Novel strategies for enhancing shotgun lipidomics for comprehensive analysis of cellular lipidomes.

Shotgun lipidomics is one of the most powerful tools in analysis of cellular lipidomes in lipidomics, which directly analyzes lipids from lipid extracts of diverse biological samples with high accuracy/precision. However, despite its great advances in high throughput analysis of cellular lipidomes, low coverage of poorly ionized lipids, especially those species in very low abundance, and some types of isomers within complex lipid extracts by shotgun lipidomics remains a huge challenge. In the past few years, many strategies have been developed to enhance shotgun lipidomics for comprehensive analysis of lipid species. Chemical derivatization represents one of the most attractive and effective strategies, already receiving considerable attention. This review focuses on novel advanced derivatization strategies for enhancing shotgun lipidomics. It is anticipated that with the development of enhanced strategies, shotgun lipidomics can make greater contributions to biological and biomedical research.

Search for Upstream Cell Volume Sensors: The Role of Plasma Membrane and Cytoplasmic Hydrogel.

The plasma membrane plays a prominent role in the regulation of cell volume by mediating selective transport of extra- and intracellular osmolytes. Recent studies show that upstream sensors of cell volume changes are mainly located within the cytoplasm that displays properties of a hydrogel and not in the plasma membrane. Cell volume changes occurring in anisosmotic medium as well as in isosmotic environment affect properties of cytoplasmic hydrogel that, in turn, trigger rapid regulatory volume increase and decrease (RVI and RVD). The downstream signaling pathways include reorganization of 2D cytoskeleton and altered composition of polyphosphoinositides located on the inner surface of the plasma membrane. In addition to its action on physico-chemical properties of cytoplasmic hydrogel, cell volume changes in anisosmotic conditions affect the ionic strength of the cytoplasm and the [Na+]i/[K+]i ratio. Elevated intracellular ionic strength evoked by long term exposure of cells to hypertonic environment resulted in the activation of TonEBP and augmented expression of genes controlling intracellular organic osmolyte levels. The role of Na+i/K+i -sensitive, Ca2+i -mediated and Ca2+i-independent mechanisms of excitation-transcription coupling in cell volume-adjustment remains unknown.

Nuclear Lipids in the Nervous System: What they do in Health and Disease.

In the last 20 years it has been widely demonstrated that cell nucleus contains neutral and polar lipids localized in nuclear membranes, nucleoli, nuclear matrix and chromatin. Nuclear lipids may show specific organization forming nuclear lipid microdomains and have both structural and functional roles. Depending on their localization, nuclear lipids play different roles such as the regulation of nuclear membrane and nuclear matrix fluidity but they also can act as platforms for vitamin and hormone function, for active chromatin anchoring, and for the regulation of gene expression, DNA duplication and transcription. Crosstalk among different kinds of lipid signalling pathways influence the physiopathology of numerous cell types. In neural cells the nuclear lipids are involved in cell proliferation, differentiation, inflammation, migration and apoptosis. Abnormal metabolism of nuclear lipids might be closely associated with tumorigenesis and neurodegenerative diseases such as Alzheimer disease and Parkinson disease among others.

Lipid compartmentalization in the endosome system.

Lipids play an essential role in the structure of the endosomal membranes as well as in their dynamic rearrangement during the transport of internalized cargoes along the endocytic pathway. In this review, we discuss the function of endosomal lipids mainly in mammalian cells, focusing on two well-known components of the lipid rafts, sphingomyelin and cholesterol, as well as on three anionic phospholipids, phosphatidylserine, polyphosphoinositides and the atypical phospholipid, bis(monoacylglycero)phosphate/lysobisphosphatidic acid. We detail the structure, metabolism, distribution and role of these lipids in the endosome system as well as their importance in pathological conditions where modification of the endosomal membrane flow can lead to various diseases such as lipid-storage diseases, myopathies and neuropathies.

The SOAR of STIM1 interacts with plasma membrane lipids to form ER-PM contact sites.

