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1.  Inscuteable Regulates the Pins-Mud Spindle Orientation Pathway 
PLoS ONE  2012;7(1):e29611.
During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud).
PMCID: PMC3254608  PMID: 22253744
2.  Deletion of Atbf1/Zfhx3 In Mouse Prostate Causes Neoplastic Lesions, Likely by Attenuation of Membrane and Secretory Proteins and Multiple Signaling Pathways 
Neoplasia (New York, N.Y.)  2014;16(5):377-389.
The ATBF1/ZFHX3 gene at 16q22 is the second most frequently mutated gene in human prostate cancer and has reduced expression or mislocalization in several types of human tumors. Nonetheless, the hypothesis that ATBF1 has a tumor suppressor function in prostate cancer has not been tested. In this study, we examined the role of ATBF1 in prostatic carcinogenesis by specifically deleting Atbf1 in mouse prostatic epithelial cells. We also examined the effect of Atbf1 deletion on gene expression and signaling pathways in mouse prostates. Histopathologic analyses showed that Atbf1 deficiency caused hyperplasia and mouse prostatic intraepithelial neoplasia (mPIN) primarily in the dorsal prostate but also in other lobes. Hemizygous deletion of Atbf1 also increased the development of hyperplasia and mPIN, indicating a haploinsufficiency of Atbf1. The mPIN lesions expressed luminal cell markers and harbored molecular changes similar to those in human PIN and prostate cancer, including weaker expression of basal cell marker cytokeratin 5 (Ck5), cell adhesion protein E-cadherin, and the smooth muscle layer marker Sma; elevated expression of the oncoproteins phospho-Erk1/2, phospho-Akt and Muc1; and aberrant protein glycosylation. Gene expression profiling revealed a large number of genes that were dysregulated by Atbf1 deletion, particularly those that encode for secretory and cell membrane proteins. The four signaling networks that were most affected by Atbf1 deletion included those centered on Erk1/2 and IGF1, Akt and FSH, NF-κB and progesterone and β-estradiol. These findings provide in vivo evidence that ATBF1 is a tumor suppressor in the prostate, suggest that loss of Atbf1 contributes to tumorigenesis by dysregulating membrane and secretory proteins and multiple signaling pathways, and provide a new animal model for prostate cancer.
PMCID: PMC4198693  PMID: 24934715
3.  Wnt/β-Catenin activation promotes prostate tumor progression in a mouse model 
Oncogene  2010;30(16):1868-1879.
Our previous studies have found that activation of Wnt/β-Catenin signaling resulted in mouse prostatic intraepithelial neoplasia (mPIN). In the large probasin promoter directed SV40-Large T-antigen (LPB-Tag) expressing mouse prostate, mPIN forms with rare areas of adenocarcinoma. Combining expression of both Wnt-signaling and Tag expression in the mouse prostate, we have studied the role of Wnt/β-Catenin signaling in the progression from mPIN to adenocarcinoma. Our results show that the prostates of mice expressing Tag alone or nuclear β-Catenin alone developed mPIN while the activation of both Tag and the Wnt/β-Catenin pathway resulted in invasive prostate adenocarcinoma. Also, Foxa2, a forkhead transcription factor, was induced by active Wnt/β-Catenin signaling; and the expression of Foxa2 was associated with the invasive phenotype in the primary prostate cancer. In the LPB-Tag/dominant active (D.A.) β-Catenin prostates, MMP7, a Wnt/β-Catenin target gene, was up-regulated. Furthermore, we also assessed AR and AR signaling pathway in these LPB-Tag/D.A. β-Catenin mice. Although β-Catenin is a well known AR co-activator in vitro, our study provides strong in vivo evidences indicating that both AR protein and the AR pathway were down-regulated in the prostate of LPB-Tag/D.A. β-Catenin mice. Histological analysis shows that prostate sections derived from the LPB-Tag/D.A. β-Catenin mice display neuroendocrine differentiation (NED) but NE cancer does not develop. Together, our findings indicate that Wnt/β-Catenin signaling plays an important role in the progression of mPIN to prostate adenocarcinoma.
PMCID: PMC3081383  PMID: 21151173
Prostate; Wnt/β-Catenin; AR; Foxa2; T-antigen
4.  Caveolin-1 Upregulation Contributes to c-Myc–Induced High-Grade Prostatic Intraepithelial Neoplasia and Prostate Cancer 
Molecular cancer research : MCR  2011;10(2):218-229.
Previously we reported caveolin-1 (Cav-1) overexpression in prostate cancer (PCa) cells and demonstrated that it promotes PCa progression. Here, we report that Cav-1 was overexpressed in 41.7% (15 of 36) of high-grade prostatic intraepithelial neoplasia (HGPIN) specimens obtained during radical prostatectomies. Positive correlations exist between Cav-1–positive (Cav-1+) HGPIN and Cav-1+ primary PCa (rho = 0.655, P< 0.0001) and between Cav-1 and c-Myc expression in HGPIN (rho = 0.41, P = 0.032). To determine whether Cav-1 cooperates with c-Myc in development of premalignant lesions and PCa in vivo, we generated transgenic mice with c-Myc overexpression driven by the ARR2PB promoter. In this ARR2PB–c-myc model, Cav-1 overexpression was found in mouse PIN (mPIN) lesions and PCa cells and was associated with a significantly higher ratio of proliferative to apoptotic labeling in mPIN lesions than in the Cav-1–negative epithelia adjacent to those lesions (10.02 vs 4.34; P = 0.007). Cav-1 overexpression was also associated with increased levels of P-Akt and VEGF-A, which were previously associated with Cav-1–induced PCa cell survival and positive-feedback regulation of cellular Cav-1 levels, respectively. In multiple PCa cell lines, Cav-1 protein (but not mRNA) was induced by c-Myc transfection, whereas VEGF siRNA transfection abrogated c-Myc–induced Cav-1 overexpression, suggesting a c-Myc–VEGF–Cav-1 signaling axis. Overall, our results suggest that Cav-1 is associated with c-Myc in the development of HGPIN and PCa. Further, Cav-1 overexpression in HGPIN is potentially a biomarker for early identification of patients who tend to develop Cav-1+ primary PCa.
