GWAS of adventitious root formation in roses identifies a putative phosphoinositide phosphatase (SAC9) for marker-assisted selection.

Rose propagation by cuttings is limited by substantial genotypic differences in adventitious root formation. To identify possible genetic factors causing these differences and to develop a marker for marker-assisted selection for high rooting ability, we phenotyped 95 cut and 95 garden rose genotypes in a hydroponic rooting system over 6 weeks. Data on rooting percentage after 3 to 6 weeks, root number, and root fresh mass were highly variable among genotypes and used in association mappings performed on genotypic information from the WagRhSNP 68 K Axiom SNP array for roses. GWAS analyses revealed only one significantly associated SNP for rooting percentage after 3 weeks. Nevertheless, prominent genomic regions/peaks were observed and further analysed for rooting percentage after 6 weeks, root number and root fresh mass. Some of the SNPs in these peak regions were associated with large effects on adventitious root formation traits. Very prominent were ten SNPs, which were all located in a putative phosphoinositide phosphatase SAC9 on chromosome 2 and showed very high effects on rooting percentage after 6 weeks of more than 40% difference between nulliplex and quadruplex genotypes. SAC9 was reported to be involved in the regulation of endocytosis and in combination with other members of the SAC gene family to regulate the translocation of auxin-efflux PIN proteins via the dephosphorylation of phosphoinositides. For one SNP within SAC9, a KASP marker was successfully derived and used to select genotypes with a homozygous allele configuration. Phenotyping these homozygous genotypes for adventitious root formation verified the SNP allele dosage effect on rooting. Hence, the presented KASP derived from a SNP located in SAC9 can be used for marker-assisted selection in breeding programs for high rooting ability in the future.

Increased Phospholipid Flux Bypasses Overlapping Essential Requirements for the Yeast Sac1p Phosphoinositide Phosphatase and ER-PM Membrane Contact Sites.

In budding yeast cells, much of the inner surface of the plasma membrane (PM) is covered with the endoplasmic reticulum (ER). This association is mediated by seven ER membrane proteins that confer cortical ER-PM association at membrane contact sites (MCSs). Several of these membrane "tether" proteins are known to physically interact with the phosphoinositide phosphatase Sac1p. However, it is unclear how or if these interactions are necessary for their interdependent functions. We find that SAC1 inactivation in cells lacking the homologous synaptojanin-like genes INP52 and INP53 results in a significant increase in cortical ER-PM MCSs. We show in sac1Δ, sac1tsinp52Δ inp53Δ, or Δ-super-tether (Δ-s-tether) cells lacking all seven ER-PM tethering genes that phospholipid biosynthesis is disrupted and phosphoinositide distribution is altered. Furthermore, SAC1 deletion in Δ-s-tether cells results in lethality, indicating a functional overlap between SAC1 and ER-PM tethering genes. Transcriptomic profiling indicates that SAC1 inactivation in either Δ-s-tether or inp52Δ inp53Δ cells induces an ER membrane stress response and elicits phosphoinositide-dependent changes in expression of autophagy genes. In addition, by isolating high-copy suppressors that rescue sac1Δ Δ-s-tether lethality, we find that key phospholipid biosynthesis genes bypass the overlapping function of SAC1 and ER-PM tethers and that overexpression of the phosphatidylserine/phosphatidylinositol-4-phosphate transfer protein Osh6 also provides limited suppression. Combined with lipidomic analysis and determinations of intracellular phospholipid distributions, these results suggest that Sac1p and ER phospholipid flux controls lipid distribution to drive Osh6p-dependent phosphatidylserine/phosphatidylinositol-4-phosphate counter-exchange at ER-PM MCSs.

The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants.