Depletion of Ca2+ from the endoplasmic reticulum (ER) causes the ER Ca2+ sensor STIM1 to form membrane contact sites (MCSs) with the plasma membrane (PM). At the ER-PM MCS, STIM1 binds to Orai channels to induce cellular Ca2+ entry. The prevailing view of this sequential process is that STIM1 interacts with the PM and with Orai1 using two separate modules: a C-terminal polybasic domain (PBD) for the interaction with PM phosphoinositides and the STIM-Orai activation region (SOAR) for the interaction with Orai channels. Here, using electron and fluorescence microscopy and protein-lipid interaction assays, we show that oligomerization of the SOAR promotes direct interaction with PM phosphoinositides to trap STIM1 at ER-PM MCSs. The interaction depends on a cluster of conserved lysine residues within the SOAR and is co-regulated by the STIM1 coil-coiled 1 and inactivation domains. Collectively, our findings uncover a molecular mechanism for formation and regulation of ER-PM MCSs by STIM1.

The Enigmatic Role of Lipids in Cilia Signaling.

Primary cilia are specialized cellular structures that project from the surface of most cell types in metazoans and mediate transduction of major signaling pathways. The ciliary membrane is contiguous with the plasma membrane, yet it exhibits distinct protein and lipid composition, which is essential for ciliary function. Diffusion barriers at the base of a cilium are responsible for establishing unique molecular composition of this organelle. Although considerable progress has been made in identifying mechanisms of ciliary protein trafficking in and out of cilia, it remains largely unknown how the distinct lipid identity of the ciliary membrane is achieved. In this mini review, I summarize recent developments in characterizing lipid composition and organization of the ciliary membrane and discuss the emerging roles of lipids in modulating activity of ciliary signaling components including ion channels and G protein-coupled receptors.

Actin sequestering protein, profilin, regulates intracellular vesicle transport in Leishmania.

Profilins are the key regulators of actin dynamics in all eukaryotic cells. However, little information is available on their biochemical properties and functions in kinetoplastids, such as Trypanosoma and Leishmania. We show here that Leishmania parasites express only one homolog of profilin (LdPfn), which catalyzes nucleotide exchange on G-actin and promotes actin polymerization at its low concentrations. However, at high concentrations, it strongly inhibits the polymerization process by sequestering actin monomers. We further demonstrate that LdPfn binds to actin in Leishmania promastigotes, by both immunofluorescence microscopy and IgG affinity chromatography. Further, we reveal that this protein besides binding to poly-null-proline motifs, also binds more efficiently to PI(3,5)P2, which is found on early or late endosomes or lysosomes, than to PI(4,5)P2 and PI(3,4,5)P3. Additionally, we show that heterozygous mutants of profilin display significantly slower growth and intracellular vesicle trafficking activity, which is reversed on episomal gene complementation. Together, these findings suggest that profilin regulates intracellular vesicle trafficking in Leishmania perhaps through its binding to polyphosphoinositides.

Polyphosphoinositides in the nucleus: Roadmap of their effectors and mechanisms of interaction.

Biomolecular interactions between proteins and polyphosphoinositides (PPIn) are essential in the regulation of the vast majority of cellular processes. Consequently, alteration of these interactions is implicated in the development of many diseases. PPIn are phosphorylated derivatives of phosphatidylinositol and consist of seven species with different phosphate combinations. PPIn signal by recruiting proteins via canonical domains or short polybasic motifs. Although their actions are predominantly documented on cytoplasmic membranes, six of the seven PPIn are present within the nucleus together with the PPIn kinases, phosphatases and phospholipases that regulate their turnover. Importantly, the contribution of nuclear PPIn in the regulation of nuclear processes has led to an increased recognition of their importance compared to their more accepted cytoplasmic roles. This review summarises our knowledge on the identification and functional characterisation of nuclear PPIn-effector proteins as well as their mode of interactions, which tend to favour polybasic motifs.

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion.

Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca(2+)-triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca(2+)-triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.