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 Dictyostelium discoideum CenB Is a Bona Fide Centrin Essential for Nuclear Architecture and Centrosome Stability

2009 ◽  
Vol 8 (8) ◽  
pp. 1106-1117 ◽  
Author(s):  
Sebastian Mana-Capelli ◽  
Ralph Gräf ◽  
Denis A. Larochelle
Keyword(s):  
Dictyostelium Discoideum ◽  
Calcium Binding ◽  
Amino Acid Level ◽  
Nuclear Architecture ◽  
Microtubule Organizing Center ◽  
Whole Cells ◽  
Ef Hand ◽  
Interphase Cells ◽  
Organizing Center ◽  
Bona Fide

ABSTRACT Centrins are a family of proteins within the calcium-binding EF-hand superfamily. In addition to their archetypical role at the microtubule organizing center (MTOC), centrins have acquired multiple functionalities throughout the course of evolution. For example, centrins have been linked to different nuclear activities, including mRNA export and DNA repair. Dictyostelium discoideum centrin B is a divergent member of the centrin family. At the amino acid level, DdCenB shows 51% identity with its closest relative and only paralog, DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a well-supported monophyletic and divergent group within the centrin family of proteins. Interestingly, fluorescently tagged versions of DdCenB were not found at the centrosome (in whole cells or in isolated centrosomes). Instead, DdCenB localized to the nuclei of interphase cells. This localization disappeared as the cells entered mitosis, although Dictyostelium cells undergo a closed mitosis in which the nuclear envelope (NE) does not break down. DdCenB knockout cells exhibited aberrant nuclear architecture, characterized by enlarged and deformed nuclei and loss of proper centrosome-nucleus anchoring (observed as NE protrusions). At the centrosome, loss of DdCenB resulted in defects in the organization and morphology of the MTOC and supernumerary centrosomes and centrosome-related bodies. The multiple defects that the loss of DdCenB generated at the centrosome can be explained by its atypical division cycle, transitioning into the NE as it divides at mitosis. On the basis of these findings, we propose that DdCenB is required at interphase to maintain proper nuclear architecture, and before delocalizing from the nucleus, DdCenB is part of the centrosome duplication machinery.

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.

A novel structural component of the Dictyostelium centrosome

1996 ◽  
Vol 109 (13) ◽  
pp. 3103-3112 ◽  
Author(s):  
A. Kalt ◽  
M. Schliwa
Keyword(s):  
Mitotic Spindle ◽  
Structural Component ◽  
Myosin Ii ◽  
Live Cells ◽  
Microtubule Organizing Center ◽  
Direct Role ◽  
Cytoplasmic Microtubule ◽  
Interphase Cells ◽  
Organizing Center

The microtubule-organizing center of D. discoideum is a nucleus-associated body (NAB) that consists of a multilayered, box-shaped core embedded in an amorphous corona from which the microtubules emerge. The composition of the NAB is still largely unresolved. Here we have examined a high molecular mass component of the NAB which was identified by a monoclonal antibody raised against isolated nucleus/NAB complexes. This antibody recognized a 350 kDa component which is immunologically related to the D. discoideum heavy chain of myosin II. The 350 kDa antigen was localized only at the NAB in interphase cells, while in mitotic cells it may also be found in the vicinity of the NAB as well as in association with the mitotic spindle. Immunogold labeling experiments showed that the protein is part of the NAB corona. This association was not destroyed by treatment with 2 M urea or 0.6 M KCl. The 350 kDa antigen was part of the thiabendazole-induced cytoplasmic microtubule-organizing centers. A direct role in the polymerization of tubulin could not be determined in an in vitro microtubule nucleation assay, whereas antibody electroporation of live cells appeared to interfere with the generation of a normal microtubule system in a subset of cells. Our observations suggest that the 350 kDa antigen is a structural component of the NAB corona which could be involved in its stabilization.

The Role of the Centrosome in Development and Progression of Breast Cancer

2001 ◽  
Vol 7 (S2) ◽  
pp. 582-583
Author(s):  
W. Lingle ◽  
J. Salisbury ◽  
S. Barrett ◽  
V. Negron ◽  
C. Whitehead
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Microtubule Organizing Center ◽  
Daughter Cells ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Close Proximity ◽  
Interphase Cells ◽  
Organizing Center

The centrosome is the major microtubule organizing center in most mammalian cells, and as such it determines the number, polarity, and spatial distribution of microtubules (MTs). Interphase MTs, together with actin and intermediate filaments, constitute the cell's cytoskeleton, which dynamically maintains cell polarity and tissue architecture. Interphase cells begin Gl of the cell cycle with one centrosome. During S phase, the centrosome duplicates concomitantly with DNA replication. Duplicated centrosomes usually remain in close proximity to one another until late G2, at which time they separate and then move during prophase to become the poles that organize the bipolar mitotic spindle. During the G2/M transition, interphase MTs depolymerize and a new population of highly dynamic mitotic MTs are nucleated at the spindle poles. The bipolar mitotic spindle apparatus constitutes the machinery that partitions and separates sister chromatids equally between two daughter cells.

scholarly journals A novel mitotic spindle pole component that originates from the cytoplasm during prophase.

