Tubby Protein Research Articles

Arabidopsis Tubby domain-containing F-box proteins positively regulate immunity by modulating PI4Kβ protein levels.

The Tubby domain, named after the TUBBY protein in mice, binds to phosphatidylinositol 4,5-bisphosphate. Arabidopsis has 11 Tubby domain-containing proteins referred to as Tubby-Like Proteins (TLPs). Of the 11 TLPs, 10 possess the N-terminal F-box domain, which can interact with SKP-like proteins and form SKP1-Cullin-F-box E3 ligase complexes. Although mice TUBBY has been extensively studied, plant TLPs' functions are scarcely detailed. In this study, we show that the Arabidopsis Tubby-like protein 6 (TLP6) and its redundant homologs, TLP1, TLP2, TLP5, and TLP10, positively regulate Arabidopsis immune responses. Furthermore, in an immunoprecipitation mass spectrometry analysis to search for ubiquitination substrates of the TLPs, we identified two redundant phosphoinositide biosynthesis enzymes, phosphatidylinositol 4-kinase β proteins (PI4Kβs), PI4Kβ1 and PI4Kβ2, as TLP interactors. Importantly, TLP6 overexpression lines fully phenocopy the phenotypes of the pi4kβ1,2 mutant, while TLP6 overexpression also leads to increased PI4Kβ2 ubiquitination and reduction in its protein level in a proteasome-dependent manner. Most significantly, TLP6 overexpression does not further enhance the autoimmunity of the pi4kβ1,2 double mutant, supporting the hypothesis that TLP6 targets the PI4Kβs for ubiquitination and degradation. Thus, our study reveals a novel mechanism where TLPs promote plant immune responses by modulating the PI4Kβs protein levels.

Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus.

Background: Acute exercise contributes to decreased feeding through leptin and interleukin/Janus kinase 2/signal transducers and activators of transcription 3 (IL-6/JAK2/STAT3) signaling. Considering the pleiotropic use of substrates by JAK2 and that JAK2 can phosphorylate the Tubby protein (TUB) in CHO-IR cells, we speculated that acute exercise can activate the IL-6/JAK2/TUB pathway to decrease food intake. Aims: We investigated whether acute exercise induced tyrosine phosphorylation and the association of TUB and JAK2 in the hypothalamus and if IL-6 is involved in this response, whether acute exercise increases the IL-6/TUB axis to regulate feeding, and if leptin has an additive effect over this mechanism. Methods: We applied a combination of genetic, pharmacological, and molecular approaches. Key findings: The in vivo experiments showed that acute exercise increased the tyrosine phosphorylation and association of JAK2/TUB in the hypothalamus, which reduced feeding. This response was dependent on IL-6. Leptin had no additive effect on this mechanism. Significance: The results of this study suggest a novel hypothalamic pathway by which IL-6 released by exercise regulates feeding and reinforces the beneficial effects of exercise.

TULP2 deletion mice exhibit abnormal outer dense fiber structure and male infertility.

Purpose Tulp2 (tubby‐like protein 2) is a member of the tubby protein family and expressed predominantly in mouse testis. Recently, it was reported that Tulp2 knockout (KO) mice exhibited disrupted sperm tail morphology; however, it remains to be determined how TULP2 deletion causes abnormal tail formation.MethodsThe authors analyzed male fertility, sperm morphology, and motility of two Tulp2 KO mouse lines that were generated using the conventional method that utilizes homologous recombination in embryonic stem (ES) cells as well as the clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) system. Furthermore, the authors observed the spermatogenesis of Tulp2 KO mice in more detail using scanning and transmission electron microscopy (SEM and TEM).ResultsBoth mouse lines of Tulp2 KO exhibited male infertility, abnormal tail morphology, and impaired sperm motility. No overt abnormalities were found in the formation of the mitochondrial sheath in Tulp2 KO mice using the freeze‐fracture method with SEM. In contrast, abnormal outer dense fiber (ODF) structure was observed in Tulp2 KO testis with TEM.ConclusionsTULP2 may play roles in the correct formation and/or maintenance of ODF, which may lead to abnormal tail morphology, impaired sperm motility, and male infertility.

