scholarly journals The Golgi apparatus remains associated with microtubule organizing centers during myogenesis.

1985 ◽  
Vol 101 (2) ◽  
pp. 630-638 ◽  
Author(s):  
A M Tassin ◽  
M Paintrand ◽  
E G Berger ◽  
M Bornens
Keyword(s):  
Golgi Apparatus ◽  
Nuclear Periphery ◽  
Spatial Relationship ◽  
Cell Types ◽  
Microtubule Depolymerization ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Microtubule Disruption ◽  
Tight Association

In vitro myogenesis involves a dramatic reorganization of the microtubular network, characterized principally by the relocalization of microtubule nucleating sites at the surface of the nuclei in myotubes, in marked contrast with the classical pericentriolar localization observed in myoblasts (Tassin, A. M., B. Maro, and M. Bornens, 1985, J. Cell Biol., 100:35-46). Since a spatial relationship between the Golgi apparatus and the centrosome is observed in most animal cells, we have decided to follow the fate of the Golgi apparatus during myogenesis by an immunocytochemical approach, using wheat germ agglutinin and an affinity-purified anti-galactosyltransferase. We show that Golgi apparatus in myotubes displays a perinuclear distribution which is strikingly different from the polarized juxtanuclear organization observed in myoblasts. As a result, the Golgi apparatus in myotubes is situated close to the microtubule organizing center (MTOC), the cis-side being situated at a fixed distance from the nuclear envelope, a situation which suggests the existence of a structural association between the Golgi apparatus and the nuclear periphery. This is supported by experiments of microtubule depolymerization by nocodazole, in which a minimal effect was observed on Golgi apparatus localization in myotubes in contrast with the dramatic scattering observed in myoblasts. In both cell types, electron microscopy reveals that microtubule disruption generates individual dictyosomes; this suggests that the connecting structures between dictyosomes are principally affected. This structural dependency of the Golgi apparatus upon microtubules is not apparently accompanied by a reverse dependency of MTOC structure or function upon Golgi apparatus activity. Golgi apparatus modification by monensin, as effective in myotubes as in myoblasts, is without apparent effect on MTOC localization or activity and on microtubule stability. The main result of our study is to show that in a cell type where the MTOC is dissociated from centrioles and where antero-posterior polarity has disappeared, the association between the Golgi apparatus and the MTOC is maintained. The significance of such a tight association is discussed.

scholarly journals The role of Aspergillus nidulans polo-like kinase PlkA in microtubule-organizing center control

2021 ◽  
Author(s):  
Xiaolei Gao ◽  
Saturnino Herrero ◽  
Valentin Wernet ◽  
Sylvia Erhardt ◽  
Oliver Valerius ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Cell Types ◽  
Spindle Pole ◽  
Receptor Protein ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Spindle Pole Bodies ◽  
Organizing Center ◽  
Ring Complex ◽  
Core Components

Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) were described in many cell types. Functional analogs of centrosomes in fungi are the spindle pole bodies (SPBs). In Aspergillus nidulans additional MTOCs were discovered at septa (sMTOC). Although the core components are conserved in both MTOCs, their composition and organization are different and dynamic. Here, we show that the polo-like kinase PlkA binds the γ-tubulin ring complex (γ-TuRC) receptor protein ApsB and contributes to targeting ApsB to both MTOCs. PlkA coordinates SPB outer plaque with sMTOC activities. PlkA kinase activity was required for astral MT formation involving ApsB recruitment. PlkA also interacted with the γ-TuRC inner plaque receptor protein PcpA. Mitosis was delayed without PlkA, and the PlkA protein was required for proper mitotic spindle morphology, although this function was independent of its catalytic activity. Our results suggest polo-like kinase as a regulator of MTOC activities and as a scaffolding unit through interaction with γ-tubulin ring complex receptors.

