One measure of career success for clinical researchers is reporting their findings in a peer-revi... more One measure of career success for clinical researchers is reporting their findings in a peer-reviewed journal. Writing a clinical manuscript that has impact and relevance to their intended audience is crucial for publication success. However, clinicians and scientists whose native language is not English may find it challenging to effectively communicate the clinical relevance of their research and they may seek help from a professional medical writer. In this article, we focus on what makes a high-quality clinical manuscript and some of the ethical issues that must be considered. Professional medical writers will benefit from understanding and applying these concepts as they assist authors in preparing a well-structured, ethically sound, and highly readable manuscript that clearly expresses the clinical relevance of their findings. Using these approaches, medical writers and their clients can be confident that the final manuscript meets the quality expectations and ethical standards of international Englishlanguage journals.
SUMMARY Writing a clinical manuscript that has impact and relevance to your intended audience is ... more SUMMARY Writing a clinical manuscript that has impact and relevance to your intended audience is crucial for publication success. Furthermore, publishing in English will increase the visibility of your research worldwide. However, effectively communicating the clinical relevance of your research in English is particularly challenging if it is not your native language. In this article, we focus on the essentials of what makes a high-quality clinical manuscript. Understanding these essentials will assist you in writing a well-structured, ethically sound, and highly readable manuscript that clearly expresses the clinical relevance of your findings. Using these approaches, you can be confident that your work adheres to the standards of international English language journals, and improve your chances of publication success.
Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. Howe... more Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. However, little is known about the mechanism by which these GTPases are regulated in response to apoptotic stimuli. Here, we demonstrate that TIAM1, a Rac-specific guanine nucleotide exchange factor, is cleaved by caspases during apoptosis. TIAM1 cleavage occurs in multiple cell lines in response to diverse apoptotic stimuli such as ceramide, Fas, and serum deprivation. Processing occurs at residue 993 of TIAM1 and removes the NH2-terminal of TIAM’s two pleckstrin homology domains, leaving a stable fragment containing the Dbl homology and COOH-terminal pleckstrin homology domains. This leads to functional inactivation of TIAM1, as determined by failure of the cleavage product to stimulate GTP loading of Rac in vivo. Furthermore, this product is defective in signaling to two independent Rac effectors, c-Jun NH2-terminal kinase and serum response factor. Finally, we demonstrate that in cells t...
ABSTRACT The TRE17 oncogene was identified by virtue of its transforming abilities in fibroblasts... more ABSTRACT The TRE17 oncogene was identified by virtue of its transforming abilities in fibroblasts, however its molecular interactions and cellular functions have remained unexplored. TRE17 contains a TBC domain that is homologous to the yeast proteins Msb3 and Msb4, which function in a pathway with Cdc42p in regulating polarized actin remodeling and secretion. This suggested that perhaps TRE17 functions downstream of Rho GTPases in mammalian cells and regulating similar activities. Thus, my thesis work has focused on providing the first insights into this novel oncogene, including its cellular localization, interactions with G-proteins, and the regulation of actin remodeling and vesicular trafficking. I demonstrate that TRE17 localizes to the plasma membrane and to an Arf6-dependent tubular recycling endosome. This localization is dynamic and requires the integrity of the cytoskeleton. A truncated TRE17 allele induces the formation of peripheral membrane protrusions and enlarged vesicular and vacuolar structures within the cytosol, indicating this may be a hyperactive allele of TRE17. I also show that TRE17 is regulated by the Rho GTPases Rac1 and Cdc42. TRE17 indirectly associates with these G-proteins in a GTP-dependent manner, and the activation of Rac or Cdc42 induces the recruitment of TRE17 to the plasma membrane. Downstream of these GTPases, TRE17 stimulates actin remodeling at the cell cortex and dynamic membrane protrusions. Further, I demonstrate that TRE17 regulates the small GTPase Arf6. TRE17 directly associates with Arf6 in a GDP-dependent manner via TRE17's TBC domain. This association stimulates Arf6 activation, possibly by acting as a chaperone to recruit inactive Arf6 to the plasma membrane. TRE17 also regulates the trafficking of Arf6-dependent cargo, such as the β1-integrin and MHC I; and cell spreading, a process that depends on the coordinate regulation of actin remodeling and vesicular trafficking. Together, these results suggest that TRE17 functions downstream of Rho GTPases to regulate actin remodeling as well as vesicular trafficking via the Arf6-dependent pathway. Since deregulation of these processes lead to the acquisition of a more migratory and invasive phenotype in cancer cells, these studies not only provide insight into the normal function of TRE17, but also into how it may contribute to tumorigensis.