The phosphatase FIG4 and the scaffold protein VAC14 function in the biosynthesis of PI(3,5)P2, a signaling lipid that inhibits the lysosomal chloride transporter ClC-7. Loss-of-function mutations of FIG4 and VAC14 reduce PI(3,5)P2 and result in lysosomal disorders characterized by accumulation of enlarged lysosomes and neurodegeneration. Similarly, a gain of function mutation of CLCN7 encoding ClC-7 also results in enlarged lysosomes. We therefore tested the ability of reduced CLCN7 expression to compensate for loss of FIG4 or VAC14. Knock-out of CLCN7 corrected lysosomal swelling and partially corrected lysosomal hyperacidification in FIG4 null cell cultures. Knockout of the related transporter CLCN6 (ClC-6) in FIG4 null cells did not affect the lysosome phenotype. In the Fig4 null mouse, reduction of ClC-7 by expression of the dominant negative CLCN7 variant p.Gly215Arg improved growth and neurological function and increased lifespan by 20%. These observations demonstrate a role for the CLCN7 chloride transporter in pathogenesis of FIG4 and VAC14 disorders. Reduction of CLCN7 provides a new target for treatment of FIG4 and VAC14 deficiencies that lack specific therapies, such as Charcot-Marie-Tooth Type 4J and Yunis-Varón syndrome.

Altered phenotypes due to genetic interaction between the mouse phosphoinositide biosynthesis genes Fig4 and Pip4k2c.

Loss-of-function mutations of FIG4 are responsible for neurological disorders in human and mouse that result from reduced abundance of the signaling lipid PI(3,5)P2. In contrast, loss-of-function mutations of the phosphoinositide kinase PIP4K2C result in elevated abundance of PI(3,5)P2. These opposing effects on PI(3,5)P2 suggested that we might be able to compensate for deficiency of FIG4 by reducing expression of PIP4K2C. To test this hypothesis in a whole animal model, we generated triallelic mice with genotype Fig 4-/-, Pip4k2c+/-; these mice are null for Fig 4 and haploinsufficient for Pip4k2c. The neonatal lethality of Fig 4 null mice in the C57BL/6J strain background was rescued by reduced expression of Pip4k2c. The lysosome enlargement characteristic of Fig 4 null cells was also reduced by heterozygous loss of Pip4k2c. The data demonstrate interaction between these two genes, and suggest that inhibition of the kinase PIPK4C2 could be a target for treatment of FIG4 deficiency disorders such as Charcot-Marie-Tooth Type 4J and Yunis-Varón Syndrome.

Chloroquine corrects enlarged lysosomes in FIG4 null cells and reduces neurodegeneration in Fig4 null mice.

Loss-of-function mutations of FIG4 impair the biosynthesis of PI(3,5)P2 and are responsible for rare genetic disorders including Yunis-Varón Syndrome and Charcot-Marie-Tooth Disease Type 4 J. Cultured cells deficient in FIG4 accumulate enlarged lysosomes with hyperacidic pH, due in part to impaired regulation of lysosomal ion channels and elevated intra-lysosomal osmotic pressure. We evaluated the effects of the FDA approved drug chloroquine, which is known to reduce lysosome acidity, on FIG4 deficient cell culture and on a mouse model. Chloroquine corrected the enlarged lysosomes in FIG4 null cells. In null mice, addition of chloroquine to the drinking water slowed progression of the disorder. Growth and mobility were dramatically improved during the first month of life, and spongiform degeneration of the nervous system was reduced. The median survival of Fig4 null mice was increased from 4 weeks for untreated mutants to 8 weeks with chloroquine treatment (p < 0.009). Chloroquine thus corrects the lysosomal swelling in cultured cells and ameliorates Fig4 deficiency in vivo. The improved phenotype of mice with complete loss of Fig4 suggests that chloroquine could be beneficial FIG2 in partial loss-of-function disorders such as Charcot-Marie-Tooth Type 4 J.

Ribosomal targeting strategy and nuclear labeling to analyze photoreceptor phosphoinositide signatures.

