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.

Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 407-422 ◽  
Author(s):  
E A Vallen ◽  
W Ho ◽  
M Winey ◽  
M D Rose
Keyword(s):  
Copy Number ◽  
Gene Dosage ◽  
Spindle Pole Body ◽  
Direct Interaction ◽  
Spindle Pole ◽  
High Copy Number ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Recessive Mutations ◽  
Organizing Center

Abstract KAR1 encodes an essential component of the yeast spindle pole body (SPB) that is required for karyogamy and SPB duplication. A temperature-sensitive mutation, kar1-delta 17, mapped to a region required for SPB duplication and for localization to the SPB. To identify interacting SPB proteins, we isolated 13 dominant mutations and 3 high copy number plasmids that suppressed the temperature sensitivity of kar1-delta 17. Eleven extragenic suppressor mutations mapped to two linkage groups, DSK1 and DSK2. The extragenic suppressors were specific for SPB duplication and did not suppress karyogamy-defective alleles. The major class, DSK1, consisted of mutations in CDC31. CDC31 is required for SPB duplication and encodes a calmodulin-like protein that is most closely related to caltractin/centrin, a protein associated with the Chlamydomonas basal body. The high copy number suppressor plasmids contained the wild-type CDC31 gene. One CDC31 suppressor allele conferred a temperature-sensitive defect in SPB duplication, which was counter-suppressed by recessive mutations in KAR1. In spite of the evidence for a direct interaction, the strongest CDC31 alleles, as well as both DSK2 alleles, suppressed a complete deletion of KAR1. However, the CDC31 alleles also made the cell supersensitive to KAR1 gene dosage, arguing against a simple bypass mechanism of suppression. We propose a model in which Kar1p helps localize Cdc31p to the SPB and that Cdc31p then initiates SPB duplication via interaction with a downstream effector.

scholarly journals Export from Pericentriolar Endocytic Recycling Compartment to Cell Surface Depends on Stable, Detyrosinated (Glu) Microtubules and Kinesin

2002 ◽  
Vol 13 (1) ◽  
pp. 96-109 ◽  
Author(s):  
Sharron X. Lin ◽  
Gregg G. Gundersen ◽  
Frederick R. Maxfield
Keyword(s):  
Elevated Temperature ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Endocytic Recycling ◽  
In Cells

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.

scholarly journals Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements

1979 ◽  
Vol 83 (3) ◽  
pp. 623-632 ◽  
Author(s):  
M Schliwa ◽  
U Euteneuer ◽  
W Herzog ◽  
K Weber
Keyword(s):  
Complete Removal ◽  
Cell System ◽  
The State ◽  
Microtubule Assembly ◽  
Microtubule Organizing Center ◽  
Functional Changes ◽  
Organizing Center ◽  
Pigment Distribution ◽  
And Function ◽  
In Cells

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

Effect of shear stress upon localization of the Golgi apparatus and microtubule organizing center in isolated cultured endothelial cells

1993 ◽  
Vol 104 (4) ◽  
pp. 1145-1153 ◽  
Author(s):  
D.E. Coan ◽  
A.R. Wechezak ◽  
R.F. Viggers ◽  
L.R. Sauvage
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Cell Migration ◽  
Golgi Apparatus ◽  
Flow Direction ◽  
Microtubule Organizing Center ◽  
Directed Cell Migration ◽  
Time Period ◽  
Significant Difference ◽  
Organizing Center

Despite substantial evidence to suggest that directed cell migration is dependent upon positioning of the Golgi apparatus (GA) and the microtubule organizing center (MTOC), some controversy exists about whether such a relationship is relevant to endothelial cells under flow. The present study was undertaken to provide an indepth investigation of the relationship between shear stress, GA/MTOC localization, cell migration and nuclear position. Bovine carotid endothelial cells were exposed to 22 or 88 dynes/cm2 for 0.5, 2, 8 or 24 h, and localization of their GA/MTOCs was determined relative to the direction of flow. In no-flow control specimens, (0, 0.5, 2, 8 and 24 h) there was no change in the equally distributed GA/MTOCs. In contrast, during the first 8 h at 88 dynes/cm2 and by 2 h at 22 dynes/cm2 there was a significant increase in the number of cells with GA/MTOCs localized upstream to flow direction. The effect was temporary, however, and by 24 h there was no significant difference between the no-flow, 22 and 88 dynes/cm2 specimens. Analysis of GA/MTOC localization with respect to the direction of cell migration determined that 72.5% of no-flow cells possessed GA/MTOCs localized to the sides of nuclei nearest the direction of migration. In contrast, 64% of the specimens shear stressed over the same time period had GA/MTOCs localized to the sides of nuclei opposite the direction of migration. These results suggest that positioning of the GA/MTOC in endothelial cells is not dependent completely upon the direction of migration.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Ultrastructural immunocytochemical localization of calmodulin in cultured cells.