PMCID: PMC3908884  PMID: 22144662
Caveolin-1; c-Myc; prostatic intraepithelial neoplasia; prostate cancer; metaplasia
5.  Membrane Targeting and Asymmetric Localization of Drosophila Partner of Inscuteable Are Discrete Steps Controlled by Distinct Regions of the Protein 
Molecular and Cellular Biology  2002;22(12):4230-4240.
Asymmetric division of neural progenitors is a key mechanism by which neuronal diversity in the Drosophila central nervous system is generated. The distinct fates of the daughter cells derived from these divisions are achieved through preferential segregation of the cell fate determinants Prospero and Numb to one of the two daughters. This is achieved by coordinating apical and basal mitotic spindle orientation with the basal cortical localization of the cell fate determinants during mitosis. A complex of apically localized proteins, including Inscuteable (Insc), Partner of Inscuteable (Pins), Bazooka (Baz), DmPar-6, DaPKC, and Gαi, is required to mediate and coordinate basal protein localization with mitotic spindle orientation. Pins, a molecule which directly interacts with Insc, is a key component required for the integrity of this complex; in the absence of Pins, other components become mislocalized or destabilized, and basal protein localization and mitotic spindle orientation are defective. Here we define the functional domains of Pins. We show that the C-terminal region containing the Gαi binding GoLoco motifs is necessary and sufficient for targeting to the neuroblast cortex, which appears to be a prerequisite for apical localization of Pins. The N-terminal tetratricopeptide repeat-containing region of Pins is required for two processes; TPR repeats 1 to 3 plus the C-terminal region are required for apical localization but are insufficient to recruit Insc to the apical cortex, whereas TPR repeats 1 to 7 plus C-terminal Pins can perform both functions. Hence, the abilities of Pins to cortically localize, to apically localize, and to restore Insc apical localization are all separable, and all three capabilities are necessary to mediate asymmetric division. Moreover, the need for N-terminal Pins can be obviated by fusing a minimal Insc functional domain with the C-terminal region of Pins; this chimeric molecule is apically localized and can fulfill the functions of both Insc and Pins.
PMCID: PMC133846  PMID: 12024035
6.  Stromal Activation Associated with Development of Prostate Cancer in Prostate-Targeted Fibroblast Growth Factor 8b Transgenic Mice12 
Neoplasia (New York, N.Y.)  2010;12(11):915-927.
Expression of fibroblast growth factor 8 (FGF-8) is commonly increased in prostate cancer. Experimental studies have provided evidence that it plays a role in prostate tumorigenesis and tumor progression. To study how increased FGF-8 affects the prostate, we generated and analyzed transgenic (TG) mice expressing FGF-8b under the probasin promoter that targets expression to prostate epithelium. Prostates of the TG mice showed an increased size and changes in stromal and epithelialmorphology progressing fromatypia and prostatic intraepithelial neoplasia (mouse PIN, mPIN) lesions to tumors with highly variable phenotype bearing features of adenocarcinoma, carcinosarcoma, and sarcoma. The development of mPIN lesions was preceded by formation of activated stroma containing increased proportion of fibroblastic cells, rich vasculature, and inflammation. The association between advancing stromal and epithelial alterations was statistically significant. Microarray analysis and validation with quantitative polymerase chain reaction revealed that expression of osteopontin and connective tissue growth factor was markedly upregulated in TG mouse prostates compared with wild type prostates. Androgen receptor staining was decreased in transformed epithelium and in hypercellular stroma but strongly increased in the sarcoma-like lesions. In conclusion, our data demonstrate that disruption of FGF signaling pathways by increased epithelial production of FGF-8b leads to strongly activated and atypical stroma, which precedes development of mPIN lesions and prostate cancer with mixed features of adenocarcinoma and sarcoma in the prostates of TG mice. The results suggest that increased FGF-8 in human prostate may also contribute to prostate tumorigenesis by stromal activation.
PMCID: PMC2978914  PMID: 21076617
7.  Development of a Reactive Stroma Associated with Prostatic Intraepithelial Neoplasia in EAF2 Deficient Mice 
PLoS ONE  2013;8(11):e79542.
ELL-associated factor 2 (EAF2) is an androgen-responsive tumor suppressor frequently deleted in advanced prostate cancer that functions as a transcription elongation factor of RNA Pol II through interaction with the ELL family proteins. EAF2 knockout mice on a 129P2/OLA-C57BL/6J background developed late-onset lung adenocarcinoma, hepatocellular carcinoma, B-cell lymphoma and high-grade prostatic intraepithelial neoplasia. In order to further characterize the role of EAF2 in the development of prostatic defects, the effects of EAF2 loss were compared in different murine strains. In the current study, aged EAF2−/− mice on both the C57BL/6J and FVB/NJ backgrounds exhibited mPIN lesions as previously reported on a 129P2/OLA-C57BL/6J background. In contrast to the 129P2/OLA-C57BL/6J mixed genetic background, the mPIN lesions in C57BL/6J and FVB/NJ EAF2−/− mice were associated with stromal defects characteristic of a reactive stroma and a statistically significant increase in prostate microvessel density. Stromal inflammation and increased microvessel density was evident in EAF2-deficient mice on a pure C57BL/6J background at an early age and preceded the development of the histologic epithelial hyperplasia and neoplasia found in the prostates of older EAF2−/− animals. Mice deficient in EAF2 had an increased recovery rate and a decreased overall response to the effects of androgen deprivation. EAF2 expression in human cancer was significantly down-regulated and microvessel density was significantly increased compared to matched normal prostate tissue; furthermore EAF2 expression was negatively correlated with microvessel density. These results suggest that the EAF2 knockout mouse on the C57BL/6J and FVB/NJ genetic backgrounds provides a model of PIN lesions associated with an altered prostate microvasculature and reactive stromal compartment corresponding to that reported in human prostate tumors.
PMCID: PMC3832612  PMID: 24260246
8.  Conditional Expression of Human 15-Lipoxygenase-1 in Mouse Prostate Induces Prostatic Intraepithelial Neoplasia: The FLiMP Mouse Model1 
Neoplasia (New York, N.Y.)  2006;8(6):510-522.