Reversible phosphorylation of phosphatidylinositol by phosphoinositide (PI) kinases and phosphatases generates seven distinct phosphoinositide phosphates, called phosphoinositides or PIPs. All seven PIPs are formed in the retina and photoreceptor cells. Around 50 genes in the mammalian genome encode PI kinases and PI phosphatases. There are no studies available on the distribution of these enzymes in the retina and photoreceptors. AIM: To employ Ribosomal Targeting Strategy and Nuclear Labeling to Analyze Phosphoinositide Signatures in rod-photoreceptor cells. METHODS: HA-tagging of ribosomal protein Rpl22 was induced with Cre-recombinase under the control of the rhodopsin promoter. Actively translating mRNAs associated with polyribosomes were isolated by immunoprecipitation with HA antibody, followed by RNA isolation and gene identification. We also isolated biotinylated-rod nuclei from NuTRAP mice under the control of the rhodopsin-Cre promoter and analyzed nuclear phosphoinositides. RESULTS: Our results indicate that the expression of class I and class III PI 3-kinase, PI4K IIIß, PI 5-kinase, PIKfyve, PI3-phosphatases, MTMR2, 4, 6, 7, 14, PI4-phosphatase, TMEM55A, PI 5-phosphatases, SYNJI, INPP5B, INPP5E, INPP5F, SKIP and other phosphatases with dual substrate specificity, PTPMT1, SCAM1, and FIG4 are highly enriched in rod photoreceptor cells compared with the retina and cone-like retina. Our analysis identified the presence of PI(4)P, PI(3,4)P2, PI(3,5)P2, and PI(4,5)P2 in the rod nuclei. CONCLUSIONS: Our studies for the first time demonstrate the expression of PI kinases, PI phosphatases, and nuclear PIPs in rod photoreceptor cells. The NuTRAP mice may be useful not only for epigenetic and transcriptomic studies but also for in vivo cell-specific lipidomics research.

SAC1 regulates autophagosomal phosphatidylinositol-4-phosphate for xenophagy-directed bacterial clearance.

Phosphoinositides are important molecules in lipid signaling, membrane identity, and trafficking that are spatiotemporally controlled by factors from both mammalian cells and intracellular pathogens. Here, using small interfering RNA (siRNA) directed against phosphoinositide kinases and phosphatases, we screen for regulators of the host innate defense response to intracellular bacterial replication. We identify SAC1, a transmembrane phosphoinositide phosphatase, as an essential regulator of xenophagy. Depletion or inactivation of SAC1 compromises fusion between Salmonella-containing autophagosomes and lysosomes, leading to increased bacterial replication. Mechanistically, the loss of SAC1 results in aberrant accumulation of phosphatidylinositol-4-phosphate [PI(4)P] on Salmonella-containing autophagosomes, thus facilitating recruitment of SteA, a PI(4)P-binding Salmonella effector protein, which impedes lysosomal fusion. Replication of Salmonella lacking SteA is suppressed by SAC-1-deficient cells, however, demonstrating bacterial adaptation to xenophagy. Our findings uncover a paradigm in which a host protein regulates the level of its substrate and impairs the function of a bacterial effector during xenophagy.

Adeno-associated virus gene therapy to the rescue for Charcot-Marie-Tooth disease type 4J.

The genetic peripheral neuropathy known as Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive mutations in the FIG4 gene. The transformational success of adeno-associated virus (AAV) gene therapy for spinal muscular atrophy has generated substantial interest in using this approach to create similar treatments for CMT. In this issue of the JCI, Presa et al. provide a preclinical demonstration of efficacy using AAV-directed gene therapy for CMT4J. The study showed a dramatic improvement in both survival and neuropathy symptoms in a severe mouse model of CMT4J after administration of AAV gene therapy at several time points. The authors' approach advances the technique for delivering treatments to individuals with CMT, for which FDA-approved therapies have not yet come to the clinic.

AAV9-mediated FIG4 delivery prolongs life span in Charcot-Marie-Tooth disease type 4J mouse model.

Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive, loss-of-function mutations in FIG4, encoding a phosphoinositol(3,5)P2-phosphatase. CMT4J patients have both neuron loss and demyelination in the peripheral nervous system, with vacuolization indicative of endosome/lysosome trafficking defects. Although the disease is highly variable, the onset is often in childhood and FIG4 mutations can dramatically shorten life span. There is currently no treatment for CMT4J. Here, we present the results of preclinical studies testing a gene-therapy approach to restoring FIG4 expression. A mouse model of CMT4J, the Fig4-pale tremor (plt) allele, was dosed with a single-stranded adeno-associated virus serotype 9 (AAV9) to deliver a codon-optimized human FIG4 sequence. Untreated, Fig4plt/plt mice have a median survival of approximately 5 weeks. When treated with the AAV9-FIG4 vector at P1 or P4, mice survived at least 1 year, with largely normal gross motor performance and little sign of neuropathy by neurophysiological or histopathological evaluation. When mice were treated at P7 or P11, life span was still significantly prolonged and peripheral nerve function was improved, but rescue was less complete. No unanticipated adverse effects were observed. Therefore, AAV9-mediated delivery of FIG4 is a well-tolerated and efficacious strategy in a mouse model of CMT4J.

