The significance of PTEN's protein phosphatase activity

Many hundreds of research papers over the last ten years have established the significance of PTEN's lipid phosphatase activity in mediating many of its effects on specific cellular processes in many different cell types, including cell growth, proliferation, survival, and migration ([Backman et al., 2002], [Iijima et al., 2002], [Leslie and Downes, 2002] and [Salmena et al., 2008]). In some cases, detailed signalling mechanisms have been identified by which these PtdInsP₃-dependent effects are manifest ([Kolsch et al., 2008], [Manning and Cantley, 2007] and [Tee and Blenis, 2005]). Further, in some settings, in vivo data from, for example genetic deletion of PTEN, relates closely with independent manipulation of the PI3K/Akt signalling pathway ([Bayascas et al., 2005], [Chen et al., 2006], [Crackower et al., 2002] and [Ma et al., 2005]). Together these studies indicate that the dominant effects of PTEN function are mediated through its regulation of PtdInsP₃-dependent signalling, but that its protein phosphatase activity also contributes in some settings. These conclusions are of great importance given the intense efforts underway to develop PI3K (EC 2.7.1.153) inhibitors as cancer therapeutics. The experiments reviewed here have firmly established that the protein phosphatase activity of PTEN plays a role in the regulation of cellular processes including migration. On the other hand, it has not been established beyond doubt that PTEN acts on substrates other than itself; no such substrates have been confidently identified and effector mechanisms for PTEN's protein phosphatase activity are currently unclear. The goal for future research must be firstly to understand the signalling mechanisms by which PTEN protein phosphatase activity acts: whether this is through identifying substrates, or working out how autodephosphorylation mediates its effects. Secondly, and critically, the significance of PTEN's protein phosphatase activity must be established in vivo. This can be achieved through relating the phenotypes intervening with both PTEN and with protein phosphatase effector pathways when they are identified, and through the generation of mouse models expressing substrate selective PTEN mutants. We should then be able to answer the important question of whether PTEN's protein phosphatase activity contributes to tumour suppression.     

Identification of novel VHL regulated genes by transcriptomic analysis of RCC10 renal carcinoma cells

Previous microarray studies have revealed a broad range of genes which are regulated by VHL and have provided much insight into how VHL may function as a tumour suppressor gene ([Wykoff et al., 2000b] and [Zatyka et al., 2002]). The current study has highlighted several genes of interest which are not currently recognised as being regulated by VHL. Of the candidate VHL regulated genes that we identified ASS was selected for further study due to its therapeutic implications. Tumours with low ASS levels display a reduced capacity to synthesise arginine, and as such are reliant on extracellular arginine for normal cellular function. Promising results in mouse xenograft models have shown that arginine deprivation may be a useful treatment strategy for these tumours. Understanding how ASS expression levels are regulated should provide insight into which tumour types would be most sensitive to treatment with arginine degrading enzymes. In this study we provide strong evidence that VHL status regulates ASS expression levels in three independent CCRCC cell backgrounds. Regulation of ASS by VHL/HIF suggests that arginine deprivation may be useful in the treatment of VHL defective CCRCCs and non-renal hypoxic tumours.     

Chemotherapeutic agents and gene expression in cardiac myocytes

It is becoming apparent that anti-cancer chemotherapies are increasingly associated with cardiac dysfunction or even congestive heart failure (Minotti et al., 2004; Eliott, 2006; Suter et al., 2004; Ren, 2005). Our data suggest that one of the contributing factors to the cardiotoxicitiy of these drugs may be the activation of the AhR-response (including the increased expression of Cyp1a1) and/or other detoxification program in cardiac myocytes themselves. The induction of such responses may have secondary effects (e.g. to increase the level of intracellular oxidative stress), which may influence the contractility or even survival of cardiac myocytes. Furthermore, the specific response of cardiac myocytes, both with respect to the metabolizing enzymes and the export channels, potentially differs from other cells (e.g. we failed to detect any increase in expression of other “classical” AhR-responsive genes, Ugt1a1 and Ugt1a6). This could account for, for example, the observation that doxoribicinol (the 13-hydroxy form of doxorubicin) accumulates in cardiac myocytes but not in hepatocytes (Del Tacca et al., 1985; Olson et al., 1988). Given the vulnerability of the heart and the almost irreparable damage that can be done by severe oxidative stress, further studies would seem to be merited specifically on the effects of chemotherapeutic agents on cardiac myocytes.     

