Lipid Tethering Research Articles

Lipid tethering of breast tumor cells reduces cell aggregation during mammosphere formation

Mammosphere assays are widely used in vitro to identify prospective cancer-initiating stem cells that can propagate clonally to form spheres in free-floating conditions. However, the traditional mammosphere assay inevitably introduces cell aggregation that interferes with the measurement of true mammosphere forming efficiency. We developed a method to reduce tumor cell aggregation and increase the probability that the observed mammospheres formed are clonal in origin. Tethering individual tumor cells to lipid anchors prevents cell drift while maintaining free-floating characteristics. This enables real-time monitoring of single tumor cells as they divide to form mammospheres. Monitoring tethered breast cancer cells provided detailed size information that correlates directly to previously published single cell tracking data. We observed that 71% of the Day 7 spheres in lipid-coated wells were between 50 and 150 μm compared to only 37% in traditional low attachment plates. When an equal mixture of MCF7-GFP and MCF7-mCherry cells were seeded, 65% of the mammospheres in lipid-coated wells demonstrated single color expression whereas only 32% were single-colored in low attachment wells. These results indicate that using lipid tethering for mammosphere growth assays can reduce the confounding factor of cell aggregation and increase the formation of clonal mammospheres.

Mycobacterial dynamin-like protein IniA mediates membrane fission

Mycobacterium tuberculosis infection remains a major threat to human health worldwide. Drug treatments against tuberculosis (TB) induce expression of several mycobacterial proteins, including IniA, but its structure and function remain poorly understood. Here, we report the structures of Mycobacterium smegmatis IniA in both the nucleotide-free and GTP-bound states. The structures reveal that IniA folds as a bacterial dynamin-like protein (BDLP) with a canonical GTPase domain followed by two helix-bundles (HBs), named Neck and Trunk. The distal end of its Trunk domain exists as a lipid-interacting (LI) loop, which binds to negatively charged lipids for membrane attachment. IniA does not form detectable nucleotide-dependent dimers in solution. However, lipid tethering indicates nucleotide-independent association of IniA on the membrane. IniA also deforms membranes and exhibits GTP-hydrolyzing dependent membrane fission. These results confirm the membrane remodeling activity of BDLP and suggest that IniA mediates TB drug-resistance through fission activity to maintain plasma membrane integrity.

Hybrid Lipids Inspired by Extremophiles and Eukaryotes Afford Serum‐Stable Membranes with Low Leakage

This paper presents a new hybrid lipid that fuses the ideas of molecular tethering of lipid tails used by archaea and the integration of cholesterol groups used by eukaryotes, thereby leveraging two strategies employed by nature to increase lipid packing in membranes. Liposomes comprised of pure hybrid lipids exhibited a 5-30-fold decrease in membrane leakage of small ions and molecules compared to liposomes that used only one strategy (lipid tethering or cholesterol incorporation) to increase membrane integrity. Molecular dynamics simulations reveal that tethering of lipid tails and integration of cholesterol both reduce the disorder in lipid tails and time-dependent variance in area per lipid within a membrane, leading to tighter lipid packing. These hybrid lipid membranes have exceptional stability in serum, yet can support functional ion channels, can serve as a substrate for phospholipase enzymes, and can be used for liposomal delivery of molecules into living cells.

Abstract 3978: Lipid tethering to enable real-time imaging of breast tumor cell cytoskeletal dynamics and rapid drug testing in free-floating metastatic microenvironments

Abstract Free-floating tumor cells located in the peripheral circulation of cancer patients, known as circulating tumor cells (CTCs), play an important role in cancer and have become key targets for studying metastasis. The presence of CTCs in the bloodstream or lymphatics correlates with decreased cancer patient survival and provides a minimally invasive method to study disease progression and treatment response. Understanding the molecular characteristics and functional properties of CTCs have been impeded by the challenges of imaging CTCs under conditions that model the free-floating microenvironments of the circulation. Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells within a microfluidic device while maintaining their free-floating character. We use cytophobic polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membrane. This approach enables high-resolution time-lapse microscopy of McTNs on viable free-floating tumor cells and real-time analysis of dynamic cellular features. In addition, tethering makes it now possible to rapidly measure drug responses of tumor cells in free-floating microenvironments to select effective therapies and avoid drugs that could inadvertently increase metastasis. The ability to image tumor cells in the absence of ECM attachment can immensely enhance our understanding of CTCs under conditions that better recapitulate the free-floating microenvironments that tumor cells encounter during metastasis. Citation Format: Kristi Riti Chakrabarti, James Andorko, Rebecca Whipple, Peipei Zhang, Elisabeth Sooklal, Christopher Jewell, Stuart Martin. Lipid tethering to enable real-time imaging of breast tumor cell cytoskeletal dynamics and rapid drug testing in free-floating metastatic microenvironments. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3978.

