Circulating blood platelets are specialized cells that prevent bleeding and minimize blood vessel injury. Large progenitor cells in the bone marrow called megakaryocytes (MKs) are the source of platelets. MKs release platelets through a series of fascinating cell biological events. During maturation, they become polyploid and accumulate massive amounts of protein and membrane. Then, in a cytoskeletal-driven process, they extend long branching processes, designated proplatelets, into sinusoidal blood vessels where they undergo fission to release platelets. Given the need for platelets in many pathological situations, understanding how this process occurs is an active area of research with important clinical applications.
In yeast, the localization of homologous recombination–associated proteins to heterochromatic regions of the genome is necessary for proper nuclear organization.
The eukaryotic genome is highly organized in the nucleus, and this organization affects various nuclear processes. However, the molecular details of higher-order organization of chromatin remain obscure. In the present study, we show that the Saccharomyces cerevisiae silenced loci HML and HMR cluster in three-dimensional space throughout the cell cycle and independently of the telomeres. Long-range HML–HMR interactions require the homologous recombination (HR) repair pathway and phosphorylated H2A (γ-H2A). γ-H2A is constitutively present at silenced loci in unperturbed cells, its localization requires heterochromatin, and it is restricted to the silenced domain by the transfer DNA boundary element. SMC proteins and Scc2 localize to the silenced domain, and Scc2 binding requires the presence of γ-H2A. These findings illustrate a novel pathway for heterochromatin organization and suggest a role for HR repair proteins in genomic organization.
ParA-dependent DNA segregation determines the cell cycle localization pattern of the pole-organizing protein PopZ by promoting its assembly into a matrix.
Bacterial cell poles constitute defined subcellular domains where numerous proteins localize, often at specific times, to affect various physiological processes. How pole recognition occurs and what governs the timing of protein localization are often unknown. In this paper, we investigate the mechanisms governing the localization of PopZ, a chromosome-anchoring protein whose unipolar to bipolar localization pattern is critical for cell cycle progression in Caulobacter crescentus. We provide evidence that polar localization of PopZ relied on its self-assembly into a higher-order structure (matrix) and that the unipolar to bipolar transition was coupled to the asymmetric distribution of ParA during the translocation of the origin-proximal ParB–parS partition complex. Collectively, our data suggest a model in which a local increase of ParA concentration promotes the assembly of a PopZ matrix precisely when and where this matrix is needed. Such coupling of protein assembly with a cell cycle–associated molecular asymmetry may represent a principle of cellular organization for controlling protein localization in both time and space.
Liprin-α2 is required for the presynaptic recruitment and turnover of RIM1 and CASK, components of the release machinery, and facilitates synaptic output by regulating synaptic vesicle pool size.
The presynaptic active zone mediates synaptic vesicle exocytosis, and modulation of its molecular composition is important for many types of synaptic plasticity. Here, we identify synaptic scaffold protein liprin-α2 as a key organizer in this process. We show that liprin-α2 levels were regulated by synaptic activity and the ubiquitin–proteasome system. Furthermore, liprin-α2 organized presynaptic ultrastructure and controlled synaptic output by regulating synaptic vesicle pool size. The presence of liprin-α2 at presynaptic sites did not depend on other active zone scaffolding proteins but was critical for recruitment of several components of the release machinery, including RIM1 and CASK. Fluorescence recovery after photobleaching showed that depletion of liprin-α2 resulted in reduced turnover of RIM1 and CASK at presynaptic terminals, suggesting that liprin-α2 promotes dynamic scaffolding for molecular complexes that facilitate synaptic vesicle release. Therefore, liprin-α2 plays an important role in maintaining active zone dynamics to modulate synaptic efficacy in response to changes in network activity.
Localized Hippo signaling at contact sites between border cells induces polarized F-actin polymerization that drives collective cell migration.