NCP2/RHD4/SAC7, SAC6 and SAC8 phosphoinositide phosphatases are required for PtdIns4P and PtdIns(4,5)P2 homeostasis and Arabidopsis development.

Phosphoinositides play important roles in plant growth and development. Several SAC domain phosphoinositide phosphatases have been reported to be important for plant development. Here, we show functional analysis of SUPPRESSOR OF ACTIN 6 (SAC6) to SAC8 in Arabidopsis, a subfamily of phosphoinositide phosphatases containing SAC-domain and two transmembrane motifs. We isolated an Arabidopsis mutant ncp2 that lacked cotyledons in seedling and embryo in pid, a background defective in auxin signaling and transport. NCP2 encodes RHD4/SAC7 phosphoinositide phosphatase. SAC6, SAC7 and SAC8 exhibit overlapping and specific expression patterns in seedling and embryo. The sac6 sac7 embryos either fail to develop into seeds, or have three or four cotyledons. The embryo development of sac7 sac8 and sac6 sac7 sac8 mutants is significantly delayed or lethal, and the seedlings are arrested at early stages. Auxin maxima are decreased in double and triple sac mutants. The contents of PtdIns4P and PtdIns(4,5)P2 in sac6 sac7 and sac7 sac8 mutants are dramatically increased. Protein trafficking of the plasma membrane (PM)-localized protein PIN1 and PIN2 from trans-Golgi network/early endosome back to PM is delayed in sac7 sac8 mutants. These results indicate that SAC6-SAC8 are essential for maintaining homeostasis of PtdIns4P and PtdIns(4,5)P2, and auxin-mediated development in Arabidopsis.

Regulation of lysosomal positioning via TMEM55B phosphorylation.

Lysosomes are dynamic organelles that are transported along microtubules bidirectionally via kinesin and dynein motor proteins. Lysosomal positioning, which is determined by the balance of the bidirectional lysosomal movement, changes under various conditions and affects lysosomal functions such as autophagy and antigen presentation. A recent study by Takemasu et al. (Phosphorylation of TMEM55B by Erk/MAPK regulates lysosomal positioning. J. Biochem. 2019; 166:175-185) has shown that phosphorylation of the transmembrane protein TMEM55B is involved in the retrograde lysosomal trafficking towards the perinuclear region. They found that TMEM55B is phosphorylated upon stimulation with various ligands and that Erk/MAPK mediates the TMEM55B phosphorylation. They have also revealed that a phosphorylation mimic mutant of TMEM55B enhances perinuclear lysosomal clustering compared to the wild-type TMEM55B. These findings suggest that TMEM55B phosphorylation by Erk/MAPK is responsible for regulating lysosomal positioning in response to external stimuli.

Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis.

Cell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non-plasma membrane PI(4,5)P2 , and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P2 phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over-activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P2 phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable.

Roles for a lipid phosphatase in the activation of its opposing lipid kinase.

Fig4 is a phosphoinositide phosphatase that converts PI3,5P2 to PI3P. Paradoxically, mutation of Fig4 results in lower PI3,5P2, indicating that Fig4 is also required for PI3,5P2 production. Fig4 promotes elevation of PI3,5P2, in part, through stabilization of a protein complex that includes its opposing lipid kinase, Fab1, and the scaffold protein Vac14. Here we show that multiple regions of Fig4 contribute to its roles in the elevation of PI3,5P2: its catalytic site, an N-terminal disease-related surface, and a C-terminal region. We show that mutation of the Fig4 catalytic site enhances the formation of the Fab1-Vac14-Fig4 complex, and reduces the ability to elevate PI3,5P2. This suggests that independent of its lipid phosphatase function, the active site plays a role in the Fab1-Vac14-Fig4 complex. We also show that the N-terminal disease-related surface contributes to the elevation of PI3,5P2 and promotes Fig4 association with Vac14 in a manner that requires the Fig4 C-terminus. We find that the Fig4 C-terminus alone interacts with Vac14 in vivo and retains some functions of full-length Fig4. Thus, a subset of Fig4 functions are independent of its phosphatase domain and at least three regions of Fig4 play roles in the function of the Fab1-Vac14-Fig4 complex.