1983 ◽  
Vol 31 (4) ◽  
pp. 445-461 ◽  
Author(s):  
M C Willingham ◽  
J Wehland ◽  
C B Klee ◽  
N D Richert ◽  
A V Rutherford ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Performic Acid ◽  
Microtubule Organizing Center ◽  
Interphase Cells ◽  
Organizing Center ◽  
Late Telophase ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules ◽  
And Function ◽  
Fibroblastic Cells

Using an antibody prepared against performic acid-treated calmodulin, we have localized calmodulin in cultured fibroblastic cells by immunofluorescence and immunoelectron microscopy. In interphase cells, calmodulin was found to be diffusely distributed throughout the cytosol. An increased amount of calmodulin was found in the pericentriolar region of interphase cells. No significant aggregation of calmodulin was found in association with microfilaments, peripheral cytoplasmic microtubules or clathrin-coated structures. Calmodulin was present in moderate amounts in microvilli, ruffles, and zeiotic blebs of the cell surface. In motitic cells, calmodulin was found concentrated in the pericentriolar region, and appeared to concentrate along radiating spindle microtubules proximal to the centrioles. Redistribution of calmodulin was seen between early and late telophase, in which the pericentriolar pattern of calmodulin in early telophase shifted to an aggregation on the intercellular bridge, with a large part of the midbody portion of the bridge being devoid of calmodulin. These results show that calmodulin is distributed throughout the cytosol, but is markedly concentrated in the region of the microtubule organizing center in interphase cells, as well as in elements of the mitotic spindle apparatus. This distribution suggests that calmodulin has a regulatory role in the organization and function of microtubules during interphase, as well as during mitosis.

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.

A protein related to brain microtubule-associated protein MAP1B is a component of the mammalian centrosome

1994 ◽  
Vol 107 (2) ◽  
pp. 601-611 ◽  
Author(s):  
J.E. Dominguez ◽  
B. Buendia ◽  
C. Lopez-Otin ◽  
C. Antony ◽  
E. Karsenti ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Microtubule Organizing Center ◽  
Microtubule Associated Proteins ◽  
Organizing Center ◽  
Associated Proteins ◽  
Pericentriolar Material ◽  
Peptide Maps

The centrosome is the main microtubule organizing center of mammalian cells. Structurally, it is composed of a pair of centrioles surrounded by a fibro-granular material (the pericentriolar material) from which microtubules are nucleated. However, the nature of centrosomal molecules involved in microtubules nucleation is still obscure. Since brain microtubule-associated proteins (MAPs) lower the critical tubulin concentration required for microtubule nucleation in tubulin solution in vitro, we have examined their possible association with centrosomes. By immunofluorescence, monoclonal and polyclonal antibodies raised against MAP1B stain the centrosome in cultured cells as well as purified centrosomes, whereas antibodies raised against MAP2 give a completely negative reaction. The MAP1B-related antigen is localized to the pericentriolar material as revealed by immunoelectron microscopy. In preparations of purified centrosomes analyzed on poly-acrylamide gels, a protein that migrates as brain MAP1B is present. After blotting on nitrocellulose, it is decorated by anti-MAP1B antibodies and the amino acid sequence of proteolytic fragments of this protein is similar to brain MAP1B. Moreover, brain MAP1B and its centrosomal counterpart share the same phosphorylation features and have similar peptide maps. These data strongly suggest that a protein homologue to MAP1B is present in centrosomes and it is a good candidate for being involved in the nucleating activity of the pericentriolar material.

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