Toxicology in vitro : an international journal published in association with BIBRA, Jan 20, 2018
Co-culture of hepatocyte and fibroblasts has shown distinct advantages in enhancing certain liver... more Co-culture of hepatocyte and fibroblasts has shown distinct advantages in enhancing certain liver specific functions and maintaining hepatic polarity. However, the utility of hepatocyte co-culture models for studies, such as drug-drug interaction studies, has not been completely elucidated. In this study the induction of Cyp1a2, Cyp2b1/2, and Cyp3a2, the three major cytochrome P450 (CYP) isoforms in the rat liver, was evaluated in randomly mixed co-cultures and micropatterned co-cultures. We found that in both co-culture configurations, the drug-induced Cyp1a2, Cyp2b1/2, Cyp3a2 mRNA and activity were suppressed relative to those in monocultured hepatocytes. Further, we observed a significant increase in TGFβ1 production in the co-cultures. Addition of 100 pg/ml TGFβ1 to hepatocyte monocultures resulted in the suppression of Cyp1a2, Cyp2b1/2, and Cyp3a2 induction. These findings implicate TGFβ1 as one of the important factors impairing drug induced CYP induction in co-cultures and su...
The de novo formation of secretory lumens plays an important role during organogenesis. It involv... more The de novo formation of secretory lumens plays an important role during organogenesis. It involves the establishment of a cellular apical pole and the elongation of luminal cavities. The molecular parameters controlling cell polarization have been heavily scrutinized. In particular, signalling from the extracellular matrix (ECM) proved essential to the proper localization of the apical pole by directed protein transport. However, little is known about the regulation of the shape and the directional development of lumen into tubes. We demonstrate that the spatial scaffolding of cells by ECM can control tube shapes and can direct their elongation. We developed a minimal organ approach comprising of hepatocyte doublets cultured in artificial microniches to precisely control the spatial organization of cellular adhesions in three dimensions. This approach revealed a mechanism by which the spatial repartition of integrin-based adhesion can elicit an anisotropic intercellular mechanical ...
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 2001
Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. Howe... more Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. However, little is known about the mechanism by which these GTPases are regulated in response to apoptotic stimuli. Here, we demonstrate that TIAM1, a Rac-specific guanine nucleotide exchange factor, is cleaved by caspases during apoptosis. TIAM1 cleavage occurs in multiple cell lines in response to diverse apoptotic stimuli such as ceramide, Fas, and serum deprivation. Processing occurs at residue 993 of TIAM1 and removes the NH(2)-terminal of TIAM's two pleckstrin homology domains, leaving a stable fragment containing the Dbl homology and COOH-terminal pleckstrin homology domains. This leads to functional inactivation of TIAM1, as determined by failure of the cleavage product to stimulate GTP loading of Rac in vivo. Furthermore, this product is defective in signaling to two independent Rac effectors, c-Jun NH(2)-terminal kinase and serum response factor. Finally, we demonstrate that in...
ABSTRACT The TRE17 oncogene was identified by virtue of its transforming abilities in fibroblasts... more ABSTRACT The TRE17 oncogene was identified by virtue of its transforming abilities in fibroblasts, however its molecular interactions and cellular functions have remained unexplored. TRE17 contains a TBC domain that is homologous to the yeast proteins Msb3 and Msb4, which function in a pathway with Cdc42p in regulating polarized actin remodeling and secretion. This suggested that perhaps TRE17 functions downstream of Rho GTPases in mammalian cells and regulating similar activities. Thus, my thesis work has focused on providing the first insights into this novel oncogene, including its cellular localization, interactions with G-proteins, and the regulation of actin remodeling and vesicular trafficking. I demonstrate that TRE17 localizes to the plasma membrane and to an Arf6-dependent tubular recycling endosome. This localization is dynamic and requires the integrity of the cytoskeleton. A truncated TRE17 allele induces the formation of peripheral membrane protrusions and enlarged vesicular and vacuolar structures within the cytosol, indicating this may be a hyperactive allele of TRE17. I also show that TRE17 is regulated by the Rho GTPases Rac1 and Cdc42. TRE17 indirectly associates with these G-proteins in a GTP-dependent manner, and the activation of Rac or Cdc42 induces the recruitment of TRE17 to the plasma membrane. Downstream of these GTPases, TRE17 stimulates actin remodeling at the cell cortex and dynamic membrane protrusions. Further, I demonstrate that TRE17 regulates the small GTPase Arf6. TRE17 directly associates with Arf6 in a GDP-dependent manner via TRE17's TBC domain. This association stimulates Arf6 activation, possibly by acting as a chaperone to recruit inactive Arf6 to the plasma membrane. TRE17 also regulates the trafficking of Arf6-dependent cargo, such as the β1-integrin and MHC I; and cell spreading, a process that depends on the coordinate regulation of actin remodeling and vesicular trafficking. Together, these results suggest that TRE17 functions downstream of Rho GTPases to regulate actin remodeling as well as vesicular trafficking via the Arf6-dependent pathway. Since deregulation of these processes lead to the acquisition of a more migratory and invasive phenotype in cancer cells, these studies not only provide insight into the normal function of TRE17, but also into how it may contribute to tumorigensis.
