It takes two to tango

PROPPINs are a family of PtdIns3P and PtdIns(3,5)P₂-binding proteins. The crystal structure now unravels the presence of two distinct phosphoinositide-binding sites at the circumference of the seven bladed β-propeller. Mutagenesis analysis of the binding sites shows that both are required for normal membrane association and autophagic activities. We identified a set of evolutionarily conserved basic and polar residues within both binding pockets, which are crucial for phosphoinositide binding. We expect that membrane association of PROPPINs is further stabilized by membrane insertions and interactions with other proteins.     

It takes T to tango

Of three recently cloned T-type voltage-gated calcium channels, alpha(1g) is most likely responsible for burst firing in thalamic relay cells. These neurons burst during various thalamocortical oscillations including absence seizures. In this issue of Neuron, Kim et al. inactivated alpha(1g), and resultant mice were deficient in relay cell bursting and resistant to GABA(B) receptor-dependent absence seizures, suggesting roles for alpha(1g) and relay cell bursting in absences.     

Gauchos and ochos: a Wee1-Cdk tango regulating mitotic entry

The kinase Wee1 has been recognized for a quarter century as a key inhibitor of Cyclin dependent kinase 1 (Cdk1) and mitotic entry in eukaryotes. Nonetheless, Wee1 regulation is not well understood and its large amino-terminal regulatory domain (NRD) has remained largely uncharted. Evidence has accumulated that cyclin B/Cdk1 complexes reciprocally inhibit Wee1 activity through NRD phosphorylation. Recent studies have identified the first functional NRD elements and suggested that vertebrate cyclin A/Cdk2 complexes also phosphorylate the NRD. A short NRD peptide, termed the Wee box, augments the activity of the Wee1 kinase domain. Cdk1/2-mediated phosphorylation of the Wee box (on T239) antagonizes kinase activity. A nearby region harbors a conserved RxL motif (RxL1) that promotes cyclin A/Cdk2 binding and T239 phosphorylation. Mutation of either T239 or RxL1 bolsters the ability of Wee1 to block mitotic entry, consistent with negative regulation of Wee1 through these sites. The region in human somatic Wee1 that encompasses RxL1 also binds Crm1, directing Wee1 export from the nucleus. These studies have illuminated important aspects of Wee1 regulation and defined a specific molecular pathway through which cyclin A/Cdk2 complexes foster mitotic entry. The complexity, speed, and importance of regulation of mitotic entry suggest that there is more to be learned.     

