Cholesterol Depletion in Adipocytes Causes Caveolae Collapse Concomitant with Proteosomal Degradation of Cavin-2 in a Switch-Like Fashion

Caveolae, little caves of cell surfaces, are enriched in cholesterol, a certain level of which is required for their structural integrity. Here we show in adipocytes that cavin-2, a peripheral membrane protein and one of 3 cavin isoforms present in caveolae from non-muscle tissue, is degraded upon cholesterol depletion in a rapid fashion resulting in collapse of caveolae. We exposed 3T3-L1 adipocytes to the cholesterol depleting agent methyl-β-cyclodextrin, which results in a sudden and extensive degradation of cavin-2 by the proteasome and a concomitant movement of cavin-1 from the plasma membrane to the cytosol along with loss of caveolae. The recovery of cavin-2 at the plasma membrane is cholesterol-dependent and is required for the return of cavin-1 from the cytosol to the cell surface and caveolae restoration. Expression of shRNA directed against cavin-2 also results in a cytosolic distribution of cavin-1 and loss of caveolae. Taken together, these data demonstrate that cavin-2 functions as a cholesterol responsive component of caveolae that is required for cavin-1 localization to the plasma membrane, and caveolae structural integrity

Functional diversification of the nematode mbd2/3 gene between Pristionchus pacificus and Caenorhabditis elegans

Abstract Background Several members of the Methyl-Binding Domain protein family link DNA methylation with chromatin remodeling complexes in vertebrates. Amongst the four classes of MBD proteins, MBD2/3 is the most highly conserved and widespread in metazoans. We have previously reported that an mbd2/3 like gene (mbd-2) is encoded in the genomes of the nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae. RNAi knock-down of mbd-2 in the two Caenorhabditis species results in varying percentages of lethality. Results Here, we report that a general feature of nematode MBD2/3 proteins seems to be the lack of a bona fide methyl-binding domain. We isolated a null allele of mbd-2 in P. pacificus and show that Ppa-mbd-2 mutants are viable, fertile and display a fully penetrant egg laying defect. This egg laying defect is partially rescued by treatment with acetylcholine or nicotine suggesting a specific function of this protein in vulval neurons. Using Yeast-two-hybrid screens, Ppa-MBD-2 was found to associate with microtubule interacting and vesicle transfer proteins. Conclusion These results imply that MBD2/3 proteins in nematodes are more variable than their relatives in insects and vertebrates both in structure and function. Moreover, nematode MBD2/3 proteins assume functions independent of DNA methylation ranging from the indispensable to the non-essential.</p

Behavioral and Immune Responses to Infection Require Gαq- RhoA Signaling in C. elegans

Following pathogen infection the hosts' nervous and immune systems react with coordinated responses to the danger. A key question is how the neuronal and immune responses to pathogens are coordinated, are there common signaling pathways used by both responses? Using C. elegans we show that infection by pathogenic strains of M. nematophilum, but not exposure to avirulent strains, triggers behavioral and immune responses both of which require a conserved Gαq-RhoGEF Trio-Rho signaling pathway. Upon infection signaling by the Gαq pathway within cholinergic motorneurons is necessary and sufficient to increase release of the neurotransmitter acetylcholine and increase locomotion rates and these behavioral changes result in C. elegans leaving lawns of M. nematophilum. In the immune response to infection signaling by the Gαq pathway within rectal epithelial cells is necessary and sufficient to cause changes in cell morphology resulting in tail swelling that limits the infection. These Gαq mediated behavioral and immune responses to infection are separate, act in a cell autonomous fashion and activation of this pathway in the appropriate cells can trigger these responses in the absence of infection. Within the rectal epithelium the Gαq signaling pathway cooperates with a Ras signaling pathway to activate a Raf-ERK-MAPK pathway to trigger the cell morphology changes, whereas in motorneurons Gαq signaling triggers behavioral responses independent of Ras signaling. Thus, a conserved Gαq pathway cooperates with cell specific factors in the nervous and immune systems to produce appropriate responses to pathogen. Thus, our data suggests that ligands for Gq coupled receptors are likely to be part of the signals generated in response to M. nematophilum infection

Phosphatidylinositol 4,5-bisphosphate and loss of PLCγ activity inhibit TRPM channels required for oscillatory Ca2+ signaling

The Caenorhabditis elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations that control muscle contractions required for defecation. Two highly Ca2+-selective transient receptor potential (TRP) melastatin (TRPM) channels, GON-2 and GTL-1, function with PLCγ in a common signaling pathway that regulates IP3-dependent intracellular Ca2+ release. A second PLC, PLCβ, is also required for IP3-dependent Ca2+ oscillations, but functions in an independent signaling mechanism. PLCγ generates IP3 that regulates IP3 receptor activity. We demonstrate here that PLCγ via hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) also regulates GON-2/GTL-1 function. Knockdown of PLCγ but not PLCβ activity by RNA interference (RNAi) inhibits channel activity ∼80%. Inhibition is fully reversed by agents that deplete PIP2 levels. PIP2 added to the patch pipette has no effect on channel activity in PLCγ RNAi cells. However, in control cells, 10 μM PIP2 inhibits whole cell current ∼80%. Channel inhibition by phospholipids is selective for PIP2 with an IC50 value of 2.6 μM. Elevated PIP2 levels have no effect on channel voltage and Ca2+ sensitivity and likely inhibit by reducing channel open probability, single-channel conductance, and/or trafficking. We conclude that hydrolysis of PIP2 by PLCγ functions in the activation of both the IP3 receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca2+ influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate IP3 receptor function. PIP2-dependent regulation of GON-2/GTL-1 may provide a mechanism to coordinate plasma membrane Ca2+ influx with PLCγ and IP3 receptor activity as well as intracellular Ca2+ store depletion