RESUMEN
Centrosomes catalyse the formation of microtubules needed to assemble the mitotic spindle apparatus1. Centrosomes themselves duplicate once per cell cycle, in a process that is controlled by the serine/threonine protein kinase PLK4 (refs. 2,3). When PLK4 is chemically inhibited, cell division proceeds without centrosome duplication, generating centrosome-less cells that exhibit delayed, acentrosomal spindle assembly4. Whether PLK4 inhibitors can be leveraged as a treatment for cancer is not yet clear. Here we show that acentrosomal spindle assembly following PLK4 inhibition depends on levels of the centrosomal ubiquitin ligase TRIM37. Low TRIM37 levels accelerate acentrosomal spindle assembly and improve proliferation following PLK4 inhibition, whereas high TRIM37 levels inhibit acentrosomal spindle assembly, leading to mitotic failure and cessation of proliferation. The Chr17q region containing the TRIM37 gene is frequently amplified in neuroblastoma and in breast cancer5-8, rendering these cancer types highly sensitive to PLK4 inhibition. We find that inactivating TRIM37 improves acentrosomal mitosis because TRIM37 prevents PLK4 from self-assembling into centrosome-independent condensates that serve as ectopic microtubule-organizing centres. By contrast, elevated TRIM37 expression inhibits acentrosomal spindle assembly through a distinct mechanism that involves degradation of the centrosomal component CEP192. Thus, TRIM37 is an essential determinant of mitotic vulnerability to PLK4 inhibition. Linkage of TRIM37 to prevalent cancer-associated genomic changes-including 17q gain in neuroblastoma and 17q23 amplification in breast cancer-may offer an opportunity to use PLK4 inhibition to trigger selective mitotic failure and provide new avenues to treatments for these cancers.
Asunto(s)
Mitosis/efectos de los fármacos , Mitosis/genética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos Par 17/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Centro Organizador de los Microtúbulos/efectos de los fármacos , Centro Organizador de los Microtúbulos/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Sulfonas/farmacología , Sulfonas/uso terapéutico , Ubiquitina/metabolismo , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cell surface area rapidly increases during mechanical and hypoosmotic stresses. Such expansion of the plasma membrane requires 'membrane reservoirs' that provide surface area and buffer membrane tension, but the sources of this membrane remain poorly understood. In principle, the flattening of invaginations and buds within the plasma membrane could provide this additional surface area, as recently shown for caveolae in animal cells. Here, we used microfluidics to study the rapid expansion of the yeast plasma membrane in protoplasts, which lack the rigid cell wall. To survive hypoosmotic stress, yeast cell protoplasts required eisosomes, protein-based structures that generate long invaginations at the plasma membrane. Both budding yeast and fission yeast protoplasts lacking eisosomes were unable to expand like wild-type protoplasts during hypoosmotic stress, and subsequently lysed. By performing quantitative fluorescence microscopy on single protoplasts, we also found that eisosomes disassembled as surface area increased. During this process, invaginations generated by eisosomes at the plasma membrane became flattened, as visualized by scanning electron microscopy. We propose that eisosomes serve as tension-dependent membrane reservoirs for expansion of yeast cells in an analogous manner to caveolae in animal cells.
