Lipid Droplets Form from Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces pombe.

Eukaryotic cells store cholesterol/sterol esters (SEs) and triacylglycerols (TAGs) in lipid droplets, which form from the contiguous endoplasmic reticulum (ER) network. However, it is not known if droplets preferentially form from certain regions of the ER over others. Here, we used fission yeast Schizosaccharomyces pombe cells where the nuclear and cortical/peripheral ER domains are distinguishable by light microscopy to show that SE-enriched lipid droplets form away from the nucleus at the cell tips, whereas TAG-enriched lipid droplets form around the nucleus. Sterols localize to the regions of the cells where droplets enriched in SEs are observed. TAG droplet formation around the nucleus appears to be a strong function of diacylglycerol (DAG) homeostasis with Cpt1p, which coverts DAG into phosphatidylcholine and phosphatidylethanolamine localized exclusively to the nuclear ER. Also, Dgk1p, which converts DAG into phosphatidic acid localized strongly to the nuclear ER over the cortical/peripheral ER. We also show that TAG more readily translocates from the ER to lipid droplets than do SEs. The results augment the standard lipid droplet formation model, which has SEs and TAGs flowing into the same nascent lipid droplet regardless of its biogenesis point in the cell.

Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast.

Cells sequester neutral lipids in bodies called lipid droplets. Thus, the formation and breakdown of the droplets are important for cellular metabolism; unfortunately, these processes are difficult to quantify. Here, we used time-lapse confocal microscopy to track the formation, movement and size changes of lipid droplets throughout the cell cycle in fission yeast Schizosaccharomyces pombe. In theory, the number of lipid droplets in these cells must increase for daughter cells to have the same number of droplets as the parent at a reference point in the cell cycle. We observed stable droplet formation events in G2 phase that were divided evenly between de novo formation of nascent droplets and fission of preexisting droplets. The observations that lipid droplet number is linked to the cell cycle and that droplets can form via fission were both new discoveries. Thus, we scrutinized each fission event for multiple signatures to eliminate possible artifacts from our microscopy. We augmented our time-lapse confocal microscopy with electron microscopy, which showed lipid droplet 'intermediates': droplets shaped like dumbbells that are potentially in transition states between two spherical droplets. Using these complementary microscopy techniques and also dynamic simulations, we show that lipid droplets can form by fission.

PEGylated nanoparticles interact with macrophages independently of immune response factors and trigger a non-phagocytic, low-inflammatory response.

Poly-ethylene-glycol (PEG)-based nanoparticles (NPs) - including cylindrical micelles (CNPs), spherical micelles (SNPs), and PEGylated liposomes (PLs) - are hypothesized to be cleared in vivo by opsonization followed by liver macrophage phagocytosis. This hypothesis has been used to explain the rapid and significant localization of NPs to the liver after administration into the mammalian vasculature. Here, we show that the opsonization-phagocytosis nexus is not the major factor driving PEG-NP - macrophage interactions. First, mouse and human blood proteins had insignificant affinity for PEG-NPs. Second, PEG-NPs bound macrophages in the absence of serum proteins. Third, lipoproteins blocked PEG-NP binding to macrophages. Because of these findings, we tested the postulate that PEG-NPs bind (apo)lipoprotein receptors. Indeed, PEG-NPs triggered an in vitro macrophage transcription program that was similar to that triggered by lipoproteins and different from that triggered by lipopolysaccharide (LPS) and group A Streptococcus. Unlike LPS and pathogens, PLs did not increase transcripts involved in phagocytosis or inflammation. High-density lipoprotein (HDL) and SNPs triggered remarkably similar mouse bone-marrow-derived macrophage transcription programs. Unlike opsonized pathogens, CNPs, SNPs, and PLs lowered macrophage autophagosome levels and either reduced or did not increase the secretion of key macrophage pro-inflammatory cytokines and chemokines. Thus, the sequential opsonization and phagocytosis process is likely a minor aspect of PEG-NP - macrophage interactions. Instead, PEG-NP interactions with (apo)lipoprotein and scavenger receptors appear to be a strong driving force for PEG-NP - macrophage binding, entry, and downstream effects. We hypothesize that the high presence of these receptors on liver macrophages and on liver sinusoidal endothelial cells is the reason PEG-NPs localize rapidly and strongly to the liver.

Lipid droplet organelle distribution in populations of dividing cells studied by simulation.

