A novel homeostatic mechanism tunes PI(4,5)P2-dependent signaling at the plasma membrane.

The lipid molecule phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] controls all aspects of plasma membrane (PM) function in animal cells, from its selective permeability to the attachment of the cytoskeleton. Although disruption of PI(4,5)P2 is associated with a wide range of diseases, it remains unclear how cells sense and maintain PI(4,5)P2 levels to support various cell functions. Here, we show that the PIP4K family of enzymes, which synthesize PI(4,5)P2 via a minor pathway, also function as sensors of tonic PI(4,5)P2 levels. PIP4Ks are recruited to the PM by elevated PI(4,5)P2 levels, where they inhibit the major PI(4,5)P2-synthesizing PIP5Ks. Perturbation of this simple homeostatic mechanism reveals differential sensitivity of PI(4,5)P2-dependent signaling to elevated PI(4,5)P2 levels. These findings reveal that a subset of PI(4,5)P2-driven functions might drive disease associated with disrupted PI(4,5)P2 homeostasis.

Gigaxonin is required for intermediate filament transport.

Gigaxonin is an adaptor protein for E3 ubiquitin ligase substrates. It is necessary for ubiquitination and degradation of intermediate filament (IF) proteins. Giant axonal neuropathy is a pathological condition caused by mutations in the GAN gene that encodes gigaxonin. This condition is characterized by abnormal accumulation of IFs in both neuronal and non-neuronal cells; however, it is unclear what causes IF aggregation. In this work, we studied the dynamics of IFs using their subunits tagged with a photoconvertible protein mEOS 3.2. We have demonstrated that the loss of gigaxonin dramatically inhibited transport of IFs along microtubules by the microtubule motor kinesin-1. This inhibition was specific for IFs, as other kinesin-1 cargoes, with the exception of mitochondria, were transported normally. Abnormal distribution of IFs in the cytoplasm can be rescued by direct binding of kinesin-1 to IFs, demonstrating that transport inhibition is the primary cause for the abnormal IF distribution. Another effect of gigaxonin loss was a more than 20-fold increase in the amount of soluble vimentin oligomers in the cytosol of gigaxonin knock-out cells. We speculate that these oligomers saturate a yet unidentified adapter that is required for kinesin-1 binding to IFs, which might inhibit IF transport along microtubules causing their abnormal accumulation.

PI(4,5)P2 diffuses freely in the plasma membrane even within high-density effector protein complexes.

The lipid phosphatidyl-D-myo-inositol-4,5-bisphosphate [PI(4,5)P2] is a master regulator of plasma membrane (PM) function. Its effector proteins regulate transport, signaling, and cytoskeletal processes that define PM structure and function. How a single type of lipid regulates so many parallel processes is unclear. We tested the hypothesis that spatially separate PI(4,5)P2 pools associate with different PM complexes. The mobility of PI(4,5)P2 was measured using biosensors by single-particle tracking. We found that PM lipids including PI(4,5)P2 diffuse rapidly (∼0.3 µm2/s) with Brownian motion, although they spend one third of their time diffusing more slowly. Surprisingly, areas of the PM occupied by PI(4,5)P2-dependent complexes did not slow PI(4,5)P2 lateral mobility. Only the spectrin and septin cytoskeletons showed reduced PI(4,5)P2 diffusion. We conclude that even structures with high densities of PI(4,5)P2 effector proteins, such as clathrin-coated pits and focal adhesions, do not corral unbound PI(4,5)P2, questioning a role for spatially segregated PI(4,5)P2 pools in organizing and regulating PM functions.

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.

A high-avidity biosensor reveals plasma membrane PI(3,4)P2 is predominantly a class I PI3K signaling product.