Polarity proteins regulate mammalian cell–cell junctions and cancer pathogenesis

Polarity pathways regulate important functions during the formation and maintenance of cell–cell junctions and during morphogenesis. In addition, cell polarity pathways are emerging as critical regulators of initiation and progression of carcinoma by functioning as tumor suppressors, downstream of oncogenes, or promoters of the metastatic process (Figure 2). It is highly likely that further analysis of cell polarity proteins and the pathways they control will identify novel biomarkers and potential drug targets for managing and treating patients with carcinoma.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

LECs go crazy in embryo development

We have reviewed studies in which LEC TFs have been used to explore totipotency via SE and regulation of the maturation phase during zygotic embryogenesis. LEC TFs are master regulators of the maturation phase, activating genes encoding seed proteins that define this phase of embryo development. Regulation of the maturation phase seems to involve a feedback loop between the LEC TFs and hormones. LEC TFs stimulate ABA levels and activate genes that repress GA levels, contributing to the high ABA to GA ratio characteristic of the maturation phase. High ABA levels in turn stimulate LEC TFs to activate seed protein genes, and the reduction in GA levels might facilitate LEC TF activity. Although the LEC TFs are master regulators of the maturation phase, LEC genes are initially expressed before the onset of the maturation phase. The cellular process that initiates the maturation phase is not known. Nor is it known how LEC TFs interact with ABA and GA at the molecular level.SE is an outstanding example of totipotency in plants. Ectopic expression of LEC genes causes vegetative or reproductive cells to change their fate and undergo somatic embryo development. LEC TFs, via LEC2, activate auxin biosynthetic enzymes, and we propose that an increase in endogenous auxin levels serves to induce SE (Figure 3). How exogenous or endogenous auxin acts as the induction signal remains to be determined. We suggest that LEC TFs enable cells to become competent to respond to the induction signal by inactivating GA and, perhaps, by increasing ABA levels (Figure 3). Thus, a potential thread between the roles of LEC TFs in the maturation phase and SE might be their involvement in controlling the ABA to GA balance. It remains to be determined whether and how ABA and GA influence embryogenic competence. Although many questions remain, substantial progress has been made in determining how the LEC TFs ‘go crazy’ during embryo development.     

Expression and function of phospholipase C in breast carcinoma

Phosphoinositide-specific phospholipase C (PLC) control the levels of their substrate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), and its hydrolysis products diacylglycerol (DAG) and Ins(1,4,5)P₃, second messengers key to growth control and cell movement. The former is modulated by breakdown of plasma membrane and nuclear phosphoinositides, while the latter is mediated by phosphoinositide-driven remodeling of the actin cytoskeleton. The roles of PLC in the etiology and progression of breast carcinoma, however, are poorly understood. Previous studies reported a correlation between PLCβ₂ expression and breast tumor grade, making PLCβ₂ a potential marker for clinical outcome (Bertagnolo et al., 2006). While over-expression of PLCβ₂ is not sufficient to induce transformation of normal breast epithelial cells, it appears to play a role in promoting cell migration (Bertagnolo et al., 2007).Here we examined the expression of this and other PLC mRNA (β₁–β₄, δ₁, δ₃ and δ₄, γ₁ and γ₂) in normal breast epithelial lines, MCF-10A, and compared that pattern to breast tumor lines MDA-MB-231 and to T47D, using real-time relative-quantification PCR. Our results show that PLCγ₁, γ₂ and δ₁ and δ3 are more highly expressed in the transformed cell lines compared to MCF-10A when normalized to mRNA encoding various house keeping proteins; whereas PLCβ₂ mRNA levels were considerably lower than other PLC subtypes, including PLCβ₁ in the metastatic lines. Examination of PLC mRNA levels from normal and cancerous human breast tissue showed a similar pattern of expression, however, when staging or tumor size was considered, PLCδ₁ and δ₃ expression were positively correlated.To test whether PLCδ₁ or δ₃ played any role in tumor cell proliferation or cell migration, we transfected cells with siRNA specifically targeting these isoforms. RNAi mediated knockdown of either PLC isoform, reduced proliferation of the MDA-MB-231 cells. Morphological changes including cell rounding, and surface blebbing and nuclear fragmentation were observed. These changes were accompanied by reductions in cell migration activities. On the other hand, PLCδ₁ knockdown failed to cause comparable morphological changes in the normal MCF-10A line, but did reduce cell proliferation and migration. Taken together, these data are consistent with the idea that PLCδ₁ and δ₃ isoforms support the growth and migration of normal and neoplastic mammary epithelial cells in vitro.     