Coriander Genomics Database: a genomic, transcriptomic, and metabolic database for coriander

Coriander (Coriandrum sativum L.), also known as cilantro, is a globally important vegetable and spice crop. Its genome and that of carrot are models for studying the evolution of the Apiaceae family. Here, we developed the Coriander Genomics Database (CGDB, http://cgdb.bio2db.com/) to collect, store, and integrate the genomic, transcriptomic, metabolic, functional annotation, and repeat sequence data of coriander and carrot to serve as a central online platform for Apiaceae and other related plants. Using these data sets in the CGDB, we intriguingly found that seven transcription factor (TF) families showed significantly greater numbers of members in the coriander genome than in the carrot genome. The highest ratio of the numbers of MADS TFs between coriander and carrot reached 3.15, followed by those for tubby protein (TUB) and heat shock factors. As a demonstration of CGDB applications, we identified 17 TUB family genes and conducted systematic comparative and evolutionary analyses. RNA-seq data deposited in the CGDB also suggest dose compensation effects of gene expression in coriander. CGDB allows bulk downloading, significance searches, genome browser analyses, and BLAST searches for comparisons between coriander and other plants regarding genomics, gene families, gene collinearity, gene expression, and the metabolome. A detailed user manual and contact information are also available to provide support to the scientific research community and address scientific questions. CGDB will be continuously updated, and new data will be integrated for comparative and functional genomic analysis in Apiaceae and other related plants.

The Caenorhabditis elegans Tubby homolog dynamically modulates olfactory cilia membrane morphogenesis and phospholipid composition.

Plasticity in sensory signaling is partly mediated via regulated trafficking of signaling molecules to and from primary cilia. Tubby-related proteins regulate ciliary protein transport; however, their roles in remodeling cilia properties are not fully understood. We find that the C. elegans TUB-1 Tubby homolog regulates membrane morphogenesis and signaling protein transport in specialized sensory cilia. In particular, TUB-1 is essential for sensory signaling-dependent reshaping of olfactory cilia morphology. We show that compromised sensory signaling alters cilia membrane phosphoinositide composition via TUB-1-dependent trafficking of a PIP5 kinase. TUB-1 regulates localization of this lipid kinase at the cilia base in part via localization of the AP-2 adaptor complex subunit DPY-23. Our results describe new functions for Tubby proteins in the dynamic regulation of cilia membrane lipid composition, morphology, and signaling protein content, and suggest that this conserved family of proteins plays a critical role in mediating cilia structural and functional plasticity.

The N-terminal homology (ENTH) domain of Epsin 1 is a sensitive reporter of physiological PI(4,5)P2 dynamics