Effects of Lipid Tethering in Extremophile-Inspired Membranes on H+/OH− Flux at Room Temperature

This work explores the proton/hydroxide permeability (PH+/OH−) of membranes that were made of synthetic extremophile-inspired phospholipids with systematically varied structural elements. A fluorescence-based permeability assay was optimized to determine the effects on the PH+/OH− through liposome membranes with variations in the following lipid attributes: transmembrane tethering, tether length, and the presence of isoprenoid methyl groups on one or both lipid tails. All permeability assays were performed in the presence of a low concentration of valinomycin (10 nM) to prevent buildup of a membrane potential without artificially increasing the measured PH+/OH−. Surprisingly, the presence of a transmembrane tether did not impact PH+/OH− at room temperature. Among tethered lipid monolayers, PH+/OH− increased with increasing tether length if the number of carbons in the untethered acyl tail was constant. Untethered lipids with two isoprenoid methyl tails led to lower PH+/OH− values than lipids with only one or no isoprenoid tails. Molecular dynamics simulations revealed a strong positive correlation between the probability of observing water molecules in the hydrophobic core of these lipid membranes and their proton permeability. We propose that water penetration as revealed by molecular dynamics may provide a general strategy for predicting proton permeability through various lipid membranes without the need for experimentation.

Potent lipidated antagonists to protease‐activated receptor 2 (PAR2)

Protease‐activated receptor‐2 (PAR2) belongs to a four‐member family of G‐Protein coupled receptors (GPCRs) that contain internal ligands exposed following exogenous or endogenous protease cleavage of the extracellular amino terminus. PAR2 is associated with a variety of inflammatory conditions, including asthma and pain. The contributions of PAR2 signaling to disease has been hindered by the lack of potent, efficacious antagonists, and their potential for biased‐ligand signaling. We recently demonstrated that lipid tethering of known PAR2 peptidomimetic agonists based on the primary trypsin cleavage sequence (SLIGRL) increased their potency > 200 fold. In this study, we used lipid tethering (hexadecyl (Hdc) group with polyethylene glycol (PEG) spacers) and heterocycle (2‐aminothiazoyl; 2‐at) substitution of hexapeptide sequence derived from the primary cleavage site of kallikreins 4/16 (SSKGRS) to elucidate novel PAR2 antagonists. Compound 562 (C562), 2‐aminothiazol‐4yl‐SKGRS‐PEG3‐Hdc blocks PAR2 Ca2+ signaling elicited via peptidomimetics (2‐at‐LIGRL‐NH2) or via asthma associated protease activation (Alternaria alternata filtrates) in cultured human bronchial epithelial cells (16HBE14o‐). This compound was a biased‐signaling antagonist in that it had no effect on mitogen activated protein kinase (MAPK) signaling, the other major signaling pathway activated via PAR2. A shortened version of C562, 2‐at‐SKGR‐PEG3‐Hdc (C595), maintained antagonistic activity against peptidomimetic activation in an in vitro physiological signaling assay (xCELLigence). C595 is closely related to the previously described potent and specific PAR2 agonist, 2‐at‐LIGR‐PEG3‐Hdc. Thus, we screened a series of potential PAR2 ligands with a heterocycle serine substitute followed by four amino acids (XXGR) and the PEG3‐Hdc lipid tether. We describe several potent agonists, and one partial agonist (C608, 2‐at‐TIGR‐PEG3‐Hdc) that also acts as a potent, specific and biased signaling antagonist of PAR2. When used in nanomolar concentrations, C608 blocked PAR2‐dependent Ca2+ signaling via protease or peptidomimetics without effects on MAPK signaling. C562, C595 and C608 are novel pharmacological tools that can be used to evaluate the physiological consequences of PAR2 full and biased ligand signaling.Support or Funding InformationThis work was primarily funded by a multi‐PI grant (NS 073664 to SB, JV and TJP) from the National Institutes of Health. Additional support for this work was from the following grants: National Institute of Health training grants (T32 HL 007249 for ANF; T32 ES 007091 for CLS), National Institutes of Health R01NS 065926 (TJP) and R01AI083403 (SB; Michael O. Daines, PI), State of Arizona Technology and Research Initiative Fund Awarded through Bio5 (JV).

Lipid tethering of breast tumor cells enables real-time imaging of free-floating cell dynamics and drug response

Free-floating tumor cells located in the blood of cancer patients, known as circulating tumor cells (CTCs), have become key targets for studying metastasis. However, effective strategies to study the free-floating behavior of tumor cells in vitro have been a major barrier limiting the understanding of the functional properties of CTCs. Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells while maintaining their free-floating character. We use polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membranes. This coating remains optically clear, allowing capture of high-resolution images and videos of McTNs on viable free-floating cells. In addition, we show that tethering allows for the real-time analysis of McTN dynamics on individual tumor cells and in response to tubulin-targeting drugs. The ability to image detached tumor cells can vastly enhance our understanding of CTCs under conditions that better recapitulate the microenvironments they encounter during metastasis.

Development and evaluation of small peptidomimetic ligands to protease-activated receptor-2 (PAR2) through the use of lipid tethering.

Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered-peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG 3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2–2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG 3-Hdc retained potency (EC50 = 2.1 nM; 1.3–3.4 nM) with improved selectivity for PAR2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG 3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20–100 nM). 2at-LI-PEG 3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260–360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG 3-Hdc and 2at-PEG 3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG 3-Hdc lacked efficacy. Minimum peptidomimetic PAR2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems.

Development of highly potent protease‐activated receptor 2 agonistsviasynthetic lipid tethering

Protease-activated receptor-2 (PAR₂) is a G-protein coupled receptor (GPCR) associated with a variety of pathologies. However, the therapeutic potential of PAR₂ is limited by a lack of potent and specific ligands. Following proteolytic cleavage, PAR₂ is activated through a tethered ligand. Hence, we reasoned that lipidation of peptidomimetic ligands could promote membrane targeting and thus significantly improve potency and constructed a series of synthetic tethered ligands (STLs). STLs contained a peptidomimetic PAR₂ agonist (2-aminothiazol-4-yl-LIGRL-NH₂) bound to a palmitoyl group (Pam) via polyethylene glycol (PEG) linkers. In a high-throughput physiological assay, these STL agonists displayed EC₅₀ values as low as 1.47 nM, representing a ∼200 fold improvement over the untethered parent ligand. Similarly, these STL agonists were potent activators of signaling pathways associated with PAR₂: EC₅₀ for Ca(2+) response as low as 3.95 nM; EC₅₀ for MAPK response as low as 9.49 nM. Moreover, STLs demonstrated significant improvement in potency in vivo, evoking mechanical allodynia with an EC₅₀ of 14.4 pmol. STLs failed to elicit responses in PAR2(-/-) cells at agonist concentrations of >300-fold their EC₅₀ values. Our results demonstrate that the STL approach is a powerful tool for increasing ligand potency at PAR₂ and represent opportunities for drug development at other protease activated receptors and across GPCRs.

Identification of the Regulatory Anionic Lipid Site in Kir Channels

Inwardly rectifying potassium channels (Kir) are regulated by multiple factors, including multiple lipids. In addition to a specific requirement for phosphatidyl-4,5-bisphosphate (PI(4,5)P2) for channel activity of Kir2.1, analysis of purified proteins reconstituted into liposomes has revealed a secondary requirement for non-specific anionic lipids, which increase PI(4,5)P2 sensitivity by ∼100 fold [Cheng et al. 2011, Biophys. J. 100, 620-628]. Recent crystal structures of eukaryotic Kir channels in complex with PI(4,5)P2 reveal a common PI(4,5)P2 binding site [Hansen et al, 2011, Nature. 477, 495-498; Whorton & MacKinnon, 2011, Cell. 147, 199-208], but they have not identified the synergistic anionic lipid site.We have performed extensive docking simulations to identify potential interaction sites of different phospholipids with Kir2.1 channels. These simulations indicate two distinct binding sites; a high affinity site that corresponds to the crystallographic PI(4,5)P2 binding pocket and a lower affinity site, involving two lysine residues further towards the periphery of the cytoplasmic domain, that may correspond to the secondary anionic lipid site. When the two lysine residues are mutated to cysteine, channel activity is essentially abolished, even in the presence of PIP2. Cysteine modification of these residues by decyl-MTS, which essentially provides a ‘lipid tether' to the residue, restores channel activity in the presence of low levels of PIP2. These results point strongly to the identified site as being the site for non-specific anionic lipid interaction, and support a model in which the anionic lipid interaction (or ‘lipid tethering') pulls the Kir domain towards the membrane, facilitating PI(4,5)P2-mediated channel opening.

Control of Inward Rectifier K Channel Activity by Lipid Tethering of Cytoplasmic Domains

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Control of Inward Rectifier K Channel Activity by Lipid Tethering of Cytoplasmic Domains

Interactions between nontransmembrane domains and the lipid membrane are proposed to modulate activity of many ion channels. In Kir channels, the so-called “slide-helix” is proposed to interact with the lipid headgroups and control channel gating. We examined this possibility directly in a cell-free system consisting of KirBac1.1 reconstituted into pure lipid vesicles. Cysteine substitution of positively charged slide-helix residues (R49C and K57C) leads to loss of channel activity that is rescued by in situ restoration of charge following modification by MTSET+ or MTSEA+, but not MTSES− or neutral MMTS. Strikingly, activity is also rescued by modification with long-chain alkyl-MTS reagents. Such reagents are expected to partition into, and hence tether the side chain to, the membrane. Systematic scanning reveals additional slide-helix residues that are activated or inhibited following alkyl-MTS modification. A pattern emerges whereby lipid tethering of the N terminus, or C terminus, of the slide-helix, respectively inhibits, or activates, channel activity. This study establishes a critical role of the slide-helix in Kir channel gating, and directly demonstrates that physical interaction of soluble domains with the membrane can control ion channel activity.