Temporal Production of the Signaling Lipid Phosphatidic Acid by Phospholipase D2 Determines the Output of Extracellular Signal-Regulated Kinase Signaling in Cancer Cells

The Ras-extracellular signal-regulated kinase (ERK) cascade is an important signaling module in cells. One regulator of the Ras-ERK cascade is phosphatidic acid (PA) generated by phospholipase D (PLD) and diacylglycerol kinase (DGK). Using a newly developed PA biosensor, PASS (phosphatidic acid biosensor with superior sensitivity), we found that PA was generated sequentially by PLD and DGK in epidermal growth factor (EGF)-stimulated HCC1806 breast cancer cells. Inhibition of PLD2, one of the two PLD members, was sufficient to eliminate most of the PA production, whereas inhibition of DGK decreased PA production only at the later stages of EGF stimulation, suggesting that PLD2 precedes DGK activation. The temporal production of PA by PLD2 is important for the nuclear activation of ERK. While inhibition of both PLD and DGK had no effect on the overall ERK activity, inhibition of PLD2 but not PLD1 or DGK blocked the nuclear ERK activity in several cancer cell lines. The decrease of active ERK in the nucleus inhibited the activation of Elk1, c-fos, and Fra1, the ERK nuclear targets, leading to decreased proliferation of HCC1806 cells. Together, these findings reveal that PA production by PLD2 determines the output of ERK in cancer cell growth factor signaling.     

A novel gene expression pathway regulated by nuclear phosphoinositides

Star-PAP is a recently identified nuclear speckle localized non-canonical poly(A) polymerase that has a functional interaction with PIPKIα, and whose activity is modulated by the PIPKIα product, PI4,5P₂. Similar to other poly(A) polymerases, such as the canonical PAPα and the non-canonical GLD2 PAP, Star-PAP resides in a large complex of proteins involved in the 3′ end formation of mRNAs (Fig. 4). The Star-PAP complex shares components with the canonical PAPα complex though it contains unique associated proteins such as PIPKIα and CKIα. The Star-PAP complex assembles into a highly stable 3′ end processing machine upon oxidative stress induction. This assembled complex shows enhanced enzyme activity and hypersensitivity to exogenous PI4,5P₂, implying that an activated Star-PAP is distinctly modified and/or contains unique factors as compared to Star-PAP purified from resting cells.     

Calcium signaling as a novel method to optimize the biosynthetic response of chondrocytes to dynamic mechanical loading

