Ulrike S. Eggert

Mechanical properties such as elastic resistance to distortion define many materials and can routinely be measured with an indenting tip as simple as one’s finger. We have miniaturized the indentation principle by 8 orders of magnitude to analyze lipid bilayer nanomechanics, which are of relevance in biology and disease progression. Our nanoscale indenting tip is designed with DNA nanotechnology to be an integral part of a molecular machine which autonomously binds to membranes, actively indents localized membrane patches, measures the membranes’ elastic response to indentation, and converts the indentation degree into an optical readout signal. Compared to serially operated macro-to-microscale indentation probes, our miniaturization strategy enables deploying millions of the indenting machines for parallel analysis of membrane elasticity. The nanoscale molecular machines are also taken up by biological cells to reveal softer inner membrane compartments compared to stiffer cytoplasmic membranes, using indentation depths of solely up to 1 nm. We envision the DNA-based nanotools to contribute to the analysis of membrane mechanics of cells as well as soft polymeric interphases.

Cells actively maintain complex lipidomes that encompass thousands of lipids; however, many of the roles of these lipids remain unexplored. Specific interactions between lipids and membrane proteins are a likely reason for lipidome complexity. Here we report the development of a technique, named lipid-trap mass spectrometry (LTMS), to systematically study lipid–protein interactions directly captured from mammalian cells. LTMS uses immunoprecipitation of GFP-tagged proteins expressed in HeLa cells, followed by lipidomic analysis of lipids bound to the GFP-tagged protein. We applied LTMS to cell division to illustrate the technique. We chose this process because membranes regulate their lipid composition as they undergo major changes during cytokinesis, and many cytokinetic proteins, including RACGAP1 and ESCRT-III components CHMP4B and CHMP2A, are membrane-associated. Using LTMS, we found that RACGAP1 and CHMP4B associate with specific lipid species in dividing compared with non-dividing cells. We expand our understanding of lipid diversity during cell division and present a general approach to explore lipid–protein interactions to further our knowledge of the roles of lipids in mammalian cells.

Cells actively maintain complex lipidomes that encompass thousands of lipids, however, many of the roles of these lipids remain unexplored. Specific interactions between lipids and membrane proteins are a likely reason for the evolutionary conservation of complex lipidomes. We report the development of a technique, named lipid-trap mass spectrometry (LTMS), to systematically study protein-lipid interactions directly captured from mammalian cells. LTMS uses immunoprecipitation of GFP-tagged proteins expressed in HeLa, followed by lipidomic analysis of lipids bound to the GFP-tagged protein. We applied LTMS to cell division to illustrate the technique. We chose this process because membranes regulate their lipid composition as they undergo major changes during cytokinesis and many cytokinetic proteins, including RACGAP1 and ESCRTIII components CHMP4B and CHMP2A, are membrane-associated. Using LTMS, we found that RACGAP1 and CHMP4B associate with specific lipid species in dividing compared to non-dividing cells. We expand our understanding of lipid diversity during cell division and present a general approach to explore lipid-protein interactions to further our understanding of the roles of lipids in mammalian cells.

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 (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with 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 recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH₃I and CD₃I respectively.

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 (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with 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 recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH₃I and CD₃I respectively.

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.

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.

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.

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.

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.