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1.
J Cell Sci ; 124(Pt 9): 1433-44, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21486941

ABSTRACT

The multifunctional structural protein 4.1R is required for assembly and maintenance of functional nuclei but its nuclear roles are unidentified. 4.1R localizes within nuclei, at the nuclear envelope, and in cytoplasm. Here we show that 4.1R, the nuclear envelope protein emerin and the intermediate filament protein lamin A/C co-immunoprecipitate, and that 4.1R-specific depletion in human cells by RNA interference produces nuclear dysmorphology and selective mislocalization of proteins from several nuclear subcompartments. Such 4.1R-deficiency causes emerin to partially redistribute into the cytoplasm, whereas lamin A/C is disorganized at nuclear rims and displaced from nucleoplasmic foci. The nuclear envelope protein MAN1, nuclear pore proteins Tpr and Nup62, and nucleoplasmic proteins NuMA and LAP2α also have aberrant distributions, but lamin B and LAP2ß have normal localizations. 4.1R-deficient mouse embryonic fibroblasts show a similar phenotype. We determined the functional effects of 4.1R-deficiency that reflect disruption of the association of 4.1R with emerin and A-type lamin: increased nucleus-centrosome distances, increased ß-catenin signaling, and relocalization of ß-catenin from the plasma membrane to the nucleus. Furthermore, emerin- and lamin-A/C-null cells have decreased nuclear 4.1R. Our data provide evidence that 4.1R has important functional interactions with emerin and A-type lamin that impact upon nuclear architecture, centrosome-nuclear envelope association and the regulation of ß-catenin transcriptional co-activator activity that is dependent on ß-catenin nuclear export.


Subject(s)
Cell Nucleus/metabolism , Centrosome/metabolism , Cytoskeletal Proteins/metabolism , Membrane Proteins/metabolism , Nuclear Envelope/metabolism , Animals , Cell Line, Tumor , Cytoskeletal Proteins/genetics , Dogs , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , Lamin Type A/genetics , Lamin Type A/metabolism , Membrane Proteins/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Transport/genetics , Protein Transport/physiology , Transcription, Genetic , beta Catenin/genetics , beta Catenin/metabolism
2.
Mol Cell Biol ; 28(7): 2283-94, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18212055

ABSTRACT

Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.


Subject(s)
Cell Cycle/physiology , Centrioles/chemistry , Centrosome/ultrastructure , Cytoskeletal Proteins/physiology , Membrane Proteins/physiology , Spindle Apparatus/ultrastructure , Anaphase/physiology , Antigens, Nuclear/analysis , Cell Cycle Proteins , Cell Line/ultrastructure , Centrosome/metabolism , Chromosome Segregation/physiology , Cytoskeletal Proteins/analysis , Cytoskeletal Proteins/deficiency , Cytoskeletal Proteins/genetics , Down-Regulation , HeLa Cells/ultrastructure , Humans , Interphase/physiology , Membrane Proteins/deficiency , Membrane Proteins/genetics , Microtubules/ultrastructure , Nuclear Matrix-Associated Proteins/analysis , Nuclear Proteins/analysis , RNA Interference , RNA, Small Interfering/pharmacology , Spindle Apparatus/chemistry , Spindle Apparatus/metabolism , Tumor Suppressor Protein p53/physiology
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