1986 ◽  
Vol 103 (5) ◽  
pp. 1863-1872 ◽  
Author(s):  
P R Sager ◽  
N L Rothfield ◽  
J M Oliver ◽  
R D Berlin
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Early Prophase ◽  
Microtubule Organizing Center ◽  
Spindle Poles ◽  
Interphase Cells ◽  
Organizing Center ◽  
Mitotic Spindle Pole ◽  
Pole Component

Several unique aspects of mitotic spindle formation have been revealed by investigation of an autoantibody present in the serum of a patient with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, schlerodacytyly, and telangiectasias) syndrome. This antibody was previously shown to label at the spindle poles of metaphase and anaphase cells and to be absent from interphase cells. We show here that the serum stained discrete cytoplasmic foci in early prophase cells and only later localized to the spindle poles. The cytoplasmic distribution of the antigen was also seen in nocodazole-arrested cells and prophase cells in populations treated with taxol. In normal and taxol-treated cells, the microtubules appeared to emanate from the cytoplasmic foci and polar stain, and in cells released from nocodazole block, microtubules regrew from antigen-containing centers. This characteristic distribution suggests that the antigen is part of a microtubule organizing center. Thus, we propose that a prophase originating polar antigen functions in spindle pole organization as a coalescing microtubule organizing center that is present only during mitosis. Characterization of the serum showed reactions with multiple proteins at 115, 110, 50, 36, 30, and 28 kD. However, affinity-eluted antibody from the 115/110-kD bands was shown to specifically label the spindle pole and cytosolic foci in prophase cells.

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.

scholarly journals The Golgi Complex Is a Microtubule-organizing Organelle

2001 ◽  
Vol 12 (7) ◽  
pp. 2047-2060 ◽  
Author(s):  
Karine Chabin-Brion ◽  
Jérôme Marceiller ◽  
Franck Perez ◽  
Catherine Settegrana ◽  
Anne Drechou ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Microtubule Assembly ◽  
Microtubule Nucleation ◽  
Hepatic Cells ◽  
Golgi Membranes ◽  
Cytoplasmic Face ◽  
Interphase Cells ◽  
Highly Correlated

We show that the Golgi complex can directly stimulate microtubule nucleation in vivo and in vitro and thus behaves as a potent microtubule-organizing organelle in interphase cells. With the use of nocodazole wash-out experiments in hepatic cells, we found that the occurrence of noncentrosomal, early stabilized microtubules is highly correlated with the subcellular localization of Golgi membranes. With the use of in vitro reconstituted microtubule assembly systems with or without cytosol, we also found that, in contrast to centrosomally attached microtubules, the distal ends of Golgi-attached microtubules are remotely stabilized in a way that requires additional cytosolic component(s). Finally, we demonstrate that Golgi-based microtubule nucleation is direct and involves a subset of γ-tubulin bound to the cytoplasmic face of the organelle.

scholarly journals Tubular early endosomal networks in AtT20 and other cells.

1991 ◽  
Vol 115 (3) ◽  
pp. 635-653 ◽  
Author(s):  
J Tooze ◽  
M Hollinshead
Keyword(s):  
Electron Microscope ◽  
Fluid Phase ◽  
Microtubule Organizing Center ◽  
Local Networks ◽  
Late Endosomes ◽  
Interphase Cells ◽  
Organizing Center ◽  
Mdck I ◽  
Att20 Cell ◽  
In Cells

Using horseradish peroxidase (HRP) as a fluid-phase endocytic tracer, we observed through the electron microscope numerous tubular endosomes with a diameter of 30-50 nm and lengths of greater than 2 microns in thick sections (0.2-0.5 microns) of AtT20 cells. These tubular endosomes are multibranching and form local networks but not a single reticulum throughout the cytoplasm. They are sometimes in continuity with vesicular endosomal structures but have not been observed in continuity with AtT20 cell late endosomes. Tubular endosomal networks are not uniformly distributed throughout the cytoplasm, but are particularly abundant in growth cones, in patches below the plasma membrane of the cell body, and surrounding the centrioles and microtubule organizing center (MTOC). Tubular endosomes at all these locations receive HRP within the first 5 min of endocytosis but approximately 30 min of endocytosis are required to load the tubular endosomal networks with HRP so that their full extent can be visualized in the electron microscope. After 10 min of endocytosis, complete unloading occurs within 30 min of chase, but between 30 and 60 min are required to chase out all the tracer from the tubular endosomes loaded to steady state during 60 min endocytosis of 10 mg/ml HRP. In interphase cells, neither the loading nor unloading of tubular endosomes depends on microtubules but in cells blocked in mitosis by depolymerization of the mitotic spindle with nocodazole, HRP does not chase out of tubular endosomes. The thread-like shape of tubular endosomes is not dependent on microtubules. Furthermore, HRP is delivered to AtT20 tubular endosomes at 20 degrees C. All these properties indicate that AtT20 cell tubular endosomes are an early endocytic compartment distinct from late endosomes. Tubular endosomes like those in AtT20 cells have been seen in cells of the following lines: PC12, HeLa, Hep2, Vero, MDCK I and II, CCL64, RK13, and NRK; they are particularly abundant in the first three lines. In contrast, tubular endosomes are sparse in 3T3 and BHK21 cells. The tubular endosomes we have observed appear to be identical to the endosomal reticulum observed in the living Hep2 cells by Hopkins, C. R., A. Gibson, H. Shipman, and K. Miller. 1990.