Phospholipase Cβ (PLCβ)-induced depletion of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) transduces a plethora of signals into cellular responses. Importance and diversity of PI(4,5)P2-dependent processes led to strong need for biosensors of physiological PI(4,5)P2 dynamics applicable in live-cell experiments. Membrane PI(4,5)P2 can be monitored with fluorescently-labelled phosphoinositide (PI) binding domains that associate to the membrane depending on PI(4,5)P2 levels. The pleckstrin homology domain of PLCδ1 (PLCδ1-PH) and the C-terminus of tubby protein (tubbyCT) are two such sensors widely used to study PI(4,5)P2 signaling. However, certain limitations apply to both: PLCδ1-PH binds cytoplasmic inositol-1,4,5-trisphosphate (IP3) produced from PI(4,5)P2 through PLCβ, and tubbyCT responses do not faithfully report on PLCβ-dependent PI(4,5)P2 dynamics. In searching for an improved biosensor, we fused N-terminal homology domain of Epsin1 (ENTH) to GFP and examined use of this construct as genetically-encoded biosensor for PI(4,5)P2 dynamics in living cells. We utilized recombinant tools to manipulate PI or Gq protein-coupled receptors (GqPCR) to stimulate PLCβ signaling and characterized PI binding properties of ENTH-GFP with total internal reflection (TIRF) and confocal microscopy. ENTH-GFP specifically recognized membrane PI(4,5)P2 without interacting with IP3, as demonstrated by dialysis of cells with the messenger through a patch pipette. Utilizing Ci-VSP to titrate PI(4,5)P2 levels, we found that ENTH-GFP had low PI(4,5)P2 affinity. Accordingly, ENTH-GFP was highly sensitive to PLCβ-dependent PI(4,5)P2 depletion, and in contrast to PLCδ1-PH, overexpression of ENTH-GFP did not attenuate GqPCR signaling. Taken together, ENTH-GFP detects minute changes of PI(4,5)P2 levels and provides an important complementation of experimentally useful reporters of PI(4,5)P2 dynamics in physiological pathways.

Tubby proteins prove their adaptability

Study reveals that Tubby family proteins help deliver GPCRs and other integral membrane proteins into cilia.

C-terminally mutated tubby protein accumulates in aggresomes.

The tubby protein (Tub), a putative transcription factor, plays important roles in the maintenance and function of neuronal cells. A splicing defect-causing mutation in the 3′-end of the tubby gene, which is predicted to disrupt the carboxy-terminal region of the Tub protein, causes maturity-onset obesity, blindness, and deafness in mice. Although this pathological Tub mutation leads to a loss of function, the precise mechanism has not yet been investigated. Here, we found that the mutant Tub proteins were mostly localized to puncta found in the perinuclear region and that the C-terminus was important for its solubility. Immunocytochemical analysis revealed that puncta of mutant Tub co-localized with the aggresome. Moreover, whereas wild-type Tub was translocated to the nucleus by extracellular signaling, the mutant forms failed to undergo such translocation. Taken together, our results suggest that the malfunctions of the Tub mutant are caused by its misfolding and subsequent localization to aggresomes.

Interactions between wheat Tubby-like and SKP1-like proteins.

Tubby proteins are highly conserved in a wide range of multicellular organisms. The Tubby gene family was first verified in obese mice. In plants, 11 Tubby genes have been identified in Arabidopsis, 14 in rice, and 11 in poplar. However, there is very little information about Tubby-like proteins in wheat. In this study, we identified four Tubby-like protein genes (TaTULP1-TaTULP4) in wheat. A comparison of the gene structure showed a conserved exon number pattern in TaTULPs, although the length of the introns differed. With the exception of TaTULP2, TaTULPs had four exons. To identify the chromosome localization of TaTULPs, BLASTn analyses were performed using the URGI database to predict the chromosomal location of TaTULP genes. TaTULP1, 2, 3 and 4 genes were localized on chromosomes 4, 5, 7 and 2. All TaTULPs harbor a Tubby domain in their C-terminal region and an F-box domain in the N terminus. We investigated protein-protein interactions between the F-box domain of TaTULPs and various wheat SKP1-like proteins (TaSKPs) using the yeast two-hybrid system. TaTULP1, TaTULP3 and TaTULP4 were found to interact with TaSKP1, TaSKP3 and TaSKP6, whereas TaTULP2 showed no interaction with TaSKP proteins. TaTULP proteins tagged with green fluorescent protein were targeted to the Golgi apparatus in plant cells. Our analysis of TaTULPs will aid in understanding the functions of TaTULPs in plants.

In vivo tracking of phosphoinositides in Drosophila photoreceptors.