Collective migration of Drosophila border cells depends on a dynamic actin cytoskeleton that is highly polarized such that it concentrates around the outer rim of the migrating cluster of cells. How the actin cytoskeleton becomes polarized in these cells to enable collective movement remains unknown. Here we show that the Hippo signaling pathway links determinants of cell polarity to polarization of the actin cytoskeleton in border cells. Upstream Hippo pathway components localize to contacts between border cells inside the cluster and signal through the Hippo and Warts kinases to polarize actin and promote border cell migration. Phosphorylation of the transcriptional coactivator Yorkie (Yki)/YAP by Warts does not mediate the function of this pathway in promoting border cell migration, but rather provides negative feedback to limit the speed of migration. Instead, Warts phosphorylates and inhibits the actin regulator Ena to activate F-actin Capping protein activity on inner membranes and thereby restricts F-actin polymerization mainly to the outer rim of the migrating cluster.
Injury-induced BMP signaling in the midgut negatively regulates intestinal stem cell division, whereas regional constitutive BMP signaling promotes copper cell differentiation.
Although much is known about injury-induced signals that increase rates of Drosophila melanogaster midgut intestinal stem cell (ISC) proliferation, it is largely unknown how ISC activity returns to quiescence after injury. In this paper, we show that the bone morphogenetic protein (BMP) signaling pathway has dual functions during midgut homeostasis. Constitutive BMP signaling pathway activation in the middle midgut mediated regional specification by promoting copper cell differentiation. In the anterior and posterior midgut, injury-induced BMP signaling acted autonomously in ISCs to limit proliferation and stem cell number after injury. Loss of BMP signaling pathway members in the midgut epithelium or loss of the BMP signaling ligand decapentaplegic from visceral muscle resulted in phenotypes similar to those described for juvenile polyposis syndrome, a human intestinal tumor caused by mutations in BMP signaling pathway components. Our data establish a new link between injury and hyperplasia and may provide insight into how BMP signaling mutations drive formation of human intestinal cancers.
Localized activation of netrin signaling induces focused F-actin formation and the protrusive force necessary for physical displacement of basement membrane during cell transmigration.
Though critical to normal development and cancer metastasis, how cells traverse basement membranes is poorly understood. A central impediment has been the challenge of visualizing invasive cell interactions with basement membrane in vivo. By developing live-cell imaging methods to follow anchor cell (AC) invasion in Caenorhabditis elegans, we identify F-actin–based invadopodia that breach basement membrane. When an invadopodium penetrates basement membrane, it rapidly transitions into a stable invasive process that expands the breach and crosses into the vulval tissue. We find that the netrin receptor UNC-40 (DCC) specifically enriches at the site of basement membrane breach and that activation by UNC-6 (netrin) directs focused F-actin formation, generating the invasive protrusion and the cessation of invadopodia. Using optical highlighting of basement membrane components, we further demonstrate that rather than relying solely on proteolytic dissolution, the AC’s protrusion physically displaces basement membrane. These studies reveal an UNC-40–mediated morphogenetic transition at the cell–basement membrane interface that directs invading cells across basement membrane barriers.
The budding yeast Wee1 kinase Swe1 restrains the metaphase-to-anaphase transition by preventing the Cdk1-dependent phosphorylation and activation of APCCdc20.
Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APCCdc20). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25+) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2ACdc55 triggers anaphase onset.
RNF111/Arkadia targets SUMOylated XPC for ubiquitylation, negatively regulating its association with damaged DNA
Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs), promoting their K48-linked ubiquitylation and degradation. Only a single mammalian STUbL, RNF4, has been identified. We show that human RNF111/Arkadia is a new STUbL, which used three adjacent SIMs for specific recognition of poly-SUMO2/3 chains, and used Ubc13–Mms2 as a cognate E2 enzyme to promote nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response.
Bacteria use DNA segregation machinery to position polarity protein.
Bacteria use DNA segregation machinery to position polarity protein.
IgSF9b forms a novel subsynaptic domain for adhesion that links to the gephyrin- and GABAA receptor–containing domain to promote inhibitory synaptic development.
Synaptic adhesion molecules regulate diverse aspects of synapse formation and maintenance. Many known synaptic adhesion molecules localize at excitatory synapses, whereas relatively little is known about inhibitory synaptic adhesion molecules. Here we report that IgSF9b is a novel, brain-specific, homophilic adhesion molecule that is strongly expressed in GABAergic interneurons. IgSF9b was preferentially localized at inhibitory synapses in cultured rat hippocampal and cortical interneurons and was required for the development of inhibitory synapses onto interneurons. IgSF9b formed a subsynaptic domain distinct from the GABAA receptor– and gephyrin-containing domain, as indicated by super-resolution imaging. IgSF9b was linked to neuroligin 2, an inhibitory synaptic adhesion molecule coupled to gephyrin, via the multi-PDZ protein S-SCAM. IgSF9b and neuroligin 2 could reciprocally cluster each other. These results suggest a novel mode of inhibitory synaptic organization in which two subsynaptic domains, one containing IgSF9b for synaptic adhesion and the other containing gephyrin and GABAA receptors for synaptic transmission, are interconnected through S-SCAM and neuroligin 2.