ABSTRACT This chapter focuses on the assays for monitoring p70S6 kinase (p70S6k) kinase and p90RS... more ABSTRACT This chapter focuses on the assays for monitoring p70S6 kinase (p70S6k) kinase and p90RSK (more commonly referred to as RSK) activation. A hallmark of the mitogenic response is an increase in protein synthesis. This increase arises from the modulation of multiple components of the translational machinery, including phosphorylation of the S6 polypeptide of the 40S ribosomal subunit. Much has been learned about the signaling pathways that mediate activation of p70S6k and RSK. One common activator of both enzymes is p21ras. However, p21ras utilizes distinct downstream effectors to elicit their activation. The development of specific antibodies against p70S6k and RSK has allowed monitoring of their activation in vivo. The availability of clean immunoblotting antibodies that detect mobility shifts is particularly useful for examining stimulation of transfected forms ofp70S6k by candidate activators. Because p70S6k protein levels can vary from sample to sample during transfections, such antibodies provide an additional control by demonstrating p70S6k activation through changes in the ratio of the four phosphorylated bands.
The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeli... more The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induc...
Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays... more Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 ...
The Rho family GTPase Cdc42 is recognized for its role in cellular proliferation and transformati... more The Rho family GTPase Cdc42 is recognized for its role in cellular proliferation and transformation. However, the mechanism by which it promotes cell cycle progression has remained undefined. Using an inducible expression system, we show that constitutively active Cdc42 (Cdc42V12) is sufficient by itself to induce anchorage-independent but not mitogen-independent growth in NIH3T3 cells. However, Cdc42V12 markedly accelerates activation of cyclin E-Cdk2 in response to mitogen. These effects were highly specific, as the kinetics of cyclin D-Cdk4 activation was unaltered. Cdc42V12 promotes Cdk2 activation by selectively inducing cyclin E expression without affecting other regulatory proteins such as the p27 Cdk inhibitor or Cdc25A. Furthermore, Cdc42V12 was able to activate a reporter gene driven by the cyclin E promoter in the absence of exogenous mitogen or adhesion. Cyclin E induction was sensitive to rapamycin but not inhibitors of mitogen-activated protein kinases, implicating p70 S6 kinase (p70S6k) as the relevant mediator. Consistent with this notion, wild type and constitutively active alleles of p70S6k were sufficient to activate the cyclin E promoter. In sum, these studies provide novel insights into the mechanism by which Cdc42 promotes G 1 progression.
In two conditioning experiments, identical procedures (previously shown to produce place preferen... more In two conditioning experiments, identical procedures (previously shown to produce place preferences for a cocaine-paired environment) were used to assess dopaminergic and behavioral activity correlates of cocaine reward conditioning and sensitization. In these experiments, animals received repeated injections of intravenous cocaine (4.2 mg / kg36) or saline (0.2 ml36) on alternating days. One group in each of these experiments ('Cocaine Cues') occupied a consistent distinctive environment during cocaine treatments and testing sessions. For the other conditioned group ('Novel'), all procedures were the same, except that the last cocaine injection was administered while animals were occupying a novel environment. During day 1 and day 6 of the cocaine treatment, behavioral activity was assessed in experiment 1 and in vivo microdialysis procedures were conducted in experiment 2. Over the course of the conditioning sessions, cocaine-induced behavioral activity (locomotion and rearing) increased significantly in the Cocaine Cues group, but not in the Novel group. In addition, cocaine-induced increases in NAcc dopamine levels were significantly greater when cocaine-experienced animals were tested in a cocaine-paired environment compared to equally experienced and cocaine-naive animals tested in a novel environment. Context-dependent behavioral sensitization is a well-documented phenomenon. The observation of a corresponding enhancement of dopamine efflux in lieu of a lengthy withdrawal period is uncommon, but can be attributed to methodological differences across studies. The present study uniquely demonstrates concurrent context-dependent potentiation of behavioral and dopaminergic responses to cocaine occurring in conjunction with cocaine reward.