Association of kinesin with the Golgi apparatus in rat hepatocytes

1994 ◽  
Vol 107 (9) ◽  
pp. 2417-2426 ◽  
Author(s):  
D.L. Marks ◽  
J.M. Larkin ◽  
M.A. McNiven
Keyword(s):  
Golgi Apparatus ◽  
Immunofluorescence Microscopy ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Immunoblot Analysis ◽  
Subcellular Fractionation ◽  
Rat Hepatocyte ◽  
Membranous Structure ◽  
Microtubule Disruption ◽  
Motor Enzymes

The Golgi apparatus is a dynamic membranous structure, which has been observed to alter its location and morphology during the cell cycle and after microtubule disruption. These dynamics are believed to be supported by a close structural interaction of the Golgi with the microtubule cytoskeleton and associated motor enzymes. One microtubule-dependent motor enzyme, kinesin, has been implicated in Golgi movement and function although direct evidence supporting this interaction is lacking. In this study, we utilized two well-characterized kinesin antibodies in conjunction with subcellular fractionation techniques, immunoblot analysis and immunofluorescence microscopy to conduct a detailed study on the association of kinesin with the Golgi and other membranous organelles in a polarized epithelial cell, the primary rat hepatocyte. We found that kinesin represents approximately 0.3% of total protein in rat liver homogenates, with approximately 30% membrane-associated and the remainder in the cytosol. Among membrane fractions, kinesin was concentrated markedly in Golgi-enriched fractions, which were prepared using two independent techniques. Kinesin was also abundant in fractions enriched in transcytotic carriers and secretory vesicles, with lower levels detected on fractions enriched in endosomes, endoplasmic reticulum, lysosomes and mitochondria. Immunofluorescence microscopy showed that kinesin is concentrated on Golgi-like structures in both primary cultured hepatocytes and rat hepatocyte-derived clone 9 cells. Double-label immunofluorescence demonstrated that kinesin staining colocalizes with the Golgi marker, alpha-mannosidase II, in both cell types. These results provide compelling evidence showing that kinesin is associated with the Golgi complex in cells and implicate this motor enzyme in Golgi structure, function and dynamics.

scholarly journals Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor.

1993 ◽  
Vol 120 (2) ◽  
pp. 325-338 ◽  
Author(s):  
B L Tang ◽  
S H Wong ◽  
X L Qi ◽  
S H Low ◽  
W Hong
Keyword(s):  
Golgi Apparatus ◽  
Brefeldin A ◽  
Cell Types ◽  
In Vitro Translation ◽  
Tubular Structures ◽  
Human Sequence ◽  
Intermediate Compartment ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

We have isolated a cDNA clone (mERD2) for the mammalian (bovine) homologue of the yeast ERD2 gene, which codes for the yeast HDEL receptor. The deduced amino acid sequence bears extensive homology to its yeast counterpart and is almost identical to a previously described human sequence. The sequence predicts a very hydrophobic protein with multiple membrane spanning domains, as confirmed by analysis of the in vitro translation product. The protein encoded by mERD2 (p23) has widespread occurrence, being present in all the cell types examined. p23 was localized to the cis-side of the Golgi apparatus and to a spotty intermediate compartment which mediates ER to Golgi transport. A majority of the intracellular staining could be accumulated in the intermediate compartment by a low temperature (15 degrees C) or brefeldin A. During recovery from these treatments, the spotty intermediate compartment staining of p23 was shifted to the perinuclear staining of the Golgi apparatus and tubular structures marked by p23 were observed. These tubular structures may serve to mediate transport between the intermediate compartment and the Golgi apparatus.