Yamashita studies how germline stem cells orient their asymmetric cell divisions.
Yamashita studies how germline stem cells orient their asymmetric cell divisions.
The combined action of dynamic microtubules and Rho signaling determines the level and asymmetric distribution of a mobile E-cadherin–Bazooka complex during the generation of a patterned epithelium.
Distributing junctional components around the cell periphery is key for epithelial tissue morphogenesis and homeostasis. We discovered that positioning of dynamic microtubules controls the asymmetric accumulation of E-cadherin. Microtubules are oriented preferentially along the dorso-ventral axis in Drosophila melanogaster embryonic epidermal cells, and thus more frequently contact E-cadherin at dorso-ventral cell–cell borders. This inhibits RhoGEF2, reducing membrane recruitment of Rho-kinase, and increasing a specific E-cadherin pool that is mobile when assayed by fluorescence recovery after photobleaching. This mobile E-cadherin is complexed with Bazooka/Par-3, which in turn is required for normal levels of mobile E-cadherin. Mobile E-cadherin–Bazooka prevents formation of multicellular rosette structures and cell motility across the segment border in Drosophila embryos. Altogether, the combined action of dynamic microtubules and Rho signaling determines the level and asymmetric distribution of a mobile E-cadherin–Bazooka complex, which regulates cell behavior during the generation of a patterned epithelium.
The GEF Tiam1 acts as a novel molecular link to the VE-cadherin–p67phox–Par3 polarity complex, leading to localized activation of Rac1 and NADPH oxidase in response to fluid flow.
Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood. Here, we identify a novel polarity complex that directs localized Rac1 activation required for downstream reactive oxygen species (ROS) production. Vav2 is required for Rac1 GTP loading, whereas, surprisingly, Tiam1 functions as an adaptor in a VE-cadherin–p67phox–Par3 polarity complex that directs localized activation of Rac1. Furthermore, loss of Tiam1 led to the disruption of redox signaling both in vitro and in vivo. Our results describe a novel molecular cascade that regulates redox signaling by the coordinated regulation of Rac1 and by linking components of the polarity complex to the NADPH oxidase.
Spatial control of EB3 phosphorylation by Aurora B during mitotic exit regulates microtubule dynamics necessary for the stabilization of focal adhesions and coordinated daughter cell spreading, as well as for midbody stabilization and efficient cytokinesis.
During mitosis, human cells round up, decreasing their adhesion to extracellular substrates. This must be quickly reestablished by poorly understood cytoskeleton remodeling mechanisms that prevent detachment from epithelia, while ensuring the successful completion of cytokinesis. Here we show that the microtubule end-binding (EB) proteins EB1 and EB3 play temporally distinct roles throughout cell division. Whereas EB1 was involved in spindle orientation before anaphase, EB3 was required for stabilization of focal adhesions and coordinated daughter cell spreading during mitotic exit. Additionally, EB3 promoted midbody microtubule stability and, consequently, midbody stabilization necessary for efficient cytokinesis. Importantly, daughter cell adhesion and cytokinesis completion were spatially regulated by distinct states of EB3 phosphorylation on serine 176 by Aurora B. This EB3 phosphorylation was enriched at the midbody and shown to control cortical microtubule growth. These findings uncover differential roles of EB proteins and explain the importance of an Aurora B phosphorylation gradient for the spatiotemporal regulation of microtubule function during mitotic exit and cytokinesis.
Cytosolic pH is a cellular signal involved both in the glucose sensing that mediates proteasome storage granule formation and in a more general mechanism for signaling carbon source exhaustion.
