Biochemical and Biophysical Research Communications
Galpha/LGN-mediated asymmetric spindle positioning does not lead to unequal cleavage of the mother cell in 3-D cultured MDCK cells
Highlights
► Apical targeting of Gαi1/LGN leads to asymmetric spindle positioning in 3-D cultured MDCK cells. ► We established a system where asymmetric spindle positioning can be consistently induced. ► Live cell time-lapse analysis of cell division in 3-D cultured MDCK cells. ► Asymmetric spindle positioning does not lead to the generation of unequal sized daughter cells.
Introduction
Cells propagate their progenies through cell division. To generate identical daughter cells, the mother cells divide symmetrically by positioning the mitotic spindle at the center of the cell. The mitotic spindle then directs central cleavage furrow formation which leads to the production of two equal sized daughter cells that inherit identical set of genetic materials and cytoplasmic components. Another type of cell division, called asymmetric cell division, is usually utilized by stem/progenitor cells to generate different daughter cells with distinct cell fates [1]. Asymmetric cell division usually results in the generation of unequal sized daughter cells.
It is generally believed that the position of mitotic spindle dictates the relative size of the daughter cells: a centrally located spindle leads to an equal cleavage, whereas an eccentrically located spindle results in an unequal cleavage, therefore generates unequal sized daughter cells [2], [3], [4]. Spindle positioning is achieved through interactions between astral microtubules and the cell cortex [5]. Recent studies suggest that receptor-independent G-protein signaling is a major regulator of spindle positioning in model systems including Caenorhabditis elegans and Drosophila [3], [6]. Cortical Gα and its binding partner GPR-1/2 (in C. elegans) or Pins (in Drosophila) are the key players for generating asymmetric spindle positioning and subsequent unequal sized daughter cells [6]. In C. elegans zygote, Gα and GPR-1/2 are enriched at the posterior cell cortex where they serve as force generators, probably through cytoplasmic dynein, to produce stronger pulling forces on astral microtubules and lead to spindle displacement towards this side of the cell [7], [8], [9]. Such asymmetric spindle positioning leads to the production of a larger anterior and a smaller posterior daughter cell. In Drosophila neuroblast, apically localized Gα and Pins are required for spindle positioning and the generation of unequal sized daughters – a larger apical daughter cell that remains to be a neuroblast and a smaller basal ganglion mother cell [10], [11], [12], [13]. In mammalian system, Gα and LGN (mammalian homologue of Pins) are also involved in regulating spindle orientation during neurogenesis, epidermal differentiation, epithelial morphogenesis and dermomyotome development [14], [15], [16], [17], [18], [19], [20]. However, whether Gα/LGN complex can direct asymmetric spindle positioning and asymmetric cleavage of the mother cell in mammalian system is not known.
Using an apically located protein Crumb3 (Crb3) as a vehicle, we have successfully targeted ectopically expressed Gαi1 and endogenous LGN to the apical cell membrane in 3-D cultured MDCK epithelial cells [20]. Here, we report that the apical Gαi1/LGN complex not only redirects the mitotic spindle orientation from perpendicular to parallel to the apical–basal axis, but also consistently positioned the metaphase spindle asymmetrically toward the apical cell membrane. Such a reproducible and biologically relevant system allowed us to test whether asymmetric spindle positioning leads to the generation of unequal sized daughter cells. By analyzing fixed cysts samples and live cell imaging of the cell division process, surprisingly, we found that Gαi1/LGN-mediated asymmetric spindle positioning does not result in asymmetric cleavage of the mother cell; instead, the mother cell eventually generates two equal sized daughter cells. Our findings challenge the general opinion that asymmetric spindle positioning leads to unequal sized daughter cells.
Section snippets
Antibodies
The following antibodies were used: mouse anti-α-tubulin (Sigma–Aldrich), rabbit anti-γ-tubulin (Invitrogen), mouse anti-β-catenin (BD); and secondary Alexa 488, Alexa 594, Alexa 680 (Invitrogen), and IRDye800 (Rockland) conjugated goat anti-mouse or rabbit antibodies. Hoechst 33342 (Invitrogen) was used for DNA staining.
Cell culture and stable cell lines
MDCK cells were cultured in DMEM supplemented with 10% fetal calf serum and penicillin/streptomycin (100 IU/ml and 100 mg/ml, respectively) at 37 °C in a humidified 5% CO2
Apical targeting of Gαi1/LGN leads to asymmetric spindle positioning during cystogenesis of MDCK cells
Organotypic 3-D culture of MDCK cells phenocopies many aspects of in vivo cystogenesis and has been used as a model system to study epithelial morphogenesis in vitro. Using such a system, we showed previously that LGN plays a critical role in directing spindle orientation during epithelial morphogenesis [20]. During cystogenesis, atypical protein kinase C-mediated apical exclusion leads to restricted lateral cortical localization of LGN which directs spindle orientation to be perpendicular to
Discussion
Mechanisms of asymmetric spindle positioning and cell division have been intensely studied in the C. elegans zygote and Drosophila neuroblast [1], [3], [22]. Although most of the identified molecules that are involved in the regulation of asymmetric cell division are conserved in the two systems, there are obvious differences between the two types of cells in the ways of producing unequal sized daughter cells. In Drosophila neuroblast, asymmetric cell division involves differential spindle
Acknowledgments
We thank Dr. Daniel Gerlich for providing the pmCherry-α-tubulin-IRES-puro2 plasmid. This work was supported by grants from National Institutes of Health (GM079506) and American Cancer Society (RSG0717601CSM) to Q. Du.
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