Tara Teslaa

Wnt signaling directs a metabolic program of glycolysis and angiogenesis in colon cancer

Much of the mechanism by which Wnt signaling drives proliferation during oncogenesis is attributed to its regulation of the cell cycle. Here, we show how Wnt/β‐catenin signaling directs another hallmark of tumorigenesis, namely Warburg metabolism. Using biochemical assays and fluorescence lifetime imaging microscopy (FLIM) to probe metabolism in vitro and in living tumors, we observe that interference with Wnt signaling in colon cancer cells reduces glycolytic metabolism and results in small, poorly perfused tumors. We identify pyruvate dehydrogenase kinase 1 (PDK1) as an important direct target within a larger gene program for metabolism. PDK1 inhibits pyruvate flux to mitochondrial respiration and a rescue of its expression in Wnt‐inhibited cancer cells rescues glycolysis as well as vessel growth in the tumor microenvironment. Thus, we identify an important mechanism by which Wnt‐driven Warburg metabolism directs the use of glucose for cancer cell proliferation and links it to vessel delivery of oxygen and nutrients.

Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade

Burkholderia pseudomallei and Burkholderia thailandensis are related pathogens that invade a variety of cell types, replicate in the cytoplasm, and spread to nearby cells. We have investigated temporal and spatial requirements for virulence determinants in the intracellular life cycle, using genetic dissection and photothermal nanoblade delivery, which allows efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells. The conserved Bsa type III secretion system (T3SSBsa) is dispensable for invasion, but is essential for escape from primary endosomes. By nanoblade delivery of B. thailandensis we demonstrate that all subsequent events in intercellular spread occur independently of T3SSBsa activity. Although intracellular movement was essential for cell–cell spread by B. pseudomallei and B. thailandensis, neither BimA-mediated actin polymerization nor the formation of membrane protrusions containing bacteria was required for B. thailandensis. Surprisingly, the cryptic (fla2) flagellar system encoded on chromosome 2 of B. thailandensis supported rapid intracellular motility and efficient cell–cell spread. Plaque formation by both pathogens was dependent on the activity of a type VI secretion system (T6SS-1) that functions downstream from T3SSBsa-mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleate giant cells (MNGCs) in multiple cell types. By infection and nanoblade delivery, we observed complete correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies showed that plaque formation represents MNGC death. Our data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles.

Pluripotent stem cell energy metabolism: an update

Recent studies link changes in energy metabolism with the fate of pluripotent stem cells (PSCs). Safe use of PSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Relative shifts in metabolism from naïve through “primed” pluripotent states to lineage‐directed differentiation place variable demands on mitochondrial biogenesis and function for cell types with distinct energetic and biosynthetic requirements. In this context, mitochondrial respiration, network dynamics, TCA cycle function, and turnover all have the potential to influence reprogramming and differentiation outcomes. Shifts in cellular metabolism affect enzymes that control epigenetic configuration, which impacts chromatin reorganization and gene expression changes during reprogramming and differentiation. Induced PSCs (iPSCs) may have utility for modeling metabolic diseases caused by mutations in mitochondrial DNA, for which few disease models exist. Here, we explore key features of PSC energy metabolism research in mice and man and the impact this work is starting to have on our understanding of early development, disease modeling, and potential therapeutic applications.

Pulsed laser triggered high speed microfluidic fluorescence activated cell sorter

We report a high speed and high purity pulsed laser triggered fluorescence activated cell sorter (PLACS) with a sorting throughput up to 20,000 mammalian cells/s with 37% sorting purity, 90% cell viability in enrichment mode, and >;90% purity in high purity mode at 1,500 cells/s or 3,000 beads/s. Fast switching (30 μs) and a small perturbation volume (~90 pL) is realized by a unique sorting mechanism in which explosive vapor bubbles are generated using focused laser pulses in a single layer microfluidic PDMS channel.

Techniques to Monitor Glycolysis

An increased flux through glycolysis supports the proliferation of cancer cells by providing additional energy in the form of ATP as well as glucose-derived metabolic intermediates for nucleotide, lipid, and protein biosynthesis. Thus, glycolysis and other metabolic pathways that control cell proliferation may represent valuable targets for therapeutic interventions and diagnostic procedures. In this context, the measurement of glucose uptake and lactate excretion by malignant cells may be useful to detect shifts in glucose catabolism, while determining the activity of rate-limiting glycolytic enzymes can provide insights into points of metabolic regulation. Moreover, metabolomic studies can be used to generate large, integrated datasets to track changes in carbon flux through glycolysis and its collateral anabolic pathways. As discussed here, these approaches can reveal and quantify the metabolic alterations that underlie malignant cell proliferation.

α-Ketoglutarate Accelerates the Initial Differentiation of Primed Human Pluripotent Stem Cells

Pluripotent stem cells (PSCs) can self-renew or differentiate from naive or more differentiated, primed, pluripotent states established by specific culture conditions. Increased intracellular α-ketoglutarate (αKG) was shown to favor self-renewal in naive mouse embryonic stem cells (mESCs). The effect of αKG or αKG/succinate levels on differentiation from primed human PSCs (hPSCs) or mouse epiblast stem cells (EpiSCs) remains unknown. We examined primed hPSCs and EpiSCs and show that increased αKG or αKG-to-succinate ratios accelerate, and elevated succinate levels delay, primed PSC differentiation. αKG has been shown to inhibit the mitochondrial ATP synthase and to regulate epigenome-modifying dioxygenase enzymes. Mitochondrial uncoupling did not impede αKG-accelerated primed PSC differentiation. Instead, αKG induced, and succinate impaired, global histone and DNA demethylation in primed PSCs. The data support αKG promotion of self-renewal or differentiation depending on the pluripotent state.

Photothermal nanoblade for large cargo delivery into mammalian cells

We report a photothermal nanoblade that utilizes a metallic nanostructure to harvest laser pulse energy and convert it into a localized explosive vapor bubble, which rapidly punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. Integrating the metallic nanostructure with a micropipette, the nanoblade generates a micron-sized membrane access port for delivering concentrated cargo (5×108 bacteria/ml) with high efficiency (46%) and cell viability (>90%) into mammalian cells. Additional biologic and inanimate cargo over 3-orders of magnitude in size including DNA, 200 nm polystyrene beads to 2 µm bacteria have been delivered into multiple cell types.

Nanoblade delivery and incorporation of quantum dot conjugates into tubulin networks in live cells.

Quantum dots (QDs) have not been used to label cytoskeleton structure of live cells owing to limitations in delivery strategies, and QDs conjugation methods and issues with nonspecific binding. We conjugated tubulin to QDs and applied the emerging method of photothermal nanoblade to deliver QD-tubulin conjugates into live Hela cells. This method will open new opportunities for cytosolic targeting of QDs in live cells.

Photothermal nanoblade for patterned cell membrane cutting

We report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized and specifically shaped explosive vapor bubble. Rapid bubble expansion and collapse punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The membrane cutting pattern is controlled by the metallic nanostructure configuration, laser pulse polarization, and energy. Highly controllable, sub-micron sized circular hole pairs to half moon-like, or cat-door shaped, membrane cuts were realized in glutaraldehyde treated HeLa cells.

Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells

mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggests metabolite profiling can help assess the quality and function of mtDNA-modified cells.