The incidence and mortality of prostate cancer (PCa) vary greatly in different geographic regions, for which lifestyle factors, such as dietary fat intake, have been implicated. Human 15-lipoxygenase-1 (h15-LO-1), which metabolizes polyunsaturated fatty acids, is a highly regulated, tissue-specific, lipid-peroxidating enzyme that functions in physiological membrane remodeling and in the pathogenesis of atherosclerosis, inflammation, and carcinogenesis. We have shown that aberrant overexpression of 15-LO-1 occurs in human PCa, particularly high-grade PCa, and in high-grade prostatic intraepithelial neoplasia (HGPIN), and that the murine orthologue is increased in SV40-based genetically engineered mouse (GEM) models of PCa, such as LADY and TRansgenic Adenocarcinoma of Mouse Prostate. To further define the role of 15-LO-1 in prostate carcinogenesis, we established a novel GEM model with targeted overexpression of h15-LO-1 in the prostate [human fifteen lipoxygenase-1 in mouse prostate (FLiMP)]. We used a Cre- mediated and a loxP-mediated recombination strategy to target h15-LO-1 specifically to the prostate of C57BL/6 mice. Wild-type (wt), FLiMP+/-, and FLiMP+/+ mice aged 7 to 21, 24 to 28, and 35 weeks were characterized by histopathology, immunohistochemistry (IHC), and DNA/RNA and enzyme analyses. Compared to wt mice, h15-LO-1 enzyme activity was increased similarly in both homozygous FLiMP+/+ and hemizygous FLiMP+/- prostates. Dorsolateral and ventral prostates of FLiMP mice showed focal and progressive epithelial hyperplasia with nuclear atypia, indicative of the definition of mouse prostatic intraepithelial neoplasia (mPIN) according to the National Cancer Institute. These foci showed increased proliferation by Ki-67 IHC. No progression to invasive PCa was noted up to 35 weeks. By IHC, h15-LO-1 expression was limited to luminal epithelial cells, with increased expression in mPIN foci (similar to human HGPIN). In summary, targeted overexpression of h15-LO-1 (a gene overexpressed in human PCa and HGPIN) to mouse prostate is sufficient to promote epithelial proliferation and mPIN development. These results support 15-LO-1 as having a role in prostate tumor initiation and as an early target for dietary or other prevention strategies. The FLiMP mouse model should also be useful in crosses with other GEM models to further define the combinations of molecular alterations necessary for PCa progression.
PMCID: PMC1601466  PMID: 16820097
Transgenic; mouse model; genetically engineered mouse model; arachidonic acid; prostate cancer
9.  Id4 deficiency attenuates prostate development and promotes PIN-like lesions by regulating androgen receptor activity and expression of NKX3.1 and PTEN 
Molecular Cancer  2013;12:67.
Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. Id4 is expressed in the normal prostate where its expression is also regulated by androgens. In this study we investigated the effect of loss of Id4 (Id4-/-) on adult prostate morphology.
Histological analysis was performed on prostates from 6-8 weeks old Id4-/-, Id4+/- and Id4+/+ mice. Expression of Id1, Sox9, Myc, androgen receptor, Akt, p-Akt, Pten and Nkx3.1 was investigated by immunohistochemistry. Androgen receptor binding on NKX3.1 promoter was studied by chromatin immuno-precipitation. Id4 was either over-expressed or silenced in prostate cancer cell lines DU145 and LNCaP respectively followed by analysis of PTEN, NKX3.1 and Sox9 expression.
Id4-/- mice had smaller prostates with fewer tubules, smaller tubule diameters and subtle mPIN like lesions. Levels of androgen receptor were similar between wild type and Id4-/- prostate. Decreased NKX3.1 expression was in part due to decreased androgen receptor binding on NKX3.1 promoter in Id4-/- mice. The increase in the expression of Myc, Sox9, Id1, Ki67 and decrease in the expression of PTEN, Akt and phospho-AKT was associated with subtle mPIN like lesions in Id4-/- prostates. Finally, prostate cancer cell line models in which Id4 was either silenced or over-expressed confirmed that Id4 regulates NKX3.1, Sox9 and PTEN.
Our results suggest that loss of Id4 attenuates normal prostate development and promotes hyperplasia/dysplasia with subtle mPIN like lesions characterized by gain of Myc and Id1 and loss of Nkx3.1 and Pten expression. One of the mechanisms by which Id4 may regulate normal prostate development is through regulating androgen receptor binding to respective response elements such as those on NKX3.1 promoter. In spite of these complex alterations, large neoplastic lesions in Id4-/- prostates were not observed suggesting the possibility of mechanisms/pathways such as loss of Akt that could restrain the formation of significant pre-cancerous lesions.
PMCID: PMC3694449  PMID: 23786676
Id4; Prostate; Androgen receptor; NKX3.1; Akt; PTEN
10.  Disruption of PPARγ signaling results in mouse prostatic intraepithelial neoplasia involving active autophagy 
Cell death and differentiation  2009;17(3):469-481.
Peroxisome proliferator-activated receptor-gamma (PPARγ) regulates the interface between cellular lipid metabolism, redox status and organelle differentiation. Conditional prostatic epithelial knockout of PPARγ in mice resulted in focal hyperplasia which developed into mouse prostatic intraepithelial neoplasia (mPIN). The grade of PIN became more severe with time. Electron microscopy (EM) showed accumulated secondary lysosomes containing cellular organelles and debris suggestive of autophagy. Consistent with this analysis the autophagy marker LC3 was found to be upregulated in areas of PIN in PPARγ KO tissues. We selectively knocked down PPARγ2 isoform in wild-type mouse prostatic epithelial cells and examined the consequences of this in a tissue recombination model. Histopathologically grafted tissues resembled the conditional PPARγ KO mouse prostates. EM studies of PPARγ- and PPARγ2-deficient epithelial cells in vitro were suggestive of autophagy, consistent with the prostatic tissue analysis. This was confirmed by examining expression of beclin-1 and LC3. Gene expression profiling in PPARγ-/γ2-deficient cells indicated a major dysregulation of cell cycle control and metabolic signaling networks related to peroxisomal and lysosomal maturation, lipid oxidation and degradation. The putative autophagic phenotypes of PPARγ-deficient cells could be rescued by re-expression of either γ1 or γ2 isoform. We conclude that disruption of PPARγ signaling results in autophagy and oxidative stress during mPIN pathogenesis.