Cellular homeostasis in the Drosophila retina requires the lipid phosphatase Sac1.

The complex functions of cellular membranes, and thus overall cell physiology, depend on the distribution of crucial lipid species. Sac1 is an essential, conserved, ER-localized phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates secretory trafficking and plasma membrane function. PI4P from multiple pools is delivered to Sac1 by oxysterol-binding protein and related proteins in exchange for other lipids and sterols, which places Sac1 at the intersection of multiple lipid distribution pathways. However, much remains unknown about the roles of Sac1 in subcellular homeostasis and organismal development. Using a temperature-sensitive allele (Sac1ts), we show that Sac1 is required for structural integrity of the Drosophila retinal floor. The ßps-integrin Myospheroid, which is necessary for basal cell adhesion, is mislocalized in Sac1ts retinas. In addition, the adhesion proteins Roughest and Kirre, which coordinate apical retinal cell patterning at an earlier stage, accumulate within Sac1ts retinal cells due to impaired endo-lysosomal degradation. Moreover, Sac1 is required for ER homeostasis in Drosophila retinal cells. Together, our data illustrate the importance of Sac1 in regulating multiple aspects of cellular homeostasis during tissue development.

Phosphatidylinositol-(4,5)-Bisphosphate Regulates Plasma Cholesterol Through LDL (Low-Density Lipoprotein) Receptor Lysosomal Degradation.

OBJECTIVE: TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of ApoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr-/- mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 (human hepatoma) cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, the TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.

The nuclear phosphoinositide response to stress.

Accumulating evidence reveals that nuclear phosphoinositides (PIs) serve as central signaling hubs that control a multitude of nuclear processes by regulating the activity of nuclear proteins. In response to cellular stressors, PIs accumulate in the nucleus and multiple PI isomers are synthesized by the actions of PI-metabolizing enzymes, kinases, phosphatases and phospholipases. By directly interacting with effector proteins, phosphoinositide signals transduce changes in cellular functions. Here we describe nuclear phosphoinositide signaling in multiple sub-nuclear compartments and summarize the literature that demonstrates roles for specific kinases, phosphatases, and phospholipases in the orchestration of nuclear phosphoinositide signaling in response to cellular stress. Additionally, we discuss the specific PI-protein complexes through which these lipids execute their functions by regulating the configuration, stability, and transcription activity of their effector proteins. Overall, our review provides a detailed landscape of the current understanding of the nuclear PI-protein interactome and its role in shaping the coordinated response to cellular stress.

A SAC Phosphoinositide Phosphatase Controls Rice Development via Hydrolyzing PI4P and PI(4,5)P2.

Phosphoinositides (PIs) as regulatory membrane lipids play essential roles in multiple cellular processes. Although the exact molecular targets of PI-dependent modulation remain largely elusive, the effects of disturbed PI metabolism could be employed to identify regulatory modules associated with particular downstream targets of PIs. Here, we identified the role of GRAIN NUMBER AND PLANT HEIGHT1 (GH1), which encodes a suppressor of actin (SAC) domain-containing phosphatase with unknown function in rice (Oryza sativa). Endoplasmic reticulum-localized GH1 specifically dephosphorylated and hydrolyzed phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Inactivation of GH1 resulted in massive accumulation of both PI4P and PI(4,5)P2, while excessive GH1 caused their depletion. Notably, superabundant PI4P and PI(4,5)P2 could both disrupt actin cytoskeleton organization and suppress cell elongation. Interestingly, both PI4P and PI(4,5)P2 inhibited actin-related protein2 and -3 (Arp2/3) complex-nucleated actin-branching networks in vitro, whereas PI(4,5)P2 showed more dramatic effects in a dose-dependent manner. Overall, the overaccumulation of PI(4,5)P2 resulting from dysfunction of SAC phosphatase possibly perturbs Arp2/3 complex-mediated actin polymerization, thereby disordering cell development. These findings imply that the Arp2/3 complex might be the potential molecular target of PI(4,5)P2-dependent modulation in eukaryotes, thereby providing insights into the relationship between PI homeostasis and plant growth and development.

