Insights into E. coli cyclopropane fatty acid synthase (CFAS) towards enantioselective carbene free biocatalytic cyclopropanation.

Cyclopropane fatty acid synthases (CFAS) are a class of S-adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene based approaches. This work describes the characterisation of E. coli CFAS enzyme (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids.  ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 from  in up to 58% conversion and 73% ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ecCFAS was found to be correlated with the electronic properties of phospholipid headgroups  and for the first time ecCFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in-silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ecCFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining pure ecCFAS with the SAM regenerating AtHMT enzyme in presence of CH3I and CD3I.

A palmitoyl transferase chemical-genetic system to map ZDHHC-specific S-acylation.

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical-genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms.

Biocompatible Magnetic Conjugated Polymer Nanoparticles for Optical and Lifetime Imaging Applications in the First Biological Window.

Conjugated polymers are organic semiconductors that can be used for fluorescence microscopy of living specimens. Here, we report the encapsulation of the bright-red-emitting conjugated polymer, poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorenylene}-alt-co-{2,5-bis(N,N'-diphenylamino)-1,4-phenylene}] (CN-FO-DPD), and superparamagnetic iron oxide nanoparticles (SPIONs) within poly(styrene-co-maleic anhydride) (PSMA) micelles. The resulting particles exhibited an emission peak at 657 nm, a fluorescence quantum yield of 21%, an average diameter of 65 nm, and a ζ potential of -30 mV. They are taken up by cells, and we describe their use in fluorescence microscopy of living Hela cells and zebrafish embryos and their associated cytotoxicity in HEK, HeLa, and HCE cells.

Removal of Stomatin, a Membrane-Associated Cell Division Protein, Results in Specific Cellular Lipid Changes.

Lipids are key constituents of all cells, which express thousands of different lipid species. In most cases, it is not known why cells synthesize such diverse lipidomes, nor what regulates their metabolism. Although it is known that dividing cells specifically regulate their lipid content and that the correct lipid complement is required for successful division, it is unclear how lipids connect with the cell division machinery. Here, we report that the membrane protein stomatin is involved in the cytokinesis step of cell division. Although it is not a lipid biosynthetic enzyme, depletion of stomatin causes cells to change their lipidomes. These changes include specific lipid species, like ether lipids, and lipid families like phosphatidylcholines. Addition of exogenous phosphatidylcholines rescues stomatin-induced defects. These data suggest that stomatin interfaces with lipid metabolism. Stomatin has multiple contacts with the plasma membrane and we identify which sites are required for its role in cell division, as well as associated lipid shifts. We also show that stomatin's mobility on the plasma membrane changes during division, further supporting the requirement for a highly regulated physical interaction between membrane lipids and this newly identified cell division protein.

Co-ordinated control of the Aurora B abscission checkpoint by PKCε complex assembly, midbody recruitment and retention.

A requirement for PKCε in exiting from the Aurora B dependent abscission checkpoint is associated with events at the midbody, however, the recruitment, retention and action of PKCε in this compartment are poorly understood. Here, the prerequisite for 14-3-3 complex assembly in this pathway is directly linked to the phosphorylation of Aurora B S227 at the midbody. However, while essential for PKCε control of Aurora B, 14-3-3 association is shown to be unnecessary for the activity-dependent enrichment of PKCε at the midbody. This localisation is demonstrated to be an autonomous property of the inactive PKCε D532N mutant, consistent with activity-dependent dissociation. The C1A and C1B domains are necessary for this localisation, while the C2 domain and inter-C1 domain (IC1D) are necessary for retention at the midbody. Furthermore, it is shown that while the IC1D mutant retains 14-3-3 complex proficiency, it does not support Aurora B phosphorylation, nor rescues division failure observed with knockdown of endogenous PKCε. It is concluded that the concerted action of multiple independent events facilitates PKCε phosphorylation of Aurora B at the midbody to control exit from the abscission checkpoint.

Hydrophobic Interactions between DNA Duplexes and Synthetic and Biological Membranes.

Equipping DNA with hydrophobic anchors enables targeted interaction with lipid bilayers for applications in biophysics, cell biology, and synthetic biology. Understanding DNA-membrane interactions is crucial for rationally designing functional DNA. Here we study the interactions of hydrophobically tagged DNA with synthetic and cell membranes using a combination of experiments and atomistic molecular dynamics (MD) simulations. The DNA duplexes are rendered hydrophobic by conjugation to a terminal cholesterol anchor or by chemical synthesis of a charge-neutralized alkyl-phosphorothioate (PPT) belt. Cholesterol-DNA tethers to lipid vesicles of different lipid compositions and charges, while PPT DNA binding strongly depends on alkyl length, belt position, and headgroup charge. Divalent cations in the buffer can also influence binding. Our MD simulations directly reveal the complex structure and energetics of PPT DNA within a lipid membrane, demonstrating that longer alkyl-PPT chains provide the most stable membrane anchoring but may disrupt DNA base paring in solution. When tested on cells, cholesterol-DNA is homogeneously distributed on the cell surface, while alkyl-PPT DNA accumulates in clustered structures on the plasma membrane. DNA tethered to the outside of the cell membrane is distinguished from DNA spanning the membrane by nuclease and sphingomyelinase digestion assays. The gained fundamental insight on DNA-bilayer interactions will guide the rational design of membrane-targeting nanostructures.