ABSTRACTIn order to monitor phosphoinositide turnover during phospholipase C (PLC)-mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, TbR332H (a mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], and the P4M domain from Legionella SidM for phosphatidylinositol 4-phosphate (PtdIns4P). Using accurately calibrated illumination, we found that only ∼50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by ∼100-fold dimmer intensities in mutants lacking Ca2+-permeable transient receptor potential (TRP) channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t½ ∼12 s) was faster than PtdIns(4,5)P2 (t½ ∼40 s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca2+- and PKC-dependent inhibition of PLC is required for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest that phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle.

Diverse Cell Type-Specific Mechanisms Localize G Protein-Coupled Receptors to Caenorhabditis elegans Sensory Cilia

The localization of signaling molecules such as G protein-coupled receptors (GPCRs) to primary cilia is essential for correct signal transduction. Detailed studies over the past decade have begun to elucidate the diverse sequences and trafficking mechanisms that sort and transport GPCRs to the ciliary compartment. However, a systematic analysis of the pathways required for ciliary targeting of multiple GPCRs in different cell types in vivo has not been reported. Here we describe the sequences and proteins required to localize GPCRs to the cilia of the AWB and ASK sensory neuron types in Caenorhabditis elegans. We find that GPCRs expressed in AWB or ASK utilize conserved and novel sequences for ciliary localization, and that the requirement for a ciliary targeting sequence in a given GPCR is different in different neuron types. Consistent with the presence of multiple ciliary targeting sequences, we identify diverse proteins required for ciliary localization of individual GPCRs in AWB and ASK. In particular, we show that the TUB-1 Tubby protein is required for ciliary localization of a subset of GPCRs, implying that defects in GPCR localization may be causal to the metabolic phenotypes of tub-1 mutants. Together, our results describe a remarkable complexity of mechanisms that act in a protein- and cell-specific manner to localize GPCRs to cilia, and suggest that this diversity allows for precise regulation of GPCR-mediated signaling as a function of external and internal context.

Insulin Phosphorylates Tyrosine Residue 464 of Tub and Translocates Tubby into the Nucleus in HIRcB Cells.

BackgroundThe tubby protein has a motif that might be relevant for its action in the insulin signaling pathway. Previous studies have indicated that tubby undergoes phosphorylation on tyrosine residues in response to several stimuli and is known to localize in the nucleus as well as in the plasma membrane. However, the relationship between phosphorylation and nuclear translocation is not well understood. Here, we report that insulin directly phosphorylates tubby, which translocates into the nucleus.MethodsThe effects of insulin on Tubby were performed with Western blot. The immunoprecipitation and confocal microscopy were performed to prove phosphorylation and nuclear translocation.ResultsMutation study reveals that tyrosine residue 464 of tubby gene (Tub) is a phosphorylation site activated by insulin. In addition, major portions of tubby protein in the plasma membrane are translocated into the nucleus after insulin treatment. Tyrosine kinase inhibitor pretreatment blocked insulin-induced tubby translocation, suggesting that phosphorylation is important for nuclear translocation. Moreover, mutant tyrosine residue 464 did not translocate into the nucleus in respond to insulin. These findings demonstrate that insulin phosphorylates tyrosine residue 464 of Tub, and this event is important for insulin-induced tubby nuclear translocation.ConclusionInsulin phosphorylates tyrosine residue 464 of Tub and translocates tubby into the nuclei of HIRcB cells.

