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1.
Cancers (Basel) ; 14(2)2022 Jan 17.
Article in English | MEDLINE | ID: mdl-35053604

ABSTRACT

Aberrations in the centrosome number and structure can readily be detected at all stages of tumor progression and are considered hallmarks of cancer. Centrosome anomalies are closely linked to chromosome instability and, therefore, are proposed to be one of the driving events of tumor formation and progression. This concept, first posited by Boveri over 100 years ago, has been an area of interest to cancer researchers. We have now begun to understand the processes by which these numerical and structural anomalies may lead to cancer, and vice-versa: how key events that occur during carcinogenesis could lead to amplification of centrosomes. Despite the proliferative advantages that having extra centrosomes may confer, their presence can also lead to loss of essential genetic material as a result of segregational errors and cancer cells must deal with these deadly consequences. Here, we review recent advances in the current literature describing the mechanisms by which cancer cells amplify their centrosomes and the methods they employ to tolerate the presence of these anomalies, focusing particularly on centrosomal clustering.

2.
Mol Biol Cell ; 23(19): 3827-37, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22918948

ABSTRACT

Dynactin is an essential part of the cytoplasmic dynein motor that enhances motor processivity and serves as an adaptor that allows dynein to bind cargoes. Much is known about dynactin's interaction with dynein and microtubules, but how it associates with its diverse complement of subcellular binding partners remains mysterious. It has been suggested that cargo specification involves a group of subunits referred to as the "pointed-end complex." We used chemical cross-linking, RNA interference, and protein overexpression to characterize interactions within the pointed-end complex and explore how it contributes to dynactin's interactions with endomembranes. The Arp11 subunit, which caps one end of dynactin's Arp1 filament, and p62, which binds Arp11 and Arp1, are necessary for dynactin stability. These subunits also allow dynactin to bind the nuclear envelope prior to mitosis. p27 and p25, by contrast, are peripheral components that can be removed without any obvious impact on dynactin integrity. Dynactin lacking these subunits shows reduced membrane binding. Depletion of p27 and p25 results in impaired early and recycling endosome movement, but late endosome movement is unaffected, and mitotic spindles appear normal. We conclude that the pointed-end complex is a bipartite structural domain that stabilizes dynactin and supports its binding to different subcellular structures.


Subject(s)
Microtubule-Associated Proteins/metabolism , Protein Subunits/metabolism , Actins/genetics , Actins/metabolism , Animals , COS Cells , Cattle , Chlorocebus aethiops , Dynactin Complex , Dyneins/metabolism , Endosomes/metabolism , Gene Knockdown Techniques , HeLa Cells , Humans , Kinetics , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Nuclear Envelope/metabolism , Protein Binding , Protein Interaction Mapping , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Transport , RNA Interference , Spindle Apparatus/metabolism , Transferrin/metabolism
3.
Tumori ; 98(1): 142-51, 2012.
Article in English | MEDLINE | ID: mdl-22495715

ABSTRACT

AIMS AND BACKGROUND: The pyruvate mimetic dichloroacetate (DCA) has been shown to induce cell death in cancer cells. A number of studies in vitro and in vivo have suggested this molecule may serve as an anticancer agent, but some cells are resistant. Here we wanted to examine the effects of DCA on cancerous and noncancerous cells grown in culture for a prolonged period of exposure and at increasing concentrations. METHODS: Six cancer cell lines (A549, SK-HEP-1, HCT116, UPCI:SCC070, HeLa and MES-SA) and three noncancerous lines (RPE, GM03349B and HEK293) were exposed to 0.5 mM DCA for seven days and cell counts were taken every day to determine viability and cell cycle progression. The same cell lines were also exposed to higher doses of DCA up to 10 mM and viability was scored. RESULTS: Five cancer cell lines showed high levels of cell death early in the trial, but three of the lines showed a second delayed increase in cell death at later stages. HCT116 cells were unaffected by 0.5 mM DCA. GM03349B and RPE cells also died when treated with DCA. At high concentrations, all cell lines exhibited high rates of death. No specific cell cycle arrest of the cells was observed. CONCLUSION: We found that there is considerable difference in the way cancer cells are affected by DCA. Some have populations that are highly resistant to treatment, while others have stronger rates of death only after prolonged exposure. We also found noncancerous cells are not all resistant to DCA, a significant finding that has not previously been observed in other in vitro DCA trials.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Growth Processes/drug effects , Dichloroacetic Acid/pharmacology , Caspases/metabolism , Cell Count , Cell Line , Cell Line, Tumor , HCT116 Cells , HeLa Cells , Humans , In Situ Nick-End Labeling , Mitotic Index
4.
Exp Gerontol ; 46(10): 827-32, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21798334

ABSTRACT

We have previously shown that copper supplementation extends the replicative life span of Saccharomyces cerevisiae when grown under conditions forcing cells to respire. We now show that copper's effect on life span is through Fet3p, a copper containing enzyme responsible for high affinity transport of iron into yeast cells. Life span extensions can also be obtained by supplementing the growth medium with 1mM ferric chloride. Extension by high iron levels is still dependent on the presence of Fet3p. Life span extension by iron or copper requires growth on media containing glycerol as the sole carbon source, which forces yeast to respire. Yeast grown on glucose containing media supplemented with iron show no extension of life span. The iron associated with cells grown in media supplemented with copper or iron is 1.4-1.8 times that of cells grown without copper or iron supplementation. As with copper supplementation, iron supplementation partially rescues the life span of superoxide dismutase mutants. Cells grown with copper supplementation display decreased production of superoxide as measured by dihydroethidium staining.


