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NuMA localization, stability, and function in spindle orientation involve 4.1 and Cdk1 interactions.

Seldin L, Poulson ND, Foote HP, Lechler T - Mol. Biol. Cell (2013)

Bottom Line: This is functionally important, as loss of 4.1/NuMA interaction results in spindle orientation defects, using two distinct assays.Inhibition of Cdk1 or mutation of a single residue in NuMA mimics this effect.This work highlights the complexity of NuMA localization and reveals the importance of NuMA cortical stability for productive force generation during spindle orientation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 Department of Dermatology, Duke University Medical Center, Durham, NC 27710.

ABSTRACT
The epidermis is a multilayered epithelium that requires asymmetric divisions for stratification. A conserved cortical protein complex, including LGN, nuclear mitotic apparatus (NuMA), and dynein/dynactin, plays a key role in establishing proper spindle orientation during asymmetric divisions. The requirements for the cortical recruitment of these proteins, however, remain unclear. In this work, we show that NuMA is required to recruit dynactin to the cell cortex of keratinocytes. NuMA's cortical recruitment requires LGN; however, LGN interactions are not sufficient for this localization. Using fluorescence recovery after photobleaching, we find that the 4.1-binding domain of NuMA is important for stabilizing its interaction with the cell cortex. This is functionally important, as loss of 4.1/NuMA interaction results in spindle orientation defects, using two distinct assays. Furthermore, we observe an increase in cortical NuMA localization as cells enter anaphase. Inhibition of Cdk1 or mutation of a single residue in NuMA mimics this effect. NuMA's anaphase localization is independent of LGN and 4.1 interactions, revealing two distinct mechanisms responsible for NuMA cortical recruitment at different stages of mitosis. This work highlights the complexity of NuMA localization and reveals the importance of NuMA cortical stability for productive force generation during spindle orientation.

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LGN binding is necessary but not sufficient for cortical NuMA recruitment, which may require association with 4.1. (A) The characterized binding regions within the NuMA protein. (B, C) Immunofluorescence analysis of endogenous NuMA localization in wild-type (WT) and LGN-knockdown keratinocytes. (D) Localization of GFP-tagged NuMA lacking the LGN-binding domain (ΔLGN BD-GFP) in wild-type cells. (E) Quantitation of NuMA cortical localization in WT and LGN-knockdown cells. n = 50 cells, p < 0.001. (F) Quantitation of cortical NuMA-GFP and NuMAΔLGN-BD-GFP localization. n = 25 cells, p < 0.0001. (G–J) Various truncation constructs of NuMA (see Construct column) tagged to GFP were transfected into wild-type cells. The amino acids spanned in each construct are specified in the Construct column. Cells were stained for endogenous LGN, and subsequent immunofluorescence analysis was performed to compare localization of these constructs with respect to cortical LGN. The Cortical column indicates whether cortical localization was detected for each construct (+, presence in; –, absence from cortex). (K) Quantitation of cortical localization of NuMA deletion constructs, as indicated. n = 25 cells for each, p < 0.0001 when comparing the 4.1-LGN BD to either the LGN BD or Δ4.1-MT BD. Scale bars, 10 μm. (L) Immunoprecipitation of GFP-tagged LGN-BD– and 4.1-LGN BD–transfected keratinocytes. Lysates were probed with anti-HA antibodies to detect associated LGN-HA. Middle blot, amounts of GFP fusion proteins in the immunoprecipitates; bottom, levels of LGN-HA in the lysates.
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Figure 2: LGN binding is necessary but not sufficient for cortical NuMA recruitment, which may require association with 4.1. (A) The characterized binding regions within the NuMA protein. (B, C) Immunofluorescence analysis of endogenous NuMA localization in wild-type (WT) and LGN-knockdown keratinocytes. (D) Localization of GFP-tagged NuMA lacking the LGN-binding domain (ΔLGN BD-GFP) in wild-type cells. (E) Quantitation of NuMA cortical localization in WT and LGN-knockdown cells. n = 50 cells, p < 0.001. (F) Quantitation of cortical NuMA-GFP and NuMAΔLGN-BD-GFP localization. n = 25 cells, p < 0.0001. (G–J) Various truncation constructs of NuMA (see Construct column) tagged to GFP were transfected into wild-type cells. The amino acids spanned in each construct are specified in the Construct column. Cells were stained for endogenous LGN, and subsequent immunofluorescence analysis was performed to compare localization of these constructs with respect to cortical LGN. The Cortical column indicates whether cortical localization was detected for each construct (+, presence in; –, absence from cortex). (K) Quantitation of cortical localization of NuMA deletion constructs, as indicated. n = 25 cells for each, p < 0.0001 when comparing the 4.1-LGN BD to either the LGN BD or Δ4.1-MT BD. Scale bars, 10 μm. (L) Immunoprecipitation of GFP-tagged LGN-BD– and 4.1-LGN BD–transfected keratinocytes. Lysates were probed with anti-HA antibodies to detect associated LGN-HA. Middle blot, amounts of GFP fusion proteins in the immunoprecipitates; bottom, levels of LGN-HA in the lysates.