In vitro drug testing requires long-term maintenance of hepatocyte liver specific functions. Hepa... more In vitro drug testing requires long-term maintenance of hepatocyte liver specific functions. Hepatocytes cultured at a higher seeding density in a sandwich configuration exhibit an increased level of liver specific functions when compared to low density cultures due to the better cell to cell contacts that promote long term maintenance of polarity and liver specific functions. However, culturing hepatocytes at high seeding densities in a standard 24-well plate poses problems in terms of the mass transport of nutrients and oxygen to the cells. In view of this drawback, we have developed a polydimethylsiloxane (PDMS) bioreactor that was able to maintain the long-term liver specific functions of a hepatocyte sandwich culture at a high seeding density. The bioreactor was fabricated with PDMS, an oxygen permeable material, which allowed direct oxygenation and perfusion to take place simultaneously. The mass transport of oxygen and the level of shear stress acting on the cells were analyzed by computational fluid dynamics (CFD). The combination of both direct oxygenation and perfusion has a synergistic effect on the liver specific function of a high density hepatocyte sandwich culture over a period of 9 days.
Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier st... more Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier structures when in extended culture. When the spheroid diameter exceeds 200 mm, cells in the inner core experience hypoxia and limited access to nutrients and drugs. Here we report the development of a thin galactosylated cellulosic sponge to culture hepatocytes in multi-well plates as 3D spheroids, and constrain them within a macroporous scaffold network to maintain spheroid size and prevent detachment. The hydrogel-based soft sponge conjugated with galactose provided suitable mechanical and chemical cues to support rapid formation of hepatocyte spheroids with a mature hepatocyte phenotype. The spheroids tethered in the sponge showed excellent maintenance of 3D cell morphology, cellecell interaction, polarity, metabolic and transporter function and/or expression. For example, cytochrome P450 (CYP1A2, CYP2B2 and CYP3A2) activities were significantly elevated in spheroids exposed to b-naphthoflavone, phenobarbital, or pregnenolone-16a-carbonitrile, respectively. The sponge also exhibits minimal drug absorption compared to other commercially available scaffolds. As the cell seeding and culture protocols are similar to various high-throughput 2D cell-based assays, this platform is readily scalable and provides an alternative to current hepatocyte platforms used in drug safety testing applications.
TBC (Tre-2/Bub2/Cdc16) domains are predicted to encode GTPase-activating proteins (GAPs) for Rab ... more TBC (Tre-2/Bub2/Cdc16) domains are predicted to encode GTPase-activating proteins (GAPs) for Rab family G proteins. While approximately 50 TBC proteins are predicted to exist in humans, little is known about their substrate specificity. Here we show that TRE17 (also called Tre-2 and USP6), a founding member of the TBC family, targets the Arf family GTPase Arf6, which regulates plasma membrane-endosome trafficking. Surprisingly, TRE17 does not function as a GAP for Arf6 but rather promotes its activation in vivo. TRE17 associates directly with Arf6 in its GDP-but not GTP-bound state. Mapping experiments pinpoint the site of interaction to the TBC domain of TRE17. Forced expression of TRE17 promotes the localization of Arf6 to the plasma membrane, leading to Arf6 activation, presumably due to facilitated access to membrane-associated guanine nucleotide exchange factors (GEFs). Furthermore, TRE17 cooperates with Arf6 GEFs to induce GTP loading of Arf6 in vivo. Finally, short interfering RNA-mediated loss of TRE17 leads to attenuated Arf6 activation. These studies identify TRE17 as a novel regulator of the Arf6-regulated plasma membrane recycling system and reveal an unexpected function for TBC domains.