Chondrocyte sensitization and desensitization to mechanical stimuli are complex phenomena that have not been fully described. In this study, we investigated the temporal response of chondrocytes to dynamic mechanical loading and whether changes in calcium signaling could be used a predictor of the biosynthetic response. Cell-seeded agarose gels pre-incubated with an intracellular \(\hbox {Ca}^{2+}\) dye (Fluo-4) were subjected to dynamic compressive loading under varying conditions (amplitude and duration). Induced changes in \(\hbox {Ca}^{2+}\) signaling were determined by confocal imaging and matrix biosynthesis by radioisotope incorporation. It was observed that chondrocytes required a minimum amount of stimulation in order to elicit an anabolic response and they quickly became insensitive to the imposed stimulus. The response appeared to be amplitude dependent and could be predicted by measuring resultant changes in \(\hbox {Ca}^{2+}\) signaling. A positive correlation between \(\hbox {Ca}^{2+}\) signaling and matrix synthesis was achieved when changes in \(\hbox {Ca}^{2+}\) signaling was expressed as a relative number of cells experiencing multiple transients. In addition, these changes in \(\hbox {Ca}^{2+}\) signaling were effective at determining optimal recovery period between successive applications of intermittent mechanical loading, in which full mechanosensitivity was achieved when \(\hbox {Ca}^{2+}\) signaling was allowed to return to baseline (control) levels. The use of \(\hbox {Ca}^{2+}\) signaling to predict the effectiveness of a particular mechanical stimulus as well as to determine optimal refractory periods appears to be advantageous over empirical-based approaches. Future work will investigate the process of \(\hbox {Ca}^{2+}\) ion sequestration into intracellular stores to elucidate potential desensitization mechanisms to dynamic mechanical loading.     

Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6

Background

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV.

Methods

We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 and the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619.

Results

OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, a blocker of DAG synthesis, inhibited acute HPV whereas {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125"}}U73343, the inactive form of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, had no effect on HPV.

Conclusion

These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.     

The secreted Alzheimer-related amyloid precursor protein fragment has an essential role in C. elegans

Mutations in the gene encoding the amyloid precursor protein (APP) or the enzymes that process APP are correlated with familial Alzheimer disease. Alzheimer disease is also associated with insulin resistance (type 2 diabetes). In our recently published study,¹ Ewald CY, Raps DA, Li C. APL-1, the Alzheimer’s Amyloid precursor protein in Caenorhabditis elegans, modulates multiple metabolic pathways throughout development. Genetics 2012; 191:493 - 507; http://dx.doi.org/10.1534/genetics.112.138768; PMID: 22466039 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] we obtained genetic evidence that the extracellular fragment of APL-1, the C. elegans ortholog of human APP, may act as a signaling molecule to modulate insulin and nuclear hormone pathways in C. elegans development. In addition, independent of insulin and nuclear hormone signaling, high levels of the extracellular fragment of APL-1 (sAPL-1) leads to a temperature-sensitive embryonic lethality, which is dependent on activity of a predicted receptor protein tyrosine phosphatase (MOA-1/R155.2). Furthermore, this embryonic lethality is enhanced by knockdown of a predicted prion-like protein (pqn-29). The precise molecular mechanisms underlying these processes remain to be determined. Here, we present hypothetical models as to how sAPL-1 signaling influences metabolic and developmental pathways. Together, with previous findings in mammals that the extracellular domain of mammalian APP (sAPP) binds to a death-receptor,² Nikolaev A, McLaughlin T, O’Leary DD, Tessier-Lavigne M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 2009; 457:981 - 9; http://dx.doi.org/10.1038/nature07767; PMID: 19225519 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] our findings support the model that sAPP signaling affects critical biological processes.     

The Linker for Activation of B Cells (LAB)/Non-T Cell Activation Linker (NTAL) Regulates Triggering Receptor Expressed on Myeloid Cells (TREM)-2 Signaling and Macrophage Inflammatory Responses Independently of the Linker for Activation of T Cells

Triggering receptor expressed on myeloid cells-2 (TREM-2) is rapidly emerging as a key regulator of the innate immune response via its regulation of macrophage inflammatory responses. Here we demonstrate that proximal TREM-2 signaling parallels other DAP12-based receptor systems in its use of Syk and Src-family kinases. However, we find that the linker for activation of T cells (LAT) is severely reduced as monocytes differentiate into macrophages and that TREM-2 exclusively uses the linker for activation of B cells (LAB encoded by the gene Lat2^−/−) to mediate downstream signaling. LAB is required for TREM-2-mediated activation of Erk1/2 and dampens proximal TREM-2 signals through a novel LAT-independent mechanism resulting in macrophages with proinflammatory properties. Thus, Lat2^−/− macrophages have increased TREM-2-induced proximal phosphorylation, and lipopolysaccharide stimulation of these cells leads to increased interleukin-10 (IL-10) and decreased IL-12p40 production relative to wild type cells. Together these data identify LAB as a critical, LAT-independent regulator of TREM-2 signaling and macrophage development capable of controlling subsequent inflammatory responses.     

