Mitotic lamin disassembly is triggered by lipid-mediated signaling

Disassembly of the nuclear lamina is a key step during open mitosis in higher eukaryotes. The activity of several kinases, including CDK1 (cyclin-dependent kinase 1) and protein kinase C (PKC), has been shown to trigger mitotic lamin disassembly, yet their precise contributions are unclear. In this study, we develop a quantitative imaging assay to study mitotic lamin B1 disassembly in living cells. We find that CDK1 and PKC act in concert to mediate phosphorylation-dependent lamin B1 disassembly during mitosis. Using ribonucleic acid interference (RNAi), we showed that diacylglycerol (DAG)-dependent PKCs triggered rate-limiting steps of lamin disassembly. RNAi-mediated depletion or chemical inhibition of lipins, enzymes that produce DAG, delayed lamin disassembly to a similar extent as does PKC inhibition/depletion. Furthermore, the delay of lamin B1 disassembly after lipin depletion could be rescued by the addition of DAG. These findings suggest that lipins activate a PKC-dependent pathway during mitotic lamin disassembly and provide evidence for a lipid-mediated mitotic signaling event.     

Molecular simulations of lipid-mediated protein-protein interactions

Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved.     

Transmodulation of BCR signaling by transduction-incompetent antigen receptors: implications for impaired signaling in anergic B cells

B cell tolerance can be maintained by functional inactivation, or anergy, wherein B cell Ag receptors (BCR) remain capable of binding Ag, but are unable to transduce signals. Although the molecular mechanisms underlying this unresponsiveness are unknown, some models of B cell anergy are characterized by disruption of proximal BCR signaling events, and by destabilization of the BCR complex. Receptor destabilization is manifest by a reduced ability to coimmunoprecipitate membrane Ig with the Ig-alpha/Ig-beta signal-transducing complex. To begin to explore the possibility that anergy is the consequence of receptor destabilization, we analyzed a panel of B lymphoma transfectants expressing constant amounts of signal-competent Ag receptors and varied amounts of a receptor with identical specificity, but bearing mutations that render it incapable of interacting with Ig-alpha/Ig-beta. This analysis revealed that coaggregation of signal-incompetent receptors prevented Ag-induced Ig-alpha and Syk phosphorylation, mobilization of Ca(2+), and the up-regulation of CD69 mediated by competent receptors. In contrast, Ag-induced Cbl and Erk phosphorylation were unaffected. Data indicate that coaggregation of destabilized receptors (as few as approximately 15% of total) with signal-competent receptors significantly affects the ability of competent receptors to transduce signals. Thus, BCR destabilization may underlie the Ag unresponsiveness of anergic B cells.     

Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex: molecular mechanisms and functional implications

Serotonergic neurotransmission in prefrontal cortex (PFC) plays a key role in regulating emotion and cognition under normal and pathological conditios. Increasing evidence suggests that serotonin receptors are involved in the complex regulation of GABAergic inhibitory transmission in PFC. Activation of postsynaptic 5-HT₂ receptors in PFC pyramidal neurons inhibits GABA_A-receptor currents via phosphorylation of GABA_A receptor γ2 subunits by RACK1-anchored PKC. In contrast, activation of postsynaptic 5-HT₄ receptors produces an activity-dependent bi-directional regulation of GABA-evoked currents in PFC pyramidal neurons, which is mediated through phosphorylation of GABA_A-receptor β subunits by anchored PKA. On the presynaptic side, GABAergic inhibition is regulated by 5-HT through the activation of 5-HT₂, 5-HT₁, and 5-HT₃ receptors on GABAergic intereneurons. These data provide a molecular and cellular mechanism for serotonin to dynamically regulate synaptic transmission and neuronal excitability in the PFC network, which may underlie the actions of many antidepressant and antipsychotic drugs.     

Transcytosis in thyroid follicle cells: Regulation and implications for thyroglobulin transport

In order to analyze quantitatively the translocation of plasma membrane during endocytosis and transcytosis and the regulation of these processes in thyroid follicle cells, the apical cell surfaces of resting and TSH-stimulated inside-out follicles were labeled with cationized ferritin. Morphometric analyses showed that the rates of endocytosis and transcytosis are TSH-dependent. More interestingly, whereas the effect of TSH on endocytosis was transient (with a maximum at 16 min), the effect on transcytosis continued to increase until the end of the experiment (i.e, 70 min). During 1 h of endocytosis, the fraction of membrane involved in transcytosis increased by a factor 4 upon TSH stimulation, corresponding to about 12% of the internalized apical plasma membrane area. Cooling to 15 degrees C slowed down, but did not block endocytosis entirely, whereas transcytosis and transfer to lysosomes were totally inhibited In order to quantitate transcytosis of thyroglobulin (TG) and to ascertain whether this molecule undergoes cleavage during transcytosis, inside-out follicles were incubated in a medium containing 3H-labeled TG in the presence of TSH; upon washing and reopening of follicles, the luminal fluid containing TG after transcytosis was found to contain about 10% of the total radioactivity taken up by follicle cells. Transcytosed TG proved to be unmodified with respect to its electrophoretic mobility. We conclude that (i) the fraction of transcytosed TG corresponds approximately to the fraction of membrane involved in this process, (ii) TG does not undergo cleavage during transcytosis, (iii) endocytosis and transcytosis are regulated by TSH but differ in their kinetics after stimulation, and (iv) transcytosis is affected by temperature in a similar way as transfer to lysosomes, suggesting the existence of a common gating step for both pathways.     