One measure of career success for clinical researchers is reporting their findings in a peer-revi... more One measure of career success for clinical researchers is reporting their findings in a peer-reviewed journal. Writing a clinical manuscript that has impact and relevance to their intended audience is crucial for publication success. However, clinicians and scientists whose native language is not English may find it challenging to effectively communicate the clinical relevance of their research and they may seek help from a professional medical writer. In this article, we focus on what makes a high-quality clinical manuscript and some of the ethical issues that must be considered. Professional medical writers will benefit from understanding and applying these concepts as they assist authors in preparing a well-structured, ethically sound, and highly readable manuscript that clearly expresses the clinical relevance of their findings. Using these approaches, medical writers and their clients can be confident that the final manuscript meets the quality expectations and ethical standards of international Englishlanguage journals.
SUMMARY Writing a clinical manuscript that has impact and relevance to your intended audience is ... more SUMMARY Writing a clinical manuscript that has impact and relevance to your intended audience is crucial for publication success. Furthermore, publishing in English will increase the visibility of your research worldwide. However, effectively communicating the clinical relevance of your research in English is particularly challenging if it is not your native language. In this article, we focus on the essentials of what makes a high-quality clinical manuscript. Understanding these essentials will assist you in writing a well-structured, ethically sound, and highly readable manuscript that clearly expresses the clinical relevance of your findings. Using these approaches, you can be confident that your work adheres to the standards of international English language journals, and improve your chances of publication success.
Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. Howe... more Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. However, little is known about the mechanism by which these GTPases are regulated in response to apoptotic stimuli. Here, we demonstrate that TIAM1, a Rac-specific guanine nucleotide exchange factor, is cleaved by caspases during apoptosis. TIAM1 cleavage occurs in multiple cell lines in response to diverse apoptotic stimuli such as ceramide, Fas, and serum deprivation. Processing occurs at residue 993 of TIAM1 and removes the NH2-terminal of TIAM’s two pleckstrin homology domains, leaving a stable fragment containing the Dbl homology and COOH-terminal pleckstrin homology domains. This leads to functional inactivation of TIAM1, as determined by failure of the cleavage product to stimulate GTP loading of Rac in vivo. Furthermore, this product is defective in signaling to two independent Rac effectors, c-Jun NH2-terminal kinase and serum response factor. Finally, we demonstrate that in cells t...
ABSTRACT The TRE17 oncogene was identified by virtue of its transforming abilities in fibroblasts... more ABSTRACT The TRE17 oncogene was identified by virtue of its transforming abilities in fibroblasts, however its molecular interactions and cellular functions have remained unexplored. TRE17 contains a TBC domain that is homologous to the yeast proteins Msb3 and Msb4, which function in a pathway with Cdc42p in regulating polarized actin remodeling and secretion. This suggested that perhaps TRE17 functions downstream of Rho GTPases in mammalian cells and regulating similar activities. Thus, my thesis work has focused on providing the first insights into this novel oncogene, including its cellular localization, interactions with G-proteins, and the regulation of actin remodeling and vesicular trafficking. I demonstrate that TRE17 localizes to the plasma membrane and to an Arf6-dependent tubular recycling endosome. This localization is dynamic and requires the integrity of the cytoskeleton. A truncated TRE17 allele induces the formation of peripheral membrane protrusions and enlarged vesicular and vacuolar structures within the cytosol, indicating this may be a hyperactive allele of TRE17. I also show that TRE17 is regulated by the Rho GTPases Rac1 and Cdc42. TRE17 indirectly associates with these G-proteins in a GTP-dependent manner, and the activation of Rac or Cdc42 induces the recruitment of TRE17 to the plasma membrane. Downstream of these GTPases, TRE17 stimulates actin remodeling at the cell cortex and dynamic membrane protrusions. Further, I demonstrate that TRE17 regulates the small GTPase Arf6. TRE17 directly associates with Arf6 in a GDP-dependent manner via TRE17's TBC domain. This association stimulates Arf6 activation, possibly by acting as a chaperone to recruit inactive Arf6 to the plasma membrane. TRE17 also regulates the trafficking of Arf6-dependent cargo, such as the β1-integrin and MHC I; and cell spreading, a process that depends on the coordinate regulation of actin remodeling and vesicular trafficking. Together, these results suggest that TRE17 functions downstream of Rho GTPases to regulate actin remodeling as well as vesicular trafficking via the Arf6-dependent pathway. Since deregulation of these processes lead to the acquisition of a more migratory and invasive phenotype in cancer cells, these studies not only provide insight into the normal function of TRE17, but also into how it may contribute to tumorigensis.