Detailed Analysis of Phospholipase C-β Activity in Living Cells

Phospholipase C-β (PLC-β) catalyzes the hydrolysis of the plasma membrane (PM) lipid phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), thereby reducing the PM concentration of its precursor PI(4)P. A recent report proposed independent PI(4,5)P2 and PI(4)P PM pools with essential physiological functions, but it remains unknown whether PLC-β targets to these pools differentially.Here, we investigated the time course and specificity of PLC-β-dependent phospholipid depletion in living CHO cells using genetically-encoded phospholipid sensors, total internal reflection fluorescence microscopy and whole cell patch clamp. PLC-β mediated PI(4,5)P2 hydrolysis was detected reliably using KCNQ (Kv7)-mediated K+ currents, the PI(4,5)P2 reporters PLCδ1-PH and Epsin ENTH, but not with the C-terminus of Tubby protein. Analogous experiments showed that activation of PLC-β did not reduce the concentration of PI(4) detected with the PI(4)P-specific reporter Osbp-PH. When PLC-β3 was heterologously overexpressed, Tubby-Cterm and Osbp-PH reported on the PLC-β-induced changes of PI(4,5)P2 and PI(4)P, respectively.In summary, we present detailed real-time analysis of PLC-β activity in living cells. Our findings indicate that phospholipid sensors may detect different phospholipid pools that are accessible to PLC-β differentially. Hence, this work supports the presence of functional phospholipid pools in living cells.This work was supported by Deutsche Forschungsgemeinschaft through SFB 593 (TP A12) to D.O.

Association of high T-cell immune infiltrate and low hemorrhage in melanoma brain metastases (MBMs) with prolonged survival.

8528 Background: Despite the poor prognosis of patients (pts) with MBM several pts have prolonged survival. We hypothesized that the heterogeneity of BrMM is determined by differences in melanoma biology and its brain microenvironment. Methods: We identified pts who have undergone craniotomy for MBMs. Evaluation entailed complete clinical information, acquisition of archived melanoma brain metastases, histopathologic analysis of hematoxylin and eosin-stained sections (n=101), whole genome expression profiling (WGEP, Illumina DASL) in 29 archived tissues. Results were validated by immunohistochemistry (IHC) or in situ hybridization (ISH). Results: In univariate analysis (log-rank) factors significantly associated with prolonged survival were high immune infiltrate (HII) plus low hemorrhage (hazard ratio, HR, 2.71, p<0.001), present melanin (1.67, p=0.03), and recursive partitioning analysis (RPA) class 1 (0.37, p<0.0001). No association between HII and use of immunotherapy prior to craniotomy was noted. Only RPA class 1 (p=0.029) and HII plus low hemorrhage (p=0.002) remained significant in Cox proportional hazards model analysis. Gene set analysis of WGEP data confirmed that Encarta pathways related with T-cell activation and differentiation were significantly associated with prolonged survival whereas Y branching of actin filaments, presenilin action in Notch and Wnt signaling, G-protein signaling through tubby protein, lissencephaly gene in neuronal migration and development are among the gene categories associated with worse survival. IHC and ISH analysis of tumor sections for various markers (n=40) showed that high peritumoral CD3+ (3.31, p=0.009), high peritumoral CD4+ (4.41, p=0.014), and high peritumoral CD8+ (3.02, p=0.030) are associated with prolonged survival whereas neither CD14+ nor FoxP3+ infiltrate, nor high melanoma expression of antigen presentation molecules are associated with survival. Conclusions: Our study is the first to document that high peritumoral T-cell infiltrates are associated with prolonged survival. High tumor hemorrhage, an adverse prognostic sign, reflects aggressive melanoma cells that migrate and invade in the brain.

Localization and Dynamics of Phosphatidylinositol 4,5-Bisphosphate (PIP2) in Adult Skeletal Muscle Fibers

