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.

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.

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.

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.

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.

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.

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.

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.

Dopamine receptor D3 regulates endocytic sorting by a Prazosin-sensitive interaction with the coatomer COPI.

Macromolecules enter cells by endocytosis and are sorted to different cellular destinations in early/sorting endosomes. The mechanism and regulation of sorting are poorly understood, although transitions between vesicular and tubular endosomes are important. We found that the antihypertensive drug Prazosin inhibits endocytic sorting by an off-target perturbation of the G protein-coupled receptor dopamine receptor D(3) (DRD3). Prazosin is also a potent cytokinesis inhibitor, likely as a consequence of its effects on endosomes. Prazosin stabilizes a normally transient interaction between DRD3 and the coatomer COPI, a complex involved in membrane transport, and shifts endosomal morphology entirely to tubules, disrupting cargo sorting. RNAi depletion of DRD3 alone also inhibits endocytic sorting, indicating a noncanonical role for a G protein-coupled receptor. Prazosin is a powerful tool for rapid and reversible perturbation of endocytic dynamics.

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.

Small molecules discovered in a pathway screen target the Rho pathway in cytokinesis.

We report the discovery of small molecules that target the Rho pathway, which is a central regulator of cytokinesis--the final step in cell division. We have developed a way of targeting a small molecule screen toward a specific pathway, which should be widely applicable to the investigation of any signaling pathway. In a chemical genetic variant of a classical modifier screen, we used RNA interference (RNAi) to sensitize cells and identified small molecules that suppressed or enhanced the RNAi phenotype. We discovered promising candidate molecules, which we named Rhodblock, and we identified the target of Rhodblock as Rho kinase. Several Rhodblocks inhibited one function of the Rho pathway in cells: the correct localization of phosphorylated myosin light chain during cytokinesis. Rhodblocks differentially perturb Rho pathway proteins in cells and can be used to dissect the mechanism of the Rho pathway during cytokinesis.

Scoring diverse cellular morphologies in image-based screens with iterative feedback and machine learning.

Many biological pathways were first uncovered by identifying mutants with visible phenotypes and by scoring every sample in a screen via tedious and subjective visual inspection. Now, automated image analysis can effectively score many phenotypes. In practical application, customizing an image-analysis algorithm or finding a sufficient number of example cells to train a machine learning algorithm can be infeasible, particularly when positive control samples are not available and the phenotype of interest is rare. Here we present a supervised machine learning approach that uses iterative feedback to readily score multiple subtle and complex morphological phenotypes in high-throughput, image-based screens. First, automated cytological profiling extracts hundreds of numerical descriptors for every cell in every image. Next, the researcher generates a rule (i.e., classifier) to recognize cells with a phenotype of interest during a short, interactive training session using iterative feedback. Finally, all of the cells in the experiment are automatically classified and each sample is scored based on the presence of cells displaying the phenotype. By using this approach, we successfully scored images in RNA interference screens in 2 organisms for the prevalence of 15 diverse cellular morphologies, some of which were previously intractable.

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.

Actin-aggregating cucurbitacins from Physocarpus capitatus.

Bioassay-guided fractionation of Physocarpus capitatus yielded two new cucurbitacins (3 and 4) along with the known cucurbitacin F (1) and dihydrocucurbitacin F (2). Preliminary mechanism of action studies indicate that the cucurbitacins cause actin aggregates and inhibit cell division.

Drug-target identification in Drosophila cells: combining high-throughout RNAi and small-molecule screens.

RNA interference (RNAi) and small-molecule approaches are synergistic on multiple levels, from technology and high-throughput screen development to target identification and functional studies. Here, we describe the RNAi screening platform that we have established and made available to the community through the Drosophila RNAi Screening Center at Harvard Medical School. We then illustrate how the combination of RNAi and small-molecule HTS can lead to effective identification of targets in drug discovery.

Small molecule screening by imaging.

Useful small molecule tools can be discovered in imaging screens that measure phenotype in single cells or small organisms. Recent examples include identification of small molecule inhibitors of processes such as cell migration, cytokinesis, mitotic spindle length determination, melanogenesis, aggresome formation, membrane transport and nuclear export. Imaging screens are currently limited by challenges in the areas of image analysis and target identification. We discuss the use of model organisms such as zebrafish in screens and review different methods of target identification. The emerging field of automated image analysis is also introduced.

Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets.

Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.

Small molecules in an RNAi world.

In this Opinion, we compare and contrast small molecules and RNAi. We discuss the advantages and disadvantages of both technologies focusing on timing, specificity, dose and therapeutic use.