One of the key questions in cell biology is how organelles are passed from parent to daughter cells. To help address this question, I used Brownian dynamics to simulate lipid droplets as model organelles in populations of dividing cells. Lipid droplets are dynamic bodies that can form both de novo and by fission, they can also be depleted. The quantitative interplay among these three events is unknown but would seem crucial for controlling droplet distribution in populations of dividing cells. Surprisingly, of the three main events studied: biogenesis, fission, and depletion, the third played the key role in maintaining droplet organelle number-and to a lesser extent volume-in populations of dividing cells where formation events would have seemed paramount. In the case of lipid droplets, this provides computational evidence that they must be sustained, most likely through contacts with the endoplasmic reticulum. The findings also agree with video microscopy experiments over much shorter timescales where droplet depletion in fission yeast cells was not observed. In general, this work shows that organelle maintenance is invaluable and lack thereof cannot necessarily be compensated for by organelle formation. This study provides a time-accurate, physical-based template for long-term cell division studies.

Interactions of Apolipoproteins with Lipid-Based Nanoparticles.

Serum proteins bind and form a dynamic protein corona around nanoparticles (NPs) that have been injected into the mammalian vasculature. Several fundamental studies have shown that apolipoproteins are prominent components of the NP corona. Since apolipoproteins control the distribution of lipoproteins, they may also control the distribution of NPs. Indeed, apolipoprotein affinity for NPs has been recently taken advantage of to deliver CRISPR reagents encapsulated in NPs to cells that express particular lipoprotein receptors. In this scenario, an apolipoprotein binds an NP and the resulting apolipoprotein-NP complex binds a cell that expresses the (apo)lipoprotein receptor. But the NP will be diverted from the target cell if it does not express the (apo)lipoprotein receptor. This may hamper NP treatment of diseases. Therefore, we must understand the kinetics of apolipoprotein-NP affinity and how apolipoprotein-NP interactions affect NP biodistribution. In this Perspective, we discuss the evolving topic of apolipoprotein-NP interactions, which is of great interest for all NP-based disease treatments. Many properties of apolipoprotein-NP complexes are yet to be determined and will have a significant impact on NP efficacy for many NP-based treatments in animal models and in the clinic.

Obesity and inflammation influence pharmacokinetic profiles of PEG-based nanoparticles.

Most patients that will be treated with soft nanoparticles (NPs) will be obese. Yet, NP testing, which begins with pharmacokinetic (PK) and toxicity studies, is carried out almost exclusively in lean rodents having healthy livers and low inflammation. To address this knowledge gap, we determined the PK and toxicity of tail-vein-injected, PEG-based cylindrical nanoparticles (CNPs) and PEGylated liposomes (PLs) as a function of obesity, liver health, and inflammation in leptin-deficient ob/ob and wild-type C57BL/6 J mice. CNPs localized faster to obese livers than to healthy livers within 24 h of injection. PLs localized faster to obese livers than to healthy livers but only 30 min post-injection. Afterwards PL localization to lean livers was higher than localization to obese livers. Overall, PL liver signal peaked ∼6 h post-injection in lean mice, ∼24 h post-injection in heavy mice, and âˆ¼ 48 h post-injection in obese mice. CNPs and PLs were non-toxic to mouse livers as assessed by histology; they reduced many cytokine and chemokine levels that were elevated by obesity. Liver macrophage depletion reduced CNP and PL liver localization as expected; liver sinusoidal endothelial cell (LSEC) depletion reduced PL liver localization but surprisingly increased CNP liver localization. The intensity of RAW264.7 macrophages was higher after CNP incubations than with PL incubations; conversely, the intensity of LSECs was higher after PL incubations than with CNP incubations. This shows the potential for key differences in NP-liver interactions. Triggering inflammation by administering lipopolysaccharide (LPS) to mice increased CNP liver localization but decreased PL liver localization. The results show that obesity and inflammation in a mouse model and in vitro affect soft PEG-based NP interaction with macrophages and LSECs, but also that these NPs can reduce pro-inflammatory pathways increased by obesity.

All-Atom Molecular Dynamics Simulations of Polyethylene Glycol (PEG) and LIMP-2 Reveal That PEG Penetrates Deep into the Proposed CD36 Cholesterol-Transport Tunnel.

