A global database of lake surface temperatures collected by in situ and satellite methods from 1985–2009

Global environmental change has influenced lake surface temperatures, a key driver of ecosystem structure and function. Recent studies have suggested significant warming of water temperatures in individual lakes across many different regions around the world. However, the spatial and temporal coherence associated with the magnitude of these trends remains unclear. Thus, a global data set of water temperature is required to understand and synthesize global, long-term trends in surface water temperatures of inland bodies of water. We assembled a database of summer lake surface temperatures for 291 lakes collected in situ and/or by satellites for the period 1985–2009. In addition, corresponding climatic drivers (air temperatures, solar radiation, and cloud cover) and geomorphometric characteristics (latitude, longitude, elevation, lake surface area, maximum depth, mean depth, and volume) that influence lake surface temperatures were compiled for each lake. This unique dataset offers an invaluable baseline perspective on global-scale lake thermal conditions as environmental change continues

A global database of lake surface temperatures collected by in situ and satellite methods from 1985–2009

Global environmental change has influenced lake surface temperatures, a key driver of ecosystem structure and function. Recent studies have suggested significant warming of water temperatures in individual lakes across many different regions around the world. However, the spatial and temporal coherence associated with the magnitude of these trends remains unclear. Thus, a global data set of water temperature is required to understand and synthesize global, long-term trends in surface water temperatures of inland bodies of water. We assembled a database of summer lake surface temperatures for 291 lakes collected in situ and/or by satellites for the period 1985–2009. In addition, corresponding climatic drivers (air temperatures, solar radiation, and cloud cover) and geomorphometric characteristics (latitude, longitude, elevation, lake surface area, maximum depth, mean depth, and volume) that influence lake surface temperatures were compiled for each lake. This unique dataset offers an invaluable baseline perspective on global-scale lake thermal conditions as environmental change continues

Directed Migration Achieved in Mammalian Cells via Ca2+ Signal Rewiring in Synthetic Protein Chimeras

Synthetic biology achieves control over cellular behavior by endogenous re-wiring. Ca2+ signals allow cells to regulate diverse processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g. VEGFR2), Ca2+ signals are generated upon ligand binding (e.g. VEGF-A). Here, firstly we engineered fusion proteins that generate a Ca2+ signal upon ligand binding by creating fusions of domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we re-wired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Next, we validated this design strategy in two systems, i.e., inflammatory cytokines such as TNFα and antibodies. A system of proteins was used: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca2+-activated RhoA (named CaRQ), VSVG and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs allowing migration towards the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, post-ganciclovir treatment, cells undergo death via the thymidine kinase suicide mechanism. Hence, this forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low pH microenvironment. Lastly, we exploited antibodies to bind their antigens exclusively in combination with the ability of the cytoplasmic domain of VEGFR2 to generate a Ca2+ signal upon oligomerization. Using protein fusions between antibody variants (i.e. nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca2+ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumour necrosis factor alpha and soluble CD14. The Ca2+ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state but instead, only required an increase in the oligomeric state. The Ca2+ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca2+ indicator or with an engineered Ca2+ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively.Ph.D

Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells

Abstract Background To achieve synthetic control over how a cell responds to other cells or the extracellular environment, it is important to reliably engineer proteins that can traffic and span the plasma membrane. Using a modular approach to assemble proteins, we identified the minimum necessary components required to engineer such membrane-spanning proteins with predictable orientation in mammalian cells. Results While a transmembrane domain (TM) fused to the N-terminus of a protein is sufficient to traffic it to the endoplasmic reticulum (ER), an additional signal peptidase cleavage site downstream of this TM enhanced sorting out of the ER. Next, a second TM in the synthetic protein helped anchor and accumulate the membrane-spanning protein on the plasma membrane. The orientation of the components of the synthetic protein were determined through measuring intracellular Ca2+ signaling using the R-GECO biosensor and through measuring extracellular quenching of yellow fluorescent protein variants by saturating acidic and salt conditions. Conclusions This work forms the basis of engineering novel proteins that span the plasma membrane to potentially control intracellular responses to extracellular conditions

Additional file 1: Figure S1. of Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells

Fluorescence images of CHO cells transfected with the plasma membrane labelled Lyn-Ceru and ER labelled STIM1-mRFP markers. CHO cells transfected with the plasma membrane labelled Lyn-Ceru showed a matte-like appearance (a, f and k) while those transfected with the ER labelled STIM1-mRFP showed a web-like fluorescence distribution (d, i and n). TM-Venus (b), TLP-Venus (g) or TLP-V-TM (l) peptides show a shift in fluorescence distribution from a wholly ER (b) to a slight (g) and then an entirely membrane appearance (l). Merged images illustrate resultant co-localization (c, e, h, j, m and o). TM: transmembrane domain TLR4, TLP: fusion of TM with signal peptidase cleavage site from human immunoglobulin K, V: Venus fluorescent protein, LC: Lyn-Ceru, STIM1: stromal interaction molecule 1. Scale bars are 10 Οm. Images are false colored: CFP, cyan; YFP, green; mRFP, red. All insets show zoomed regions (4x) of structures in dotted rectangles. All experiments were repeated at least 3 times. (PDF 9198 kb

Antibody-Based Fusion Proteins Allow Ca²⁺ Rewiring to Most Extracellular Ligands

The Ca²⁺ signaling toolkit is the set of proteins used by living systems to generate and respond to Ca²⁺ signals. The selective expression of these proteins in particular tissues, cell types and subcellular locations allows the Ca²⁺ signal to regulate a diverse set of cellular processes. Through synthetic biology, the Ca²⁺ signaling toolkit can be expanded beyond the natural repertoire to potentially allow a non-natural ligand to control downstream cellular processes. To realize this potential, we exploited the ability of an antibody to bind its antigen exclusively in combination with the ability of the cytoplasmic domain of vascular endothelial growth factor receptor 2 (VEGFR2) to generate a Ca²⁺ signal upon oligomerization. Using protein fusions between antibody variants (<i>i.e.</i>, nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca²⁺ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumor necrosis factor alpha and soluble CD14. The Ca²⁺ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state, but instead only required an increase in the oligomeric state. The Ca²⁺ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca²⁺ indicator or with an engineered Ca²⁺ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively

Autonomous Cell Migration to CSF1 Sources <i>via</i> a Synthetic Protein-Based System

Inflammatory lesions, often seen in diseases such as rheumatoid arthritis, atherosclerosis and cancer, feature an acidic (<i>i.e.</i>, low pH) microenvironment rampant with cytokines, such as CSF1. For potential therapeutic intervention targeted at these CSF1 sources, we have assembled a system of four proteins inside a cell (<i>i.e.</i>, HEK293) that initially had no natural CSF1-seeking ability. This system included a newly engineered CSF1 chimera receptor (named CSF1Rchi), the previously engineered Ca²⁺ activated RhoA (<i>i.e.</i>, CaRQ), vesicular stomatitis virus glycoprotein G (VSVG) and thymidine kinase (TK). The binding of CSF1 to the CSF1Rchi generated a Ca²⁺ signal that activated CaRQ-mediated cellular blebbing, allowing autonomous cell migration toward the CSF1 source. Next, the VSVG protein allowed these engineered cells to fuse with the CSF1 source cells, upon low pH induction. Finally, these cells underwent death postganciclovir treatment, <i>via</i> the TK suicide mechanism. Hence, this protein system could potentially serve as the basis of engineering a cell to target inflammatory lesions in diseases featuring a microenvironment with high levels of CSF1 and low pH

Additional file 2: Figure S2. of Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells

Line graph showing changes in distribution of plasma membrane labelled Lyn-Ceru compared with ER-labelled STIM1-mRFP, TM-Venus and TLP-Venus in CHO cells, post cyclohexamide treatment. CHO cells transfected with the plasma membrane labelled Lyn-Ceru and the ER labelled STIM1-mRFP, TM-Venus or TLP-Venus. Cells were imaged initially and after 1, 4 and 12 h after incubation with 10 μg/mL cyclohexamide. Y-axis shows PCC of the STIM1-mRFP, TM-Venus or TLP-Venus with Lyn-Ceru. TM: transmembrane domain TLR4, TLP: fusion of TM with signal peptidase cleavage site from human immunoglobulin K, V: Venus fluorescent protein, LC: Lyn-Ceru, STIM1: stromal interaction molecule 1. Error bars indicate s.d. Star indicates significance of p < 0.05. (PDF 238 kb

Engineering Synthetic Proteins to Generate Ca²⁺ Signals in Mammalian Cells

The versatility of Ca²⁺ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (<i>e.g.</i>, VEGFR2), Ca²⁺ signals are generated upon binding their ligand(s) (<i>e.g.</i>, VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca²⁺ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca²⁺ signals upon stimulation with various extracellular stimuli (<i>e.g.</i>, rapamycin, EDTA or extracellular free Ca²⁺). By coupling these chimeric proteins that generate Ca²⁺ signals with proteins that respond to Ca²⁺ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca²⁺. Thus, using this design strategy, it is possible to engineer proteins to generate a Ca²⁺ signal to rewire a wide range of extracellular stimuli to a wide range of Ca²⁺-activated processes