VEGF-A121a binding to Neuropilins – A concept revisited

All known splice isoforms of vascular endothelial growth factor A (VEGF-A) can bind to the receptor tyrosine kinases VEGFR-1 and VEGFR-2. We focus here on VEGF-A121a and VEGF-A165a, two of the most abundant VEGF-A splice isoforms in human tissue^{1 Kut C, Mac Gabhann F, Popel AS. Where is VEGF in the body? A meta-analysis of VEGF distribution in cancer. Br J Cancer. 2007;97:978-85. doi:10.1038/sj.bjc.6603923. PMID:17912242.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]}, and their ability to bind the Neuropilin co-receptors NRP1 and NRP2. The Neuropilins are key vascular, immune, and nervous system receptors on endothelial cells, neuronal axons, and regulatory T cells respectively. They serve as co-receptors for the Plexins in Semaphorin binding on neuronal and vascular endothelial cells, and for the VEGFRs in VEGF binding on vascular and lymphatic endothelial cells, and thus regulate the initiation and coordination of cell signaling by Semaphorins and VEGFs.^{2 Guo HF, Vander Kooi CW. Neuropilin Functions as an Essential Cell Surface Receptor. J Biol Chem. 2015;290:29120-6. doi:10.1074/jbc.R115.687327. PMID:26451046.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]} There is conflicting evidence in the literature as to whether only heparin-binding VEGF-A isoforms – that is, isoforms with domains encoded by exons 6 and/or 7 plus 8a – bind to Neuropilins on endothelial cells. While it is clear that VEGF-A165a binds to both NRP1 and NRP2, published studies do not all agree on the ability of VEGF-A121a to bind NRPs. Here, we review and attempt to reconcile evidence for and against VEGF-A121a binding to Neuropilins. This evidence suggests that, in vitro, VEGF-A121a can bind to both NRP1 and NRP2 via domains encoded by exons 5 and 8a; in the case of NRP1, VEGF-A121a binds with lower affinity than VEGF-A165a. In in vitro cell culture experiments, both NRP1 and NRP2 can enhance VEGF-A121a-induced phosphorylation of VEGFR2 and downstream signaling including proliferation. However, unlike VEGFA-165a, experiments have shown that VEGF-A121a does not ‘bridge’ VEGFR2 and NRP1, i.e. it does not bind both receptors simultaneously at their extracellular domain. Thus, the mechanism by which Neuropilins potentiate VEGF-A121a-mediated VEGFR2 signaling may be different from that for VEGF-A165a. We suggest such an alternate mechanism: interactions between NRP1 and VEGFR2 transmembrane (TM) and intracellular (IC) domains.     

Tyr(less) kinase signaling during mitosis

Tyrosine phosphorylation is rare, representing only about 0.5% of phosphorylations in the cell under basal conditions. While mitogenic tyrosine kinase signaling has been extensively explored, the role of phosphotyrosine signaling across the cell cycle and in particular during mitosis is poorly understood.

Two recent, independent studies tackled this question from different angles to reveal exciting new insights into the role of this modification during cell division. Caron et al.¹ Caron D, Byrne DP, Thebault P, Soulet D, Landry CR, Eyers PA, Elowe S. Mitotic phosphotyrosine network analysis reveals that tyrosine phosphorylation regulates Polo-like kinase 1 (PLK1). Sci Signal 2016; 9:rs14; PMID:27965426; http://dx.doi.org/10.1126/scisignal.aah3525[Crossref], [PubMed], [Web of Science ®] [Google Scholar] exploited mitotic phosphoproteomics data sets to determine the extent of mitotic tyrosine phosphorylation, and St-Denis et al.² St-Denis N, Gupta GD, Lin ZY, Gonzalez-Badillo B, Veri AO, Knight JD, Rajendran D, Couzens AL, Currie KW, Tkach JM, et al. Phenotypic and interaction profiling of the human phosphatases identifies diverse mitotic regulators. Cell Rep 2016; 17:2488-501; PMID:27880917; http://dx.doi.org/10.1016/j.celrep.2016.10.078[Crossref], [PubMed], [Web of Science ®] [Google Scholar] identified protein tyrosine phosphatases from all subfamilies as regulators of mitotic progression or spindle formation. These studied collectively revealed that tyrosine phosphorylation may play a more prominent and active role in mitotic progression than previously appreciated.     