PMCID: PMC2821953  PMID: 19834493
Peroxisome proliferator-activated receptor gamma (γ), PPARγ; Peroxisome proliferator response element, PPRE; Peroxisome; Lysosome; Mitochondria; Autophagy; Fatty acid oxidation; Oxidative Stress; Differentiation; Prostatic Intraepithelial Neoplasia, PIN
11.  Distinct roles of Gαi and Gβ13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions 
The Journal of Cell Biology  2003;162(4):623-633.
The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprising of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Gαi; in addition, Gβ13F is also required. However, the role of Gαi and the hierarchical relationship between the G protein subunits and apical components are not well defined. Here we describe the isolation of Gαi mutants and show that Gαi and Gβ13F play distinct roles. Gαi is required for Pins to localize to the cortex, and the effects of loss of Gαi or pins are highly similar, supporting the idea that Pins/Gαi act together to mediate various aspects of neuroblast asymmetric division. In contrast, Gβ13F appears to regulate the asymmetric localization/stability of all apical components, and Gβ13F loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, our results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts.
PMCID: PMC2173805  PMID: 12925708
neuroblast; asymmetric division; astral microtubules; heterotrimeric G proteins; Drosophila
12.  Emergence of tissue polarization from synergy of intracellular and extracellular auxin signaling 
Here, we provide a novel mechanistic framework for cell polarization during auxin-driven plant development that combines intracellular auxin signaling for regulation of expression of PINFORMED (PIN) auxin efflux transporters and the theoretical assumption of extracellular auxin signaling for regulation of PIN subcellular dynamics.The competitive utilization of auxin signaling component in the apoplast might account for the elusive mechanism for cell-to-cell communication for tissue polarization.Computer model simulations faithfully and robustly recapitulate experimentally observed patterns of tissue polarity and asymmetric auxin distribution during formation and regeneration of vascular systems, and during the competitive regulation of shoot branching by apical dominance.Our model generated new predictions that could be experimentally validated, highlighting a mechanistically conceivable explanation for the PIN polarization and canalization of the auxin flow in plants.
A key question of developmental biology relates to a fundamental issue in cell and tissue polarities, namely, how an individual cell in a polarized tissue senses the polarities of its neighbors and its position within tissue. In plant development, this issue is of pronounced importance, because plants have a remarkable ability to redefine cell and tissue polarities in different developmental programs, such as embryogenesis, postembryonic organogenesis, vascular tissue formation, and tissue regeneration (Kleine-Vehn and Friml, 2008).
A polar, cell-to-cell transport of the small signaling molecule auxin in conjunction with local auxin biosynthesis determines auxin gradients during embryonic and postembryonic development, giving positional cues for primordia formation, organ patterning, and tropistic growth (Friml et al, 2002; Benková et al, 2003; Reinhardt et al, 2003; Heisler et al, 2005; Scarpella et al, 2006; Dubrovsky et al, 2008). Over the past decades, theoretical models proposed that auxin acts as a polarizing cue in the center of a positive feedback mechanisms for auxin transport that has a key role in synchronized polarity rearrangements. However, the mechanistic basis for such a feedback loop between auxin and its own transport remains to a large extent elusive.
The direction of auxin transport largely depends on the polar subcellular localization of PINFORMED (PIN) proteins at the plasma membrane (Petrášek et al, 2006; Wiśniewska et al, 2006). These proteins recycle between the plasma membrane and intracellular endosomal compartments (Geldner et al, 2001; Dhonukshe et al, 2007), and their recycling modulates PIN-dependent auxin efflux rates and enable rapid changes in PIN polarity (Dubrovsky et al, 2008; Kleine-Vehn et al, 2008a). Nevertheless, the molecular basis for PIN polarization in plants remains unknown.
To gain new mechanistic insights in the hypothetical feedback mechanisms governing PIN polarization, several theoretical studies (Mitchison, 1980; Sachs, 1981; Rolland-Lagan and Prusinkiewicz, 2005; Jönsson et al, 2006; Smith et al, 2006; Merks et al, 2007; Bayer et al, 2009; Kramer, 2009) have been carried out. These models suggest that auxin promotes its own transport by modulating the amount of PIN proteins at the plasma membrane by incorporating either not yet identified flux gradient-based component (Mitchison, 1980; Rolland-Lagan and Prusinkiewicz, 2005; Bayer et al, 2009; Kramer, 2009) or an unknown short-range intercellular signal-transmitting auxin concentrations of its direct neighbors (Jönsson et al, 2006; Smith et al, 2006; Merks et al, 2007; Bayer et al, 2009; Sahlin et al, 2009).
Here, we propose a feedback driven, biologically plausible model for PIN polarization and auxin transport that introduces the combination of intracellular and extracellular auxin signaling pathways as a unified approach for tissue polarization in plants. Our computer model is based on chemiosmotic hypothesis (Goldsmith et al, 1981; Figure 1A) and integrates up-to-date experimental data, such as auxin feedback on PIN expression (Peer et al, 2004; Heisler et al, 2005) via a nuclear auxin signaling pathway (Chapman and Estelle, 2009; Figure 1B), auxin carrier recycling auxin (Dubrovsky et al, 2008; Kleine-Vehn et al, 2008a; Figure 1C), and auxin feedback on PIN endocytosis (Paciorek et al, 2005) via novel hypothetical, yet plausible, assumption of extracellular auxin perception (Figure 1D).
