Necroptosis is associated with Rab27-independent expulsion of extracellular vesicles containing RIPK3 and MLKL.

Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector-mixed lineage kinase domain like (MLKL)-was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. Assessing EV number by nanoparticle tracking analysis revealed an increased number of EVs released during necroptosis. To evaluate the nature of such vesicles, we performed a newly adapted, highly sensitive mass spectrometry-based proteomics on EVs released by healthy or necroptotic cells. Compared to EVs released by healthy cells, EVs released during necroptosis contained a markedly higher number of unique proteins. Receptor interacting protein kinase-3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, mouse embryonic fibroblasts (MEFs) derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis-associated EVs were sensitive to Ca2+ depletion or lysosomal disruption. Neither treatment affected the RIPK3-mediated MLKL phosphorylation. An unbiased screen using RIPK3 immunoprecipitation-mass spectrometry on necroptotic EVs led to the identification of Rab11b in RIPK3 immune-complexes. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.

Nanoparticle tracking analysis and statistical mixture distribution analysis to quantify nanoparticle-vesicle binding.

Nanoparticle tracking analysis (NTA) is a single particle tracking technique that in principle provides a more direct measure of particle size distribution compared to dynamic light scattering (DLS). Here, we demonstrate how statistical mixture distribution analysis can be used in combination with NTA to quantitatively characterize the amount and extent of particle binding in a mixture of nanomaterials. The combined approach is used to study the binding of gold nanoparticles to two types of phospholipid vesicles, those containing and lacking the model ion channel peptide gramicidin A. This model system serves as both a proof of concept for the method and a demonstration of the utility of the approach in studying nano-bio interactions. Two diffusional models (Stokes-Einstein and Kirkwood-Riseman) were compared in the determination of particle size, extent of binding, and nanoparticle:vesicle binding ratios for each vesicle type. The combination of NTA and statistical mixture distributions is shown to be a useful method for quantitative assessment of the extent of binding between particles and determination of binding ratios.

Membrane Remodeling and Stimulation of Aggregation Following α-Synuclein Adsorption to Phosphotidylserine Vesicles.

α-Synuclein is an intrinsically disordered protein abundant in presynaptic terminals in neurons and in synaptic vesicles. α-Synuclein's interaction with lipid bilayers is important not only for its normal physiological function but also in its pathological aggregation and deposition as Lewy bodies in Parkinson's disease. α-Synuclein binds preferentially to lipids with acidic head groups and to high-curvature vesicles and can modulate membrane curvature. The relationship between the protein's role as a membrane curvature sensor and generator and the role of membranes in facilitating its aggregation remains unknown. We investigated the interaction of α-synuclein with vesicles of 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) or 1,2-dilauroyl-sn-glycero-3-phospho-l-serine (DLPS). Using nanoparticle tracking along with electron microscopy, we demonstrate that α-synuclein induces extensive vesicle disruption and membrane remodeling into discoids, tubules, and ribbons with DLPS vesicles but not DOPS. Coarse-grained molecular dynamics simulations revealed that adsorption of α-synuclein to DLPS but not DOPS vesicles induced vesicle elongation and redistribution of protein to regions of higher curvature, a process that could drive protein aggregation. In agreement with this hypothesis, DLPS but not DOPS strongly stimulated α-synuclein aggregation. Our results provide new insights into the critical contribution of bilayer stability in the membrane response to α-synuclein adsorption and in stimulation of aggregation.

Evaluation of Nanoparticle Tracking Analysis for the Detection of Rod-Shaped Particles and Protein Aggregates.

Nanoparticle tracking analysis (NTA) is an important technique for measuring hydrodynamic size of globular biological particles including liposomes and viruses. Less attention has been paid to NTA of rod-like particles, despite their considerable interest. For example, amyloid fibrils and protofibrils are protein aggregates with rod-like morphology, diameters of 2-15 nm, and lengths from 50 nm to 1 µm, and linked to diseases including Alzheimer's and Parkinson's. We used NTA to measure the concentration and hydrodynamic size of gold nanorods (10 nm diameter, 35-250 nm length) and myosin (2 nm diameter, 160 nm length), as models of rod-like particles. Measured hydrodynamic diameters of gold nanorods were consistent with theoretical calculations, as long as particle concentration and solution conditions were controlled. Myosin monomers were invisible by NTA, but a small population of aggregates was detected. We combined NTA results with other light scattering data to gain insight into number and size distribution of protein solutions containing both monomer and aggregates. Finally, we demonstrated the utility of NTA and its limitations by characterizing aggregates of alpha-synuclein. Of note is the use of NTA to detect a change in morphology from compact to elongated by analyzing the ratio of hydrodynamic size to intensity.

Maspin binds to cardiolipin in mitochondria and triggers apoptosis.

A central question in cell biology is how cells respond to stress signals and biochemically regulate apoptosis. One critical pathway involves the change of mitochondrial function and release of cytochrome c to initiate apoptosis. In response to apoptotic stimuli, we found that maspin-a noninhibitory member of the serine protease inhibitor superfamily-translocates from the cytosol to mitochondria and binds to cardiolipin in the inner mitochondrial membrane. Biolayer interferometry assay revealed that recombinant maspin binds cardiolipin with an apparent Kd,of ∼15.8 µM and competes with cytochrome c (apparent Kd of ∼1.31 µM) for binding to cardiolipin-enriched membranes. A hydrophobic, lysine-rich domain in maspin consists of 27 aa, is located at position 268-294, and is responsible for the interaction of this protein with cardiolipin. Depletion of cardiolipin in cells significantly prevents maspin binding to the inner mitochondrial membrane and decreases cytochrome c release and apoptosis. Alteration to maspin's cardiolipin binding domain changes its ability to bind cardiolipin, and tumor cells expressing this mutant have a low frequency of apoptosis. We propose a model of apoptosis in which maspin binds to cardiolipin, displaces cytochrome c from the membrane, and facilitates its release to the cytoplasm.-Mahajan, N., Hoover, B., Rajendram, M., Shi, H. Y., Kawasaki, K., Weibel, D. B., Zhang, M. Maspin binds to cardiolipin in mitochondria and triggers apoptosis.

A comparison of DNA compaction by arginine and lysine peptides: a physical basis for arginine rich protamines.

Protamines are small, highly positively charged peptides used to package DNA at very high densities in sperm nuclei. Tight DNA packing is considered essential for the minimization of DNA damage by mutagens and reactive oxidizing species. A striking and general feature of protamines is the almost exclusive use of arginine over lysine for the positive charge to neutralize DNA. We have investigated whether this preference for arginine might arise from a difference in DNA condensation by arginine and lysine peptides. The forces underlying DNA compaction by arginine, lysine, and ornithine peptides are measured using the osmotic stress technique coupled with X-ray scattering. The equilibrium spacings between DNA helices condensed by lysine and ornithine peptides are significantly larger than the interhelical distances with comparable arginine peptides. The DNA surface-to-surface separation, for example, is some 50% larger with polylysine than with polyarginine. DNA packing by lysine rich peptides in sperm nuclei would allow much greater accessibility to small molecules that could damage DNA. The larger spacing with lysine peptides is caused by both a weaker attraction and a stronger short-range repulsion relative to that of the arginine peptides. A previously proposed model for binding of polyarginine and protamine to DNA provides a convenient framework for understanding the differences between the ability of lysine and arginine peptides to assemble DNA.