Sex chromosomes evolved many times independently in many different organisms [1]. According to the currently accepted model, X and Y chromosomes evolve from a pair of autosomes via a series of inversions leading to stepwise expansion of a nonrecombining region on the Y chromosome (NRY) and the consequential degeneration of genes trapped in the NRY [2]. Our results suggest that plants represent an exception to this rule as a result of their unique life-cycle that includes alteration of diploid and haploid generations and widespread haploid expression of genes in plant gametophytes [3]. Using a new high-throughput approach, we identified over 400 new genes expressed from X and Y chromosomes in Silene latifolia, a plant that evolved sex chromosomes about 10 million years ago. Y-linked genes show faster accumulation of amino-acid replacements and loss of expression, compared to X-linked genes. These degenerative processes are significantly less pronounced in more constrained genes and genes that are likely exposed to haploid-phase selection. This may explain why plants retain hundreds of expressed Y-linked genes despite millions of years of Y chromosome degeneration, whereas animal Y chromosomes are almost completely degenerate.
Vesicle transport is essential for the movement of proteins, lipids and other molecules between membrane compartments within the cell. The role of the class VI myosins in vesicular transport is especially intriguing because they are the only class that has been shown to move "backwards" towards the minus end of actin filaments1. Myosin VI is found in distinct intracellular locations and implicated in processes such as endocytosis2,3, exocytosis, maintenance of Golgi morphology4,5 and cell movement6. We have shown that the C-terminal tail is the key targeting region and have identified three binding sites: a WWY motif for Dab2 binding, a RRL motif for GIPC/Optineurin binding and a site that binds specifically and with high affinity (K d 0.3 μM) to PIP 2 -containing liposomes. This is the first demonstration that myosin VI binds lipid membranes. Lipid binding induces a large structural change in the tail (31% increase in helicity) and when associated with lipid vesicles it can dimerise. In vivo targeting and recruitment of myosin VI to clathrin-coated structures (CCSs) at the plasma membrane is mediated by Dab2 and PIP 2 binding.Dab2 is a myosin VI binding partner present on endocytic CCSs at the plasma membrane7,8. To establish whether binding to Dab2 is involved in targeting myosin VI to CCSs we tested an extensive series of myosin VI tail deletion fragments and point mutants using the mammalian 2-hybrid assay7. A relatively conservative single amino acid change from a tryptophan to a leucine (W1184L, WWY→WLY) was found to abolish myosin VI binding to Dab2 (Fig. 1a). 'Pull down' experiments using GST-tagged wild type myosin VI tail or tail containing the WWY→WLY mutation together with in vitro translated Dab2 confirmed the identity of the Dab2 binding site (Fig. 1c). To check whether the Dab2 binding site was essential for targeting myosin VI to CCSs in vivo we over-expressed GFPtagged mutant tail constructs in HeLa cells. We observed that myosin VI containing a mutated Dab2 binding site (WWY→WLY) was not targeted to CCSs (Fig. 1d,e). It was previously shown2,8 that the presence of a large insert just before the globular C-terminal domain (Fig. S1) in conjunction with Dab2 binding was also required for targeting myosin VI to CCSs at the plasma membrane.GIPC is another myosin VI binding partner9 that is found in both clathrin-coated10 and uncoated endocytic vesicles3. To test the involvement of GIPC in targeting myosin VI to CCSs we mapped the GIPC binding site on the myosin VI tail by deletion and alanine scanning mutagenesis. We observed that the mutation RRL to AAA in the C-terminal tail Correspondence should be addressed to: J.K-J (e-mail: jkj@mrc-lmb.cam.ac.uk). (Fig. 1b) abolished GIPC binding, but had no effect on Dab2 binding (Fig. 1a,b). Optineurin, a myosin VI binding protein associated with the Golgi complex and secretion, also specifically binds to the RRL binding site in the myosin VI tail4. Although phosphorylation of the threonines in the TINT sequence 15 residues upstream of RRL (Fig. S1b...
Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.
Summary Myosin VI is an actin-based retrograde motor protein, which plays a crucial role in both endocytic and secretory membrane trafficking pathways. Myosin VI’s targeting to and function in these intracellular pathways is mediated by a number of specific binding partners. In this paper we have identified a new myosin VI binding partner, Lemur tyrosine kinase 2 (LMTK2), which is the first transmembrane protein and kinase that directly binds to myosin VI. LMTK2 binds to the WWY site in the C-terminal myosin VI tail, the same site as the endocytic adaptor protein Dab2. When either myosin VI or LMTK2 is depleted by siRNA, the transferrin receptor (TfR) is trapped in swollen endosomes and tubule formation in the endocytic recycling pathway is dramatically reduced, showing that both proteins are required for the transport of cargo such as the TfR from early endosomes to the endocytic recycling compartment.
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