It takes two to tango to the melanosome

The role of clathrin adaptor proteins in sorting cargo in the biosynthetic and recycling routes is an area of intense research. In this issue, Delevoye et al. (2009. J. Cell Biol. doi:10.1083/jcb.200907122) show that a close interaction between the clathrin adaptor AP-1 and a kinesin motor KIF13A is essential for delivering melanogenic enzymes from recycling endosomes to nascent melanosomes and for organelle biogenesis.Melanosomes, along with platelet-dense granules and lung type II alveolar cell lamellar bodies, are lysosome-related organelles (LROs), compartments that originate from endosomes but are distinct from and usually coexist with lysosomes (Fig. 1). The most characteristic features of melanosomes are their ability to synthesize and store melanin and their presence in specialized pigmented cells such as skin melanocytes and iris and retinal pigment epithelial cells (Raposo and Marks, 2007; Wasmeier et al., 2008). In this issue, Delevoye et al. (see p. 247) report a melanogenic role for the clathrin adaptor AP-1 that involves interactions between the adaptor and the plus end kinesin motor KIF13A. An impressive set of data support a scenario in which the adaptor and the motor tightly interact, like in tango, to position donor recycling endosomes (REs) near nascent melanosomes at the cell periphery and to generate tubulovesicular intermediates that deliver newly synthesized pigmenting enzymes to melanosomes.Open in a separate windowFigure 1.Role of clathrin adaptor proteins in melanosome biogenesis. Post-Golgi trafficking routes of three melanosome cargoes (Pmel17, tyrosinase, and Tyrp1) in melanocytes are shown. Newly synthesized Pmel17 is transported to the limiting membrane and intraluminal vesicles of stage I melanosomes/early sorting endosomes via the plasma membrane. This process (depicted by a question mark) might involve clathrin and AP-2. From these EEA1-positive vacuolar endosomes, Pmel17 is sorted away from the late endosome/multivesicular body pathway into stage II melanosomes. Little is known as to how the enzymes essential for melanin synthesis, tyrosinase and Tyrp1, are sorted from the TGN to early REs, and it is likely that clathrin and its adaptors are involved in this process. Tyrosinase, which binds both AP-1 and -3, is transported to stage III melanosomes from tubular regions of REs, containing Tf/TfR and Rab11, by two distinct routes: one regulated by AP-3 and the other regulated by BLOC-1, BLOC-2, and perhaps AP-1. However, Tyrp1 binds only AP-1 and not AP-3, indicating a divergence of sorting mechanisms between tyrosinase and Tyrp1. Delevoye et al. (2009) now show that AP-1 interacts with the kinesin motor KIF13A to transport recycling endosomal domains to the melanocytic cell periphery. The close apposition of Tyrp1-containing tubules with melanosomes allows cargo transfer and biogenesis of stage III and IV melanosomes. Although Tf is found in these peripheral endosomal tubules, there appears to be a filtering mechanism that sorts it out before the tubules fuse with melanosomes. It is likely, although not yet confirmed, that BLOC-1 and -2 act in concert with AP-1 to transport Tyrp1. The tissue-specific Rabs, Rab32 and Rab38, might function in any or all of these pathways.Extensive studies have shown that melanosome biogenesis occurs in two waves that correspond to four morphologically distinct stages (Fig. 1; Marks and Seabra, 2001; Raposo and Marks, 2007). The first wave (stages I and II) is the formation of immature, pigment-free ellipsoidal melanosomes from vacuolar domains of early sorting endosomes. This process requires Pmel17, an integral membrane protein that likely reaches sorting endosomes by clathrin-dependent endocytosis from the plasma membrane. Upon proteolysis in the sorting endosomes/stage I melanosomes, Pmel17 forms intraluminal proteinaceous fibrils with characteristics of amyloid. The second wave starts with the post-Golgi transport of enzymes involved in melanin synthesis such as tyrosinase and tyrosinase-related protein 1 (Tyrp1) to nascent melanosomes. Melanin deposition occurs on Pmel17 fibrils and leads to the biogenesis of mature (stages III and IV) melanosomes. The clathrin adaptors AP-1 and -3 have partially redundant functions in sorting cargo proteins to melanosomes. Melanosomal cargo proteins have dileucine motifs that are recognized differentially by AP-1 and -3 in post-Golgi endosomes (Huizing et al., 2001; Theos et al., 2005). Nascent tyrosinase is found in distinct endosomal buds that contain either AP-3 or -1 in normal melanocytes and loss of AP-3 results only in a partial mislocalization of the enzyme. As these adaptors also mediate sorting from endosomes to other compartments, additional machinery, such as biogenesis of LRO complex 1 (BLOC-1), BLOC-2, and the tissue-specific small GTPases Rab32 and Rab38, regulate cargo delivery to melanosomes. Mutations in components of this melanosomal targeting machinery result in a variety of well-studied pigmentation defects in humans and animals such as Hermansky–Pudlak syndrome (Wei, 2006).Delevoye et al. (2009) show that knockdown of AP-1 in melanocytic MNT-1 cells decreases melanin content, demonstrating that AP-1 has a role in melanogenesis. Only late-stage (III/IV) melanosomes are decreased in number; unpigmented (stage I/II) melanosomes are unaffected, indicating that AP-1 functions selectively in the second wave of melanosome biogenesis. In AP-1–depleted cells, the melanosome cargo protein Tyrp1 is retained in vacuolar endosomes in a manner similar to that seen in BLOC-1–deficient melanocytes (Setty et al., 2007). Using immunofluorescence to monitor markers of various endosomal compartments, Delevoye et al. (2009) show that AP-1 performs its melanogenic function in early REs. Interestingly, additional data show that AP-1–containing REs have a peripheral distribution in MNT-1 cells, which is strikingly different from the perinuclear localization observed in other cells. Furthermore, siRNA-mediated knockdown of AP-1, but not of AP-3, relocates RE to a pericentriolar location.How might AP-1 influence endosome position? One possibility is by its association with the plus end–directed kinesin motor KIF13A (Fig. 1). Nakagawa et al. (2000) have previously shown that a subunit of AP-1 binds the C-terminal domain of KIF13A, mediating TGN to plasma membrane transport of the mannose 6-phosphate receptor. Indeed, Delevoye et al. (2009) show that KIF13A partially colocalizes with AP-1 in MNT-1 cells and coimmunoprecipitates with both AP-1 and Tyrp1. Furthermore, knockdown of KIF13A replicates the phenotype seen with AP-1 depletion: pericentriolar clustering of RE, accumulation of Tyrp1 in vacuolar endosomes, and reduction in mature melanosomes and melanin content. Delevoye et al. (2009) go on to show that the peripheral RE localization facilitates sorting of melanosomal proteins but decreases the efficiency of transferrin (Tf) receptor (TfR) recycling to the plasma membrane. They also show the converse; i.e., the pericentriolar localization of RE decreases the efficiency of melanosomal targeting and increases the efficiency of TfR recycling. Thus, the position of REs, determined by the interaction between a clathrin adaptor and a kinesin, is key for specific sorting functions of this organelle (like TfR recycling) and also regulates the biogenesis of another organelle (the melanosome). This is a novel and exciting finding and is an emerging theme in cell biology. It was recently reported that AP-1 interacts with another plus end–directed kinesin, KIF5, which helps transport endosomes to the cell periphery (Schmidt et al., 2009).The next question that Delevoye et al. (2009) approach is what is the nature of the carriers that transport melanosomal proteins from peripheral REs to immediately adjacent stage III/IV melanosomes? Live imaging experiments showed a dynamic network of Tf-containing RE tubules that extend and retract, making contact with melanosomes for at least 30 s. Double-tilt 3D electron tomography of thick (350–400 nm) sections of cells preserved by high pressure freezing and freeze substitution, a technique recently adapted to the study of melanosomes by Hurbain et al. (2008), revealed that some of these tubular elements are continuous with the melanosomal limiting membrane and that their lumens are often connected. Collectively, these results indicate that peripheral RE domains serve to deliver biosynthetic cargo to maturing melanosomes by the coordinated actions of AP-1 and KIF13A and that the mechanism involves tubular connections rather than vesicular transport (Fig. 1).The study by Delevoye et al. (2009) beautifully demonstrates the power of carefully chosen morphological and live imaging techniques, in combination with siRNA-mediated knockdown of molecules under study, to elucidate important details of cellular sorting processes. As always, several questions emerge from their results. Does this type of mechanism also operate in perinuclear REs, which were recently shown to cooperate with adjacent TGN in biosynthetic trafficking to the plasma membrane (Cancino et al., 2007; Gravotta et al., 2007)? Do newly synthesized melanosomal enzymes move from the TGN to REs using vesicular trafficking and clathrin adaptors or, rather, result from “maturation” of REs from the TGN? What is the role of clathrin in melanosome maturation? Are AP-1 and KIF13A essential for tubulogenesis from REs as the authors speculate? How are RE proteins (e.g., TfR) prevented from incorporating into melanosomes through the tubular connections? What is the mechanism that regulates docking and fusion of RE tubules with melanosomes? Likely, Rab32 and Rab38 participate in this process, as these proteins localize to tubulovesicular endosomal structures, and their loss causes mislocalization of tyrosinase and Tyrp1 (Wasmeier et al., 2006), but the SNAREs (if any) that participate in the mechanism are still unknown. Lastly, another intriguing aspect of this study is how adaptors sort proteins by differential recognition of dileucine motifs. Tyrp1 also has a dileucine motif that exclusively binds AP-1, but not AP-3, in melanocytic cells (Theos et al., 2005), whereas tyrosinase has dileucine motifs that bind AP-1 and -3, indicating that not all dileucine motifs are equal in the eyes of the adaptor.     

