The Engineer as Economist: Sewers and the Making of the Water Consumer in Colonial Cairo, 1890.

Modern sewage systems were once a cutting-edge innovation that transformed how people consumed water. This article examines a debate among a group of British engineers involved in a sewage system scheme in late nineteenth-century Cairo, when Egypt was under British colonial rule. Assessing the project's economic feasibility, the engineers came to different conclusions regarding the future users of the system and, by extension, future consumers of water. Reconstructing the debate sets up a dialogue between engineering and economics to show that engineers could be economists too. This debate represents a type of economic analysis that public works engineers pioneered in the mid-nineteenth century. The engineers reached novel conclusions by centering consumers as the foundation for calculating economic realities.

Stabilization of the RAS:PDE6D Complex Is a Novel Strategy to Inhibit RAS Signaling.

RAS is a major anticancer drug target which requires membrane localization to activate downstream signal transduction. The direct inhibition of RAS has proven to be challenging. Here, we present a novel strategy for targeting RAS by stabilizing its interaction with the prenyl-binding protein PDE6D and disrupting its localization. Using rationally designed RAS point mutations, we were able to stabilize the RAS:PDE6D complex by increasing the affinity of RAS for PDE6D, which resulted in the redirection of RAS to the cytoplasm and the primary cilium and inhibition of oncogenic RAS/ERK signaling. We developed an SPR fragment screening and identified fragments that bind at the KRAS:PDE6D interface, as shown through cocrystal structures. Finally, we show that the stoichiometric ratios of KRAS:PDE6D vary in different cell lines, suggesting that the impact of this strategy might be cell-type-dependent. This study forms the foundation from which a potential anticancer small-molecule RAS:PDE6D complex stabilizer could be developed.

PINK1 drives production of mtDNA-containing extracellular vesicles to promote invasiveness.

The cystine-glutamate antiporter, xCT, supports a glutathione synthesis program enabling cancer cells to cope with metabolically stressful microenvironments. Up-regulated xCT, in combination with glutaminolysis, leads to increased extracellular glutamate, which promotes invasive behavior by activating metabotropic glutamate receptor 3 (mGluR3). Here we show that activation of mGluR3 in breast cancer cells activates Rab27-dependent release of extracellular vesicles (EVs), which can transfer invasive characteristics to "recipient" tumor cells. These EVs contain mitochondrial DNA (mtDNA), which is packaged via a PINK1-dependent mechanism. We highlight mtDNA as a key EV cargo necessary and sufficient for intercellular transfer of invasive behavior by activating Toll-like receptor 9 in recipient cells, and this involves increased endosomal trafficking of pro-invasive receptors. We propose that an EV-mediated mechanism, through which altered cellular metabolism in one cell influences endosomal trafficking in other cells, is key to generation and dissemination of pro-invasive microenvironments during mammary carcinoma progression.

The Structural and Biochemical Characterization of UNC119B Cargo Binding and Release Mechanisms.

Two paralogs of the guanine dissociation inhibitor-like solubilizing factors UNC119, UNC119A and UNC119B, are present in the human genome. UNC119 binds to N-myristoylated proteins and masks the hydrophobic lipid from the hydrophilic cytosol, facilitating trafficking between different membranes. Two classes of UNC119 cargo proteins have been classified: low affinity cargoes, released by the Arf-like proteins ARL2 and ARL3, and high affinity cargoes, which are specifically released by ARL3 and trafficked to either the primary cilium or the immunological synapse. The UNC119 homologues have reported differences in functionality, but the structural and biochemical bases for these differences are unknown. Using myristoylated peptide binding and release assays, we show that peptides sharing the previously identified UNC119A high affinity motif show significant variations of binding affinities to UNC119B of up to 427-fold. Furthermore, we solve the first two crystal structures of UNC119B, one in complex with the high affinity cargo peptide of LCK and a second one in complex with the release factor ARL3. Using these novel structures, we identify a stretch of negatively charged amino acids unique to UNC119B that may undergo a conformational change following binding of a release factor which we propose as an additional release mechanism specific to UNC119B.

CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation.

The Scar/WAVE complex drives actin nucleation during cell migration. Interestingly, the same complex is important in forming membrane ruffles during macropinocytosis, a process mediating nutrient uptake and membrane receptor trafficking. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A, has not been characterized. Here, we implicate CYRI-A as a key regulator of macropinosome formation and integrin internalization. We find that CYRI-A is transiently recruited to nascent macropinosomes, dependent on PI3K and RAC1 activity. CYRI-A recruitment precedes RAB5A recruitment but follows sharply after RAC1 and actin signaling, consistent with it being a local inhibitor of actin polymerization. Depletion of both CYRI-A and -B results in enhanced surface expression of the α5ß1 integrin via reduced internalization. CYRI depletion enhanced migration, invasion, and anchorage-independent growth in 3D. Thus, CYRI-A is a dynamic regulator of macropinocytosis, functioning together with CYRI-B to regulate integrin trafficking.