Toxicology in vitro : an international journal published in association with BIBRA, Jan 20, 2018
Co-culture of hepatocyte and fibroblasts has shown distinct advantages in enhancing certain liver... more Co-culture of hepatocyte and fibroblasts has shown distinct advantages in enhancing certain liver specific functions and maintaining hepatic polarity. However, the utility of hepatocyte co-culture models for studies, such as drug-drug interaction studies, has not been completely elucidated. In this study the induction of Cyp1a2, Cyp2b1/2, and Cyp3a2, the three major cytochrome P450 (CYP) isoforms in the rat liver, was evaluated in randomly mixed co-cultures and micropatterned co-cultures. We found that in both co-culture configurations, the drug-induced Cyp1a2, Cyp2b1/2, Cyp3a2 mRNA and activity were suppressed relative to those in monocultured hepatocytes. Further, we observed a significant increase in TGFβ1 production in the co-cultures. Addition of 100 pg/ml TGFβ1 to hepatocyte monocultures resulted in the suppression of Cyp1a2, Cyp2b1/2, and Cyp3a2 induction. These findings implicate TGFβ1 as one of the important factors impairing drug induced CYP induction in co-cultures and su...
The de novo formation of secretory lumens plays an important role during organogenesis. It involv... more The de novo formation of secretory lumens plays an important role during organogenesis. It involves the establishment of a cellular apical pole and the elongation of luminal cavities. The molecular parameters controlling cell polarization have been heavily scrutinized. In particular, signalling from the extracellular matrix (ECM) proved essential to the proper localization of the apical pole by directed protein transport. However, little is known about the regulation of the shape and the directional development of lumen into tubes. We demonstrate that the spatial scaffolding of cells by ECM can control tube shapes and can direct their elongation. We developed a minimal organ approach comprising of hepatocyte doublets cultured in artificial microniches to precisely control the spatial organization of cellular adhesions in three dimensions. This approach revealed a mechanism by which the spatial repartition of integrin-based adhesion can elicit an anisotropic intercellular mechanical ...
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 2001
Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. Howe... more Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. However, little is known about the mechanism by which these GTPases are regulated in response to apoptotic stimuli. Here, we demonstrate that TIAM1, a Rac-specific guanine nucleotide exchange factor, is cleaved by caspases during apoptosis. TIAM1 cleavage occurs in multiple cell lines in response to diverse apoptotic stimuli such as ceramide, Fas, and serum deprivation. Processing occurs at residue 993 of TIAM1 and removes the NH(2)-terminal of TIAM's two pleckstrin homology domains, leaving a stable fragment containing the Dbl homology and COOH-terminal pleckstrin homology domains. This leads to functional inactivation of TIAM1, as determined by failure of the cleavage product to stimulate GTP loading of Rac in vivo. Furthermore, this product is defective in signaling to two independent Rac effectors, c-Jun NH(2)-terminal kinase and serum response factor. Finally, we demonstrate that in...
ABSTRACT The TRE17 oncogene was identified by virtue of its transforming abilities in fibroblasts... more ABSTRACT The TRE17 oncogene was identified by virtue of its transforming abilities in fibroblasts, however its molecular interactions and cellular functions have remained unexplored. TRE17 contains a TBC domain that is homologous to the yeast proteins Msb3 and Msb4, which function in a pathway with Cdc42p in regulating polarized actin remodeling and secretion. This suggested that perhaps TRE17 functions downstream of Rho GTPases in mammalian cells and regulating similar activities. Thus, my thesis work has focused on providing the first insights into this novel oncogene, including its cellular localization, interactions with G-proteins, and the regulation of actin remodeling and vesicular trafficking. I demonstrate that TRE17 localizes to the plasma membrane and to an Arf6-dependent tubular recycling endosome. This localization is dynamic and requires the integrity of the cytoskeleton. A truncated TRE17 allele induces the formation of peripheral membrane protrusions and enlarged vesicular and vacuolar structures within the cytosol, indicating this may be a hyperactive allele of TRE17. I also show that TRE17 is regulated by the Rho GTPases Rac1 and Cdc42. TRE17 indirectly associates with these G-proteins in a GTP-dependent manner, and the activation of Rac or Cdc42 induces the recruitment of TRE17 to the plasma membrane. Downstream of these GTPases, TRE17 stimulates actin remodeling at the cell cortex and dynamic membrane protrusions. Further, I demonstrate that TRE17 regulates the small GTPase Arf6. TRE17 directly associates with Arf6 in a GDP-dependent manner via TRE17's TBC domain. This association stimulates Arf6 activation, possibly by acting as a chaperone to recruit inactive Arf6 to the plasma membrane. TRE17 also regulates the trafficking of Arf6-dependent cargo, such as the β1-integrin and MHC I; and cell spreading, a process that depends on the coordinate regulation of actin remodeling and vesicular trafficking. Together, these results suggest that TRE17 functions downstream of Rho GTPases to regulate actin remodeling as well as vesicular trafficking via the Arf6-dependent pathway. Since deregulation of these processes lead to the acquisition of a more migratory and invasive phenotype in cancer cells, these studies not only provide insight into the normal function of TRE17, but also into how it may contribute to tumorigensis.
