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Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2(+)) and neural plate border zone (pax3(+)), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5' regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show direct in vivo evidence of YAP's role in regulating pax3 neural crest expression.

Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis.

Background Unraveling the signaling pathways responsible for the establishment of a metastatic phenotype in carcinoma cells is critically important for understanding the pathology of cancer. The acquisition of cell motility is a key property of metastatic tumor cells and is a prerequisite for invasion. Rho GTPases regulate actin cytoskeleton reorganization and the cellular responses required for cell motility and invasion. Diacylglycerol kinase ζ (DGKζ), an enzyme that phosphorylates diacylglycerol to yield phosphatidic acid, regulates the activity of the Rho GTPases Rac1 and RhoA. DGKζ mRNA is highly expressed in several different colon cancer cell lines, as well as in colon cancer tissue relative to normal colonic epithelium, and thus may contribute to the metastatic process. Methods To investigate potential roles of DGKζ in cancer metastasis, a cellular, isogenic model of human colorectal cancer metastatic transition was used. DGKζ protein levels, Rac1 and RhoA activity, and PAK phosphorylation were measured in the non-metastatic SW480 adenocarcinoma cell line and its highly metastatic variant, the SW620 line. The effect of DGKζ silencing on Rho GTPase activity and invasion through Matrigel-coated Transwell inserts was studied in SW620 cells. Invasiveness was also measured in PC-3 prostate cancer and MDA-MB-231 breast cancer cells depleted of DGKζ. Results DGKζ protein levels were elevated approximately 3-fold in SW620 cells compared to SW480 cells. There was a concomitant increase in active Rac1 in SW620 cells, as well as substantial increases in the expression and phosphorylation of the Rac1 effector PAK1. Similarly, RhoA activity and expression were increased in SW620 cells. Knockdown of DGKζ expression in SW620 cells by shRNA-mediated silencing significantly reduced Rac1 and RhoA activity and attenuated the invasiveness of SW620 cells in vitro . DGKζ silencing in highly metastatic MDA-MB-231 breast cancer cells and PC-3 prostate cancer cells also significantly attenuated their invasiveness. Conclusion Elevated DGKζ expression contributes to increased Rho GTPase activation and the enhanced motility of metastatic cancer cells. These findings warrant further investigation of the clinical relevance of DGKζ upregulation in colon and other cancers. Interfering with DGKζ function could provide a means of inhibiting invasion and metastasis.

Doc number: 208 Abstract Background: Unraveling the signaling pathways responsible for the establishment of a metastatic phenotype in carcinoma cells is critically important for understanding the pathology of cancer. The acquisition of cell motility is a key property of metastatic tumor cells and is a prerequisite for invasion. Rho GTPases regulate actin cytoskeleton reorganization and the cellular responses required for cell motility and invasion. Diacylglycerol kinase ζ (DGKζ), an enzyme that phosphorylates diacylglycerol to yield phosphatidic acid, regulates the activity of the Rho GTPases Rac1 and RhoA. DGKζ mRNA is highly expressed in several different colon cancer cell lines, as well as in colon cancer tissue relative to normal colonic epithelium, and thus may contribute to the metastatic process. Methods: To investigate potential roles of DGKζ in cancer metastasis, a cellular, isogenic model of human colorectal cancer metastatic transition was used. DGKζ protein levels, Rac1 and RhoA activity, and PAK phosphorylation were measured in the non-metastatic SW480 adenocarcinoma cell line and its highly metastatic variant, the SW620 line. The effect of DGKζ silencing on Rho GTPase activity and invasion through Matrigel-coated Transwell inserts was studied in SW620 cells. Invasiveness was also measured in PC-3 prostate cancer and MDA-MB-231 breast cancer cells depleted of DGKζ. Results: DGKζ protein levels were elevated approximately 3-fold in SW620 cells compared to SW480 cells. There was a concomitant increase in active Rac1 in SW620 cells, as well as substantial increases in the expression and phosphorylation of the Rac1 effector PAK1. Similarly, RhoA activity and expression were increased in SW620 cells. Knockdown of DGKζ expression in SW620 cells by shRNA-mediated silencing significantly reduced Rac1 and RhoA activity and attenuated the invasiveness of SW620 cells in vitro . DGKζ silencing in highly metastatic MDA-MB-231 breast cancer cells and PC-3 prostate cancer cells also significantly attenuated their invasiveness. Conclusion: Elevated DGKζ expression contributes to increased Rho GTPase activation and the enhanced motility of metastatic cancer cells. These findings warrant further investigation of the clinical relevance of DGKζ upregulation in colon and other cancers. Interfering with DGKζ function could provide a means of inhibiting invasion and metastasis.