PIP2 is a precursor of important second messengers, and by itself is a direct modulator of the activity of ion channels and transporters. We investigated the localization and dynamic changes in PIP2 levels in live adult muscle fibers by expressing two PIP2 sensors: an EGFP construct of the pleckstrin homology (PH) domain of the phospholipase delta1 subunit (PH-EGFP), and a construct of the PIP2 binding domain of the tubby protein (EGFP-tubby). EGFP-tubby has higher affinity and specificity for PIP2 than PH-EGFP, and has been used to sequester PIP2 in the plasma membrane. Their respective plasmids (∗) were transfected by in vivo electroporation of FDB muscles. Two-photon laser scanning microscopy (TPLSM) shows that both EGFP-tubby and PH-EGFP are efficiently expressed in muscle fibers and that they are distributed in a double-banded pattern indicating localization at the transverse tubular system (TTS) membranes (in addition to the sarcolemma). Peak/baseline ratio analysis of TPLSM images suggests that there is a larger proportion of EGFP-tubby than PH-EGFP associated with the TTS membranes. Although the expression of PH-EGFP does not change neither the voltage-dependence nor the amplitude of Ca2+ release signals detected with Rhod-5N, the expression of EGFP-tubby apparently left-shifts their voltage-dependence by >10 mV. To further investigate the localization and dynamics of PIP2 in the TTS, we performed fluorescence resonance energy transfer (FRET) studies. The voltage-dependence of FRET signals, based on the translocation of the lipophilic anion dipicrylamine (DPA), shows that the EGFP tags of both PH-EGFP and EGFP-tubby are anchored within ∼6-9 nm of the TTS membrane. (∗)The plasmids were kindly provided to us by Dr. Tamas Balla, NICHD, NIH. This work was supported by NIH grants AR047664, AR041802, and AR054816.

Microarray Analysis of Differentially Expressed Genes in the Brains of Tubby Mice

The tubby mouse is characterized by progressive retinal and cochlear degeneration and late-onset obesity. These phenotypes are caused by a loss-of-function mutation in the tub gene and are shared with several human syndromes, suggesting the importance of tubby protein in central nervous system (CNS) functioning. Although evidence suggests that tubby may act as a transcription factor mediating G-protein coupled receptor (GPCR) signaling, any downstream gene regulated by tubby has yet to be identified. To explore potential target genes of tubby with region-specific transcription patterns in the brain, we performed a microarray analysis using the cerebral cortex and hypothalamus of tubby mice. We also validated the changes of gene expression level observed with the microarray analysis using real-time RT-PCR. We found that expression of erythroid differentiation factor 1 (Erdr1) and caspase 1 (Casp1) increased, while p21-activated kinase 1 (Pak1) and cholecystokinin 2 receptor (Cck2r) expression decreased in the cerebral cortex of tubby mice. In the hypothalamic region, Casp 1 was up-regulated and micro-crystallin (CRYM) was down-regulated. Based on the reported functions of the differentially expressed genes, these individual or grouped genes may account for the phenotype of tubby mice. We discussed how altered expression of genes in tubby mice might be understood as the underlying mechanism behind tubby phenotypes.

Temporal profiling of changes in phosphatidylinositol 4,5‐bisphosphate, inositol 1,4,5‐trisphosphate and diacylglycerol allows comprehensive analysis of phospholipase C‐initiated signalling in single neurons1

Phosphatidylinositol 4,5-bisphosphate (PIP2) fulfils vital signalling roles in an array of cellular processes, yet until recently it has not been possible selectively to visualize real-time changes in PIP2 levels within living cells. Green fluorescent protein (GFP)-labelled Tubby protein (GFP-Tubby) enriches to the plasma membrane at rest and translocates to the cytosol following activation of endogenous Gαq/11-coupled muscarinic acetylcholine receptors in both SH-SY5Y human neuroblastoma cells and primary rat hippocampal neurons. GFP-Tubby translocation is independent of changes in cytosolic inositol 1,4,5-trisphosphate and instead reports dynamic changes in levels of plasma membrane PIP2. In contrast, enhanced GFP (eGFP)-tagged pleckstrin homology domain of phospholipase C (PLCδ1) (eGFP-PH) translocation reports increases in cytosolic inositol 1,4,5-trisphosphate. Comparison of GFP-Tubby, eGFP-PH and the eGFP-tagged C12 domain of protein kinase C-γ [eGFP-C1(2); to detect diacylglycerol] allowed a selective and comprehensive analysis of PLC-initiated signalling in living cells. Manipulating intracellular Ca2+ concentrations in the nanomolar range established that GFP-Tubby responses to a muscarinic agonist were sensitive to intracellular Ca2+ up to 100–200 nM in SH-SY5Y cells, demonstrating the exquisite sensitivity of agonist-mediated PLC activity within the range of physiological resting Ca2+ concentrations. We have also exploited GFP-Tubby selectively to visualize, for the first time, real-time changes in PIP2 in hippocampal neurons.