The heart of our extracellular receptor-based polarization (ERP) mechanism is the competitive utilization of auxin receptors in the intercellular space that allows a direct and simple cell-to-cell communication scheme. In our model, auxin binds to its extracellular receptor in the concentration-dependent manner and induces signal to modulate PIN protein abundance at the plasma membrane (Figure 1D). The direct mode of the signal transfer involves temporal immobilization of recruited receptors to the plasma membrane, which is reflected by reduced diffusion of receptors involved in auxin signaling (Figure 1D). This competitive utilization mechanism enables cell-to-cell communication in our model, leading to receptor enrichment at the site of higher auxin concentration (Figure 1D). The PIN polarization and polar auxin transport in our model both depend on and contribute to the establishment of differential auxin signaling in the cell wall. This feedback loop leads ultimately to the alignment of PIN polarization within a tissue.
We demonstrated the plausibility of the ERP model for various processes, including de novo vascularization, venation patterning, and tissue regeneration in computer simulations performed with only minimal initial assumptions, a discrete auxin source, and a distal sink. The ERP model reproduces the very detailed PIN polarization events that occur during primary vein initiation (Scarpella et al, 2006), such as basal PIN1 polarity in provascular cells, transient adverse PIN1 polarization in neighboring cells during the alignment of tissue polarization, and inner-lateral polarity displayed by the tissues surrounding a conductive auxin channel (Figure 3). Additionally, the ERP model generates high auxin concentration and high auxin flux simultaneously in emerging veins, revising the classical canalization models (Mitchison, 1980; Rolland-Lagan and Prusinkiewicz, 2005). Importantly, all our model simulations support the claim that the ERP model represents the first single approach that faithfully reproduces PIN polarization, both with the auxin gradient (basal PIN1 polarity in provascular cells) and against the auxin gradient (transient adverse PIN1 polarization in neighboring cells surrounding the provascular bundle), as well as producing the corresponding auxin distribution patterns during auxin canalization.
The proposed model introduces the extracellular auxin signaling pathway, which is crucial to account for coordinated PIN polarization and auxin distribution during venation patterning in plants. The putative candidate for extracellular auxin receptor is auxin-binding protein 1 (ABP1), which resides in the lumen of the endoplasmic reticulum and is secreted to the cell wall (Napier et al, 2002; Tromas et al, 2009) where it is physiologically active (Leblanc et al, 1999; Steffens et al, 2001). Additionally, auxin inhibits clathrin-dependent PIN internalization via binding to ABP1 (Robert et al, 2010). Thus, we speculate that the extracellular fraction of ABP1 (or additionally yet to be identified ABPs) could correspond to the common pool of extracellular auxin receptors in the ERP model. A future challenge will be to test whether the ERP model unifies complex PIN polarization and auxin distribution patterns in embryogenesis, root system maintenance, and de novo organ formation.
Plant development is exceptionally flexible as manifested by its potential for organogenesis and regeneration, which are processes involving rearrangements of tissue polarities. Fundamental questions concern how individual cells can polarize in a coordinated manner to integrate into the multicellular context. In canalization models, the signaling molecule auxin acts as a polarizing cue, and feedback on the intercellular auxin flow is key for synchronized polarity rearrangements. We provide a novel mechanistic framework for canalization, based on up-to-date experimental data and minimal, biologically plausible assumptions. Our model combines the intracellular auxin signaling for expression of PINFORMED (PIN) auxin transporters and the theoretical postulation of extracellular auxin signaling for modulation of PIN subcellular dynamics. Computer simulations faithfully and robustly recapitulated the experimentally observed patterns of tissue polarity and asymmetric auxin distribution during formation and regeneration of vascular systems and during the competitive regulation of shoot branching by apical dominance. Additionally, our model generated new predictions that could be experimentally validated, highlighting a mechanistically conceivable explanation for the PIN polarization and canalization of the auxin flow in plants.
PMCID: PMC3018162  PMID: 21179019
auxin; canalization; cell polarity; PIN proteins
13.  Tuberous Sclerosis Complex 1: An Epithelial Tumor Suppressor Essential to Prevent Spontaneous Prostate Cancer in Aged Mice 
Cancer research  2010;70(21):8937-8947.
The phosphoinositide 3-kinase (PI3K) pathway regulates mammalian cell growth, survival, and motility and plays a major pathogenetic role in human prostate cancer (PCa). However, the oncogenic contributions downstream of the PI3K pathway made by mammalian target of rapamycin complex 1 (mTORC1)–mediated cell growth signal transduction in PCa have yet to be elucidated in detail. Here, we engineered constitutive mTORC1 activation in prostate epithelium by a conditional genetic deletion of tuberous sclerosis complex 1 (Tsc1), a potent negative regulator of mTORC1 signaling. Epithelial inactivation was not immediately tumorigenic, but Tsc1-deficient mice developed prostatic intraepithelial neoplasia (mPIN) in lateral and anterior prostates by 6 months of age, with increasing disease penetrance over time. Lateral prostate lesions in 16- to 22-month-old mutant mice progressed to two types of more advanced lesions, adenomatous gland forming lesion (Type 1) and atypical glands embedded in massively expanded reactive stroma (Type 2). Both Type 1 and Type 2 lesions contained multiple foci of microinvasive carcinoma. Epithelial neoplastic and atypical stromal lesions persisted despite 4 weeks of RAD001 chemotherapy. Rapalogue resistance was not due to AKT or extracellular signal-regulated kinase 1/2 activation. Expression of the homeobox gene Nkx3.1 was lost in Tsc1-deficient mPIN, and it cooperated with TSC1 loss in mPIN initiation in doubly mutant Tsc1:Nkx3.1 prostatic epithelial knockout mice. Thus, TSC1 inactivation distal to PI3K and AKT activation is sufficient to activate a molecular signaling cascade producing prostatic neoplasia and focal carcinogenesis.
PMCID: PMC3064856  PMID: 20940396
14.  Canoe binds RanGTP to promote PinsTPR/Mud-mediated spindle orientation 
The Journal of Cell Biology  2011;195(3):369-376.
The scaffolding protein Canoe regulates spindle orientation by binding to RanGTP and recruiting RanGTP and Mud to the cell cortex.
