The Par3-like polarity protein Par3L is essential for mammary stem cell maintenance - PubMed (original) (raw)
The Par3-like polarity protein Par3L is essential for mammary stem cell maintenance
Yongliang Huo et al. Nat Cell Biol. 2014 Jun.
Erratum in
- Nat Cell Biol. 2014 Jul;16(7):716
Abstract
The Par polarity proteins play key roles in asymmetric division of Drosophila melanogaster stem cells; however, whether the same mechanisms control stem cells in mammals is controversial. Although necessary for mammary gland morphogenesis, Par3 is not essential for mammary stem cell function. We discovered that, instead, a previously uncharacterized protein, Par3-like (Par3L), is vital for mammary gland stem cell maintenance. Par3L function has been mysterious because, unlike Par3, it does not interact with atypical protein kinase C or the Par6 polarity protein. We found that Par3L is expressed by multipotent stem cells in the terminal end buds of murine mammary glands. Ablation of Par3L resulted in rapid and profound stem cell loss. Unexpectedly, Par3L, but not Par3, binds to the tumour suppressor protein Lkb1 and inhibits its kinase activity. This interaction is key for the function of Par3L in mammary stem cell maintenance. Our data reveal insights into a link between cell polarity proteins and stem cell survival, and uncover a biological function for Par3L.
Figures
Figure 1
Par3L is required for mammary gland regeneration. (a, b) immuno-staining shows that Par3L is expressed specifically at the apical junctions of luminal epithelial cells (marked with CK8, red) in mature mammary ducts and in cap cells of the terminal end buds of developing mammary glands. (c) Par3L depletion results in abnormal terminal end bud morphology. (d) Carmine alum staining of whole mammary glands shows that Par3L depletion impairs mammary gland regeneration (e) quantification of regeneration, as % of fat pads showing any ductal outgrowth. (f) depletion of Par3L significantly reduces the extent of outgrowth into the fat pads. This phenotype could be reversed by over-expression of human Par3L, which is resistant to the Par3L shRNAs. (g) partial depletion of Par3L by shPar3L#1 significantly reduces mammary ductal tree side branching. This phenotype could be rescued by over-expression of human Par3L. Graph shows mean ± SD. n represents the number of mammary glands examined in each experiment. Statistical significance was calculated for each experimental group compared to the shLuc control group; p values were calculated by the Student t test.
Figure 2
Par3L is essential for mammary gland stem cell maintenance. (a) Lentivirus-transduced (GFP+) colonies grown from single cells in Matrigel, first passage. (b) serial colony-forming assay shows that Par3L depletion decreases the number of colony forming cells. Graph shows mean ± SD for 3 experimental replicates (independent transductions). (c) competition assay demonstrates that Par3L depletion impairs MRU maintenance in a cell autonomous manner. Equal numbers of red (control) and green (shPar3, top panel, or control, bottom panel) were mixed, injected into cleared fat pads and allowed to regenerate for 6 weeks.
Figure 3
Loss of Par3L causes mammary stem cell loss through apoptosis and/or failure of self-renewal. (a) representative images of the staining of tertiary colonies. Par3L depletion decreases the percentage of CK14+CK8+ double positive cells, increased cleave Caspase-3+ cells, but does not change the Ki67+ cell ratio. (b to d) Quantification of Ki67+ cells,(b), cleaved Caspase-3+ cells (c), and CK14+CK8+ cells, (d). n represents the number of colonies scored. Original source data for (c) is provided in Supplementary Table 1. (e) Staining of mature mammary gland ducts regenerated from mammary cells expressing shPar3L#1 or shLuc control. The ducts have normal myoepithelial and luminal epithelial organization. Partial depletion of Par3L has no effects on cleaved Caspase-3 or Ki67 staining in the ducts. (f) Quantification of Ki67 and cleaved Caspase-3 staining in the mammary gland ducts. Graph shows mean ± SEM. n represents the number of mammary glands that were scored. Statistical significance was calculated for each experimental group compared to the shLuc control group. p values were calculated by Student t test.