An ARF GTPase module promoting invasion and metastasis through regulating phosphoinositide metabolism.

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.

ARL3 activation requires the co-GEF BART and effector-mediated turnover.

The ADP-ribosylation factor-like 3 (ARL3) is a ciliopathy G-protein which regulates the ciliary trafficking of several lipid-modified proteins. ARL3 is activated by its guanine exchange factor (GEF) ARL13B via an unresolved mechanism. BART is described as an ARL3 effector which has also been implicated in ciliopathies, although the role of its ARL3 interaction is unknown. Here, we show that, at physiological GTP:GDP levels, human ARL3GDP is weakly activated by ARL13B. However, BART interacts with nucleotide-free ARL3 and, in concert with ARL13B, efficiently activates ARL3. In addition, BART binds ARL3GTP and inhibits GTP dissociation, thereby stabilising the active G-protein; the binding of ARL3 effectors then releases BART. Finally, using live cell imaging, we show that BART accesses the primary cilium and colocalises with ARL13B. We propose a model wherein BART functions as a bona fide co-GEF for ARL3 and maintains the active ARL3GTP, until it is recycled by ARL3 effectors.

ARL3, a small GTPase with a functionally conserved role in primary cilia and immune synapses.

The primary cilium and the immunological synapse are both specialized functional plasma membrane domains that share several similarities. Signalling output of membrane domains is regulated, spatially and temporally, by segregating and focusing lipids and proteins. ARL3, a small GTPase, plays a major role in concentrating lipid-modified proteins in both the immunological synapse and the primary cilia. Here in this review we will introduce the role of ARL3 in health and disease and its role in polarizing signalling at the primary cilia and immunological synapses.

Structural Basis of CYRI-B Direct Competition with Scar/WAVE Complex for Rac1.

Rac1 is a major regulator of actin dynamics, with GTP-bound Rac1 promoting actin assembly via the Scar/WAVE complex. CYRI competes with Scar/WAVE for interaction with Rac1 in a feedback loop regulating actin dynamics. Here, we reveal the nature of the CYRI-Rac1 interaction, through crystal structures of CYRI-B lacking the N-terminal helix (CYRI-BΔN) and the CYRI-BΔN:Rac1Q61L complex, providing the molecular basis for CYRI-B regulation of the Scar/WAVE complex. We reveal CYRI-B as having two subdomains - an N-terminal Rac1 binding subdomain with a unique Rac1-effector interface and a C-terminal Ratchet subdomain that undergoes conformational changes induced by Rac1 binding. Finally, we show that the CYRI protein family, CYRI-A and CYRI-B can produce an autoinhibited hetero- or homodimers, adding an additional layer of regulation to Rac1 signaling.

Spatiotemporal Regulation of Signaling: Focus on T Cell Activation and the Immunological Synapse.

In a signaling network, not only the functions of molecules are important but when (temporal) and where (spatial) those functions are exerted and orchestrated is what defines the signaling output. To temporally and spatially modulate signaling events, cells generate specialized functional domains with variable lifetime and size that concentrate signaling molecules, enhancing their transduction potential. The plasma membrane is a key in this regulation, as it constitutes a primary signaling hub that integrates signals within and across the membrane. Here, we examine some of the mechanisms that cells exhibit to spatiotemporally regulate signal transduction, focusing on the early events of T cell activation from triggering of T cell receptor to formation and maturation of the immunological synapse.

Bacterial delivery of the anti-tumor azurin-like protein Laz to glioblastoma cells.

Salmonella typhimurium VNP-20009 (VNP) is a non-pathogenic attenuated strain, which, as a facultative anaerobe, preferentially accumulates in hypoxic regions of solid tumors. Here, VNP was utilized as a delivery vehicle of the anti-tumor protein Lipidated azurin, Laz, which is produced by the meningitis-causing bacterium Neisseria meningitides. In brain cancer cells, Laz has been demonstrated to induce apoptosis through an interaction with the tumor suppressor protein p53. In this study, the laz gene, including its signal sequence, was cloned downstream of a hypoxia inducible promoter (HIP-1), before being electroporated into VNP. Successful ectopic expression and export of the Laz protein by VNP under hypoxic conditions were confirmed by Western blot analysis of the cell-free culture medium. Effective expression of Laz by VNP was investigated in two glioblastoma cell lines: LN-229 and U-373, with the latter line carrying a mutated version of p53; as well as in the breast cancer line MCF-7. Cytotoxicity of the VNP-Laz was assessed by determining the fluorescence of the apoptotic marker caspases 3/7. Compared to the purified Laz, VNP-Laz, significantly induced apoptosis in MCF-7, LN-229 and, to a much lower extent in U-373 cells, suggesting a p53-linked mechanism. Our results might represent a new approach of targeted gene delivery and suggest a potential application in brain tumor therapy.