Mentions: In addition to the cytoskeleton, other protein–protein interactions are required for cortical NuMA localization. NuMA has a number of motifs that mediate direct interactions with other proteins (Figure 2A). The best-characterized of these is LGN, which is essential for NuMA localization in a wide array of cell types, including keratinocytes (Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006; Williams et al., 2011). We first verified these findings using a previously published LGN short hairpin RNA (shRNA)–knockdown approach (Williams et al., 2011). LGN levels were greatly reduced in knockdown cells as compared with controls (Supplemental Figure S1B). In keratinocytes, NuMA was clearly and specifically lost at the cell cortex upon LGN knockdown; however, its spindle pole localization was not affected (Figure 2, C and E). Consistent with this result, a NuMA mutant lacking the LGN-binding domain (LGN BD) was also unable to localize to the cell cortex, despite its successful recruitment to spindle poles (Figure 2, D and F).


NuMA localization, stability, and function in spindle orientation involve 4.1 and Cdk1 interactions.

Seldin L, Poulson ND, Foote HP, Lechler T - Mol. Biol. Cell (2013)

LGN binding is necessary but not sufficient for cortical NuMA recruitment, which may require association with 4.1. (A) The characterized binding regions within the NuMA protein. (B, C) Immunofluorescence analysis of endogenous NuMA localization in wild-type (WT) and LGN-knockdown keratinocytes. (D) Localization of GFP-tagged NuMA lacking the LGN-binding domain (ΔLGN BD-GFP) in wild-type cells. (E) Quantitation of NuMA cortical localization in WT and LGN-knockdown cells. n = 50 cells, p < 0.001. (F) Quantitation of cortical NuMA-GFP and NuMAΔLGN-BD-GFP localization. n = 25 cells, p < 0.0001. (G–J) Various truncation constructs of NuMA (see Construct column) tagged to GFP were transfected into wild-type cells. The amino acids spanned in each construct are specified in the Construct column. Cells were stained for endogenous LGN, and subsequent immunofluorescence analysis was performed to compare localization of these constructs with respect to cortical LGN. The Cortical column indicates whether cortical localization was detected for each construct (+, presence in; –, absence from cortex). (K) Quantitation of cortical localization of NuMA deletion constructs, as indicated. n = 25 cells for each, p < 0.0001 when comparing the 4.1-LGN BD to either the LGN BD or Δ4.1-MT BD. Scale bars, 10 μm. (L) Immunoprecipitation of GFP-tagged LGN-BD– and 4.1-LGN BD–transfected keratinocytes. Lysates were probed with anti-HA antibodies to detect associated LGN-HA. Middle blot, amounts of GFP fusion proteins in the immunoprecipitates; bottom, levels of LGN-HA in the lysates.
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Related In: Results  -  Collection