ABSTRACT This chapter focuses on the assays for monitoring p70S6 kinase (p70S6k) kinase and p90RS... more ABSTRACT This chapter focuses on the assays for monitoring p70S6 kinase (p70S6k) kinase and p90RSK (more commonly referred to as RSK) activation. A hallmark of the mitogenic response is an increase in protein synthesis. This increase arises from the modulation of multiple components of the translational machinery, including phosphorylation of the S6 polypeptide of the 40S ribosomal subunit. Much has been learned about the signaling pathways that mediate activation of p70S6k and RSK. One common activator of both enzymes is p21ras. However, p21ras utilizes distinct downstream effectors to elicit their activation. The development of specific antibodies against p70S6k and RSK has allowed monitoring of their activation in vivo. The availability of clean immunoblotting antibodies that detect mobility shifts is particularly useful for examining stimulation of transfected forms ofp70S6k by candidate activators. Because p70S6k protein levels can vary from sample to sample during transfections, such antibodies provide an additional control by demonstrating p70S6k activation through changes in the ratio of the four phosphorylated bands.
The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeli... more The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induc...
Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays... more Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 ...
The Rho family GTPase Cdc42 is recognized for its role in cellular proliferation and transformati... more The Rho family GTPase Cdc42 is recognized for its role in cellular proliferation and transformation. However, the mechanism by which it promotes cell cycle progression has remained undefined. Using an inducible expression system, we show that constitutively active Cdc42 (Cdc42V12) is sufficient by itself to induce anchorage-independent but not mitogen-independent growth in NIH3T3 cells. However, Cdc42V12 markedly accelerates activation of cyclin E-Cdk2 in response to mitogen. These effects were highly specific, as the kinetics of cyclin D-Cdk4 activation was unaltered. Cdc42V12 promotes Cdk2 activation by selectively inducing cyclin E expression without affecting other regulatory proteins such as the p27 Cdk inhibitor or Cdc25A. Furthermore, Cdc42V12 was able to activate a reporter gene driven by the cyclin E promoter in the absence of exogenous mitogen or adhesion. Cyclin E induction was sensitive to rapamycin but not inhibitors of mitogen-activated protein kinases, implicating p70 S6 kinase (p70S6k) as the relevant mediator. Consistent with this notion, wild type and constitutively active alleles of p70S6k were sufficient to activate the cyclin E promoter. In sum, these studies provide novel insights into the mechanism by which Cdc42 promotes G 1 progression.
In two conditioning experiments, identical procedures (previously shown to produce place preferen... more In two conditioning experiments, identical procedures (previously shown to produce place preferences for a cocaine-paired environment) were used to assess dopaminergic and behavioral activity correlates of cocaine reward conditioning and sensitization. In these experiments, animals received repeated injections of intravenous cocaine (4.2 mg / kg36) or saline (0.2 ml36) on alternating days. One group in each of these experiments ('Cocaine Cues') occupied a consistent distinctive environment during cocaine treatments and testing sessions. For the other conditioned group ('Novel'), all procedures were the same, except that the last cocaine injection was administered while animals were occupying a novel environment. During day 1 and day 6 of the cocaine treatment, behavioral activity was assessed in experiment 1 and in vivo microdialysis procedures were conducted in experiment 2. Over the course of the conditioning sessions, cocaine-induced behavioral activity (locomotion and rearing) increased significantly in the Cocaine Cues group, but not in the Novel group. In addition, cocaine-induced increases in NAcc dopamine levels were significantly greater when cocaine-experienced animals were tested in a cocaine-paired environment compared to equally experienced and cocaine-naive animals tested in a novel environment. Context-dependent behavioral sensitization is a well-documented phenomenon. The observation of a corresponding enhancement of dopamine efflux in lieu of a lengthy withdrawal period is uncommon, but can be attributed to methodological differences across studies. The present study uniquely demonstrates concurrent context-dependent potentiation of behavioral and dopaminergic responses to cocaine occurring in conjunction with cocaine reward.
