus. At interphase, the Golgi has a restricted pericentriolar localization. During mitosis, it fragments into small vesicles that are dispersed throughout the cytoplasm until telophase, when they again coalesce near the centrosome. These observations have suggested that the Golgi complex utilizes a dynein-like motor to mediate its transport from the cell periphery towards the minus ends of microtubules, located at the centrosome. We utilized semi-intact cells to study the interaction of the Golgi complex with the microtubule apparatus. We show here that Golgi complexes can enter semi-intact cells and associate stably with cytoplasmic constituents. Stable association, termed here "Golgi capture," requires ATP hydrolysis and intact microtubules, and occurs maximally at physiological temperature in the presence of added cytosolic proteins. Once translocated into the semi-intact cell cytoplasm, exogenous Golgi complexes display a distribution similar to endogenous Golgi complexes, near the microtubule-organizing center. The process of Golgi capture requires cytoplasmic tubulin, and is abolished if cytoplasmic dynein is immunodepleted from the cytosol. Cytoplasmic dynein, prepared from CHO cell cytosol, restores Golgi capture activity to reactions carried out with dynein immuno-depleted cytosol. These results indicate that cytoplasmic dynein can interact with isolated Golgi complexes, and participate in their accumulation near the centrosomes of semi-intact, recipient cells. Thus, cytoplasmic dynein appears to play a role in determining the subcellular localization of the Golgi complex.

Calmodulin and the contractile vacuole complex in mitotic cells of Dictyostelium discoideum

1993 ◽  
Vol 104 (4) ◽  
pp. 1119-1127 ◽  
Author(s):  
Q. Zhu ◽  
T. Liu ◽  
M. Clarke
Keyword(s):  
Dictyostelium Discoideum ◽  
Golgi Complex ◽  
Contractile Vacuole ◽  
Microtubule Organizing Center ◽  
Golgi Membranes ◽  
Golgi Staining ◽  
Interphase Cells ◽  
Organizing Center ◽  
Tubulin Antibodies ◽  
Mitotic Cells

In amoebae of the eukaryotic microorganism Dictyostelium discoideum, calmodulin is greatly enriched on membranes of the contractile vacuole complex, an osmoregulatory organelle. Antibodies specific for Dictyostelium calmodulin were used in the present study to immunolocalize the contractile vacuole complex in relation to the Golgi complex (detected with wheat germ agglutinin) and the microtubule organizing center (MTOC, detected with anti-tubulin antibodies). Cells were examined throughout the cell cycle. Double-staining experiments indicated that the contractile vacuole complex extended to the MTOC in interphase cells, usually, but not always, overlapping the Golgi complex. In metaphase and anaphase cells, the Golgi staining became diffuse, suggesting dispersal of Golgi membranes. In the same mitotic cells, anti-calmodulin antibodies labeled numerous small cortical vacuoles, indicating that the contractile vacuole complex had also become dispersed. When living mitotic cells were examined, the small cortical vacuoles were seen to be active, implying that all parts of the Dictyostelium contractile vacuole complex possess the ability to accumulate fluid and fuse with the plasma membrane. In contrast to observations reported for other types of cells, anti-calmodulin antibodies did not label the mitotic spindle in Dictyostelium. Despite this difference in localization, it is possible that vacuole-associated calmodulin in Dictyostelium cells and spindle-associated calmodulin in larger eukaryotic cells might perform a similar function, namely, regulating calcium levels.