Class I phosphoinositide 3-OH kinase (PI3K) signaling is central to animal growth and metabolism, and pathological disruption of this pathway affects cancer and diabetes. However, the specific spatial/temporal dynamics and signaling roles of its minor lipid messenger, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), are not well understood. This owes principally to a lack of tools to study this scarce lipid. Here we developed a high-sensitivity genetically encoded biosensor for PI(3,4)P2, demonstrating high selectivity and specificity of the sensor for the lipid. We show that despite clear evidence for class II PI3K in PI(3,4)P2-driven function, the overwhelming majority of the lipid accumulates through degradation of class I PI3K-produced PIP3 However, we show that PI(3,4)P2 is also subject to hydrolysis by the tumor suppressor lipid phosphatase PTEN. Collectively, our results show that PI(3,4)P2 is potentially an important driver of class I PI3K-driven signaling and provides powerful new tools to begin to resolve the biological functions of this lipid downstream of class I and II PI3K.

SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes.

Gradients of PtdIns4P between organelle membranes and the endoplasmic reticulum (ER) are thought to drive counter-transport of other lipids via non-vesicular traffic. This novel pathway requires the SAC1 phosphatase to degrade PtdIns4P in a 'cis' configuration at the ER to maintain the gradient. However, SAC1 has also been proposed to act in 'trans' at membrane contact sites, which could oppose lipid traffic. It is therefore crucial to determine which mode SAC1 uses in living cells. We report that acute inhibition of SAC1 causes accumulation of PtdIns4P in the ER, that SAC1 does not enrich at membrane contact sites, and that SAC1 has little activity in 'trans', unless a linker is added between its ER-anchored and catalytic domains. The data reveal an obligate 'cis' activity of SAC1, supporting its role in non-vesicular lipid traffic and implicating lipid traffic more broadly in inositol lipid homeostasis and function.

PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER-PM contact sites.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)-resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2 Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2 Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase-mediated conversion to PI(4,5)P2 Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.

TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes.

Lysosomal distribution is linked to the role of lysosomes in many cellular functions, including autophagosome degradation, cholesterol homeostasis, antigen presentation, and cell invasion. Alterations in lysosomal positioning contribute to different human pathologies, such as cancer, neurodegeneration, and lysosomal storage diseases. Here we report the identification of a novel mechanism of lysosomal trafficking regulation. We found that the lysosomal transmembrane protein TMEM55B recruits JIP4 to the lysosomal surface, inducing dynein-dependent transport of lysosomes toward the microtubules minus-end. TMEM55B overexpression causes lysosomes to collapse into the cell center, whereas depletion of either TMEM55B or JIP4 results in dispersion toward the cell periphery. TMEM55B levels are transcriptionally upregulated following TFEB and TFE3 activation by starvation or cholesterol-induced lysosomal stress. TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome-lysosome fusion. Overall our data suggest that the TFEB/TMEM55B/JIP4 pathway coordinates lysosome movement in response to a variety of stress conditions.

The DREAM complex mediates GIST cell quiescence and is a novel therapeutic target to enhance imatinib-induced apoptosis.

Gastrointestinal stromal tumors (GIST) can be successfully treated with imatinib mesylate (Gleevec); however, complete remissions are rare and patients frequently achieve disease stabilization in the presence of residual tumor masses. The clinical observation that discontinuation of treatment can lead to tumor progression suggests that residual tumor cells are, in fact, quiescent and, therefore, able to re-enter the cell-division cycle. In line with this notion, we have previously shown that imatinib induces GIST cell quiescence in vitro through the APC(CDH1)-SKP2-p27(Kip1) signaling axis. Here, we provide evidence that imatinib induces GIST cell quiescence in vivo and that this process also involves the DREAM complex, a multisubunit complex that has recently been identified as an additional key regulator of quiescence. Importantly, inhibition of DREAM complex formation by depletion of the DREAM regulatory kinase DYRK1A or its target LIN52 was found to enhance imatinib-induced cell death. Our results show that imatinib induces apoptosis in a fraction of GIST cells while, at the same time, a subset of cells undergoes quiescence involving the DREAM complex. Inhibition of this process enhances imatinib-induced apoptosis, which opens the opportunity for future therapeutic interventions to target the DREAM complex for more efficient imatinib responses.