Polyethylene glycol (PEG) is the most prominent clinically administered synthetic polymer. For example, over 300 million people have been administered PEGylated liposome vaccines for SARS-CoV-2. PEG is used in mammals because it has low affinity for most proteins and vice versa. However, this makes it difficult to study the few interactions with proteins that PEG has. On the atomistic level, there are two PEG-protein structures: (1) PEG-LIMP-2 and (2) PEG-αPEG. In the first structure, two monomers of a 1.5 kDa PEG polymer (PEG2) had electron density deep in the postulated cholesterol transport tunnel of LIMP-2, a lysosomal cholesterol transport protein and member of the CD36 super family of proteins. It is unclear how PEG entered this tunnel. In the second structure, PEG wrapped around a surface-exposed tryptophan on its antibody. Since tryptophan is a rare residue, it is unclear if this PEG-Trp interaction is ubiquitous. To gain deeper mechanistic insight into PEG-protein interactions, we surrounded the LIMP-2 apo structure with 13 PEG chains of 10 monomers each (PEG10), water, and KCl and simulated the system using NAMD. One of the 13 chains penetrated LIMP-2 and came within 3 Å of PEG2. This was possible because of the strong hydrogen bonding between multiple oxygens along PEG10 and Arg192 but, most importantly, the clamping of the tertiary structure on PEG10. Clamping stabilized the movements of PEG10, and the leading oxygen of PEG10 was able to penetrate LIMP-2 and head toward to the position occupied by PEG2. Phe383 appears to act as a gate for objects to move through this cavity, which continues to the basal/membrane side of LIMP-2. Of all residues, PEG10 molecules had the most sustained interactions with lysine and arginine because of their strong hydrogen-bonding capabilities. These results show that the oxygens of PEG bind residues with high hydrogen bonding capabilities. However, the PEG-protein interaction is likely to be transient unless groups of resides can clamp down on PEG or a cavity that at least part of the PEG chain can enter is in close proximity to lower PEG's entropy.

Tetra-Primer Amplification-Refractory Mutation System (ARMS)-PCR for Genotyping Mouse Leptin Gene Mutation.

Due to spontaneous deficiency in leptin, ob/ob mice are one of the most commonly used experimental animal models in diabetes research. In this study, we reported a quick and easy-to-conduct genotyping method using tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to differentiate mice with a mutated allele from the wild-type genotype. The amplicon patterns of different genotypes are clearly visible and distinguishable on 1.5% agarose gel. This method can serve as a valuable tool to differentiate genotypes for breeding purposes, to maintain animal colonies, control the available space in the animal facility, and identify appropriate individuals for animal experiments.

Elongated PEO-based nanoparticles bind the high-density lipoprotein (HDL) receptor scavenger receptor class B I (SR-BI).

Targeting cell-surface receptors with nanoparticles (NPs) is a crucial aspect of nanomedicine. Here, we show that soft, flexible, elongated NPs with poly-ethylene-oxide (PEO) exteriors and poly-butadiene (PBD) interiors - PEO-PBD filomicelles - interact directly with the major high-density lipoprotein (HDL) receptor and SARS-CoV-2 uptake factor, SR-BI. Filomicelles have a ~ 6-fold stronger interaction with reconstituted SR-BI than PEO-PBD spheres. HDL, and the lipid transport inhibitor, BLT-1, both block the uptake of filomicelles by macrophages and Idla7 cells, the latter are constitutively expressing SR-BI (Idla7-SR-BI). Co-injections of HDL and filomicelles into wild-type mice reduced filomicelle signal in the liver and increased filomicelle plasma levels. The same was true with SCARB1-/- mice. SR-BI binding is followed by phagocytosis for filomicelle macrophage entry, but only SR-BI is needed for entry into Idla7-SR-BI cells. PEO-PBD spheres did not interact strongly with SR-BI in the above experiments. The results show elongated PEO-based NPs can bind cells via cooperativity among SR-BI receptors on cell surfaces.

Molecular dynamics simulations of Arp2/3 complex activation.

Actin-related protein 2 and 3 (Arp2/3) complex forms a dendritic network of actin filaments during endocytosis and cellular locomotion by nucleating branches on the sides of preexisting actin filaments. Reconstructions of electron tomograms of branch junctions show how Arp2/3 complex anchors the branch, with Arp2 and Arp3 serving as the first two subunits of the branch. Our aim was to characterize the massive conformational change that moves Arp2 ∼30 Å from its position in crystal structures of inactive Arp2/3 complex to its position in branch junctions. Starting with the inactive crystal structure, we used atomistic-scale molecular dynamics simulations to drive Arp2 toward the position observed in branch junctions. When we applied forces to Arp2 while restraining Arp3, one block of structure (Arp2, subunit ARPC1, the globular domain of ARPC4 and ARPC5) rotated counterclockwise by 30° around a pivot point in an α-helix of ARPC4 (Glu8¹-Asn¹°°) to align Arp2 next to Arp3 in a second block of structure including ARPC3 and the globular domains of ARPC2. This active structure buried more surface area than the inactive conformation. The complex was stable in all simulations. In most simulations, collisions of subdomain 2 of Arp2 with Arp3 impeded the movement of Arp2.

