RESUMEN
Proper centrosome number and function relies on the accurate assembly of centrioles, barrel-shaped structures that form the core duplicating elements of the organelle. The growth of centrioles is regulated in a cell cycle-dependent manner; while new daughter centrioles elongate during the S/G2/M phase, mature mother centrioles maintain their length throughout the cell cycle. Centriole length is controlled by the synchronized growth of the microtubules that ensheathe the centriole barrel. Although proteins exist that target the growing distal tips of centrioles, such as CP110 and Cep97, these proteins are generally thought to suppress centriolar microtubule growth, suggesting that distal tips may also contain unidentified counteracting factors that facilitate microtubule polymerization. Currently, a mechanistic understanding of how distal tip proteins balance microtubule growth and shrinkage to either promote daughter centriole elongation or maintain centriole length is lacking. Using a proximity-labeling screen in Drosophila cells, we identified Cep104 as a novel component of a group of evolutionarily conserved proteins that we collectively refer to as the distal tip complex (DTC). We found that Cep104 regulates centriole growth and promotes centriole elongation through its microtubule-binding TOG domain. Furthermore, analysis of Cep104 null flies revealed that Cep104 and Cep97 cooperate during spermiogenesis to align spermatids and coordinate individualization. Lastly, we mapped the complete DTC interactome and showed that Cep97 is the central scaffolding unit required to recruit DTC components to the distal tip of centrioles.
Asunto(s)
Centriolos , Proteínas Asociadas a Microtúbulos , Masculino , Animales , Centriolos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Drosophila/metabolismo , Centrosoma/metabolismo , Espermatogénesis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
Polo-like kinase 4 (Plk4) is the master-regulator of centriole assembly, and cell cycle-dependent regulation of its activity maintains proper centrosome number. During most of the cell cycle, Plk4 levels are nearly undetectable due to its ability to autophosphorylate and trigger its own ubiquitin-mediated degradation. However, during mitotic exit, Plk4 forms a single aggregate on the centriole surface to stimulate centriole duplication. Whereas most Polo-like kinase family members are monomeric, Plk4 is unique because it forms homodimers. Notably, Plk4 trans-autophosphorylates a degron near its kinase domain, a critical step in autodestruction. While it is thought that the purpose of homodimerization is to promote trans-autophosphorylation, this has not been tested. Here, we generated separation-of-function Plk4 mutants that fail to dimerize and show that homodimerization creates a binding site for the Plk4 activator, Asterless. Surprisingly, however, Plk4 dimer mutants are catalytically active in cells, promote centriole assembly, and can trans-autophosphorylate through concentration-dependent condensate formation. Moreover, we mapped and then deleted the weak-interacting regions within Plk4 that mediate condensation and conclude that dimerization and condensation are not required for centriole assembly. Our findings suggest that Plk4 dimerization and condensation function simply to down-regulate Plk4 and suppress centriole overduplication.
Asunto(s)
Proteínas de Ciclo Celular , Centriolos , Centriolos/metabolismo , Dimerización , Línea Celular , Proteínas de Ciclo Celular/metabolismo , Centrosoma/metabolismo , FosforilaciónRESUMEN
Barrier-to-autointegration factor (BAF/BANF) is a nuclear lamina protein essential for nuclear integrity, chromatin structure, and genome stability. Whereas complete loss of BAF causes lethality in multiple organisms, the A12T missense mutation of the BANF1 gene in humans causes a premature aging syndrome, called Néstor-Guillermo Progeria Syndrome (NGPS). Here, we report the first in vivo animal investigation of progeroid BAF, using CRISPR editing to introduce the NGPS mutation into the endogenous Drosophila baf gene. Progeroid BAF adults are born at expected frequencies, demonstrating that this BAF variant retains some function. However, tissue homeostasis is affected, supported by studies of the ovary, a tissue that depends upon BAF for stem cell survival and continuous oocyte production. We find that progeroid BAF causes defects in germline stem cell mitosis that delay anaphase progression and compromise chromosome segregation. We link these defects to decreased recruitment of centromeric proteins of the kinetochore, indicating dysfunction of cenBAF, a localized pool of dephosphorylated BAF produced by Protein Phosphatase PP4. We show that DNA damage increases in progenitor germ cells, which causes germ cell death due to activation of the DNA damage transducer kinase Chk2. Mitotic defects appear widespread, as aberrant chromosome segregation and increased apoptosis occur in another tissue. Together, these data highlight the importance of BAF in establishing centromeric structures critical for mitosis. Further, these studies link defects in cenBAF function to activation of a checkpoint that depletes progenitor reserves critical for tissue homeostasis, aligning with phenotypes of NGPS patients.
Asunto(s)
Drosophila , Progeria , Animales , Femenino , Humanos , Drosophila/metabolismo , Progeria/genética , Progeria/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Nucleares/metabolismo , Centrómero/metabolismo , Homeostasis/genéticaRESUMEN
Polo-like kinase 4 (Plk4) is the master regulator of centriole assembly. Several evolutionarily conserved mechanisms strictly regulate Plk4 abundance and activity to ensure cells maintain a proper number of centrioles. In this issue of Genes & Development, Phan et al. (pp. 718-736) add to this growing list by describing a new mechanism of control that restricts Plk4 translation through competitive ribosome binding at upstream open reading frames (uORFs) in the mature Plk4 mRNA. Fascinatingly, this mechanism is especially critical in the development of primordial germ cells in mice that are transcriptionally hyperactive and thus exquisitely sensitive to Plk4 mRNA regulation.