Regulated spindle orientation maintains epithelial tissue integrity and stem cell asymmetric cell division. In Drosophila melanogaster neural stem cells (neuroblasts), the scaffolding protein Canoe (Afadin/Af-6 in mammals) regulates spindle orientation, but its protein interaction partners and mechanism of action are unknown. In this paper, we use our recently developed induced cell polarity system to dissect the molecular mechanism of Canoe-mediated spindle orientation. We show that a previously uncharacterized portion of Canoe directly binds the Partner of Inscuteable (Pins) tetratricopeptide repeat (TPR) domain. The Canoe–PinsTPR interaction recruits Canoe to the cell cortex and is required for activation of the PinsTPR-Mud (nuclear mitotic apparatus in mammals) spindle orientation pathway. We show that the Canoe Ras-association (RA) domains directly bind RanGTP and that both the CanoeRA domains and RanGTP are required to recruit Mud to the cortex and activate the Pins/Mud/dynein spindle orientation pathway.
PMCID: PMC3206335  PMID: 22024168
15.  Developmental expression of three small GTPases in the mouse eye 
Molecular Vision  2007;13:1144-1153.
The small GTPases function as "molecular switches" by binding and releasing GTP to mediate downstream signaling effects. The Rho-family of GTPases is central in modulating cell differentiation and cytoskeletal changes. Since eye development requires comprehensive morphogenetic movements and extensive cellular differentiation, we hypothesize that different small GTPases may play important roles during morphogenesis of eye development. To explore this possibility, we examined the expression patterns of three major Rho-GTPases: RhoA, Rac1, and Cdc42 in embryonic, postnatal (one day after birth), and adult (two-month old) mouse eye.
Various ocular tissues were collected from embryonic, postnatal, and adult C57BL/6 mice. Western blots were conducted using total proteins extracted from cornea, retina, lens epithelial cells, and lens fiber cells of the adult mice or different fractions of rat lenses. Immunohistochemistry (IHC) was performed with 6 μm thick sections cut through the eye ball region of 11.5 pc, 14.5 pc, 17.5 pc, postnatal, and adult mice. Parallel controls were run using the rabbit preimmune and GTPase-specific antibodies blocked with saturating levels of corresponding peptide antigen.
In the embryonic mouse eye, RhoA and Cdc42 expressions were initially detectable in all three compartments at 11.5 pc. However, Rac1 became easily detectable in these compartments at 14.5 pc. Increased levels of RhoA, Rac1, and Cdc42 were detected in the three compartments at 17.5 pc and the strongest signals for RhoA, Rac1, and Cdc42 were observed in the primary lens fiber cells at 17.5 pc. In the postnatal mouse eye, the three small GTPases were significantly expressed in both endothelial and epithelial cells of mouse cornea, epithelial cells of the ocular lens, photoreceptors, horizontal/amacrine/Muller's cells, and some ganglian cells of the retina. Much lower level of expression was observed in the corneal stroma fibroblasts, lens fiber cells, and the inner and outer plexiform layers of the mouse retina. In the adult mouse eye, all three Rho-GTPases were expressed in corneal epithelial cells and retina. However, only RhoA protein was detected in corneal endothelial cells and Rac1 protein detected in the ocular lens.
The strong expression of the three small GTPases in the cornea, lens, and retina of mouse eye at embryonic 17.5 pc and postnatal stage suggests their important functions for the morphogenesis of the different compartments of the mouse eye. Particularly, high levels of expression of RhoA, Rac1, and Cdc42 in embryonic lens fiber cells suggest their involvement in differentiation of primary lens fiber cells. In the adult mouse eye, all three Rho-GTPases seem to be involved in differentiation of corneal epithelial cells and retina, however, RhoA alone may be required for endothelial cell differentiation and Rac1 likely plays an important role in supporting continuous lens growth and maintenance of lens transparency.
PMCID: PMC2779149  PMID: 17653061
16.  Building cortical polarity in a cell line: identification of an Aurora-A/PinsLINKER spindle orientation pathway 
Cell  2009;138(6):1150-1163.
Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. Here we develop a method to construct cortical polarity in a normally unpolarized cell line, and use this method to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals)-dependent spindle orientation. We identify a previously unrecognized evolutionarily-conserved Pins domain (PinsLINKER) that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized PinsTPR domain has no function alone, but placing the PinsTPR in cis to the PinsLINKER gives dynein-dependent precise spindle orientation. This "induced cortical polarity" assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity.
PMCID: PMC2789599  PMID: 19766567
17.  A Joint Effect of New Western Diet and Retinoid X Receptor Alpha Prostate-Specific Knockout with Development of High Grade Prostatic Intraepithelial Neoplasia in Mice - A Preliminary Study 
The Prostate  2012;72(10):1052-1059.
The “New Western-style Diet” (NWD) characterized by high in fat and low in fiber, vitamin D, calcium and methyl donors - are considered as a risk factor for prostate cancer. Previous studies have shown that premalignant lesions of human prostate have decreased expression of the Retinoid X Receptor alpha (RXRα). This study was to determine the effect of diet in RXRα knockout mice in developing high grade prostate intraepithelial neoplasia (mPIN).
Male mice (N = 54) with or without the RXRα prostate null mutation were fed either NWD or AIN-76A control diet for 10 months; prostates were harvested at 11 months of age and examined for prostate mPIN.
mPIN was seen in 79% of RXRα prostate null mice fed NWD (n=19), 30.8% RXRα prostate null mice fed AIN-76A (n=13), 42.9% RXRα wild type mice fed NWD (n=14), and 12.5% RXRα wild type mice fed AIN-76A (n=8). Unconditional Logistic analysis showed a significant joint effect of NWD and RXRα status in developing mPIN 26.3 (95% CI: 2.5-280), but interaction was not significant owing to the small sample size 1.6 (0.09-27.7, p=0.7441).
This study provides preliminary data to support a joint RXRα-diet effect in prostate carcinogenesis.
