Proteome of Stored RBC Membrane and Vesicles from Heterozygous Beta Thalassemia Donors

Genetic characteristics of blood donors may impact the storability of blood products. Despite higher basal stress, red blood cells (RBCs) from eligible donors that are heterozygous for beta-thalassemia traits (βThal+) possess a differential nitrogen-related metabolism, and cope better with storage stress compared to the control. Nevertheless, not much is known about how storage impacts the proteome of membrane and extracellular vesicles (EVs) in βThal+. For this purpose, RBC units from twelve βThal+ donors were studied through proteomics, immunoblotting, electron microscopy, and functional ELISA assays, versus units from sex- and aged-matched controls. βThal+ RBCs exhibited less irreversible shape modifications. Their membrane proteome was characterized by different levels of structural, lipid raft, transport, chaperoning, redox, and enzyme components. The most prominent findings include the upregulation of myosin proteoforms, arginase-1, heat shock proteins, and protein kinases, but the downregulation of nitrogen-related transporters. The unique membrane proteome was also mirrored, in part, to that of βThal+ EVs. Network analysis revealed interesting connections of membrane vesiculation with storage and stress hemolysis, along with proteome control modulators of the RBC membrane. Our findings, which are in line with the mild but consistent oxidative stress these cells experience in vivo, provide insight into the physiology and aging of stored βThal+ RBCs.

A Small Molecule Fluorogenic Probe for the Detection of Sphingosine in Living Cells

ABSTRACTThe single-chained sphingolipid sphingosine is an essential structural lipid and signaling molecule. Abnormal sphingosine metabolism is observed in several diseases, including cancer, diabetes, and Alzheimer’s. Despite its biological importance, there are a lack of tools for detecting sphingosine in living cells. This is likely due to the broader challenge of developing highly selective and live-cell compatible affinity probes for hydrophobic lipid species. In this work, we have developed a small molecule fluorescent turn-on probe for labeling sphingosine in living cells. This probe utilizes a selective reaction between sphingosine and salicylaldehyde esters to fluorescently label sphingosine molecules. We demonstrate that this probe exhibits a dose-dependent response to sphingosine and is able to detect endogenous pools of sphingosine. Using our probe, we successfully detected sphingosine accumulation in live Niemann-Pick type C1 (NPC1) patient cells, a lipid transport disorder in which increased sphingosine mediates disease progression. This work provides a simple and accessible method for the detection of sphingosine and should facilitate study of this critical signaling lipid in biology and disease.

Probing the subcellular distribution of phosphatidylinositol reveals a surprising lack at the plasma membrane

The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids’ role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.

Probing the Subcellular Distribution of Phosphatidylinositol Reveals a Surprising Lack at the Plasma Membrane

The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids’ role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn have been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we have used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared to the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.SummaryZewe et al develop approaches to map the subcellular distribution of the major phospholipid, phosphatidylinositol (PI), revealing that the lipid is present in most membranes except for plasma membrane, where it is mainly found as PI4P and PI(4,5)P2.

Size-dependent formation of membrane nanotubes: continuum modeling and molecular dynamics simulations

With continuum theory and molecular dynamics simulations we demonstrated that the lipid membrane upon extraction exhibits size- and tension-dependent mechanical behaviors, and different structural lipid rearrangements in different leaflets.

Does PtdIns(4,5)P2 concentrate so it can multi-task?

Ptdns(4,5)P2 is a minor structural lipid of the plasma membrane (PM), but a master regulator of PM function. Serving either as a substrate for the generation of second messengers, or more commonly as a ligand triggering protein recruitment or activation, it regulates most aspects of PM function. Understanding how this relatively simple biological macromolecule can regulate such a vast array of different functions in parallel, is the key to understanding the biology of the PM as a whole, in both health and disease. In this review, potential mechanisms are discussed that might explain how a lipid can separately regulate so many protein complexes. The focus is on the spatial distribution of the lipid molecules, their metabolism and their interactions. Open questions that still need to be resolved are highlighted, as are potential experimental approaches that might shed light on the mechanisms at play. Moreover, the broader question is raised as to whether PtdIns(4,5)P2 should be thought of as a bona fide signalling molecule or more of a simple lipid cofactor or perhaps both, depending on the context of the particular function in question.