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

Efficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP's Cdc42 and Rac interacting binding ("CRIB") motif has been thought to be essential for its activation. However, we show that the CRIB motif's biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought-Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

Hypoxic cancer-associated fibroblasts increase NCBP2-AS2/HIAR to promote endothelial sprouting through enhanced VEGF signaling.

Intratumoral hypoxia causes the formation of dysfunctional blood vessels, which contribute to tumor metastasis and reduce the efficacy of therapeutic treatments. Blood vessels are embedded in the tumor stroma of which cancer-associated fibroblasts (CAFs) constitute a prominent cellular component. We found that hypoxic human mammary CAFs promoted angiogenesis in CAF-endothelial cell cocultures in vitro. Mass spectrometry-based proteomic analysis of the CAF secretome unraveled that hypoxic CAFs contributed to blood vessel abnormalities by altering their secretion of various pro- and anti-angiogenic factors. Hypoxia induced pronounced remodeling of the CAF proteome, including proteins that have not been previously related to this process. Among those, the uncharacterized protein NCBP2-AS2 that we renamed HIAR (hypoxia-induced angiogenesis regulator) was the protein most increased in abundance in hypoxic CAFs. Silencing of HIAR abrogated the pro-angiogenic and pro-migratory function of hypoxic CAFs by decreasing secretion of the pro-angiogenic factor VEGFA and consequently reducing VEGF/VEGFR downstream signaling in the endothelial cells. Our study has identified a regulator of angiogenesis and provides a map of hypoxia-induced molecular alterations in mammary CAFs.

ARL3 Mutations Cause Joubert Syndrome by Disrupting Ciliary Protein Composition.

Joubert syndrome (JBTS) is a genetically heterogeneous autosomal-recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997-109106128, UCSC Genome Browser hg 19), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP ribosylation factor-like GTPase 3 (ARL3), is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo.

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions.

Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a 'local inhibitor' as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.

The Ciliary Machinery Is Repurposed for T Cell Immune Synapse Trafficking of LCK.

Upon engagement of the T cell receptor with an antigen-presenting cell, LCK initiates TCR signaling by phosphorylating its activation motifs. However, the mechanism of LCK activation specifically at the immune synapse is a major question. We show that phosphorylation of the LCK activating Y394, despite modestly increasing its catalytic rate, dramatically focuses LCK localization to the immune synapse. We describe a trafficking mechanism whereby UNC119A extracts membrane-bound LCK by sequestering the hydrophobic myristoyl group, followed by release at the target membrane under the control of the ciliary ARL3/ARL13B. The UNC119A N terminus acts as a "regulatory arm" by binding the LCK kinase domain, an interaction inhibited by LCK Y394 phosphorylation, thus together with the ARL3/ARL13B machinery ensuring immune synapse focusing of active LCK. We propose that the ciliary machinery has been repurposed by T cells to generate and maintain polarized segregation of signals such as activated LCK at the immune synapse.

Maintaining protein composition in cilia.

The primary cilium is a sensory organelle that is vital in regulating several signalling pathways. Unlike most organelles cilia are open to the rest of the cell, not enclosed by membranes. The distinct protein composition is crucial to the function of cilia and many signalling proteins and receptors are specifically concentrated within distinct compartments. To maintain this composition, a mechanism is required to deliver proteins to the cilium whilst another must counter the entropic tendency of proteins to distribute throughout the cell. The combination of the two mechanisms should result in the concentration of ciliary proteins to the cilium. In this review we will look at different cellular mechanisms that play a role in maintaining the distinct composition of cilia, including regulation of ciliary access and trafficking of ciliary proteins to, from and within the cilium.

Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium.

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates ß-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis.

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity.

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion.

A fully automated procedure for the parallel, multidimensional purification and nucleotide loading of the human GTPases KRas, Rac1 and RalB.

Small GTPases regulate many key cellular processes and their role in human disease validates many proteins in this class as desirable targets for therapeutic intervention. Reliable recombinant production of GTPases, often in the active GTP loaded state, is a prerequisite for the prosecution of drug discovery efforts. The preparation of these active forms can be complex and often constricts the supply to the reagent intensive techniques used in structure base drug discovery. We have established a fully automated, multidimensional protein purification strategy for the parallel production of the catalytic G-domains of KRas, Rac1 and RalB GTPases in the active form. This method incorporates a four step chromatography purification with TEV protease-mediated affinity tag cleavage and a conditioning step that achieves the activation of the GTPase by exchanging GDP for the non-hydrolyzable GTP analogue GMPPnP. We also demonstrate that an automated method is efficient at loading of KRas with mantGDP for application in a SOS1 catalysed fluorescent nucleotide exchange assay. In comparison to more conventional manual workflows the automated method offers marked advantages in method run time and operator workload. This reduces the bottleneck in protein production while generating products that are highly purified and effectively loaded with nucleotide analogues.