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Figure 2: LGN binding is necessary but not sufficient for cortical NuMA recruitment, which may require association with 4.1. (A) The characterized binding regions within the NuMA protein. (B, C) Immunofluorescence analysis of endogenous NuMA localization in wild-type (WT) and LGN-knockdown keratinocytes. (D) Localization of GFP-tagged NuMA lacking the LGN-binding domain (ΔLGN BD-GFP) in wild-type cells. (E) Quantitation of NuMA cortical localization in WT and LGN-knockdown cells. n = 50 cells, p < 0.001. (F) Quantitation of cortical NuMA-GFP and NuMAΔLGN-BD-GFP localization. n = 25 cells, p < 0.0001. (G–J) Various truncation constructs of NuMA (see Construct column) tagged to GFP were transfected into wild-type cells. The amino acids spanned in each construct are specified in the Construct column. Cells were stained for endogenous LGN, and subsequent immunofluorescence analysis was performed to compare localization of these constructs with respect to cortical LGN. The Cortical column indicates whether cortical localization was detected for each construct (+, presence in; –, absence from cortex). (K) Quantitation of cortical localization of NuMA deletion constructs, as indicated. n = 25 cells for each, p < 0.0001 when comparing the 4.1-LGN BD to either the LGN BD or Δ4.1-MT BD. Scale bars, 10 μm. (L) Immunoprecipitation of GFP-tagged LGN-BD– and 4.1-LGN BD–transfected keratinocytes. Lysates were probed with anti-HA antibodies to detect associated LGN-HA. Middle blot, amounts of GFP fusion proteins in the immunoprecipitates; bottom, levels of LGN-HA in the lysates.
Mentions: In addition to the cytoskeleton, other protein–protein interactions are required for cortical NuMA localization. NuMA has a number of motifs that mediate direct interactions with other proteins (Figure 2A). The best-characterized of these is LGN, which is essential for NuMA localization in a wide array of cell types, including keratinocytes (Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006; Williams et al., 2011). We first verified these findings using a previously published LGN short hairpin RNA (shRNA)–knockdown approach (Williams et al., 2011). LGN levels were greatly reduced in knockdown cells as compared with controls (Supplemental Figure S1B). In keratinocytes, NuMA was clearly and specifically lost at the cell cortex upon LGN knockdown; however, its spindle pole localization was not affected (Figure 2, C and E). Consistent with this result, a NuMA mutant lacking the LGN-binding domain (LGN BD) was also unable to localize to the cell cortex, despite its successful recruitment to spindle poles (Figure 2, D and F).

Bottom Line: This is functionally important, as loss of 4.1/NuMA interaction results in spindle orientation defects, using two distinct assays.Inhibition of Cdk1 or mutation of a single residue in NuMA mimics this effect.This work highlights the complexity of NuMA localization and reveals the importance of NuMA cortical stability for productive force generation during spindle orientation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 Department of Dermatology, Duke University Medical Center, Durham, NC 27710.

ABSTRACT
The epidermis is a multilayered epithelium that requires asymmetric divisions for stratification. A conserved cortical protein complex, including LGN, nuclear mitotic apparatus (NuMA), and dynein/dynactin, plays a key role in establishing proper spindle orientation during asymmetric divisions. The requirements for the cortical recruitment of these proteins, however, remain unclear. In this work, we show that NuMA is required to recruit dynactin to the cell cortex of keratinocytes. NuMA's cortical recruitment requires LGN; however, LGN interactions are not sufficient for this localization. Using fluorescence recovery after photobleaching, we find that the 4.1-binding domain of NuMA is important for stabilizing its interaction with the cell cortex. This is functionally important, as loss of 4.1/NuMA interaction results in spindle orientation defects, using two distinct assays. Furthermore, we observe an increase in cortical NuMA localization as cells enter anaphase. Inhibition of Cdk1 or mutation of a single residue in NuMA mimics this effect. NuMA's anaphase localization is independent of LGN and 4.1 interactions, revealing two distinct mechanisms responsible for NuMA cortical recruitment at different stages of mitosis. This work highlights the complexity of NuMA localization and reveals the importance of NuMA cortical stability for productive force generation during spindle orientation.

Show MeSH
Related in: MedlinePlus