PMCID: PMC4321893  PMID: 22314496
prostate cancer; Retinoid X Receptor α; PIN; New Western Diet; Prostate-specific gene knockout
18.  The Maize PIN Gene Family of Auxin Transporters 
Auxin is a key regulator of plant development and its differential distribution in plant tissues, established by a polar cell to cell transport, can trigger a wide range of developmental processes. A few members of the two families of auxin efflux transport proteins, PIN-formed (PIN) and P-glycoprotein (ABCB/PGP), have so far been characterized in maize. Nine new Zea mays auxin efflux carriers PIN family members and two maize PIN-like genes have now been identified. Four members of PIN1 (named ZmPIN1a–d) cluster, one gene homologous to AtPIN2 (ZmPIN2), three orthologs of PIN5 (ZmPIN5a–c), one gene paired with AtPIN8 (ZmPIN8), and three monocot-specific PINs (ZmPIN9, ZmPIN10a, and ZmPIN10b) were cloned and the phylogenetic relationships between early-land plants, monocots, and eudicots PIN proteins investigated, including the new maize PIN proteins. Tissue-specific expression patterns of the 12 maize PIN genes, 2 PIN-like genes and ZmABCB1, an ABCB auxin efflux carrier, were analyzed together with protein localization and auxin accumulation patterns in normal conditions and in response to drug applications. ZmPIN gene transcripts have overlapping expression domains in the root apex, during male and female inflorescence differentiation and kernel development. However, some PIN family members have specific tissue localization: ZmPIN1d transcript marks the L1 layer of the shoot apical meristem and inflorescence meristem during the flowering transition and the monocot-specific ZmPIN9 is expressed in the root endodermis and pericycle. The phylogenetic and gene structure analyses together with the expression pattern of the ZmPIN gene family indicate that subfunctionalization of some maize PINs can be associated to the differentiation and development of monocot-specific organs and tissues and might have occurred after the divergence between dicots and monocots.
PMCID: PMC3355596  PMID: 22639639
PIN-formed; auxin efflux carriers; kernel development; inflorescences; monocots; polar auxin transport; Zea mays
19.  A mouse model of heterogeneous, c-MYC-initiated prostate cancer with loss of Pten and p53 
Oncogene  2011;31(3):322-332.
Human tumors are heterogeneous and evolve through a dynamic process of genetic mutation and selection. During this process, the effects of a specific mutation on the incipient cancer cell may dictate the nature of subsequent mutations that can be tolerated or selected for, affecting the rate at which subsequent mutations occur. Here we have used a new mouse model of prostate cancer that recapitulates several salient features of the human disease to examine the relative rates in which the remaining wild type alleles of Pten and p53 tumor suppressor genes are lost. In this model, focal overexpression of c-MYC in a few prostate luminal epithelial cells provokes a mild proliferative response. In the context of compound Pten/p53 heterozygosity, c-MYC-initiated cells progress to prostatic intraepithelial neoplasia (mPIN) and adenocarcinoma lesions with marked heterogeneity within the same prostate glands. Using Laser Capture Microdissection and gene copy number analyses, we found that the frequency of Pten loss was significantly higher than that of p53 loss in mPIN but not invasive carcinoma lesions. c-MYC overexpression, unlike Pten loss, did not activate the p53 pathway in transgenic mouse prostate cells, explaining the lack of selective pressure to lose p53 in the c-MYC-overexpressing cells. This model of heterogeneous prostate cancer based on alterations in genes relevant to the human disease may be useful for understanding pathogenesis of the disease and testing new therapeutic agents.
PMCID: PMC3179816  PMID: 21685943
c-MYC; Pten; p53; prostate cancer; rate of mutations
20.  Klf5 Deletion Promotes Pten Deletion–Initiated Luminal-Type Mouse Prostate Tumors through Multiple Oncogenic Signaling Pathways12 
Neoplasia (New York, N.Y.)  2014;16(11):883-899.
Krüppel-like factor 5 (KLF5) regulates multiple biologic processes. Its function in tumorigenesis appears contradictory though, showing both tumor suppressor and tumor promoting activities. In this study, we examined whether and how Klf5 functions in prostatic tumorigenesis using mice with prostate-specific deletion of Klf5 and phosphatase and tensin homolog (Pten), both of which are frequently inactivated in human prostate cancer. Histologic analysis demonstrated that when one Pten allele was deleted, which causes mouse prostatic intraepithelial neoplasia (mPIN), Klf5 deletion accelerated the emergence and progression of mPIN. When both Pten alleles were deleted, which causes prostate cancer, Klf5 deletion promoted tumor growth, increased cell proliferation, and caused more severe morphologic and molecular alterations. Homozygous deletion of Klf5 was more effective than hemizygous deletion. Unexpectedly, while Pten deletion alone expanded basal cell population in a tumor as reported, Klf5 deletion in the Pten-null background clearly reduced basal cell population while expanding luminal cell population. Global gene expression profiling, pathway analysis, and experimental validation indicate that multiple mechanisms could mediate the tumor-promoting effect of Klf5 deletion, including the up-regulation of epidermal growth factor and its downstream signaling molecules AKT and ERK and the inactivation of the p15 cell cycle inhibitor. KLF5 also appears to cooperate with several transcription factors, including CREB1, Sp1, Myc, ER and AR, to regulate gene expression. These findings validate the tumor suppressor function of KLF5. They also yield a mouse model that shares two common genetic alterations with human prostate cancer—mutation/deletion of Pten and deletion of Klf5.
PMCID: PMC4240924  PMID: 25425963
21.  Conditional Transgenic Expression of PIM1 Kinase in Prostate Induces Inflammation-Dependent Neoplasia 
PLoS ONE  2013;8(4):e60277.
