Nuclear Localization of C-Terminal Domains of the Kinesin-like Protein MKLP-1

doi:10.1006/bbrc.1999.0952

Biochemical and Biophysical Research Communications

Volume 260, Issue 3, 14 July 1999, Pages 605-608

https://doi.org/10.1006/bbrc.1999.0952 Get rights and content

Abstract

The successful execution of mitosis in mammalian cells requires the activities of numerous kinesin-like proteins. The $M$ itotic $K$ inesin- $L$ ike $P$ rotein- $1$ (MKLP-1) localizes to the spindle equator and is believed to participate in the separation of spindle poles during anaphase B. Injection of antibodies against MKLP-1 into dividing cells results in cell cycle arrest, suggesting that MKLP-1 is essential for mitosis. To further characterize MKLP-1, constructs encoding C-terminal domains of MKLP-1 were expressed as fusions to the green fluorescent protein and localized in HeLa cells. All constructs localized to the nucleus indicating the presence of at least one nuclear localization sequence in the C-terminus of the protein. C-terminal domains of MKLP-1 expressed in insect cells also localized to the nucleus as shown by subcellular fractionation. These proteins remained tightly associated with the nucleus following both detergent and salt extraction, suggesting a tight interaction with a component of the nucleus.

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Citation Excerpt :
This reduction of the apical membrane is accomplished through release of the midbody after cellular divisions, along with extraneous membrane components, into the developing ventricle (Marzesco et al., 2005). The mitotic kinesin-like protein (MKLP-1) has been shown to be required for formation of the midbody in mammalian cells, as well as being required for cytokinesis (Liu and Erikson, 2007; Deavours and Walker, 1999; Matuliene and Kuriyama, 2002; Kuriyama et al., 2002), for these reasons we used MKLP to label midbody particles. hESC neural differentiation protocols have been extensively developed and characterized by many groups, and the protocols range from long term differentiations using stromal feeders and exogenous factors, such as BMP4 (Nat et al., 2007; Pankratz et al., 2007; Dhara et al., 2008; Schulz et al., 2003) to a short term protocol (Bajpai et al., 2009; Curchoe et al., 2010; Cimadamore et al., 2009), previously characterized by our group, that can yield multi-potent neural stem cells that could be appropriate for use in cell based therapies due to the elimination of animal components and expensive exogenous factors.
The in vitro neuralization of hESCs has been widely used to generate central and peripheral nervous system components from neural precursors (Bajpai et al., 2009; Curchoe et al., 2010), most often through an intermediate “rosette” stage. Here we confirm that hESC derived neuro-epithelial rosettes express many characteristics of the developing embryonic neural plate (Aaku-Saraste et al., 1996), characterized by expression of the tight junction proteins ZO-1 and N-Cadherin. Moreover, neuro-epithelial rosettes display a characteristic acetylated alpha tubulin cytoskeletal arrangement (similar to that observed in the developing embryonic neural plate) (Bhattacharyya et al., 1994).
Demonstrated here for the first time MKLP was observed in a hESC model system. We found MKLP expression in small particles in between mitotic spindles, large particles aggregating in the lumen of neuroepithelial rosettes, and we did not observe MKLP in the nucleus of hESC derived neural precursors as previously described in the HeLa cell line. We observed MKLP + particles in aggregations in the lumen of “early” rosette structures. Furthermore, we observed that MKLP⁺ particle aggregations can also be lost from the lumens of hESC derived neuro-epithelial rosettes, similar to a phenomenon observed in the developing neural tube in vivo (Marzesco et al., 2005). We determined that this loss of MKLP⁺ particles occurs from “late” as opposed to “early” stage neuro-epithelial rosettes (characterized by junction type).
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Regular ArticleNuclear Localization of C-Terminal Domains of the Kinesin-like Protein MKLP-1

Abstract

Cell

Curr. Biol.

Proc. Natl. Acad. Sci. USA

Nature

J. Cell Biol.

Regular Article
Nuclear Localization of C-Terminal Domains of the Kinesin-like Protein MKLP-1