Asunto(s)
Protoplastos/metabolismo , Levaduras/metabolismo , Animales , Membrana Celular/metabolismo , Modelos BiológicosRESUMEN
Eisosomes are multiprotein structures that generate linear invaginations at the plasma membrane of yeast cells. The core component of eisosomes, the BAR domain protein Pil1, generates these invaginations through direct binding to lipids including phosphoinositides. Eisosomes promote hydrolysis of phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) by functioning with synaptojanin, but the cellular processes regulated by this pathway have been unknown. Here, we found that PI(4,5)P2 regulation by eisosomes inhibits the cell integrity pathway, a conserved MAPK signal transduction cascade. This pathway is activated by multiple environmental conditions including osmotic stress in the fission yeast Schizosaccharomyces pombe. Activation of the MAPK Pmk1 was impaired by mutations in the phosphatidylinositol (PI) 5-kinase Its3, but this defect was suppressed by removal of eisosomes. Using fluorescent biosensors, we found that osmotic stress induced the formation of PI(4,5)P2 clusters that were spatially organized by eisosomes in both fission yeast and budding yeast cells. These cortical clusters contained the PI 5-kinase Its3 and did not assemble in the its3-1 mutant. The GTPase Rho2, an upstream activator of Pmk1, also co-localized with PI(4,5)P2 clusters under osmotic stress, providing a molecular link between these novel clusters and MAPK activation. Our findings have revealed that eisosomes regulate activation of MAPK signal transduction through the organization of cortical lipid-based microdomains.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Orgánulos/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Schizosaccharomyces/metabolismo , Presión Osmótica , Schizosaccharomyces/enzimologíaRESUMEN
Stable compartments of the plasma membrane promote a wide range of cellular functions. In yeast cells, cytosolic structures called eisosomes generate prominent cortical invaginations of unknown function. Through a series of genetic screens in fission yeast, we found that the eisosome proteins Pil1 and Sle1 function with the synaptojanin-like lipid phosphatase Syj1 and its ligand Tax4. This genetic pathway connects eisosome function with the hydrolysis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] in cells. Defects in PI(4,5)P2 regulation led to eisosome defects, and we found that the core eisosome protein Pil1 can bind to and tubulate liposomes containing PI(4,5)P2. Mutations in components of the Pil1-Sle1-Syj1-Tax4 pathway suppress the growth and morphology defects of TORC2 mutants, indicating that eisosome-dependent regulation of PI(4,5)P2 feeds into signal transduction pathways. We propose that the geometry of membrane invaginations generates spatial and temporal signals for lipid-mediated signaling events in cells.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Liposomas , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/metabolismo , Transporte de Proteínas , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington's disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms. IMPORTANCE: Polyglutamine (polyQ) proteins encoded by repetitive CAG DNA sequences serve a variety of normal biological functions. Yet some proteins with abnormally expanded polyQ regions cause neurodegeneration through unknown mechanisms. To study how distinct cellular environments modulate polyQ aggregation and toxicity, we expressed CAG-expanded huntingtin fragments in Schizosaccharomyces pombe In stark contrast to many other eukaryotes, S. pombe is uniquely devoid of proteins containing long polyQ tracts. Our results show that S. pombe cells, despite their low content of endogenous polyQ proteins, exhibit striking and unexpected resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the emergence and expansion of polyQ domains in eukaryotic evolution.
RESUMEN
Cytoskeletal polymers are organized into a wide variety of higher-order structures in cells. The yeast BAR domain protein Pil1 self-assembles into tubules in vitro, and forms linear polymers at cortical eisosomes in cells. In the fission yeast S. pombe, over-expressed Pil1 forms thick rods that detach from the plasma membrane. In this study, we used thin-section electron microscopy to determine the ultrastructure of these cytoplasmic Pil1 rods. We found that cytoplasmic rods contained crosslinked Pil1 tubules that displayed regular, hexagonal spacing. These bundles were stained by filipin, a sterol-binding fluorescent dye, suggesting that they contained lipids. Cytoplasmic Pil1 rods were present but less abundant in sle1Δ and fhn1Δ mutant cells. We also found that endogenous Pil1 formed thick rods under saturated growth conditions. Taken together, our findings suggest the presence of cellular mechanisms that assemble Pil1 tubules into higher-order structures.