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

1997 ◽  
Vol 45 (10) ◽  
pp. 1351-1363 ◽  
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.

scholarly journals Radial F-actin Organization During Early Neuronal Development

2018 ◽  
Author(s):  
Durga Praveen Meka ◽  
Robin Scharrenberg ◽  
Bing Zhao ◽  
Theresa König ◽  
Irina Schaefer ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Super Resolution ◽  
Actin Dynamics ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Actin Organization ◽  
Organizing Center ◽  
Super Resolution Microscopy ◽  
Radial Organization

AbstractThe centrosome is thought to be the major neuronal microtubule-organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin-organizing center (Farina et al., 2016), raising the possibility that neuronal development may, in addition, require a centrosome-based actin radial organization. Here we report, using super-resolution microscopy and live-cell imaging, F-actin organization around the centrosome with dynamic F-actin aster-like structures with F-actin fibers extending and retracting actively. Photoconversion/photoactivation experiments and molecular manipulations of F-actin stability reveal a robust flux of somatic F-actin towards the cell periphery. Finally, we show that somatic F-actin intermingles with centrosomal PCM-1 satellites. Knockdown of PCM-1 and disruption of centrosomal activity not only affect F-actin dynamics near the centrosome but also in distal growth cones. Collectively the data show a radial F-actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers; hence sustaining initial neuronal development.

scholarly journals Visualization of the intracellular behavior of HIV in living cells

2002 ◽  
Vol 159 (3) ◽  
pp. 441-452 ◽  
Author(s):  
David McDonald ◽  
Marie A. Vodicka ◽  
Ginger Lucero ◽  
Tatyana M. Svitkina ◽  
Gary G. Borisy ◽  
...  
Keyword(s):  
Life Cycle ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Microtubule Organizing Center ◽  
Microtubule Network ◽  
Organizing Center ◽  
Infected Cells ◽  
Resolution Imaging ◽  
Transcription Complexes ◽  
Hiv 1

To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to determine the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resolution imaging of microtubule-associated structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.

scholarly journals Coupled ER to Golgi Transport Reconstituted with Purified Cytosolic Proteins

1997 ◽  
Vol 139 (5) ◽  
pp. 1097-1108 ◽  
Author(s):  
Charles Barlowe
Keyword(s):  
Golgi Complex ◽  
Intact Cell ◽  
Vesicle Fusion ◽  
Intact Cells ◽  
Vesicle Budding ◽  
Vesicle Docking ◽  
Golgi Membranes ◽  
Golgi Transport ◽  
A Cell ◽  
A Subunit

A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-α-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-α-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.

scholarly journals Kinetics and intermediates of marginal band reformation: evidence for peripheral determinants of microtubule organization.

1984 ◽  
Vol 99 (1) ◽  
pp. 70s-75s ◽  
Author(s):  
M Miller ◽  
F Solomon
Keyword(s):  
Cell Types ◽  
Microtubule Assembly ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Microtubule Organization ◽  
Mature Erythrocyte ◽  
The Reformation ◽  
Marginal Band ◽  
Organizing Center

The microtubules of the mature erythrocyte of the chicken are confined to a band at the periphery. Whole-mount electron microscopy after extraction reveals that the number of microtubules in each cell is almost the same. All the microtubules can be depolymerized by incubation in the cold, and the marginal band can be quantitatively and qualitatively reformed by return to 39 degrees C. These properties allow the reformation of the marginal band to be treated as an in vivo microtubule assembly reaction. The kinetics of this reaction and the intermediates detected during reformation suggest a mechanism of microtubule organization that is distinct from that observed in other cell types. Apparently only one or two growing microtubule ends are available for assembly--assembly is only detected at the cell periphery, even at early times--and there is no evidence of the participation of a microtubule-organizing center.

scholarly journals In AtT20 and HeLa cells brefeldin A induces the fusion of tubular endosomes and changes their distribution and some of their endocytic properties.

1992 ◽  
Vol 118 (4) ◽  
pp. 813-830 ◽  
Author(s):  
J Tooze ◽  
M Hollinshead
Keyword(s):  
Hela Cells ◽  
Brefeldin A ◽  
Cell Types ◽  
Morphological Evidence ◽  
Detectable Effect ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Open Network ◽  
Early Endosomes ◽  
Organizing Center

