Identification and immunolocalization of cytoplasmic dynein in <i>dictyostelium</i>

Koonce, Michael P.; McIntosh, J. Richard

doi:10.1002/cm.970150108

Cited by 73 publications

(43 citation statements)

References 48 publications

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“…The cells were allowed to attach to acid-cleaned coverslips overnight in a 8.5-cm Petri dish, and then they were fixed according to published protocols (Koonce and McIntosh, 1990). Briefly, the cells were fixed at room temperature with 2.5% formaldehyde in a 1,4-piperazinediethanesulfonic acid (PIPES)-EGTA buffer for 3 min followed by 1% formaldehyde in dehydrated methanol at Ϫ10°C for 5 min.…”

Section: Immunostaining and Microscopy Of Fixed Cellsmentioning

confidence: 99%

The Localization of Inner Centromeric Protein (INCENP) at the Cleavage Furrow Is Dependent on Kif12 and Involves Interactions of the N Terminus of INCENP with the Actin Cytoskeleton

Chen

Lakshmikanth

Spudich

et al. 2007

MBoC

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The inner centromeric protein (INCENP) and other chromosomal passenger proteins are known to localize on the cleavage furrow and to play a role in cytokinesis. However, it is not known how INCENP localizes on the furrow or whether this localization is separable from that at the midbody. Here, we show that the association of Dictyostelium INCENP (DdINCENP) with the cortex of the cleavage furrow involves interactions with the actin cytoskeleton and depends on the presence of the kinesin-6 -related protein Kif12. We found that Kif12 is found on the central spindle and the cleavage furrow during cytokinesis. Kif12 is not required for the redistribution of DdINCENP from centromeres to the central spindle. However, in the absence of Kif12, DdINCENP fails to localize on the cleavage furrow. Domain analysis indicates that the N terminus of DdINCENP is necessary and sufficient for furrow localization and that it binds directly to the actin cytoskeleton. Our data suggest that INCENP moves from the central spindle to the furrow of a dividing cell by a Kif12-dependent pathway. Once INCENP reaches the equatorial cortex, it associates with the actin cytoskeleton where it then concentrates toward the end of cytokinesis.

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Section: Immunostaining and Microscopy Of Fixed Cellsmentioning

confidence: 99%

The Localization of Inner Centromeric Protein (INCENP) at the Cleavage Furrow Is Dependent on Kif12 and Involves Interactions of the N Terminus of INCENP with the Actin Cytoskeleton

Chen

Lakshmikanth

Spudich

et al. 2007

MBoC

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“…The purified fusion protein was injected into rabbits to raise polyclonal antiDdINCENP antibodies (Cocalico Biologicals, Reamstown, PA). For the Western blot analysis of DdINCENP, whole cell lysates of 1 ϫ 10 6 cells were loaded in each lane and separated on a 6% SDS-PAGE gel (Koonce and McIntosh, 1990).…”

Section: Western Blot Analysismentioning

confidence: 99%

“…Then they were fixed according to the protocol published by Dr. McIntosh (Koonce and McIntosh, 1990). Briefly, the cells were fixed at room temperature with 2.5% formaldehyde in a PIPES-EGTA buffer for 3 min followed by 1% formaldehyde in dehydrated methanol at Ϫ10°C for 5 min.…”

Section: Immunostaining and Microscopy Of Fixed Cellsmentioning

confidence: 99%

Contractile Ring-independent Localization of DdINCENP, a Protein Important for Spindle Stability and Cytokinesis

Chen

Lozanne

2006

MBoC

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“…6A). The DdM7-talinA complex sedimented more slowly than the 20 S dynein complex (32) (Fig. 6A) and was estimated to sediment at ϳ14 S. The sedimentation of each protein in the absence of the other was examined to rule out the possibility that the individual proteins both happen to sediment at 14 S due to homodimerization or to a highly elongated structure.…”

Section: Fig 2 the Ddm7 Tail Binds To Talinamentioning

confidence: 99%

Identification of a Myosin VII-Talin Complex

Tuxworth¹,

Stephens

Ryan

et al. 2005

Journal of Biological Chemistry

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Myosin VII (M7) plays a role in adhesion in both Dictyostelium and mammalian cells where it is a component of a complex of proteins that serve to link membrane receptors to the underlying actin cytoskeleton. The nature of this complex is not fully known, prompting a search for M7-binding proteins. Co-immunoprecipitation experiments reveal that Dictyostelium M7 (DdM7) interacts with talinA, an actin-binding protein with a known role in cell-substrate adhesion. No additional proteins are observed in the immunoprecipitate, indicating that the interaction is direct. The Nterminal region of the DdM7 tail that lies between the region of predicted coil and the first MyTH4 domain is found to harbor the talinA binding site. Localization experiments reveal that talinA does not serve as a membrane receptor for DdM7 and vice versa. These findings reveal that talinA is a major DdM7 binding partner and suggest that their interaction induces a conformational change in each that, in combination with membrane receptor binding, promotes the assembly of a high avidity receptor complex essential for adhesion of the cell to substrata.A diverse range of cellular movements is driven by members of the myosin superfamily (1). Myosins generate movement along actin filaments in an ATP-dependent manner and are characterized by the presence of a conserved motor domain, typically located at the N terminus, and a highly divergent C-terminal tail region. The class VII myosins (M7) 1 are among the most widely expressed of the myosin family members, having been identified in organisms ranging from Dictyostelium to human. The M7 tail domain contains a tandem repeat of two MyTH4/FERM domains separated by an SH3 domain. In many cases, the tail begins with a region of predicted coiledcoil. In vertebrates, M7a is expressed almost exclusively in cells that possess specialized actin structures, most notably in the sensory hair cells of the ear in mice and zebrafish (2, 3). Loss of M7 function results in significant defects in all organisms where examined (3-6). Mouse and zebrafish M7a mutants have defects in hearing that are due to disorganization of the actin-filled stereocilia of critical sensory cells (3, 7). Consistent with these observations, M7a is localized to regions of the stereocilia where links between adjacent stereocilia are formed and current evidence suggests that M7a is critical for providing a connection between the actin cytoskeleton and these extracellular links via interaction with vezatin and harmonin, proteins that interact directly or indirectly with cadherins (8, 9).The lower eukaryote Dictyostelium discoideum expresses a single M7, DdM7, an initially surprising finding given that this organism does not possess any highly specialized actin-based structures. DdM7 is localized at the plasma membrane where it is transiently found in actively extending actin-rich regions of the cell, such as the leading edge and filopodial tips. Dictyostelium lacking DdM7 adhere poorly to surfaces, lack calciumdependent cell-cell adhesion, are defec...

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Identification and immunolocalization of cytoplasmic dynein in dictyostelium

Cited by 73 publications

References 48 publications

The Localization of Inner Centromeric Protein (INCENP) at the Cleavage Furrow Is Dependent on Kif12 and Involves Interactions of the N Terminus of INCENP with the Actin Cytoskeleton

The Localization of Inner Centromeric Protein (INCENP) at the Cleavage Furrow Is Dependent on Kif12 and Involves Interactions of the N Terminus of INCENP with the Actin Cytoskeleton

Contractile Ring-independent Localization of DdINCENP, a Protein Important for Spindle Stability and Cytokinesis

Identification of a Myosin VII-Talin Complex

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