Extracellular signal-regulated kinase 3 (ERK3) promotes cell migration and tumor metastasis in multiple cancer types, including lung cancer. The extracellular-regulated kinase 3 protein has a unique structure. In addition to the N-terminal kinase domain, ERK3 includes a central conserved in extracellular-regulated kinase 3 and ERK4 (C34) domain and an extended C-terminus. However, relatively little is known regarding the role(s) of the C34 domain. A yeast two-hybrid assay using extracellular-regulated kinase 3 as bait identified diacylglycerol kinase ζ (DGKζ) as a binding partner. DGKζ was shown to promote migration and invasion in some cancer cell types, but its role in lung cancer cells is yet to be described. The interaction of extracellular-regulated kinase 3 and DGKζ was confirmed by co-immunoprecipitation and in vitro binding assays, consistent with their co-localization at the periphery of lung cancer cells. The C34 domain of ERK3 was sufficient for binding to DGKζ, while extracellular-regulated kinase 3 bound to the N-terminal and C1 domains of DGKζ. Surprisingly, in contrast to extracellular-regulated kinase 3, DGKζ suppresses lung cancer cell migration, suggesting DGKζ might inhibit ERK3-mediated cell motility. Indeed, co-overexpression of exogenous DGKζ and extracellular-regulated kinase 3 completely blocked the ability of ERK3 to promote cell migration, but DGKζ did not affect the migration of cells with stable ERK3 knockdown. Furthermore, DGKζ had little effect on cell migration induced by overexpression of an ERK3 mutant missing the C34 domain, suggesting DGKζ requires this domain to prevent ERK3-mediated increase in cell migration. In summary, this study has identified DGKζ as a new binding partner and negative regulator of extracellular-regulated kinase 3 in controlling lung cancer cell migration.

We report long-cavity (60.5 λ) GaN-based vertical-cavity surface-emitting lasers with a topside monolithic GaN concave mirror, a buried tunnel junction current aperture, and a bottomside nanoporous GaN distributed Bragg reflector. Under pulsed operation, a VCSEL with a 9 µm aperture had a threshold current density of 6.6 kA/cm2, a differential efficiency of 0.7%, and a maximum output power of 290 µW for a lasing mode at 411 nm and a divergence angle of 8.4°. Under CW operation, the threshold current density increased to 7.3 kA/cm2, the differential efficiency decreased to 0.4%, and a peak output power of 130 µW was reached at a current density of 23 kA/cm2.

Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase [zeta] (DGK[zeta]), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGK[zeta] is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGK[zeta] was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGK[zeta]-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGK[zeta]-null cells were smaller than those found in wild type cells. Rescue of this defect required DGK[zeta] catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGK[zeta] mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGK[zeta] functions in concert with Rac1 to regulate macropinocytosis.

Lipid droplets (LDs) are organelles that are necessary for eukaryotic and prokaryotic metabolism and energy storage. They have a unique structure consisting of a spherical phospholipid monolayer encasing neutral lipids such as triacylglycerol (TAG). LDs have garnered increased interest for their implications in disease and for drug delivery applications. Consequently, there is an increased need for tools to study their structure, composition, and dynamics in biological contexts. In this work, we utilize CHARMM‐GUI Membrane Builder to simulate and analyze LDs with and without a plant LD protein, oleosin. The results show that Membrane Builder can generate biologically relevant all‐atom LD systems with relatively short equilibration times using a new TAG library having optimized headgroup parameters. TAG molecules originally inserted into a lipid bilayer aggregate in the membrane center, forming a TAG‐only core flanked by two monolayers. The TAG‐only core thickness stably grows with increasing TAG mole fraction. A 70 % TAG system has a core that is thick enough to house oleosin without its interactions with the distal leaflet or disruption of its secondary structure. We hope that Membrane Builder can aid in the future study of LD systems, including their structure and dynamics with and without proteins. CHARMM‐GUI Membrane Builder can be used to build biologically relevant simulations of lipid droplet models with and without proteins using an extensive library of triacylglycerol lipids. Membrane Builder generates lipid droplet models with easily tunable sizes and compositions, while keeping equilibration times relatively short. These features make it a prime tool for the computational study of lipid droplets.

Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2.sup.+) and neural plate border zone (pax3.sup.+ ), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5' regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show direct in vivo evidence of YAP's role in regulating pax3 neural crest expression.