scholarly journals The Centrosome and the Primary Cilium: The Yin and Yang of a Hybrid Organelle

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 701 ◽  
Author(s):  
Joukov ◽  
De Nicolo
Keyword(s):  
Primary Cilium ◽  
Primary Cilia ◽  
Microtubule Assembly ◽  
Microtubule Organizing Center ◽  
Environmental Signals ◽  
Intracellular Signals ◽  
Interphase Cells ◽  
Organizing Center ◽  
Pericentriolar Material ◽  
Yin And Yang

Centrosomes and primary cilia are usually considered as distinct organelles, although both are assembled with the same evolutionary conserved, microtubule-based templates, the centrioles. Centrosomes serve as major microtubule- and actin cytoskeleton-organizing centers and are involved in a variety of intracellular processes, whereas primary cilia receive and transduce environmental signals to elicit cellular and organismal responses. Understanding the functional relationship between centrosomes and primary cilia is important because defects in both structures have been implicated in various diseases, including cancer. Here, we discuss evidence that the animal centrosome evolved, with the transition to complex multicellularity, as a hybrid organelle comprised of the two distinct, but intertwined, structural-functional modules: the centriole/primary cilium module and the pericentriolar material/centrosome module. The evolution of the former module may have been caused by the expanding cellular diversification and intercommunication, whereas that of the latter module may have been driven by the increasing complexity of mitosis and the requirement for maintaining cell polarity, individuation, and adhesion. Through its unique ability to serve both as a plasma membrane-associated primary cilium organizer and a juxtanuclear microtubule-organizing center, the animal centrosome has become an ideal integrator of extracellular and intracellular signals with the cytoskeleton and a switch between the non-cell autonomous and the cell-autonomous signaling modes. In light of this hypothesis, we discuss centrosome dynamics during cell proliferation, migration, and differentiation and propose a model of centrosome-driven microtubule assembly in mitotic and interphase cells. In addition, we outline the evolutionary benefits of the animal centrosome and highlight the hierarchy and modularity of the centrosome biogenesis networks.

scholarly journals Microtubule-Nucleus Interactions in Dictyostelium discoideum Mediated by Central Motor Kinesins

2009 ◽  
Vol 8 (5) ◽  
pp. 723-731 ◽  
Author(s):  
Irina Tikhonenko ◽  
Dilip K. Nag ◽  
Douglas N. Robinson ◽  
Michael P. Koonce
Keyword(s):  
Cell Division ◽  
Rna Interference ◽  
Cell Growth ◽  
Cell Viability ◽  
Dictyostelium Discoideum ◽  
Eukaryotic Cells ◽  
Mitotic Spindles ◽  
Microtubule Organizing Center ◽  
Null Cell ◽  
Organizing Center

ABSTRACT Kinesins are a diverse superfamily of motor proteins that drive organelles and other microtubule-based movements in eukaryotic cells. These motors play important roles in multiple events during both interphase and cell division. Dictyostelium discoideum contains 13 kinesin motors, 12 of which are grouped into nine families, plus one orphan. Functions for 11 of the 13 motors have been previously investigated; we address here the activities of the two remaining kinesins, both isoforms with central motor domains. Kif6 (of the kinesin-13 family) appears to be essential for cell viability. The partial knockdown of Kif6 with RNA interference generates mitotic defects (lagging chromosomes and aberrant spindle assemblies) that are consistent with kinesin-13 disruptions in other organisms. However, the orphan motor Kif9 participates in a completely novel kinesin activity, one that maintains a connection between the microtubule-organizing center (MTOC) and nucleus during interphase. kif9 null cell growth is impaired, and the MTOC appears to disconnect from its normally tight nuclear linkage. Mitotic spindles elongate in a normal fashion in kif9 − cells, but we hypothesize that this kinesin is important for positioning the MTOC into the nuclear envelope during prophase. This function would be significant for the early steps of cell division and also may play a role in regulating centrosome replication.

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