MAPK signaling specificity: it takes two to tango

A surprisingly limited repertoire of core signaling pathways generates an enormous diversity of responses, often in a cell type-specific manner. Even within one cell, a single pathway might yield two different responses depending on the input signal. In budding yeast, a mitogen-activated protein kinase (MAPK) module seems to transmit both mating pheromone and invasive growth signals in the same yeast cell. We discuss recent insights into mechanisms of differential MAPK activation in this system.     

Replication stress and cancer: It takes two to tango

Problems arising during DNA replication require the activation of the ATR–CHK1 pathway to ensure the stabilization and repair of the forks, and to prevent the entry into mitosis with unreplicated genomes. Whereas the pathway is essential at the cellular level, limiting its activity is particularly detrimental for some cancer cells. Here we review the links between replication stress (RS) and cancer, which provide a rationale for the use of ATR and Chk1 inhibitors in chemotherapy. First, we describe how the activation of oncogene-induced RS promotes genome rearrangements and chromosome instability, both of which could potentially fuel carcinogenesis. Next, we review the various pathways that contribute to the suppression of RS, and how mutations in these components lead to increased cancer incidence and/or accelerated ageing. Finally, we summarize the evidence showing that tumors with high levels of RS are dependent on a proficient RS-response, and therefore vulnerable to ATR or Chk1 inhibitors.     

It takes two transposons to tango: transposable-element-mediated chromosomal rearrangements

Transposable elements (TEs) promote various chromosomal rearrangements more efficiently, and often more specifically, than other cellular processes(1-3). One explanation of such events is homologous recombination between multiple copies of a TE present in a genome. Although this does occur, strong evidence from a number of TE systems in bacteria, plants and animals suggests that another mechanism - alternative transposition - induces a large proportion of TE-associated chromosomal rearrangements. This paper reviews evidence for alternative transposition from a number of unrelated but structurally similar TEs. The similarities between alternative transposition and V(D)J recombination are also discussed, as is the use of alternative transposition as a genetic tool.