In vitro drug testing requires long-term maintenance of hepatocyte liver specific functions. Hepa... more In vitro drug testing requires long-term maintenance of hepatocyte liver specific functions. Hepatocytes cultured at a higher seeding density in a sandwich configuration exhibit an increased level of liver specific functions when compared to low density cultures due to the better cell to cell contacts that promote long term maintenance of polarity and liver specific functions. However, culturing hepatocytes at high seeding densities in a standard 24-well plate poses problems in terms of the mass transport of nutrients and oxygen to the cells. In view of this drawback, we have developed a polydimethylsiloxane (PDMS) bioreactor that was able to maintain the long-term liver specific functions of a hepatocyte sandwich culture at a high seeding density. The bioreactor was fabricated with PDMS, an oxygen permeable material, which allowed direct oxygenation and perfusion to take place simultaneously. The mass transport of oxygen and the level of shear stress acting on the cells were analyzed by computational fluid dynamics (CFD). The combination of both direct oxygenation and perfusion has a synergistic effect on the liver specific function of a high density hepatocyte sandwich culture over a period of 9 days.
Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier st... more Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier structures when in extended culture. When the spheroid diameter exceeds 200 mm, cells in the inner core experience hypoxia and limited access to nutrients and drugs. Here we report the development of a thin galactosylated cellulosic sponge to culture hepatocytes in multi-well plates as 3D spheroids, and constrain them within a macroporous scaffold network to maintain spheroid size and prevent detachment. The hydrogel-based soft sponge conjugated with galactose provided suitable mechanical and chemical cues to support rapid formation of hepatocyte spheroids with a mature hepatocyte phenotype. The spheroids tethered in the sponge showed excellent maintenance of 3D cell morphology, cellecell interaction, polarity, metabolic and transporter function and/or expression. For example, cytochrome P450 (CYP1A2, CYP2B2 and CYP3A2) activities were significantly elevated in spheroids exposed to b-naphthoflavone, phenobarbital, or pregnenolone-16a-carbonitrile, respectively. The sponge also exhibits minimal drug absorption compared to other commercially available scaffolds. As the cell seeding and culture protocols are similar to various high-throughput 2D cell-based assays, this platform is readily scalable and provides an alternative to current hepatocyte platforms used in drug safety testing applications.
TBC (Tre-2/Bub2/Cdc16) domains are predicted to encode GTPase-activating proteins (GAPs) for Rab ... more TBC (Tre-2/Bub2/Cdc16) domains are predicted to encode GTPase-activating proteins (GAPs) for Rab family G proteins. While approximately 50 TBC proteins are predicted to exist in humans, little is known about their substrate specificity. Here we show that TRE17 (also called Tre-2 and USP6), a founding member of the TBC family, targets the Arf family GTPase Arf6, which regulates plasma membrane-endosome trafficking. Surprisingly, TRE17 does not function as a GAP for Arf6 but rather promotes its activation in vivo. TRE17 associates directly with Arf6 in its GDP-but not GTP-bound state. Mapping experiments pinpoint the site of interaction to the TBC domain of TRE17. Forced expression of TRE17 promotes the localization of Arf6 to the plasma membrane, leading to Arf6 activation, presumably due to facilitated access to membrane-associated guanine nucleotide exchange factors (GEFs). Furthermore, TRE17 cooperates with Arf6 GEFs to induce GTP loading of Arf6 in vivo. Finally, short interfering RNA-mediated loss of TRE17 leads to attenuated Arf6 activation. These studies identify TRE17 as a novel regulator of the Arf6-regulated plasma membrane recycling system and reveal an unexpected function for TBC domains.
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Papers by Jeffrey Robens