Phosphorylation of TMEM55B by Erk/MAPK regulates lysosomal positioning.

TMEM55B is first identified as phosphatidylinositol-4,5-P24-phosphatases (PtdIns-4,5-P24-phosphatases) that catalyse dephosphorylation of PtdIns-4,5-P2 to PtdIns-5-P. We demonstrate for the first time that TMEM55B is phosphorylated by Erk/MAPK and that this mechanism participates in regulation of lysosomal clustering. Exposure of RAW264.7 macrophages to various stimuli induces phosphorylation of TMEM55B on Ser76 and Ser169, sites corresponding to consensus sequences (PX(S/T)P) for phosphorylation by MAPK. Of these stimuli, Toll-like receptor ligands most strongly induce TMEM55B phosphorylation, and this is blocked by the MEK1/2 inhibitor U0126. However, phosphorylation does not impact intrinsic phosphatase activity of TMEM55B. TMEM55B has recently been implicated in starvation induced lysosomal translocation. Amino acid starvation induces perinuclear lamp1 clustering in RAW264.7 macrophages, which was attenuated by shRNA-mediated knock-down or CRISPR/Cas9-mediated knock-out of TMEM55B. Cells exposed to U0126 also exhibit attenuated lamp1 clustering. Overexpression of TMEM55B but not TMEM55A notably enhances lamp1 clustering, with TMEM55B mutants (lacking phosphorylation sites or mimicking the phosphorylated state) exhibiting lower and higher efficacies (respectively) than wild-type TMEM55B. Collectively, results suggest that phosphorylation of TMEM55B by Erk/MAPK impacts lysosomal dynamics.

Investigation of candidate genes of non-syndromic cleft lip with or without cleft palate, using both case-control and family-based association studies.

OBJECTIVE: Non-syndromic cleft of the lip and/or palate (NSCL/P) is one of the most common polygenic diseases. In this study, both case-control and family-based association study were used to confirm whether the Single Nucleotide Polymorphisms (SNPs) were associated with NSCL/P. METHODS: A total of 37 nuclear families and 189 controls were recruited, whose blood DNA was extracted and subjected to genotyping of SNPs of 27 candidate genes by polymerase chain reaction-improved multiple ligase detection reaction technology (PCR-iMLDR). Case-control statistical analysis was performed using the SPSS 19.0. Haplotype Relative Risk (HRR), transmission disequilibrium test (TDT), and Family-Based Association Test (FBAT) were used to test for over-transmission of the target alleles in case-parent trios. The gene-gene interactions on NSCL/P were analyzed by Unphased-3.1.4. RESULTS: In case-control statistical analysis, only C14orf49 chr14_95932477 had statistically significant on genotype model (P = .03) and allele model (P = .03). Seven SNPs had statistically significant on TDT. None of 26 alleles has association with NSCL/P on FBAT. Some SNPs had haplotype-haplotype interactions and genotype-genotype interactions. CONCLUSION: C14orf49 chr14_95932477 was significantly different between cases and controls on genotype model and allele model by case-control design. Seven SNPs were significantly different on HRR. Four SNPs were significantly different on TDT.

CRISPR knockout screen implicates three genes in lysosome function.

Defective biosynthesis of the phospholipid PI(3,5)P2 underlies neurological disorders characterized by cytoplasmic accumulation of large lysosome-derived vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. We first demonstrated that the enlarged vacuoles that accumulate in fibroblasts lacking FIG4, a PI(3,5)P2 biosynthetic factor, have a hyperacidic pH compared to normal cells'. We then carried out a genome-wide knockout screen in human HAP1 cells for accumulation of acidic vesicles by FACS sorting. A pilot screen captured fifteen genes, including VAC14, a previously identified cause of endolysosomal vacuolization. Three genes not previously associated with lysosome dysfunction were selected to validate the screen: C10orf35, LRRC8A, and MARCH7. We analyzed two clonal knockout cell lines for each gene. All of the knockout lines contained enlarged acidic vesicles that were positive for LAMP2, confirming their endolysosomal origin. This assay will be useful in the future for functional evaluation of patient variants in these genes, and for a more extensive genome-wide screen for genes required for endolysosome function. This approach may also be adapted for drug screens to identify small molecules that rescue endolysosomal vacuolization.