We have studied the effects of brefeldin A (BFA) on the tubular endosomes in AtT20 and HeLa cells (Tooze, J., and M. Hollinshead. 1991. J. Cell Biol. 115:635-653) by electron microscopy of cells labeled with three endocytic tracers, HRP, BSA-gold, and transferrin conjugated to HRP, and by immunofluorescence microscopy. For the latter we used antibodies specific for transferrin receptor, and, in the case of AtT20 cells, also antibodies specific for synaptophysin. In HeLa cells BFA at concentrations ranging from 1 micrograms to 10 micrograms/ml causes the dispersed patches of network of preexisting tubular early endosomes to be incorporated within 5 min into tubules approximately 50 nm in diameter but up to 40-50 microns long. These long, straight tubular endosomes are aligned along microtubules; they branch relatively infrequently to form an open network or reticulum extending from the cell periphery to the microtubule organizing center (MTOC). As the incubation with BFA is prolonged beyond 5 min, a steady state is reached in which many tubules are located in a dense network enclosing the centrioles, with branches extending in a more open network to the periphery. This effect of BFA, which is fully reversed within 15-30 min of washing out, is inhibited by pre-incubating the cells with sodium azide and 2-deoxy-D-glucose. In AtT20 cells BFA at 5 micrograms/ml or above causes the same sorts of changes, preexisting tubular endosomes are recruited into a more continuous endosomal network, and there is a massive accumulation of this network around the MTOC. Maintenance of the BFA-induced endosomal reticulum in both cell types is dependent upon the integrity of microtubules. In AtT20 cells BFA at 1 microgram/ml has no detectable effect on the early endosomal system but the Golgi stacks are converted to clusters of tubules and vesicles that remain in the region of the MTOC during prolonged incubations. Therefore, the Golgi apparatus in these cells is more sensitive to BFA than the early endosomes. The morphological evidence suggests that all the tubular early endosomes in BFA-treated HeLa and AtT20 cells are linked together in a single reticulum. Consistent with this, incubations as short as 1-3 min with 10 or 20 mg/ml HRP in the medium result in the entire endosomal reticulum in most of the BFA-treated cells being filled with HRP reaction product.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Microtubule-organizing center formation at telomeres induces meiotic telomere clustering

2013 ◽  
Vol 200 (4) ◽  
pp. 385-395 ◽  
Author(s):  
Masashi Yoshida ◽  
Satoshi Katsuyama ◽  
Kazuki Tateho ◽  
Hiroto Nakamura ◽  
Junpei Miyoshi ◽  
...  
Keyword(s):  
Chromosome Pairing ◽  
Light Chain ◽  
Homologous Chromosome ◽  
Cytoplasmic Dynein ◽  
Dynein Light Chain ◽  
Microtubule Organizing Center ◽  
Homologous Chromosome Pairing ◽  
Microtubule Motors ◽  
Microtubule Motor ◽  
Organizing Center

During meiosis, telomeres cluster and promote homologous chromosome pairing. Telomere clustering requires the interaction of telomeres with the nuclear membrane proteins SUN (Sad1/UNC-84) and KASH (Klarsicht/ANC-1/Syne homology). The mechanism by which telomeres gather remains elusive. In this paper, we show that telomere clustering in fission yeast depends on microtubules and the microtubule motors, cytoplasmic dynein, and kinesins. Furthermore, the γ-tubulin complex (γ-TuC) is recruited to SUN- and KASH-localized telomeres to form a novel microtubule-organizing center that we termed the “telocentrosome.” Telocentrosome formation depends on the γ-TuC regulator Mto1 and on the KASH protein Kms1, and depletion of either Mto1 or Kms1 caused severe telomere clustering defects. In addition, the dynein light chain (DLC) contributes to telocentrosome formation, and simultaneous depletion of DLC and dynein also caused severe clustering defects. Thus, the telocentrosome is essential for telomere clustering. We propose that telomere-localized SUN and KASH induce telocentrosome formation and that subsequent microtubule motor-dependent aggregation of telocentrosomes via the telocentrosome-nucleated microtubules causes telomere clustering.

scholarly journals Associations of elements of the Golgi apparatus with microtubules.