SOME BIOLOGICAL PROBLEMS IN CANCER BIOCHEMISTRY

1960 ◽  
Vol 38 (4) ◽  
pp. 425-433 ◽  
Author(s):  
Louis Siminovitch ◽  
Arthur Axelrad
Keyword(s):  
Normal Tissue ◽  
Normal Growth ◽  
Cell Types ◽  
Biological Properties ◽  
Cellular Level ◽  
Animal Cells ◽  
Biochemical Basis ◽  
Normal Tissues ◽  
Ascites Tumors

Understanding of the cancer process in chemical terms has been seriously hampered by the difficulty of interpreting results of biochemical comparisons between masses of tumor and of normal tissue. Normal tissue consists of a variety of cell types and tumors may originate from one or more of these. As whole masses, therefore, normal tissues cannot serve as adequate controls for experiments on any single tumor. Tumor cell populations, even those arising from a single cell type, are themselves cytogenetically and continually undergoing changes during growth (progression). It is thus difficult, if not impossible, to separate the relevant from the irrelevant biochemical features of malignancy.Progress in this field requires means of dealing with the problem of biological heterogeneity. Several biochemical approaches that are free from the hazards of heterogeneity and which have already yielded valuable results, or appear promising, are indicated. These include: (1) The use of ascites tumors for studying the biochemical machinery of cells. No normal tissue exists, however, that could serve as satisfactory control. (2) Biochemical comparisons between pairs of tumor lines which differ by only one inherited characteristic of malignancy. These might reveal a biochemical basis for the biological properties of tumor cells with different degrees of malignancy. (3) Elucidation of normal growth-controlling mechanisms between cells, e.g. action of hormones at the cellular level, and within cells, e.g. mechanism of feed-back control of enzymes and metabolic pathways. (4) Further research into the biochemistry of plant tumor induction in vitro. Here biochemical changes associated with inherited changes leading to nutritional autonomy and uncontrolled growth have already been demonstrated. (5) Studies on the biochemical events during induction of malignancy by viruses in clonal cultures of animal cells in vitro. These could serve as useful models of the whole process of carcinogenesis.

scholarly journals Aphid-Symbiotic Bacteria Cultured in Insect Cell Lines

2005 ◽  
Vol 71 (8) ◽  
pp. 4833-4839 ◽  
Author(s):  
A. C. Darby ◽  
S. M. Chandler ◽  
S. C. Welburn ◽  
A. E. Douglas
Keyword(s):  
Cell Lines ◽  
Insect Cell ◽  
Cell Types ◽  
Symbiotic Bacteria ◽  
Rrna Gene ◽  
Animal Cells ◽  
Genomic Studies ◽  
Insect Cell Lines ◽  
Cultivation In Vitro

ABSTRACT The cells and tissues of many aphids contain bacteria known as “secondary symbionts,” which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names “Candidatus Consessoris aphidicola” for T type and “Candidatus Adiaceo aphidicola” for U type.

scholarly journals Cell-type specialization is encoded by specific chromatin topologies

Nature ◽  
2021 ◽  
Author(s):  
Warren Winick-Ng ◽  
Alexander Kukalev ◽  
Izabela Harabula ◽  
Luna Zea-Redondo ◽  
Dominik Szabó ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Cell Function ◽  
Nuclear Periphery ◽  
3D Structure ◽  
Cell Types ◽  
Brain Cells ◽  
Sensory Receptor ◽  
Specific Cell ◽  
Cell Type

AbstractThe three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1–3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi4–6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive ‘melting’ of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.

SOME BIOLOGICAL PROBLEMS IN CANCER BIOCHEMISTRY

1960 ◽  
Vol 38 (1) ◽  
pp. 425-433
Author(s):  
Louis Siminovitch ◽  
Arthur Axelrad
Keyword(s):  
Normal Tissue ◽  
Normal Growth ◽  
Cell Types ◽  
Biological Properties ◽  
Cellular Level ◽  
Animal Cells ◽  
Biochemical Basis ◽  
Normal Tissues ◽  
Ascites Tumors