Figure 4
Par3L is necessary for colony forming activity by s-SHIP GFP+ cells. GFP+ cells from 6 week-old s-SHIP GFP transgenic mice were FACS sorted to homogeneity and infected by mApple-shLuc control or mApple-shPar3L#2 virus. (a, d) serial colony-forming assay shows that Par3L depletion decreases the number of colony forming cells. All mApple-positive colonies were counted and presented in the bar graph. Graph represents mean ± SD for 3 technical replicates (independent transductions of GFP+ cells from a single mouse). Original source data provided in Supplementary Table 1. (b) representative images of the staining of tertiary colonies for CK8, CK14. (c) staining for cleaved Caspase-3. (e) the ratio of GFP+ colonies over mApple positive colonies indicates shPar3L#2 over-expression causes GFP+ stem cell loss. Graph represents mean ± SD for 3 technical replicates (independent transductions of GFP+ cells from a single mouse). (f) Quantification of GFP, mApple, and CK8 triple-positive or GFP, mApple, and CK14 triple-positive cells. Par3L depletion significantly decreases the number of CK14 positive cells but not CK8 positive cells. Error bars represent mean ± SEM (n shown on figure). Statistical significance was calculated for each experimental group compared to the shLuc control group. p values were calculated by Student t test. (g) Quantification of cleaved Caspase-3+ cells. Par3L depletion significantly increased cleaved Caspase-3+ cells. Graph shows mean ± SEM. n represents the number of colonies that were scored. Statistical significance was calculated for ShPar3L#2 compared to the shLuc control group. p values were calculated by Student t test.
Figure 5
Par3L maintains mammary stem cells through interaction with LKB1. (a) Par3L interacts with LKB1 but not aPKC; while Par3 interacts with aPKC, but not LKB1. About 2% of LKB1 was captured by Par3L while only ~0.02% LKB1 precipitated with Par3. (b) The endogenous interaction between Par3L and LKB1 was confirmed by co-immunoprecipitation using mouse kidney lysates, which express relatively high levels of Par3L. (c) The region on Par3L required for LKB1 binding was mapped between PDZ2 and PDZ3 domains. Replacement of this region of Par3L with the corresponding region of Par3 disrupts the interaction between LKB1 and Par3L. All full scan images are presented in supplementary figure 6. (d, e) Mammary gland regeneration assay. Wild type human Par3L can rescue mammary gland regeneration from Par3L-depleted mammary cells, while the LKB1 binding-deficient Par3L mutant is unable to do so. (f) representative images of the staining of tertiary colonies for CK8, CK14, and cleaved Caspase-3. (g, h) quantification of CK14+CK8+ dual-positive (g) and cleaved Caspase-3+ (h) cells in in vitro serial colony forming assay. Wild type human Par3L, but not the mutant Par3L, was able to reverse the Par3L depletion effects. Graph shows mean ± SEM. n represents the number of colonies that were scored. Statistical significance was calculated for each experimental group compared to the shLuc control group. p values were calculated by Student t test.
Figure 6
Par3L maintains mammary stem cells by suppressing LKB1 activity. (a) Par3L inhibits LKB1 activity in HEK293T cells. Wild type or mutant Par3L was over-expressed in HEK293T cells and phospho-AMPK was blotted as an indicator for LKB1 activity. The full scan images are presented in supplementary figure 6. (b) Wild type Par3L reduced the phospho-AMPK level to about 40% of the control level, while mutant Par3L reduced phospho-AMPK level to about 70% of the control level. Graph shows means and ± SEM for 3 independent experiments.. p values were calculated by paired Student t test. (c) mammary cells from female C3H mice were infected by vector control or LKB1-2A-Stradα polycistronic expression virus and subjected to the serial colony-forming assay. (d) LKB1 over-expression decreases the number of colony forming cells. Graph shows mean ± SD for 3 experimental replicates (independent transductions). Original source data provided in Supplementary Table 1. (e) representative images of the staining of tertiary colonies for CK8, CK14, and cleaved Caspase-3. (f) Quantification of cleaved Caspase-3+ cells in the in vitro serial colony-forming assay. LKB1 over-expression significantly increases cleaved Caspase-3+ cells comparing to the vector control group. Graphs show mean ± SEM. n represents the number of colonies that were scored. Statistical significance was calculated for each experimental group compared to the vector control group. p values were calculated by unpaired Student t test.
References
- Knoblich JA. Mechanisms of asymmetric stem cell division. Cell. 2008;132:583–597. - PubMed
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