Regulation of signaling interactions and receptor endocytosis in growing blood vessels

Blood vessels and the lymphatic vasculature are extensive tubular networks formed by endothelial cells that have several indispensable functions in the developing and adult organism. During growth and tissue regeneration but also in many pathological settings, these vascular networks expand, which is critically controlled by the receptor EphB4 and the ligand ephrin-B2. An increasing body of evidence links Eph/ephrin molecules to the function of other receptor tyrosine kinases and cell surface receptors. In the endothelium, ephrin-B2 is required for clathrin-dependent internalization and full signaling activity of VEGFR2, the main receptor for vascular endothelial growth factor. In vascular smooth muscle cells, ephrin-B2 antagonizes clathrin-dependent endocytosis of PDGFRβ and controls the balanced activation of different signal transduction processes after stimulation with platelet-derived growth factor. This review summarizes the important roles of Eph/ephrin molecules in vascular morphogenesis and explains the function of ephrin-B2 as a molecular hub for receptor endocytosis in the vasculature.     

Regulation of signaling interactions and receptor endocytosis in growing blood vessels

Blood vessels and the lymphatic vasculature are extensive tubular networks formed by endothelial cells that have several indispensable functions in the developing and adult organism. During growth and tissue regeneration but also in many pathological settings, these vascular networks expand, which is critically controlled by the receptor EphB4 and the ligand ephrin-B2. An increasing body of evidence links Eph/ephrin molecules to the function of other receptor tyrosine kinases and cell surface receptors. In the endothelium, ephrin-B2 is required for clathrin-dependent internalization and full signaling activity of VEGFR2, the main receptor for vascular endothelial growth factor. In vascular smooth muscle cells, ephrin-B2 antagonizes clathrin-dependent endocytosis of PDGFRβ and controls the balanced activation of different signal transduction processes after stimulation with platelet-derived growth factor. This review summarizes the important roles of Eph/ephrin molecules in vascular morphogenesis and explains the function of ephrin-B2 as a molecular hub for receptor endocytosis in the vasculature.     

Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers

The oligomerization state of the ADP/ATP carrier is an important issue in understanding the mechanism underlying nucleotide exchange across the inner mitochondrial membrane. The first high resolution structure obtained in the presence of carboxyatractyloside revealed a large cavity formed within a monomer in which the inhibitor is strongly bound. Whereas the protein-protein interactions implicated in the first crystal form are not biologically relevant, the new crystal form described herein, highlights favorable protein-protein interactions. The interactions are mediated by endogenous cardiolipins, which are tightly bound to the protein, two cardiolipins being sandwiched between the monomers on the matrix side. The putative dimerization interface evidenced here is consistent with other structural, biochemical or functional data published so far.     

Regulation of active and quiescent somatic stem cells by Notch signaling

Somatic stem/progenitor cells actively proliferate and give rise to different types of mature cells (active state) in embryonic tissues while they are mostly dormant (quiescent state) in many adult tissues. Notch signaling is known to regulate both active and quiescent states of somatic stem cells, but how it regulates these different states is unknown. Recent studies revealed that the Notch effector Hes1 is expressed differently during the active and quiescent states during neurogenesis and myogenesis: high in the quiescent state and oscillatory in the active state. When the Hes1 expression level is high, both Ascl1 and MyoD expression are continuously suppressed. By contrast, when Hes1 expression oscillates, it periodically represses expression of the neurogenic factor Ascl1 and the myogenic factor MyoD, thereby driving Ascl1 and MyoD oscillations. High levels of Hes1 and the resultant Ascl1 suppression promote the quiescent state of neural stem cells, while Hes1 oscillation-dependent Ascl1 oscillations regulate their active state. Similarly, in satellite cells of muscles, known adult muscle stem cells, high levels of Hes1 and the resultant MyoD suppression seem to promote their quiescent state, while Hes1 oscillation-dependent MyoD oscillations activate their proliferation and differentiation. Therefore, the expression dynamics of Hes1 is a key regulatory mechanism of generating and maintaining active/quiescent stem cell states.     