Understanding of the cancer process in chemical terms has been seriously hampered by the difficulty of interpreting results of biochemical comparisons between masses of tumor and of normal tissue. Normal tissue consists of a variety of cell types and tumors may originate from one or more of these. As whole masses, therefore, normal tissues cannot serve as adequate controls for experiments on any single tumor. Tumor cell populations, even those arising from a single cell type, are themselves cytogenetically and continually undergoing changes during growth (progression). It is thus difficult, if not impossible, to separate the relevant from the irrelevant biochemical features of malignancy.Progress in this field requires means of dealing with the problem of biological heterogeneity. Several biochemical approaches that are free from the hazards of heterogeneity and which have already yielded valuable results, or appear promising, are indicated. These include: (1) The use of ascites tumors for studying the biochemical machinery of cells. No normal tissue exists, however, that could serve as satisfactory control. (2) Biochemical comparisons between pairs of tumor lines which differ by only one inherited characteristic of malignancy. These might reveal a biochemical basis for the biological properties of tumor cells with different degrees of malignancy. (3) Elucidation of normal growth-controlling mechanisms between cells, e.g. action of hormones at the cellular level, and within cells, e.g. mechanism of feed-back control of enzymes and metabolic pathways. (4) Further research into the biochemistry of plant tumor induction in vitro. Here biochemical changes associated with inherited changes leading to nutritional autonomy and uncontrolled growth have already been demonstrated. (5) Studies on the biochemical events during induction of malignancy by viruses in clonal cultures of animal cells in vitro. These could serve as useful models of the whole process of carcinogenesis.

scholarly journals Role of microtubules in the organization and localization of the Golgi apparatus.

1984 ◽  
Vol 99 (1) ◽  
pp. 113s-118s ◽  
Author(s):  
I V Sandoval ◽  
J S Bonifacino ◽  
R D Klausner ◽  
M Henkart ◽  
J Wehland
Keyword(s):  
Golgi Apparatus ◽  
Close Association ◽  
A549 Cells ◽  
Cell Periphery ◽  
Microtubule Depolymerization ◽  
Microtubule Organizing Center ◽  
Normal Medium ◽  
Large Numbers ◽  
Alpha Beta ◽  
Tubulin Paracrystals

Normal interphase PtK2 and A549 cells display long microtubules radiating from the microtubule-organizing center (MTOC) to the plasma membrane. Both MTOC and Golgi apparatus are contained in the same perinuclear area. Treatment of cells with 1 microM colcemid for 2 h results in microtubule depolymerization and fragmentation of the Golgi apparatus into elements scattered throughout the cytoplasm. Both normal microtubules and the Golgi apparatus assemble again following removal of colcemid. Injection of the alpha, beta-nonhydrolyzable GTP analog, guanosine 5'(alpha, beta-methylene)diphosphate [pp(CH2)pG], into interphase cells growing in normal medium results in the formation of microtubule bundles resistant to colcemid and prevents the fragmentation of the Golgi apparatus. Injection of pp(CH2)pG into cells incubated with colcemid results in substitution of tubulin ribbons for microtubules and has no effect on the Golgi-derived elements scattered throughout the cytoplasm. Removal of colcemid 1 h after the injection of pp(CH2)pG results in polymerization of large numbers of short, single randomly oriented microtubules, whereas the Golgi apparatus remains fragmented. Treatment of cells with 10 microM taxol for 3 h results both in polymerization of microtubule bundles without relation to the MTOC in the cell periphery and fragmentation of the Golgi apparatus. The Golgi-derived fragments are present exclusively in regions of the peripheral cytoplasm enriched in microtubules. The codistribution of microtubules and Golgi elements can be reversed in taxol-treated cells by injection of a monoclonal (YL 1/2) antibody reacting specifically with the tyrosylated form of alpha-tubulin. Cells incubated with colcemid after treatment with taxol have large numbers of Golgi-derived elements in close association with colcemid-resistant microtubule bundles. Incubation of cells with 50 microM vinblastine for 90 min results in microtubule dissembly, formation of tubulin paracrystals, and fragmentation of the Golgi apparatus into elements without relation to the tubulin paracrystals.

