Supplementary Methods, Figures 1 - 9, Table 1 from Small GTPase RhoE/Rnd3 Is a Critical Regulator of Notch1 Signaling
Zehua ZhuKristina TodorovaKevin C. LeeJun WangEun‐Jeong KwonIvan KehayovHyung-Gu KimVihren N. KolevG. Paolo DottoSam W. LeeAnna Mandinova
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<p>PDF file - 557KB, Supplementary Methods Suppl. Table 1. Primers used for real time and conventional RT-PCR . Suppl. Figure S1. RhoE expression is down-regulated in skin SCC tumors. Suppl. Figure S2. Notch activation does not induce expression of members of the Rho GTPase family of proteins. Suppl. Figure S3. Endogenous activation of Notch signaling induces the expression of Hey2. Suppl. Figure S4. Depletion of RhoE inhibits downstream Notch target genes. Suppl. Figure S5. Real-time RT-PCR analysis of mRNA isolated from primary keratinocytes. Suppl. Figure S6. Levels of RhoE and Keratin1 are increased during keratinocyte differentiation. Suppl. Figure S7. RhoE ablation suppresses growth and commitment to differentiation. Suppl. Figure S8. Negative control for RhoE and Notch1 staining in SCC4 cells. Suppl. Figure S9. Depletion of RhoE abrogates the binding between NIIC and importin Beta1.</p>Keywords:
miR-155
Small GTPase
A highly conserved member of the Rho family of small GTPases, Cdc42 functions as the "master regulator of cell polarity." It has been reported that for proper establishment and maintenance of cell polarity, Cdc42 regulates and requires vesicle trafficking. Importantly, we recently discovered that in budding yeast, vesicle trafficking also controls the localization and function of Bem3, a GTPase activating protein for Cdc42. Specifically, we observed that Bem3 partitioned between the plasma membrane and an internal membrane-bound compartment. This Bem3-containing compartment was present during extended periods of apical growth, required actin tracks for trafficking to polarized sites and functioned as a recycling station that was positioned at the junction of endocytic and secretory pathways. Strikingly, many of these features are reminiscent of the Spitzenkörper, a dynamic structure involved in polarized growth during hyphal development in several filamentous fungi. Furthermore, Bem3 was not merely a passive cargo but actively recruited the secretory Rab GTPase Sec4 to this Spitzenkörper-like compartment. Importantly, this function of Bem3 was independent of its GAP activity. Our work demonstrates the existence of a complementary regulation between Bem3, a regulator of Cdc42 signaling and Sec4, a key component of the secretory machinery.
Rab
CDC42
Cell polarity
Compartment (ship)
Small GTPase
Vesicular Transport Proteins
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Rap1 belongs to the Ras family of small GTPases, which are involved in a multitude of cellular signal transduction pathways and have extensively been linked to cancer biogenesis and metastasis. The small GTPase is activated in response to various extracellular and intracellular cues. Rap1 has conserved functions in Dictyostelium discoideum amoeba and mammalian cells, which are important for cell polarity, substrate and cell-cell adhesion and other processes that involve the regulation of cytoskeletal dynamics. Moreover, our recent study has shown that Rap1 is required for the formation of the replication-permissive vacuole of an intracellular bacterial pathogen. Here we review the function and regulation of Rap1 in these distinct processes, and we discuss the underlying signal transduction pathways.
Rap1
Small GTPase
CDC42
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Master regulator
Small GTPase
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<p>PDF file - 557KB, Supplementary Methods Suppl. Table 1. Primers used for real time and conventional RT-PCR . Suppl. Figure S1. RhoE expression is down-regulated in skin SCC tumors. Suppl. Figure S2. Notch activation does not induce expression of members of the Rho GTPase family of proteins. Suppl. Figure S3. Endogenous activation of Notch signaling induces the expression of Hey2. Suppl. Figure S4. Depletion of RhoE inhibits downstream Notch target genes. Suppl. Figure S5. Real-time RT-PCR analysis of mRNA isolated from primary keratinocytes. Suppl. Figure S6. Levels of RhoE and Keratin1 are increased during keratinocyte differentiation. Suppl. Figure S7. RhoE ablation suppresses growth and commitment to differentiation. Suppl. Figure S8. Negative control for RhoE and Notch1 staining in SCC4 cells. Suppl. Figure S9. Depletion of RhoE abrogates the binding between NIIC and importin Beta1.</p>
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Small GTPase
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Autophagy is an evolutionarily conserved process that enables catabolic and degradative pathways. These pathways commonly depend on vesicular transport controlled by Rabs, small GTPases inactivated by TBC/RabGAPs. The Rac1 effector TBC/RabGAP Armus (TBC1D2A) is known to inhibit Rab7, a key regulator of lysosomal function. However, the precise coordination of signaling and intracellular trafficking that regulates autophagy is poorly understood. We find that overexpression of Armus induces the accumulation of enlarged autophagosomes, while Armus depletion significantly delays autophagic flux. Upon starvation-induced autophagy, Rab7 is transiently activated. This spatiotemporal regulation of Rab7 guanosine triphosphate/guanosine diphosphate cycling occurs by Armus recruitment to autophagosomes via interaction with LC3, a core autophagy regulator. Interestingly, autophagy potently inactivates Rac1. Active Rac1 competes with LC3 for interaction with Armus and thus prevents its appropriate recruitment to autophagosomes. The precise coordination between Rac1 and Rab7 activities during starvation suggests that Armus integrates autophagy with signaling and endocytic trafficking.
Small GTPase
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<p>PDF file - 557KB, Supplementary Methods Suppl. Table 1. Primers used for real time and conventional RT-PCR . Suppl. Figure S1. RhoE expression is down-regulated in skin SCC tumors. Suppl. Figure S2. Notch activation does not induce expression of members of the Rho GTPase family of proteins. Suppl. Figure S3. Endogenous activation of Notch signaling induces the expression of Hey2. Suppl. Figure S4. Depletion of RhoE inhibits downstream Notch target genes. Suppl. Figure S5. Real-time RT-PCR analysis of mRNA isolated from primary keratinocytes. Suppl. Figure S6. Levels of RhoE and Keratin1 are increased during keratinocyte differentiation. Suppl. Figure S7. RhoE ablation suppresses growth and commitment to differentiation. Suppl. Figure S8. Negative control for RhoE and Notch1 staining in SCC4 cells. Suppl. Figure S9. Depletion of RhoE abrogates the binding between NIIC and importin Beta1.</p>
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The small GTPase Rap1 controls the actin cytoskeleton by regulating Rho GTPase signaling. We recently established that the Rap1 effectors Radil and Rasip1, together with the Rho GTPase activating protein ArhGAP29, mediate Rap1-induced inhibition of Rho signaling in the processes of epithelial cell spreading and endothelial barrier function. Here, we show that Rap1 induces the independent translocations of Rasip1 and a Radil-ArhGAP29 complex to the plasma membrane. This results in the formation of a multimeric protein complex required for Rap1-induced inhibition of Rho signaling and increased endothelial barrier function. Together with the previously reported spatiotemporal control of the Rap guanine nucleotide exchange factor Epac1, these findings elucidate a signaling pathway for spatiotemporal control of Rho signaling that operates by successive protein translocations to and complex formation at the plasma membrane.
Rap1
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