Apolipoprotein D modulates arachidonic acid signaling in cultured cells: implications for psychiatric disorders

Deficiencies in arachidonic acid (AA) parameters have been reported in schizophrenic patients. AA is a primary binding ligand for apolipoprotein D (apoD), which is increased in response to antipsychotic drug treatment and elevated in subjects with schizophrenia and bipolar disorder. In this study, we investigated whether apoD might modulate AA signaling/mobilization in cultured embryonic kidney (HEK) 293T cells. Immunofluorescent labeling revealed both cytosolic and membrane-bound expression of apoD protein in apoD-transfected cells. In cells expressing apoD, phorbal 12-myristate 13-acetate-induced AA release was inhibited compared to controls and membrane levels of AA were elevated, as indicated by the amount of AA maximally incorporated into membrane phospholipids. In addition, exogenous apoD added directly to the incubation media prevented cellular uptake of free [3H]AA. These results suggest that apoD acts to stabilize membrane-associated AA by preventing release and sequestering free AA in the cell. These actions of apoD may be beneficial to psychiatric patients.     

Color expression in experimentally regrown feathers of an overwintering migratory bird: implications for signaling and seasonal interactions

Plumage coloration in birds plays a critical role in communication and can be under selection throughout the annual cycle as a sexual and social signal. However, for migratory birds, little is known about the acquisition and maintenance of colorful plumage during the nonbreeding period. Winter habitat could influence the quality of colorful plumage, ultimately carrying over to influence sexual selection and social interactions during the breeding period. In addition to the annual growth of colorful feathers, feather loss from agonistic interactions or predator avoidance could require birds to replace colorful feathers in winter or experience plumage degradation. We hypothesized that conditions on the wintering grounds of migratory birds influence the quality of colorful plumage. We predicted that the quality of American redstart (Setophaga ruticilla) tail feathers regrown after experimental removal in Jamaica, West Indies, would be positively associated with habitat quality, body condition, and testosterone. Both yearling (SY) and adult (ASY) males regrew feathers with lower red chroma, suggesting reduced carotenoid content. While we did not observe a change in hue in ASY males, SY males shifted from yellow to orange plumage resembling experimentally regrown ASY feathers. We did not observe any effects of habitat, testosterone, or mass change. Our results demonstrate that redstarts are limited in their ability to adequately replace colorful plumage, regardless of habitat, in winter. Thus, feather loss on the nonbreeding grounds can affect social signals, potentially negatively carrying over to the breeding period.     

Regulation and function of triacylglycerol lipases in cellular metabolism

The ability to store energy in the form of energy-dense TAG (triacylglycerol) and to mobilize these stores rapidly during times of low carbohydrate availability (fasting or famine) or during heightened metabolic demand (exercise or cold-stress) is a highly conserved process essential for survival. Today, in the presence of nutrient excess and sedentary lifestyles, the regulation of this pathway is viewed as an important therapeutic target for disease prevention, as elevated circulating fatty acids in obesity contribute to many aspects of the metabolic syndrome including hepatic steatosis, atherosclerosis and insulin resistance. In the present review, we discuss the metabolic regulation and function of TAG lipases with a focus on HSL (hormone-sensitive lipase), ATGL (adipose triacylglycerol lipase) and newly identified members of the lipolytic proteome.     

Regulation of EGF signaling by cell polarity in MDCK kidney epithelial cells.

Although the presence of a dominant basolateral sorting signal ensures that the majority of newly synthesized epidermal growth factor (EGF) receptors are delivered directly to the basolateral surface in polarized epithelial cells, a fraction of the receptors are also delivered to the apical surface. Similar to most basolateral membrane proteins, the EGF receptor has an additional signal(s) that selectively targets molecules lacking a dominant basolateral signal to the apical surface. Although the physiological relevance of signal hierarchy is not known, alternative targeting may occur in different epithelial cell types or during development. The goal of this study, therefore, was to determine the effect of membrane domain location on EGF receptor function, focusing on EGF-induced MAP kinase signaling and DNA synthesis. Whereas ligand responsiveness was restricted to the basolateral domain in Madin-Darby canine kidney (MDCK) cells expressing a normal complement of receptors, apical ligand was effective if apical receptor density was increased by overexpression of an exogenous wild-type human gene. Unexpectedly, cells expressing apically localized, cytoplasmically truncated receptors, which behave as dominant negative mutations in other cell types, were also responsive to apical EGF. The cytoplasmically truncated molecules appear to have at least two effects: first, to increase the local concentration of ligand at the apical cell surface; and second, to facilitate activation of the relatively few native EGF receptors normally located at the apical surface. These results indicate that cell context is a critical determinant of receptor mutant protein phenotype.     

Regulation of IGF-I receptor signaling in tumor cells.

Signals from the IGF-IR and other members of the IR family contribute to the growth, survival, adhesion, and motility of tumor cells. These signals are initiated through recruitment of adapter proteins including the IRS family and Shc proteins, and are mediated through the PI3-kinase, mitogen activated protein (MAP) kinase and stress-activated protein kinase (SAPK) pathways. Regulation of signaling responses from the IGF-IR involves the actions of regulatory adapter proteins including RACK1 and Grb10 that recruit or sequester cytoplasmic proteins, and the actions of phosphatases including tyrosine PTP-1B, PTEN, and PP2A. This review focuses on the signaling pathways that are activated by the IGF-IR in tumor cells, the mechanisms of regulation of these pathways by adapter proteins and phosphatases, and how modulation of IGF-IR signaling could contribute to cancer progression.