Human epidermal stem cell differentiation is modulated by specific lipid subspecies.

While the lipids of the outer layers of mammalian epidermis and their contribution to barrier formation have been extensively described, the role of individual lipid species in the onset of keratinocyte differentiation remains unknown. A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. A small interfering RNA screen of 258 lipid-modifying enzymes identified two genes that on knockdown induced epidermal differentiation: ELOVL1, encoding elongation of very long-chain fatty acids protein 1, and SLC27A1, encoding fatty acid transport protein 1. By intersecting lipidomic datasets from suspension-induced differentiation and knockdown keratinocytes, we pinpointed candidate bioactive lipid subspecies as differentiation regulators. Several of these-ceramides and glucosylceramides-induced differentiation when added to primary keratinocytes in culture. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment.

Membrane and organelle dynamics during cell division.

During division, eukaryotic cells undergo a dramatic, complex and coordinated remodelling of their cytoskeleton and membranes. For cell division to occur, chromosomes must be segregated and new cellular structures, such as the spindle apparatus, must be assembled. Pre-existing organelles, such as the nuclear envelope, endoplasmic reticulum and Golgi apparatus, must be disassembled or remodelled, distributed and reformed. Smaller organelles such as mitochondria as well as cytoplasmic content must also be properly distributed between daughter cells. This mixture of organelles and cytoplasm is bound by a plasma membrane that is itself subject to remodelling as division progresses. The lipids resident in these different membrane compartments play important roles in facilitating the division process. In recent years, we have begun to understand how membrane remodelling is coordinated during division; however, there is still much to learn. In this Review, we discuss recent insights into how these important cellular events are performed and regulated.

RNAi Transfection Results in Lipidome Changes.

RNAi experiments are ubiquitously used in cell biology and are achieved by transfection of small interfering RNAs (siRNAs) into cells using a transfection reagent. These results in knock-down of proteins of interest, and the phenotypic consequences are then analyzed. It is reported here that two common RNA interference (RNAi) transfection reagents, DharmaFECT 1 and INTERFERin, in mock transfections using non-targeting siRNAs, cause alterations in the lipidome of HeLa cells. Some lipids change in response to both, presumably chemically different, transfection reagents, while other lipid species change only in response to one of the reagents. While the functional implications of these lipidomic alterations remain to be investigated, the authors' experiments suggest that it is important to use appropriate mock transfection controls during RNAi experiments, ideally complemented by an orthogonal perturbation, especially when investigating membrane-associated phenomena.

Cellular imaging using emission-tuneable conjugated polymer nanoparticles.

New materials that exhibit tuneable optical properties, notable emission across the visible spectrum, are of immense interest to biologists as they present a broad palette of colours from a single imaging agent that can be utilised in biological detection. Such a flexible system, when combined with the advantages of using conjugated polymer nanoparticles in cell imaging results in a widely useful medical diagnostic system. Here, we describe tuneable emission observed through oxidation of a conjugated polymer followed by the formation of nanoparticles and their subsequent use in cell imaging.

Lipid Cell Biology: A Focus on Lipids in Cell Division.

Cells depend on hugely diverse lipidomes for many functions. The actions and structural integrity of the plasma membrane and most organelles also critically depend on membranes and their lipid components. Despite the biological importance of lipids, our understanding of lipid engagement, especially the roles of lipid hydrophobic alkyl side chains, in key cellular processes is still developing. Emerging research has begun to dissect the importance of lipids in intricate events such as cell division. This review discusses how these structurally diverse biomolecules are spatially and temporally regulated during cell division, with a focus on cytokinesis. We analyze how lipids facilitate changes in cellular morphology during division and how they participate in key signaling events. We identify which cytokinesis proteins are associated with membranes, suggesting lipid interactions. More broadly, we highlight key unaddressed questions in lipid cell biology and techniques, including mass spectrometry, advanced imaging, and chemical biology, which will help us gain insights into the functional roles of lipids.

Capping protein regulates actin dynamics during cytokinetic midbody maturation.

