The formation of autophagosomes mediating the sequestration of cytoplasmic materials is the central step of autophagy. Several phosphoinositides, which are signaling molecules on the membrane, are involved in autophagy. However, it is not always clear whether these phosphoinositides act directly at the site of autophagosome formation, or indirectly via the regulation of other steps or pathways. To address this question, we used a set of phosphoinositide probes to systematically examine their potential presence on autophagosomal membranes in yeast (Saccharomyces cerevisiae). We verified the specificity of these probes using mutant cells deficient in the production of the corresponding phosphoinositides. We then examined starved yeast cells co-expressing a phosphoinositide probe together with an autophagosomal membrane marker, 2Katushka2S-Atg8. Our data revealed that PtdIns(4,5)P2 and PtdIns(3,5)P2 were mainly present on the plasma membrane and vacuolar membrane, respectively. We observed only occasional co-localization between the PtdIns(4)P probe and Atg8, some of which may represent the transient passage of a PtdIns(4)P-containing structure near the autophagosomal membrane. In contrast, substantial colocalization of the PtdIns(3)P probe with Atg8 was observed. Taken together, our data indicate that only PtdIns(3)P is present in a substantial amount on the autophagosomal membrane. For other phosphoinositides involved in autophagy, either their presence on the autophagosomal membrane is very transient, or they act on other cellular membranes to regulate autophagy.

Adaptive immune responses comprise the activation of T cells by peptide antigens that are presented by proteins of the Major Histocompatibility Complex (MHC) on the surface of an antigen-presenting cell. As a consequence of the T cell receptor interacting productively with a certain peptide-MHC complex, a specialized cell-cell junction known as the immunological synapse forms and is accompanied by changes in the spatiotemporal patterning and function of intracellular signaling molecules. Key modifications occurring at the cytoplasmic leaflet of the plasma and internal membranes in activated T cells comprise lipid switches that affect the binding and distribution of proteins within or near the lipid bilayer. Here, we describe two major classes of lipid switches that act at this critical water/membrane interface. Phosphoinositides are derived from phosphatidylinositol, an amphiphilic molecule that contains two fatty acid chains and a phosphate group that bridges the glycerol backbone to the carbohydrate inositol. The inositol ring can be variably (de-)phosphorylated by dedicated kinases and phosphatases, thereby creating phosphoinositide signatures that define the composition and properties of signaling molecules, molecular complexes, or whole organelles. Palmitoylation refers to the reversible attachment of the fatty acid palmitate to a substrate protein\'s cysteine residue. DHHC enzymes, named after the four conserved amino acids in their active site, catalyze this post-translational modification and thereby change the distribution of proteins at, between, and within membranes. T cells utilize these two types of molecular switches to adjust their properties to an activation process that requires changes in motility, transport, secretion, and gene expression.

Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.

The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.

In the last three decades, the presence of phospholipids in the nucleus has been shown and thoroughly investigated. A considerable amount of interest has been raised about nuclear inositol lipids, mainly because of their role in signaling acting. Here, we review the main issues of nuclear phospholipid localization and the role of nuclear inositol lipids and their related enzymes in cellular signaling, both in physiological and pathological conditions.

Synaptojanin-1 (SJ1) is a major neuronal-enriched PI(4, 5)P2 4- and 5-phosphatase implicated in the shedding of endocytic factors during endocytosis. A mutation (R258Q) that impairs selectively its 4-phosphatase activity causes Parkinsonism in humans and neurological defects in mice (SJ1RQKI mice). Studies of these mice showed, besides an abnormal assembly state of endocytic factors at synapses, the presence of dystrophic nerve terminals selectively in a subset of nigro-striatal dopamine (DA)-ergic axons, suggesting a special lability of DA neurons to the impairment of SJ1 function. Here we have further investigated the impact of SJ1 on DA neurons using iPSC-derived SJ1 KO and SJ1RQKI DA neurons and their isogenic controls. In addition to the expected enhanced clustering of endocytic factors in nerve terminals, we observed in both SJ1 mutant neuronal lines increased cilia length. Further analysis of cilia of SJ1RQDA neurons revealed abnormal accumulation of the Ca2+ channel Cav1.3 and of ubiquitin chains, suggesting a defect in the clearing of ubiquitinated proteins at the ciliary base, where a focal concentration of SJ1 was observed. We suggest that SJ1 may contribute to the control of ciliary protein dynamics in DA neurons, with implications on cilia-mediated signaling.

Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.

Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for direct lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER transmembrane contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for the maintenance of PI(4)P and PI(4,5)P2 levels at the plasma membrane, but not for PI(4)P homeostasis in the Golgi and endosomal compartments. Importantly, we show that VAPA regulates the dynamics of focal adhesions (FA) through its MSP domain, is essential to stabilize and anchor ventral ER-PM contact sites to FA, and mediates microtubule-dependent FA disassembly. To conclude, our results reveal unknown functions for VAPA-mediated membrane contact sites during cell motility and provide a dynamic picture of ER-PM contact sites connection with FA mediated by VAPA.

The lipid phosphatidylinositol 4-phosphate (PI4P) plays a master regulatory role at Golgi membranes, orchestrating membrane budding, non-vesicular lipid transport and membrane organization. It follows that harmonious Golgi function requires strictly maintained PI4P homeostasis. One of the most abundant PI4P effector proteins is the oxysterol binding protein (OSBP), a lipid transfer protein that exchanges trans-Golgi PI4P for ER cholesterol. Although this protein consumes PI4P as part of its lipid anti-porter function, whether it actively contributes to Golgi PI4P homeostasis has been questioned. Here, we employed a series of acute and chronic genetic manipulations, together with orthogonal targeting of OSBP, to interrogate its control over Golgi PI4P abundance. Modulating OSBP levels at ER:Golgi membrane contact sites produces reciprocal changes in PI4P levels. Additionally, we observe that OSBP has a high capacity for PI4P turnover, even at orthogonal organelle membranes. However, despite also visiting the plasma membrane, endogenous OSBP makes no impact on PI4P levels in this compartment. We conclude that OSBP is a major determinant of Golgi PI4P homeostasis.

Vacuolar ATPases (V-ATPases), proton pumps composed of 16 subunits, are necessary for a variety of cellular functions. Subunit \"a\" has four isoforms, a1-a4, each with a distinct cellular location. We identified a phosphoinositide (PIP) interaction motif, KXnK(R)IK(R), conserved in all four isoforms, and hypothesize that a/PIP interactions regulate V-ATPase recruitment/retention to different organelles. Among the four isoforms, a2 is enriched on Golgi with a2 mutations in the PIP motif resulting in cutis laxa. We hypothesize that the hydrophilic N-terminal (NT) domain of a2 contains a lipid-binding domain, and mutations in this domain prevent interaction with Golgi-enriched PIPs, resulting in cutis laxa. We recreated the cutis laxa-causing mutation K237_V238del, and a double mutation in the PIP-binding motif, K237A/V238A. Circular dichroism confirmed that there were no protein structure alterations. Pull-down assays with PIP-enriched liposomes revealed that wildtype a2NT preferentially binds phosphatidylinositol 4-phosphate (PI(4)P), while mutants decreased binding to PI(4)P. In HEK293 cells, wildtype a2NT was localized to Golgi and co-purified with microsomal membranes. Mutants reduced Golgi localization and membrane association. Rapamycin depletion of PI(4)P diminished a2NT-Golgi localization. We conclude that a2NT is sufficient for Golgi retention, suggesting the lipid-binding motif is involved in V-ATPase targeting and/or retention. Mutational analyses suggest a molecular mechanism underlying how a2 mutations result in cutis laxa.