RESUMEN
Ploidy variation is found in contexts as diverse as solid tumors, drug resistance in fungal infection, and normal development. Altering chromosome or genome copy number supports adaptation to fluctuating environments but is also associated with fitness defects attributed to protein imbalances. Both aneuploidy and polyploidy can arise from multinucleate states after failed cytokinesis or cell fusion. The consequences of ploidy variation in syncytia are difficult to predict because protein imbalances are theoretically buffered by a common cytoplasm. We examined ploidy in a naturally multinucleate fungus, Ashbya gossypii. Using integrated lac operator arrays, we found that chromosome number varies substantially among nuclei sharing a common cytoplasm. Populations of nuclei range from 1N to >4N, with different polyploidies in the same cell and low levels of aneuploidy. The degree of ploidy variation increases as cells age. In response to cellular stress, polyploid nuclei diminish and haploid nuclei predominate. These data suggest that mixed ploidy is tolerated in these syncytia; however, there may be costs associated with variation as stress homogenizes the genome content of nuclei. Furthermore, the results suggest that sharing of gene products is limited, and thus there is incomplete buffering of ploidy variation despite a common cytosol.
Asunto(s)
Ascomicetos/genética , Ascomicetos/citología , Ascomicetos/crecimiento & desarrollo , Núcleo Celular/genética , Segregación Cromosómica , Cromosomas Fúngicos/genética , ADN de Hongos/genética , Proteínas Fúngicas/fisiología , Dosificación de Gen , Genes Fúngicos , Proteínas Mad2/fisiología , Poliploidía , Estrés FisiológicoRESUMEN
The plasma membrane contains both dynamic and static microdomains. Given the growing appreciation of cortical microdomains in cell biology, it is important to determine the organizational principles that underlie assembly of compartmentalized structures at the plasma membrane. The fission yeast plasma membrane is highly compartmentalized by distinct sets of cortical nodes, which control signaling for cell cycle progression and cytokinesis. The mitotic inhibitor Skb1 localizes to a set of cortical nodes that provide spatial control over signaling for entry into mitosis. However, it has been unclear whether these nodes contain other proteins and how they might be organized and tethered to the plasma membrane. Here we show that Skb1 forms nodes by interacting with the novel protein Slf1, which is a limiting factor for node formation in cells. Using quantitative fluorescence microscopy and in vitro assays, we demonstrate that Skb1-Slf1 nodes are megadalton structures that are anchored to the membrane by a lipid-binding region in the Slf1 C-terminus. We propose a mechanism for higher-order node formation by Skb1 and Slf1, with implications for macromolecular assemblies in diverse cell types.
Asunto(s)
Metiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Membrana Celular/metabolismo , Cromatografía Liquida , Secuencia Conservada , Microdominios de Membrana/metabolismo , Metiltransferasas/química , Metiltransferasas/fisiología , Microscopía Fluorescente , Modelos Moleculares , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/fisiología , Schizosaccharomyces/química , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/fisiología , Espectrometría de Masas en TándemRESUMEN
The cortical cytoskeleton mediates a range of cellular activities such as endocytosis, cell motility, and the maintenance of cell rigidity. Traditional polymers, including actin, microtubules, and septins, contribute to the cortical cytoskeleton, but additional filament systems may also exist. In yeast cells, cortical structures called eisosomes generate specialized domains termed MCCs to cluster specific proteins at sites of membrane invaginations. Here we show that the core eisosome protein Pil1 forms linear cortical filaments in fission yeast cells and that purified Pil1 assembles into filaments in vitro. In cells, Pil1 cortical filaments are excluded from regions of cell growth and are independent of the actin and microtubule cytoskeletons. Pil1 filaments assemble slowly at the cell cortex and appear stable by time-lapse microscopy and fluorescence recovery after photobleaching. This stability does not require the cell wall, but Pil1 and the transmembrane protein Fhn1 colocalize and are interdependent for localization to cortical filaments. Increased Pil1 expression leads to cytoplasmic Pil1 rods that are stable and span the length of cylindrical fission yeast cells. We propose that Pil1 is a novel component of the yeast cytoskeleton, with implications for the role of filament assembly in the spatial organization of cells.