During cytokinesis, a cleavage furrow generated by actomyosin ring contraction is restructured into the midbody, a platform for the assembly of the abscission machinery that controls the final separation of daughter cells. The polymerization state of F-actin is important during assembly, ingression, disassembly, and closure of the contractile ring and for the cytoskeletal remodeling that accompanies midbody formation and progression to abscission. Actin filaments must be cleared from the abscission sites before the final cut can take place. Although many conserved proteins interact with and influence the polymerization state of actin filaments, it is poorly understood how they regulate cytokinesis in higher eukaryotes. We report here that the actin capping protein (CP), a barbed end actin binding protein, participates in the control of actin polymerization during later stages of cytokinesis in human cells. Cells depleted of CP furrow and form early midbodies, but they fail cytokinesis. Appropriate recruitment of the ESCRT-III abscission machinery to the midbody is impaired, preventing the cell from progressing to the abscission stage. To generate actin filaments of optimal length, different actin nucleators, such as formins, balance CP's activity. Loss of actin capping activity leads to excessive accumulation of formin-based linear actin filaments. Depletion of the formin FHOD1 results in partial rescue of CP-induced cytokinesis failure, suggesting that it can antagonize CP activity during midbody maturation. Our work suggests that the actin cytoskeleton is remodeled in a stepwise manner during cytokinesis, with different regulators at different stages required for successful progression to abscission.

Ammonia Induces Autophagy through Dopamine Receptor D3 and MTOR.

Hyperammonemia is frequently seen in tumor microenvironments as well as in liver diseases where it can lead to severe brain damage or death. Ammonia induces autophagy, a mechanism that tumor cells may use to protect themselves from external stresses. However, how cells sense ammonia has been unclear. Here we show that culture medium alone containing Glutamine can generate milimolar of ammonia at 37 degrees in the absence of cells. In addition, we reveal that ammonia acts through the G protein-coupled receptor DRD3 (Dopamine receptor D3) to induce autophagy. At the same time, ammonia induces DRD3 degradation, which involves PIK3C3/VPS34-dependent pathways. Ammonia inhibits MTOR (mechanistic target of Rapamycin) activity and localization in cells, which is mediated by DRD3. Therefore, ammonia has dual roles in autophagy: one to induce autophagy through DRD3 and MTOR, the other to increase autophagosomal pH to inhibit autophagic flux. Our study not only adds a new sensing and output pathway for DRD3 that bridges ammonia sensing and autophagy induction, but also provides potential mechanisms for the clinical consequences of hyperammonemia in brain damage, neurodegenerative diseases and tumors.

Synthesis of (+)-Omphadiol and (+)-Pyxidatol C.

The synthesis of (+)-omphadiol and (+)-pyxidatol C was achieved through two independent strategies. For the synthesis of (+)-omphadiol, dicyclopentadienone was used as the workbench on which the three contiguous stereocenters of the cyclopentane could be introduced selectively. These include a tertiary alcohol and a selective protonation of an enolate. A ring-closing metathesis and a cyclopropanation concluded the synthesis. For the synthesis of pyxidatol C, we used the epoxide derived from (R)-linalool that was transformed to cyclopentane 23. After chain extension, another ring-closing metathesis followed by oxidation state changes and finally a cyclopropanation led to (+)-pyxidatol C.

A comparative LC-MS based profiling approach to analyze lipid composition in tissue culture systems.

Although lipids participate in many cellular processes both as signaling and structural molecules, our understanding of the roles of individual lipids as well as global changes in lipid composition are limited. Here we describe an LC-MS based method to identify lipids that change in a biological process. This method describes the isolation of lipids from tissue culture cells, sample preparation for LC-MS, the LC-MS run, and the subsequent data processing steps to compare the global lipid profiles and identify species that are enhanced or depleted. Identifying lipids that change is the first step towards functional studies to unravel their roles.

Lipids in cell biology: how can we understand them better?

Lipids are a major class of biological molecules and play many key roles in different processes. The diversity of lipids is on the same order of magnitude as that of proteins: cells express tens of thousands of different lipids and hundreds of proteins to regulate their metabolism and transport. Despite their clear importance and essential functions, lipids have not been as well studied as proteins. We discuss here some of the reasons why it has been challenging to study lipids and outline technological developments that are allowing us to begin lifting lipids out of their "Cinderella" status. We focus on recent advances in lipid identification, visualization, and investigation of their biophysics and perturbations and suggest that the field has sufficiently advanced to encourage broader investigation into these intriguing molecules.

Dividing cells regulate their lipid composition and localization.

Although massive membrane rearrangements occur during cell division, little is known about specific roles that lipids might play in this process. We report that the lipidome changes with the cell cycle. LC-MS-based lipid profiling shows that 11 lipids with specific chemical structures accumulate in dividing cells. Using AFM, we demonstrate differences in the mechanical properties of live dividing cells and their isolated lipids relative to nondividing cells. In parallel, systematic RNAi knockdown of lipid biosynthetic enzymes identified enzymes required for division, which highly correlated with lipids accumulated in dividing cells. We show that cells specifically regulate the localization of lipids to midbodies, membrane-based structures where cleavage occurs. We conclude that cells actively regulate and modulate their lipid composition and localization during division, with both signaling and structural roles likely. This work has broader implications for the active and sustained participation of lipids in basic biology.