Tubby protein in human lymphocytes from normal weight and obese subjects

Objectives: The presence of tubby protein in human lymphocytes was investigated and their electrophoretic mobility property between normal weight and obese subjects was compared. Design and methods: 2-DE proteome map of lymphocytes has been generated and western blot analysis was conducted using anti-tub polyclonal antibody. Results and conclusions: We found the presence of tubby protein both in normal weight and in obese subjects; however in the latter an isoelectric point shift toward the acidic end was observed.

PtdIns(4,5)P2 Functions at the Cleavage Furrow during Cytokinesis

Phosphoinositides play important roles in regulating the cytoskeleton and vesicle trafficking, potentially important processes at the cleavage furrow. However, it remains unclear which, if any, of the phosphoinositides play a role during cytokinesis. A systematic analysis to determine if any of the phosphoinositides might be present or of functional importance at the cleavage furrow has not been published. Several studies hint at a possible role for one or more phosphoinositides at the cleavage furrow. The best of these are genetic data identifying mutations in phosphoinositide-modifying enzymes (a PtdIns(4)P-5-kinase in S. pombe and a PI-4-kinase in D. melanogaster) that interfere with cytokinesis. The genetic nature of these experiments leaves questions as to how direct may be their contribution to cytokinesis. Here we show that a single phosphoinositide, PtdIns(4,5)P2, specifically accumulates at the furrow. Interference with PtdIns(4,5)P2 interferes with adhesion of the plasma membrane to the contractile ring at the furrow. Finally, four distinct interventions to specifically interfere with PtdIns(4,5)P2 each impair cytokinesis. We conclude that PtdIns(4,5)P2 is present at the cleavage furrow and is required for normal cytokinesis at least in part because of a role in adhesion between the contractile ring and the plasma membrane.

Down-regulated expression of agouti-related protein (AGRP) mRNA in the hypothalamic arcuate nucleus of hyperphagic and obese tub/tub mice

A mutation in the mouse tub gene causes a phenotype characterized by maturity-onset obesity, blindness and deafness. The role of the intact tubby protein and the pathogenesis resulting in the phenotype of tub/ tub mice remain largely unknown. In this study, we have investigated whether obese tub/ tub mice exhibit altered expression levels for agouti-related protein (AGRP) or glutamic acid decarboxylase-65 (GAD65) in body weight-regulating neurons of the hypothalamic arcuate nucleus. In situ hybridization revealed that AGRP, but not GAD65 mRNA levels, were significantly lower in obese tub/ tub mice as compared to tub/+ mice. The lower levels of AGRP mRNA in the arcuate nucleus of tub/ tub mice were paralleled by lower fluorescence intensity and numbers of AGRP- and neuropeptide Y (NPY)-immunoreactive (ir) nerve fibers and terminals in the arcuate, ventromedial, dorsomedial hypothalamic nuclei and perifornical and lateral hypothalamic areas. No obvious differences in GAD65-ir nerve fibers and terminals could be detected. Measurements of daily food intake revealed that tub/ tub mice displayed progressively higher food consumption as compared to lean tub/+ littermates over a 15-day observation period. When moved to an unfamiliar environment, e.g. a novel cage, daily food intake was initially lower in tub/ tub mice than in tub/+ mice suggesting that tub/ tub mice may be more sensitive to psychogenic stress. The results together show that tub/ tub mice are hyperphagic and exhibit, within the hypothalamic arcuate nucleus, a depressed expression of neuropeptides involved in the regulation of feeding behavior.