Lipid Droplets in Disease.

Lipid droplets (LDs) are a crucial part of lipid storage; thus, they are important players in a variety of diseases that are affected by lipid imbalances such as obesity, fatty liver disease, type 2 diabetes, Alzheimer's disease, cardiovascular disease, and cancer [...].

Lipid Droplets: Formation to Breakdown.

One of the most exciting areas of cell biology during the last decade has been the study of lipid droplets. Lipid droplets allow cells to store non-polar molecules such as neutral lipids in specific compartments where they are sequestered from the aqueous environment of the cell yet can be accessed through regulated mechanisms. These structures are highly conserved, appearing in organisms throughout the phylogenetic tree. Until somewhat recently, lipid droplets were widely regarded as inert, however progress in the field has continued to demonstrate their vast roles in a number of cellular processes in both mitotic and post-mitotic cells. No doubt the increase in the attention given to lipid droplet research is due to their central role in current pressing human diseases such as obesity, type-2 diabetes, and atherosclerosis. This review provides a mechanistic timeline from neutral lipid synthesis through lipid droplet formation and size augmentation to droplet breakdown.

Molecular links among non-biodegradable nanoparticles, reactive oxygen species, and autophagy.

For nanoparticles to be successful in combating diseases in the clinic in the 21st century and beyond, they must localize to target areas of the body and avoid damaging non-target, healthy tissues. Both soft and stiff, bio-degradable and non-biodegradable nanoparticles are anticipated to be used to this end. It has been shown that stiff, non-biodegradable nanoparticles cause reactive oxygen species (ROS) generation and autophagy in a variety of cell lines in vitro. Both responses can lead to significant remodeling of the cytosol and even apoptosis. Thus these are crucial cellular functions to understand. Improved assays have uncovered crucial roles of the Akt/mTOR signaling pathway in both ROS generation and autophagy initiation after cells have internalized stiff, non-biodegradable nanoparticles over varying geometries in culture. Of particular - yet unresolved - interest is how these nanoparticles cause the activation of these pathways. This article reviews the most recent advances in nanoparticle generation of ROS and autophagy initiation with a focus on stiff, non-biodegradable technologies. We provide experimental guidelines to the reader for fleshing out the effects of their nanoparticles on the above pathways with the goal of tuning nanoparticle design.

The protein and neutral lipid composition of lipid droplets isolated from the fission yeast, Schizosaccharomyces pombe.

Lipid droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer with bound proteins. Much of the information on lipid droplet function comes from proteomic and lipodomic studies that identify the components of droplets isolated from organisms throughout the phylogenetic tree. Here, we add to that important inventory by reporting lipid droplet factors from the fission yeast, Schizosaccharomyces pombe. Unique to this study was the fact that cells were cultured in three different environments: 1) late log growth phase in glucose-based media, 2) stationary phase in glucosebased media, and 3) late log growth phase in media containing oleic acid. We confirmed colocalization of major factors with lipid droplets using live-cell fluorescent microscopy. We also analyzed droplets from each of the three conditions for sterol ester (SE) and triacylglycerol (TAG) content, along with their respective fatty acid compositions. We identified a previously undiscovered lipid droplet protein, Vip1p, which affects droplet size distribution. The results provide further insight into the workings of these ubiquitous organelles.

Isolation of cellular lipid droplets: two purification techniques starting from yeast cells and human placentas.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method-- density gradient centrifugation--is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques. In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins. In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions. The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Particle shape effects in vitro and in vivo.

Particles that have the potential to deliver imaging agents and drugs to cells and tissue now have many different shapes and sizes. This diversity in particle shape could provide new options for potential treatments of diseases because geometry affects biodistribution. However, the myriad of particle shapes now available increases the number of variables or parameters that must be taken into consideration for the drug delivery field to understand particle-cell interactions. This is especially true when the shape of a particle is a tunable parameter along with particle chemistry, charge, and hydrophobicity. Here we review the impact of shape on particle-cell interactions in vitro and the ramifications of different particle geometries on circulation, biodistribution, localization to tumors, and toxicology in rodents.