Integrin manipulation to improve regeneration

After central nervous system (CNS) insults, such as spinal cord injury or traumatic brain injury, neurons encounter a complex microenvironment where mechanisms that promote regeneration compete with inhibitory processes. Sprouting and axonal re-growth are key components of functional recovery, but are often counteracted by inhibitory molecules. Several strategies are being pursued whereby these inhibitory molecules are either being neutralized with blocking antibodies, with enzymatic degradation or downstream signaling events are being interfered with. Two recent studies¹ Tan CL, Andrews MR, Kwok JC, Heintz TG, Gumy LF, Fässler R, et al. Kindlin-1 enhances axon growth on inhibitory chondroitin sulfate proteoglycans and promotes sensory axon regeneration. J Neurosci 2012; 32:7325 - 35; http://dx.doi.org/10.1523/JNEUROSCI.5472-11.2012; PMID: 22623678 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar]^,2 Tan CL, Kwok JC, Patani R, Ffrench-Constant C, Chandran S, Fawcett JW. Integrin activation promotes axon growth on inhibitory chondroitin sulfate proteoglycans by enhancing integrin signaling. J Neurosci 2011; 31:6289 - 95; http://dx.doi.org/10.1523/JNEUROSCI.0008-11.2011; PMID: 21525268 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] show that activating integrin signaling in dorsal root ganglion (DRG) neurons renders them able to overcome inhibitory signals, and could possibly lead to new strategies to improve neuronal regeneration.     

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.Lipids are fundamental building blocks of all cells and play many important and varied roles. They are key components of the plasma membrane and other cellular compartments, including the nuclear membrane, the endoplasmic reticulum, the Golgi apparatus, and trafficking vesicles such as endosomes and lysosomes. The lipid composition of different organelles, cell types, and ultimately tissues can vary substantially, suggesting that different lipids are required for different functions (Saghatelian et al., 2006 ; Klose et al., 2013 ). Mammalian cells express tens of thousands of different lipid species and use hundreds of proteins to synthesize, metabolize, and transport them. Although the complexity and diversity of lipids approach those of proteins, we have a much poorer understanding of their functions, making lipids in many ways the “Cinderellas” of cell biology. We discuss here recent advances, and challenges, in investigating how these molecules contribute to the many biological processes in which they participate.Like proteins, lipids can have structural (e.g., by stabilizing different membrane curvatures) or signaling roles. Posttranslational lipidation of proteins (e.g., palmitoylation or farnesylation) and carbohydrate-linked lipids (glycolipids) are also important examples of cellular lipid pools. It is clear that higher-order organization is key to most lipid functions, and they are believed to assemble into signaling platforms that contain both lipids and proteins (Kusumi et al., 2012 ). Some lipid microdomains are termed lipid rafts, and much ongoing effort is being focused on defining their parameters (Simons and Sampaio, 2011 ; Suzuki et al., 2012 ; Klotzsch and Schutz, 2013 ). Because of this focus on lipid rafts, we have a better understanding of the properties of proposed raft lipids (e.g., sphingomyelins and cholesterol) than of many other lipid species. Much of what we know about lipids has come from studying synthetic membranes with specific lipid compositions. Although model membranes usually consist of very few (<10) lipid species, they have been useful for understanding the biophysical properties of lipids. At the other end of the spectrum, lipids have been implicated in various diseases, with cholesterol metabolism being a prominent example. We are now getting to a point at which we can address the roles of lipids in the middle of the spectrum—in cells.