In this issue of Chemistry & Biology, Antal and colleagues describe how phosphorylation optimizes the signaling range of protein kinase C (PKC) isoforms. Priming of these enzymes regulates intramolecular conformational changes, which reduces access to their diacylglycerol (DAG) binding C1 domains.

•Phosphoinositides have unique patterns of cellular distribution.•Tools are available to manipulate real-time cellular levels of phosphoinositides.•Phosphoinositides mediate peripheral protein recruitment to cellular membranes.•Some mechanisms of phosphoinositide binding have been thoroughly investigated.•New methods are necessary to monitor coincidence detection of phosphoinositides.Anionic lipids act as signals for the recruitment of proteins containing cationic clusters to biological membranes. A family of anionic lipids known as the phosphoinositides (PIPs) are low in abundance, yet play a critical role in recruitment of peripheral proteins to the membrane interface. PIPs are mono-, bis-, or trisphosphorylated derivatives of phosphatidylinositol (PI) yielding seven species with different structure and anionic charge. The differential spatial distribution and temporal appearance of PIPs is key to their role in communicating information to target proteins. Selective recognition of PIPs came into play with the discovery that the substrate of protein kinase C termed pleckstrin possessed the first PIP binding region termed the pleckstrin homology (PH) domain. Since the discovery of the PH domain, more than ten PIP binding domains have been identified including PH, ENTH, FYVE, PX, and C2 domains. Representative examples of each of these domains have been thoroughly characterized to understand how they coordinate PIP headgroups in membranes, translocate to specific membrane docking sites in the cell, and function to regulate the activity of their full-length proteins. In addition, a number of novel mechanisms of PIP-mediated membrane association have emerged, such as coincidence detection–specificity for two distinct lipid headgroups. Other PIP-binding domains may also harbor selectivity for a membrane physical property such as charge or membrane curvature. This review summarizes the current understanding of the cellular distribution of PIPs and their molecular interaction with peripheral proteins.

Cellular membranes are composed of hundreds of different lipids, ion channels, receptors and scaffolding complexes that act as signalling and trafficking platforms for processes fundamental to life. Cellular signalling and membrane trafficking are often regulated by peripheral proteins, which reversibly interact with lipid molecules in highly regulated spatial and temporal fashions. In most cases, one or more modular lipid-binding domain(s) mediate recruitment of peripheral proteins to specific cellular membranes. These domains, of which more than 10 have been identified since 1989, harbour structurally selective lipid-binding sites. Traditional in vitro and in vivo studies have elucidated how these domains coordinate their cognate lipids and thus how the parent proteins associate with membranes. Cellular activities of peripheral proteins and subsequent physiological processes depend upon lipid binding affinities and selectivity. Thus, the development of novel sensitive and quantitative tools is essential in furthering our understanding of the function and regulation of these proteins. As this field expands into new areas such as computational biology, cellular lipid mapping, single molecule imaging, and lipidomics, there is an urgent need to integrate technologies to detail the molecular architecture and mechanisms of lipid signalling. This review surveys emerging cellular and in vitro approaches for studying protein–lipid interactions and provides perspective on how integration of methodologies directs the future development of the field.

The Ebola virus causes severe hemorrhagic fever and has a mortality rate that can be as high as 90%, yet no vaccines or approved therapeutics, to our knowledge, are available. To replicate and egress the infected host cell the Ebola virus uses VP40, its major matrix protein to assemble at the inner leaflet of the plasma membrane. The assembly and budding of VP40 from the plasma membrane of host cells seem still poorly understood. We investigated the assembly and egress of VP40 at the plasma membrane of human cells using single-particle tracking. Our results demonstrate that actin coordinates the movement and assembly of VP40, a critical step in viral egress. These findings underscore the ability of single-molecule techniques to investigate the interplay of VP40 and host proteins in viral replication.

Ebola virus, from the Filoviridae family has a high fatality rate in humans and nonhuman primates and to date, to the best of our knowledge, has no FDA approved vaccines or therapeutics. Viral protein 40 (VP40) is the major Ebola virus matrix protein that regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of VP40 membrane binding that are important for viral release remain to be elucidated. In this study, we used fluorescence quenching of a tryptophan on the membrane-binding interface with brominated lipids along with mutagenesis of VP40 to understand the depth of membrane penetration into lipid bilayers. Experimental results indicate that VP40 penetrates 8.1 Å into the hydrocarbon core of the plasma membrane bilayer. VP40 also induces substantial changes to membrane curvature as it tubulates liposomes and induces vesiculation into giant unilamellar vesicles, effects that are abrogated by hydrophobic mutations. This is a critical step in viral egress as cellular assays demonstrate that hydrophobic residues that penetrate deeply into the plasma membrane are essential for plasma membrane localization and virus-like particle formation and release from cells.

Ebola virus assembles and buds from the inner leaflet of the plasma membrane of mammalian cells, which is primarily attributed to its major matrix protein VP40. Oligomerization of VP40 has been shown to be essential to the life cycle of the virus including formation of virions from infected cells. To date, VP40 oligomerization has mainly been assessed by chemical cross-linking following cell fractionation studies with VP40 transfected cells. This has made it difficult to discern the spatial and temporal dynamics of VP40 oligomerization. To gain a better understanding of the VP40 assembly and oligomerization process in live cells, we have employed real-time imaging of enhanced green fluorescent protein tagged VP40. Here, we use both confocal and total internal reflection microscopy coupled with number and brightness analysis to show that VP40 oligomers are localized on the plasma membrane and are highly enriched at sites of membrane protrusion, consistent with sites of viral budding. These filamentous plasma membrane protrusion sites harbor VP40 hexamers, octamers, and higher order oligomers. Consistent with previous reports, abrogation of VP40 oligomerization through mutagenesis greatly diminished VP40 egress and also abolished membrane protrusion sites enriched with VP40. In sum, real-time single-molecule imaging of fluorescently labeled Ebola VP40 is able to resolve the spatial and temporal dynamics of VP40 oligomerization.