Key structural features of the actin filament Arp2/3 complex branch junction revealed by molecular simulation.

We investigated the structure, properties and dynamics of the actin filament branch junction formed by actin-related protein (Arp) 2/3 complex using all-atom molecular dynamics (MD) simulations based on a model fit to a reconstruction from electron tomograms. Simulations of the entire structure consisting of 31 protein subunits together with solvent molecules containing ∼3 million atoms were performed for an aggregate time of 175 ns. One 75-ns simulation of the original reconstruction was compared to two 50-ns simulations of alternate structures, showing that the hypothesized branch junction structure is very stable. Our simulations revealed that the interface between Arp2/3 complex and the mother actin filament features a large number of salt bridges and hydrophobic contacts, many of which are dynamic and formed/broken on the timescale of the simulation. The simulations suggest that the DNase binding loops in Arp3, and possibly Arp2, form stabilizing contacts with the mother filament. Unbiased comparison of models sampled from the MD simulation trajectory with the primary experimental electron tomography data identified regions were snapshots from the simulation provide atomic details of the model structures and also pinpoints regions where the initial modeling based on the electron tomogram reconstruction may be suboptimal.

Nucleotide-mediated conformational changes of monomeric actin and Arp3 studied by molecular dynamics simulations.

Members of the actin family of proteins exhibit different biochemical properties when ATP, ADP-P(i), ADP, or no nucleotide is bound. We used molecular dynamics simulations to study the effect of nucleotides on the behavior of actin and actin-related protein 3 (Arp3). In all of the actin simulations, the nucleotide cleft stayed closed, as in most crystal structures. ADP was much more mobile within the cleft than ATP, despite the fact that both nucleotides adopt identical conformations in actin crystal structures. The nucleotide cleft of Arp3 opened in most simulations with ATP, ADP, and no bound nucleotide. Deletion of a C-terminal region of Arp3 that extends beyond the conserved actin sequence reduced the tendency of the Arp3 cleft to open. When the Arp3 cleft opened, we observed multiple instances of partial release of the nucleotide. Cleft opening in Arp3 also allowed us to observe correlated movements of the phosphate clamp, cleft mouth, and barbed-end groove, providing a way for changes in the nucleotide state to be relayed to other parts of Arp3. The DNase binding loop of actin was highly flexible regardless of the nucleotide state. The conformation of Ser14/Thr14 in the P1 loop was sensitive to the presence of the gamma-phosphate, but other changes observed in crystal structures were not correlated with the nucleotide state on nanosecond timescales. The divalent cation occupied three positions in the nucleotide cleft, one of which was not previously observed in actin or Arp2/3 complex structures. In sum, these simulations show that subtle differences in structures of actin family proteins have profound effects on their nucleotide-driven behavior.

Shape effects of filaments versus spherical particles in flow and drug delivery.

Interaction of spherical particles with cells and within animals has been studied extensively, but the effects of shape have received little attention. Here we use highly stable, polymer micelle assemblies known as filomicelles to compare the transport and trafficking of flexible filaments with spheres of similar chemistry. In rodents, filomicelles persisted in the circulation up to one week after intravenous injection. This is about ten times longer than their spherical counterparts and is more persistent than any known synthetic nanoparticle. Under fluid flow conditions, spheres and short filomicelles are taken up by cells more readily than longer filaments because the latter are extended by the flow. Preliminary results further demonstrate that filomicelles can effectively deliver the anticancer drug paclitaxel and shrink human-derived tumours in mice. Although these findings show that long-circulating vehicles need not be nanospheres, they also lend insight into possible shape effects of natural filamentous viruses.

Polymeric worm micelles as nano-carriers for drug delivery.

Nanoscale carriers of active compounds, especially drugs, need not be spherical in shape. Worm micelles as blends of degradable polylactic acid (PLA) and inert block copolymer amphiphiles were prepared for controlled release and initial study of carrier transport through nano-porous media. The loading capacity of a typical hydrophobic drug, Triamterene, and the release of hydrophobic dyes were evaluated together with morphological changes of the micelles. Degradation of PLA by hydrolysis led to the self-shortening of worms and a clear transition towards spherical micelles, correlating with the release of hydrophobic dyes. Perhaps equally important for application is the flexibility of worm micelles, which we show allows them to penetrate nanoporous gels where 100 nm sized vesicles cannot enter. Such gels have served as tissue models, and so the results here collectively suggest a new class of hydrophobic drug nano-carriers that are capable of tissue permeation as well as controlled release.