KIF4 helps mitotic chromosomes get in shape

DNA Topoisomerase IIα (topoIIα) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoIIα expression can be silenced in all cells by the addition of tetracycline. TopoIIα-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoIIα fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoIIα as an indicator for endogenous topoIIα dynamics, our analyses provide new evidence that topoIIα plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoIIβ and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.

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Faculty Opinions recommendation of Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717973616.793470193 ◽

2013 ◽

Author(s):

Edward Kipreos

Keyword(s):

Mitotic Chromosomes ◽

Topoisomerase Iiα

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Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes.

The Journal of Cell Biology ◽

10.1083/jcb.114.2.285 ◽

1991 ◽

Vol 114 (2) ◽

pp. 285-294 ◽

Cited By ~ 58

Author(s):

L Wordeman ◽

E R Steuer ◽

M P Sheetz ◽

T Mitchison

Keyword(s):

Indirect Immunofluorescence ◽

Motor Protein ◽

Chromosome Translocation ◽

Mitotic Chromosomes ◽

Cell Biol ◽

Microtubule Motor ◽

Specific Antigens ◽

Microtubule Motor Protein

We have used indirect immunofluorescence in combination with correlative EM to subdivide the mammalian kinetochore into two domains based on the localization of specific antigens. We demonstrate here that the fibrous corona on the distal face of the kinetochore plate contains tubulin (previously shown by Mitchison, T. J., and M. W. Kirschner. 1985. J. Cell Biol. 101:755-765) and the minus end-directed, ATP-dependent microtubule motor protein, dynein; whereas a 50-kD CREST antigen is located internal to these components in the kinetochore. Tubulin and dynein can be extracted from the kinetochore by 150 mM KI, leaving other, as yet uncharacterized, components of the kinetochore corona intact. Microtubules and tubulin subunits will associate with kinetochores in vitro after extraction with 150 mM KI, suggesting that other functionally significant, corona-associated molecules remain unextracted. Our results suggest that the corona region of the kinetochore contains the machinery for chromosome translocation along microtubules.

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Centaurin-α1 and KIF13B kinesin motor protein interaction in ARF6 signalling

Biochemical Society Transactions ◽

10.1042/bst0331279 ◽

2005 ◽

Vol 33 (6) ◽

pp. 1279-1281 ◽

Cited By ~ 8

Author(s):

V. Kanamarlapudi

Keyword(s):

Membrane Trafficking ◽

Motor Protein ◽

Small Gtpases ◽

Nucleotide Exchange ◽

Kinesin Motor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Gtpase Activating Proteins ◽

Gtp Binding ◽

Cytoskeletal Dynamics

The ARF (ADP-ribosylation factor) family of small GTPases regulate intracellular membrane trafficking by cycling between an inactive GDP- and an active GTP-bound form. Among the six known mammalian ARFs (ARF1–ARF6), ARF6 is the least conserved and plays critical roles in membrane trafficking and cytoskeletal dynamics near the cell surface. Since ARFs have undetectable levels of intrinsic GTP binding and hydrolysis, they are totally dependent on extrinsic GEFs (guanine nucleotide-exchange factors) for GTP binding and GAPs (GTPase-activating proteins) for GTP hydrolysis. We have recently isolated a novel KIF (kinesin) motor protein (KIF13B) that binds to centaurin-α1, an ARF6GAP that binds to the second messenger PIP3 [PtdIns(3,4,5)P3]. KIFs transport intracellular vesicles and recognize their cargo by binding to proteins (receptors) localized on the surface of the cargo vesicles. Identification of centaurin-α1 as a KIF13B interactor suggests that KIF13B may transport ARF6 and/or PIP3 using centaurin-α1 as its receptor. This paper reviews the studies carried out to assess the interaction and regulation of centaurin-α1 by KIF13B.

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Human Topoisomerase IIα: Targeting to Subchromosomal Sites of Activity during Interphase and Mitosis

Molecular Biology of the Cell ◽

10.1091/mbc.e03-08-0558 ◽

2004 ◽

Vol 15 (5) ◽

pp. 2388-2400 ◽

Cited By ~ 23

Author(s):

Marta Agostinho ◽

José Rino ◽

José Braga ◽

Fernando Ferreira ◽

Soren Steffensen ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Longitudinal Axis ◽

Vital Role ◽

Dependent Manner ◽

Mitotic Chromosomes ◽

Topoisomerase Iiα ◽

Catalytically Active ◽

Splicing Speckles ◽

Cycle Dependent ◽

Subcellular Resolution

Mammalian topoisomerase IIα (topo IIα) plays a vital role in the removal of topological complexities left on DNA during S phase. Here, we developed a new assay to selectively identify sites of catalytic activity of topo IIα with subcellular resolution. We show that topo IIα activity concentrates at replicating heterochromatin in late S in a replication-dependent manner and at centric heterochromatin during G2 and M phases. Inhibitor studies indicate that this cell cycle-dependent concentration over heterochromatin is sensitive to chromatin structure. We further show that catalytically active topo IIα concentrates along the longitudinal axis of mitotic chromosomes. Finally, we found that catalytically inert forms of the enzyme localize predominantly to splicing speckles in a dynamic manner and that this pool is differentially sensitive to changes in the activities of topo IIα itself and RNA polymerase II. Together, our data implicate several previously unsuspected activities in the partitioning of the enzyme between sites of activity and putative depots.

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