The Pim proteins are a family of highly homologous protein serine/threonine kinases that have been found to be overexpressed in cancer. Elevated levels of Pim1 kinase were first discovered in human leukemia and lymphomas. However, more recently Pim1 was found to be increased in solid tumors, including pancreatic and prostate cancers, and has been proposed as a prognostic marker. Although the Pim kinases have been identified as oncogenes in transgenic models, they have weak transforming abilities on their own. However, they have been shown to greatly enhance the ability of other genes or chemical carcinogens to induce tumors. To explore the role of Pim1 in prostate cancer, we generated conditional Pim1 transgenic mice, expressed Pim1 in prostate epithelium, and analyzed the contribution of PIM1 to neoplastic initiation and progression. Accordingly, we explored the effect of PIM1 overexpression in 3 different settings: upon hormone treatment, during aging, and in combination with the absence of one Pten allele. We have found that Pim1 overexpression increased the severity of mouse prostate intraepithelial neoplasias (mPIN) moderately in all three settings. Furthermore, Pim1 overexpression, in combination with the hormone treatment, increased inflammation surrounding target tissues leading to pyelonephritis in transgenic animals. Analysis of senescence induced in these prostatic lesions showed that the lesions induced in the presence of inflammation exhibited different behavior than those induced in the absence of inflammation. While high grade prostate preneoplastic lesions, mPIN grades III and IV, in the presence of inflammation did not show any senescence markers and demonstrated high levels of Ki67 staining, untreated animals without inflammation showed senescence markers and had low levels of Ki67 staining in similar high grade lesions. Our data suggest that Pim1 might contribute to progression rather than initiation in prostate neoplasia.
PMCID: PMC3614961  PMID: 23565217
22.  MYC Overexpression Induces Prostatic Intraepithelial Neoplasia and Loss of Nkx3.1 in Mouse Luminal Epithelial Cells 
PLoS ONE  2010;5(2):e9427.
Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate[1]. However, prior studies have not determined precisely when, and in which cell types, MYC is induced. Using immunohistochemistry (IHC) to localize MYC expression in Lo-MYC transgenic mice, we show that morphological and molecular alterations characteristic of high grade PIN arise in luminal epithelial cells as soon as MYC overexpression is detected. These changes include increased nuclear and nucleolar size and large scale chromatin remodeling. Mouse PIN cells retained a columnar architecture and abundant cytoplasm and appeared as either a single layer of neoplastic cells or as pseudo-stratified/multilayered structures with open glandular lumina—features highly analogous to human high grade PIN. Also using IHC, we show that the onset of MYC overexpression and PIN development coincided precisely with decreased expression of the homeodomain transcription factor and tumor suppressor, Nkx3.1. Virtually all normal appearing prostate luminal cells expressed high levels of Nkx3.1, but all cells expressing MYC in PIN lesions showed marked reductions in Nkx3.1, implicating MYC as a key factor that represses Nkx3.1 in PIN lesions. To determine the effects of less pronounced overexpression of MYC we generated a new line of mice expressing MYC in the prostate under the transcriptional control of the mouse Nkx3.1 control region. These “Super-Lo-MYC” mice also developed PIN, albeit a less aggressive form. We also identified a histologically defined intermediate step in the progression of mouse PIN into invasive adenocarcinoma. These lesions are characterized by a loss of cell polarity, multi-layering, and cribriform formation, and by a “paradoxical” increase in Nkx3.1 protein. Similar histopathological changes occurred in Hi-MYC mice, albeit with accelerated kinetics. Our results using IHC provide novel insights that support the contention that MYC overexpression is sufficient to transform prostate luminal epithelial cells into PIN cells in vivo. We also identified a novel histopathologically identifiable intermediate step prior to invasion that should facilitate studies of molecular pathway alterations occurring during early progression of prostatic adenocarcinomas.
PMCID: PMC2828486  PMID: 20195545
23.  The M3 phosphorylation motif has been functionally conserved for intracellular trafficking of long-looped PIN-FORMEDs in the Arabidopsis root hair cell 
BMC Plant Biology  2013;13:189.
PIN-FORMED (PIN) efflux carriers contribute to polar auxin transport and plant development by exhibiting dynamic and diverse asymmetrical localization patterns in the plasma membrane (PM). Phosphorylation of the central hydrophilic loop (HL) of PINs has been implicated in the regulation of PIN trafficking. Recently, we reported that a phosphorylatable motif (M3) in the PIN3-HL is necessary for the polarity, intracellular trafficking, and biological functions of PIN3. In this study, using the root hair system for PIN activity assay, we investigated whether this motif has been functionally conserved among long-HL PINs.
Root hair-specific overexpression of wild-type PIN1, 2, or 7 greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of M3 phosphorylation-defective PIN mutants failed to inhibit root hair growth. Consistent with this root hair phenotype, the PM localization of M3 phosphorylation-defective PIN1 and PIN7 was partially disrupted, resulting in less auxin efflux and restoration of root hair growth. Partial formation of brefeldin A-compartments in these phosphorylation-mutant PIN lines also suggested that their PM targeting was partially disrupted. On the other hand, compared with the PIN1 and PIN7 mutant proteins, M3-phosphorylation-defective PIN2 proteins were almost undetectable. However, the mutant PIN2 protein levels were restored by wortmannin treatment almost to the wild-type PIN2 level, indicating that the M3 motif of PIN2, unlike that of other PINs, is implicated in PIN2 trafficking to the vacuolar lytic pathway.
These results suggest that the M3 phosphorylation motif has been functionally conserved to modulate the intracellular trafficking of long-HL PINs, but its specific function in trafficking has diverged among PIN members.
PMCID: PMC4222813  PMID: 24274232
Auxin; Auxin transport; Hydrophilic loop (of PINs); Phosphorylation; PIN-FORMED (PIN); Protein trafficking; Root hair
24.  Differential functions of G protein and Baz–aPKC signaling pathways in Drosophila neuroblast asymmetric division 
The Journal of Cell Biology  2004;164(5):729-738.
Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical–basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Gγ1 subunit of heterotrimeric G protein, which produces Gγ1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gβ13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gβ13F to the membrane, indicating an essential role of cortical Gγ1–Gβ13F signaling in asymmetric divisions. In Gγ1 and Gβ13F mutant NBs, Pins–Gαi, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka–atypical PKC–Par6–Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gβγ and other mutants indicates a predominant role of Partner of Inscuteable–Gαi in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.
PMCID: PMC2172166  PMID: 14981094
epithelium; cell polarity; heterotrimeric G protein; spindle orientation; Drosophila melanogaster
25.  Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions 
Nature  2008;456(7224):962-966.
Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication—a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family1 are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms2–7. Polar PIN localization determines the direction of intercellular auxin flow8, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.
PMCID: PMC2692841  PMID: 18953331

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