The 26S proteasome is the major protein degradation machinery of the cell and is regulated at many levels. One mode of regulation involves accumulation of proteasomes in proteasome storage granules (PSGs) upon glucose depletion. Using a systematic robotic screening approach in yeast, we identify trans-acting proteins that regulate the accumulation of proteasomes in PSGs. Our dataset was enriched for subunits of the vacuolar adenosine triphosphatase (V-ATPase) complex, a proton pump required for vacuole acidification. We show that the impaired ability of V-ATPase mutants to properly govern intracellular pH affects the kinetics of PSG formation. We further show that formation of other protein aggregates upon carbon depletion also is triggered in mutants with impaired activity of the plasma membrane proton pump and the V-ATPase complex. We thus identify cytosolic pH as a specific cellular signal involved both in the glucose sensing that mediates PSG formation and in a more general mechanism for signaling carbon source exhaustion.
In mammalian cells, the phospho-dependent interaction between BRCA1 and CtIP is not required for homology-directed DNA repair or tumor suppression.
The CtIP protein facilitates homology-directed repair (HDR) of double-strand DNA breaks (DSBs) by initiating DNA resection, a process in which DSB ends are converted into 3′-ssDNA overhangs. The BRCA1 tumor suppressor, which interacts with CtIP in a phospho-dependent manner, has also been implicated in DSB repair through the HDR pathway. It was recently reported that the BRCA1–CtIP interaction is essential for HDR in chicken DT40 cells. To examine the role of this interaction in mammalian cells, we generated cells and mice that express Ctip polypeptides (Ctip-S326A) that fail to bind BRCA1. Surprisingly, isogenic lines of Ctip-S326A mutant and wild-type cells displayed comparable levels of HDR function and chromosomal stability. Although Ctip-S326A mutant cells were modestly sensitive to topoisomerase inhibitors, mice expressing Ctip-S326A polypeptides developed normally and did not exhibit a predisposition to cancer. Thus, in mammals, the phospho-dependent BRCA1–CtIP interaction is not essential for HDR-mediated DSB repair or for tumor suppression.
Live-cell imaging of the exocyst subunit Sec8 reveals how the protein’s spatiotemporal dynamics correlate with its roles in vesicle fusion.
Tethers play ubiquitous roles in membrane trafficking and influence the specificity of vesicle attachment. Unlike soluble N-ethyl-maleimide–sensitive fusion attachment protein receptors (SNAREs), the spatiotemporal dynamics of tethers relative to vesicle fusion are poorly characterized. The most extensively studied tethering complex is the exocyst, which spatially targets vesicles to sites on the plasma membrane. By using a mammalian genetic replacement strategy, we were able to assemble fluorescently tagged Sec8 into the exocyst complex, which was shown to be functional by biochemical, trafficking, and morphological criteria. Ultrasensitive live-cell imaging revealed that Sec8-TagRFP moved to the cell cortex on vesicles, which preferentially originated from the endocytic recycling compartment. Surprisingly, Sec8 remained with vesicles until full dilation of the fusion pore, supporting potential coupling with SNARE fusion machinery. Fluorescence recovery after photobleaching analysis of Sec8 at cell protrusions revealed that a significant fraction was immobile. Additionally, Sec8 dynamically repositioned to the site of membrane expansion, suggesting that it may respond to local cues during early cell polarization.
Dietary cholesterol levels control follicle stem cell proliferation in the Drosophila ovary via regulation of Hedgehog protein localization.
A healthy diet improves adult stem cell function and delays diseases such as cancer, heart disease, and neurodegeneration. Defining molecular mechanisms by which nutrients dictate stem cell behavior is a key step toward understanding the role of diet in tissue homeostasis. In this paper, we elucidate the mechanism by which dietary cholesterol controls epithelial follicle stem cell (FSC) proliferation in the fly ovary. In nutrient-restricted flies, the transmembrane protein Boi sequesters Hedgehog (Hh) ligand at the surface of Hh-producing cells within the ovary, limiting FSC proliferation. Upon feeding, dietary cholesterol stimulates S6 kinase–mediated phosphorylation of the Boi cytoplasmic domain, triggering Hh release and FSC proliferation. This mechanism enables a rapid, tissue-specific response to nutritional changes, tailoring stem cell divisions and egg production to environmental conditions sufficient for progeny survival. If conserved in other systems, this mechanism will likely have important implications for studies on molecular control of stem cell function, in which the benefits of low calorie and low cholesterol diets are beginning to emerge.