Purification and characterization of ensconsin, a novel microtubule stabilizing protein

1994 ◽  
Vol 107 (10) ◽  
pp. 2839-2849 ◽  
Author(s):  
J.C. Bulinski ◽  
A. Bossler
Keyword(s):  
Biochemical Characterization ◽  
Cell Types ◽  
Molar Ratio ◽  
Purification And Characterization ◽  
Protein Map ◽  
Tight Association ◽  
The Stability

In previous studies (Bulinski and Borisy (1979). Proc. Nat. Acad. Sci. 76, 293–297; Weatherbee et al. (1980). Biochemistry 19, 4116–4123) a microtubule-associated protein (MAP) of M(r) approximately 125,000 was identified as a prominent MAP in HeLa cells. We set out to perform a biochemical characterization of this protein, and to determine its in vitro functions and in vivo distribution. We determined that, like the assembly-promoting MAPs, tau, MAP2 and MAP4, the 125 kDa MAP was both proteolytically sensitive and thermostable. An additional property of this MAP; namely, its unusually tight association with a calcium-insensitive population of MTs in the presence of taxol, was exploited in devising an efficient purification strategy. Because of the MAP's tenacious association with a stable population of MTs, and because it appeared to contribute to the stability of this population of MTs in vitro, we have named this protein ensconsin. We examined the binding of purified ensconsin to MTs; ensconsin exhibited binding that saturated its MT binding sites at an approximate molar ratio of 1:6 (ensconsin:tubulin). Unlike other MAPs characterized to date, ensconsin's binding to MTs was insensitive to moderate salt concentrations (< or = 0.6 M). We further characterized ensconsin in immunoblotting experiments using mouse polyclonal anti-ensconsin antibodies and antibodies reactive with previously described MAPs, such as high molecular mass tau isoforms, dynamin, STOP, CLIP-170 and kinesin. These experiments demonstrated that ensconsin is distinct from other proteins of similar M(r) that may be present in association with MTs. Immunofluorescence with anti-ensconsin antibodies demonstrated that ensconsin was detectable in association with most or all of the MTs of several lines of human epithelial, fibroblastic and muscle cells; its in vivo properties and distribution, especially in response to drug or other treatments of cells, were found to be different from those of MAP4, the predominant MAP found in these cell types. We conclude that ensconsin, a MAP found in a variety of human cells, is biochemically - and perhaps functionally - distinct from other MAPs present in non-neuronal cells.

scholarly journals Cytoskeletal Tubulin Competes with Actin to Increase Deformability of Metastatic Melanoma Cells

2020 ◽  
Author(s):  
Ghodeejah Higgins ◽  
Jade Peres ◽  
Tamer Abdalrahman ◽  
Muhammad H Zaman ◽  
Dirk M Lang ◽  
...  
Keyword(s):  
Cell Migration ◽  
Growth Phase ◽  
Cell Types ◽  
Melanoma Cells ◽  
Cell Stiffness ◽  
Disease Stage ◽  
Microtubule Disruption ◽  
Membrane Protrusions ◽  
Vertical Growth Phase

ABSTRACTThe formation of membrane protrusions during migration is reliant upon the cells’ cytoskeletal structure and stiffness. It has been reported that actin disruption blocks protrusions and decreases cell stiffness whereas microtubule disruption blocks protrusion but increases stiffness in several cell types. In melanoma, cell migration is of concern as this cancer spreads unusually rapidly during early tumour development. The aim of this study was to characterise motility, structural properties and stiffness of human melanoma cells at radial growth phase (RGP), vertical growth phase (VGP), and metastatic stage (MET) in two-dimensional in vitro environments. Wound assays, western blotting and mitochondrial particle tracking were used to assess cell migration, cytoskeletal content and intracellular fluidity. Our results indicate that cell motility increase with increasing disease stage. Despite their different motility, RGP and VGP cells exhibit similar fluidity, actin and tubulin levels. MET cells, however, display increased fluidity which was associated with increased actin and tubulin content. Our findings demonstrate an interplay between actin and microtubule activity and their role in increasing motility of cells while minimizing cell stiffness at advanced disease stage. In earlier disease stages, cell stiffness may however not serve as an indicator of migratory capabilities.

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