1984 ◽  
Vol 99 (3) ◽  
pp. 1092-1100 ◽  
Author(s):  
A A Rogalski ◽  
S J Singer
Keyword(s):  
Golgi Apparatus ◽  
Cultured Cells ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
Free Microtubules ◽  
Vesicular Stomatitis ◽  
In Cells

The intracellular spatial relationships between elements of the Golgi apparatus (GA) and microtubules in interphase cells have been explored by double immunofluorescence microscopy. By using cultured cells infected with the temperature-sensitive Orsay-45 mutant of vesicular stomatitis virus and a temperature shift-down protocol, we visualized functional elements of the GA by immunolabeling of the G protein of the virus that was arrested in the GA during its intracellular passage to the plasma membrane 13 min after the temperature shift-down. Complete disassembly of the cytoplasmic microtubules by nocodazole at the nonpermissive temperature before the temperature shift led to the dispersal of the GA elements, from their normal compact perinuclear configuration close to the microtubule-organizing center (MTOC) into the cell periphery. Washout of the nocodazole that led to the reassembly of the microtubules from the MTOC also led to the recompaction of the GA elements to their normal configuration. During this recompaction process, GA elements were seen in close lateral apposition to microtubules. In cells treated with nocodazole followed by taxol, an MTOC developed, but most of the microtubules were free of the MTOC and were assembled into bundles in the cell periphery. Under these circumstances, the GA elements that had been dispersed into the cell periphery by the nocodazole treatment remained dispersed despite the presence of an MTOC. In cells treated directly with taxol, free microtubules were seen in the cytoplasm in widely different, bundled configurations from one cell to another, but, in each case, elements of the GA appeared to be associated with one of the two end regions of the microtubule bundles, and to be uncorrelated with the locations of the vimentin intermediate filaments in these cells. These results are interpreted to suggest two types of associations of elements of the GA with microtubules: one lateral, and the other (more stable) end-on. The end-on association is suggested to involve the minus-end regions of microtubules, and it is proposed that this accounts for the GA-MTOC association in normal cells.

scholarly journals Severe Tryptophan Starvation Blocks Onset of Conventional Persistence and Reduces Reactivation of Chlamydia trachomatis

2007 ◽  
Vol 75 (11) ◽  
pp. 5105-5117 ◽  
Author(s):  
Ralf M. Leonhardt ◽  
Seung-Joon Lee ◽  
Paula B. Kavathas ◽  
Peter Cresswell
Keyword(s):  
Chlamydia Trachomatis ◽  
Parasitophorous Vacuole ◽  
Bacterial Pathogen ◽  
Gamma Interferon ◽  
Human Cells ◽  
Tryptophan Depletion ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
Innate Immune Mechanism

ABSTRACT The intracellular survival of the bacterial pathogen Chlamydia trachomatis depends on protein synthesis by the microbe soon after internalization. Pharmacologic inhibition of bacterial translation inhibits early trafficking of the parasitophorous vacuole (inclusion) to the microtubule-organizing center (MTOC) and promotes its fusion with lysosomes, which is normally blocked by Chlamydia. Depletion of cellular tryptophan pools by gamma interferon-inducible indoleamine-2,3-dioxygenase (IDO) is believed to be the major innate immune mechanism controlling C. trachomatis infection in human cells, an action to which the bacteria can respond by converting into a nonreplicating but highly reactivatable persistent state. However, whether severe IDO-mediated tryptophan starvation can be sufficient to fully arrest the chlamydial life cycle and thereby counteract the onset of persistence is unknown. Here we demonstrate that at low exogenous tryptophan concentrations a substantial fraction of C. trachomatis bacteria fail to traffic to the MTOC or to switch into the conventional persistent state in gamma interferon-induced human cells. The organisms stay scattered in the cell periphery, do not retain infectivity, and display only low transcriptional activity. Importantly, the rate at which these aberrant Chlamydia bacteria become reactivated upon replenishment of cellular tryptophan pools is substantially lower. Thus, severe tryptophan depletion in cells with high IDO activity affects chlamydial development more rigorously than previously described.

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