Subject(s)
Cation Transport Proteins/genetics , Cell Respiration/genetics , Copper/metabolism , Gene Expression Regulation, Fungal/genetics , Iron/pharmacology , Mitochondria/metabolism , Animals , Biological Transport/genetics , Cation Transport Proteins/metabolism , Cell Respiration/physiology , Copper/pharmacology , Culture Media , Glycerol/pharmacology , Iron/metabolism , Life Expectancy , Mitochondria/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/genetics
5.
Nat Cell Biol ; 8(3): 264-70, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16474384

ABSTRACT

Microtubule-associated proteins (MAPs) use particular microtubule-binding domains that allow them to interact with microtubules in a manner specific to their individual cellular functions. Here, we have identified a highly basic microtubule-binding domain in the p150 subunit of dynactin that is only present in the dynactin members of the CAP-Gly family of proteins. Using single-particle microtubule-binding assays, we found that the basic domain of dynactin moves progressively along microtubules in the absence of molecular motors - a process we term 'skating'. In contrast, the previously described CAP-Gly domain of dynactin remains firmly attached to a single point on microtubules. Further analyses showed that microtubule skating is a form of one-dimensional diffusion along the microtubule. To determine the cellular function of the skating phenomenon, dynein and the dynactin microtubule-binding domains were examined in single-molecule motility assays. We found that the basic domain increased dynein processivity fourfold whereas the CAP-Gly domain inhibited dynein motility. Our data show that the ability of the basic domain of dynactin to skate along microtubules is used by dynein to maintain longer interactions for each encounter with microtubules.


Subject(s)
Dyneins/physiology , Microtubule-Associated Proteins/physiology , Microtubules/physiology , Amino Acid Sequence , Animals , Chickens , Dynactin Complex , Molecular Motor Proteins/physiology , Molecular Sequence Data , Protein Structure, Tertiary , Protein Subunits/physiology , Protein Transport , Sequence Homology, Amino Acid
6.
Science ; 307(5706): 127-9, 2005 Jan 07.
Article in English | MEDLINE | ID: mdl-15637283

ABSTRACT

Most tumor cells are characterized by increased genomic instability and chromosome segregational defects, often associated with hyperamplification of the centrosome and the formation of multipolar spindles. However, extra centrosomes do not always lead to multipolarity. Here, we describe a process of centrosomal clustering that prevented the formation of multipolar spindles in noncancer cells. Noncancer cells needed to overcome this clustering mechanism to allow multipolar spindles to form at a high frequency. The microtubule motor cytoplasmic dynein was a critical part of this coalescing machinery, and in some tumor cells overexpression of the spindle protein NuMA interfered with dynein localization, promoting multipolarity.


Subject(s)
Centrosome/physiology , Dyneins/metabolism , Spindle Apparatus/physiology , Antigens, Nuclear , Cell Cycle Proteins , Cell Line , Cell Line, Tumor , Demecolcine/pharmacology , Dynactin Complex , Humans , Microtubule-Associated Proteins/metabolism , Nuclear Matrix-Associated Proteins , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Small Interfering/metabolism , Transfection
7.
Mol Biol Cell ; 14(12): 5089-97, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14565986

ABSTRACT

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.


Subject(s)
Dyneins/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Animals , COS Cells , Cell Compartmentation/physiology , Chlorocebus aethiops , Dynactin Complex , Endocytosis/physiology , Endosomes/metabolism , Green Fluorescent Proteins , HeLa Cells , Humans , Luminescent Proteins , Microscopy, Fluorescence , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Protein Subunits/metabolism
8.
J Cell Biol ; 159(2): 245-54, 2002 Oct 28.
Article in English | MEDLINE | ID: mdl-12391026

ABSTRACT

Centrosomal dynactin is required for normal microtubule anchoring and/or focusing independently of dynein. Dynactin is present at centrosomes throughout interphase, but dynein accumulates only during S and G2 phases. Blocking dynein-based motility prevents recruitment of dynactin and dynein to centrosomes and destabilizes both centrosomes and the microtubule array, interfering with cell cycle progression during mitosis. Destabilization of the centrosomal pool of dynactin does not inhibit dynein-based motility or dynein recruitment to centrosomes, but instead causes abnormal G1 centriole separation and delayed entry into S phase. The correct balance of centrosome-associated dynactin subunits is apparently important for satisfaction of the cell cycle mechanism that monitors centrosome integrity before centrosome duplication and ultimately governs the G1 to S transition. Our results suggest that, in addition to functioning as a microtubule anchor, dynactin contributes to the recruitment of important cell cycle regulators to centrosomes.


Subject(s)
Cell Cycle/physiology , Centrioles/metabolism , Dyneins/metabolism , Microtubule-Associated Proteins/metabolism , Animals , COS Cells , Dynactin Complex , Dyneins/genetics , G1 Phase/physiology , G2 Phase/physiology , Gene Expression/physiology , Interphase/physiology , Microtubule-Associated Proteins/genetics , Mitosis/physiology , Protein Binding/physiology , S Phase/physiology
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