Asunto(s)
Proteínas de Ciclo Celular , Centriolos , Animales , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The microenvironment of solid tumors is dynamic and frequently contains pockets of low oxygen levels (hypoxia) surrounded by oxygenated tissue. Indeed, a compromised vasculature is a hallmark of the tumor microenvironment, creating both spatial gradients and temporal variability in oxygen availability. Notably, hypoxia associates with increased metastasis and poor survival in patients. Therefore, to aid therapeutic decisions and better understand hypoxia's role in cancer progression, it is critical to identify endogenous biomarkers of hypoxia to spatially phenotype oncogenic lesions in human tissue, whether precancerous, benign, or malignant. Here, we characterize the glucose transporter GLUT3/SLC2A3 as a biomarker of hypoxic prostate epithelial cells and prostate tumors. Transcriptomic analyses of non-tumorigenic, immortalized prostate epithelial cells revealed a highly significant increase in GLUT3 expression under hypoxia. Additionally, GLUT3 protein increased 2.4-fold in cultured hypoxic prostate cell lines and was upregulated within hypoxic regions of xenograft tumors, including two patient-derived xenografts (PDX). Finally, GLUT3 out-performs other established hypoxia markers; GLUT3 staining in PDX specimens detects 2.6-8.3 times more tumor area compared to a mixture of GLUT1 and CA9 antibodies. Therefore, given the heterogeneous nature of tumors, we propose adding GLUT3 to immunostaining panels when trying to detect hypoxic regions in prostate samples.
RESUMEN
The centrosome is a tiny cytoplasmic organelle that organizes and constructs massive molecular machines to coordinate diverse cellular processes. Due to its many roles during both interphase and mitosis, maintaining centrosome homeostasis is essential to normal health and development. Centrosome instability, divergence from normal centrosome number and structure, is a common pathognomonic cellular state tightly associated with cancers and other genetic diseases. As novel connections are investigated linking the centrosome to disease, it is critical to understand the breadth of centrosome functions to inspire discovery. In this review, we provide an introduction to normal centrosome function and highlight recent discoveries that link centrosome instability to specific disease states.
Asunto(s)
Centrosoma/fisiología , Inestabilidad Cromosómica/genética , Animales , Enfermedades Genéticas Congénitas/genética , Humanos , Interfase/genética , Mitosis/genética , Neoplasias/genética , Orgánulos/genéticaRESUMEN
Anticancer agents often cause drug-induced tetraploidy (DIT) in cancer cells. DIT is not only a mechanism of inherited drug resistance, but proliferating DIT cells can produce progeny with increased ploidy or aneuploid genomes that drive aggressive disease. Here, we explore combinatorial therapeutic strategies for either preventing or eliminating DIT cells.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Segregación Cromosómica/efectos de los fármacos , Linfoma no Hodgkin/tratamiento farmacológico , Tetraploidía , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Inestabilidad Cromosómica/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Humanos , Linfoma no Hodgkin/genéticaRESUMEN
During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL. Here, we show that binding of Ana2 to the Sas4 G-box enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2-G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation.
Asunto(s)
Proteínas de Ciclo Celular/genética , Centriolos/genética , Proteínas de Drosophila/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Ciclo Celular/genética , Línea Celular , Drosophila melanogaster/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Asociadas a Microtúbulos/genética , Fosforilación/genética , Unión Proteica/genéticaRESUMEN
Localized, nonindolent prostate cancer (PCa) is characterized by large-scale genomic rearrangements, aneuploidy, chromothripsis, and other forms of chromosomal instability (CIN), yet how this occurs remains unclear. A well-established mechanism of CIN is the overproduction of centrosomes, which promotes tumorigenesis in various mouse models. Therefore, we developed a single-cell assay for quantifying centrosomes in human prostate tissue. Surprisingly, centrosome loss-which has not been described in human cancer-was associated with PCa progression. By chemically or genetically inducing centrosome loss in nontumorigenic prostate epithelial cells, mitotic errors ensued, producing aneuploid, and multinucleated cells. Strikingly, transient or chronic centrosome loss transformed prostate epithelial cells, which produced highly proliferative and poorly differentiated malignant tumors in mice. Our findings suggest that centrosome loss could create a cellular crisis with oncogenic potential in prostate epithelial cells.
Asunto(s)
Centrosoma/metabolismo , Inestabilidad Genómica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Aneuploidia , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Fragmentación del ADN , Células Epiteliales/patología , Humanos , Masculino , Ratones , Mitosis/genética , Próstata/patologíaRESUMEN
Defects in mitotic spindle orientation (MSO) disrupt the organization of stem cell niches impacting tissue morphogenesis and homeostasis. Mutations in centrosome genes reduce MSO fidelity, leading to tissue dysplasia and causing several diseases such as microcephaly, dwarfism, and cancer. Whether these mutations perturb spindle orientation solely by affecting astral microtubule nucleation or whether centrosome proteins have more direct functions in regulating MSO is unknown. To investigate this question, we analyzed the consequences of deregulating Plk4 (the master centriole duplication kinase) activity in Drosophila asymmetrically dividing neural stem cells. We found that Plk4 functions upstream of MSO control, orchestrating centriole symmetry breaking and consequently centrosome positioning. Mechanistically, we show that Plk4 acts through Spd2 phosphorylation, which induces centriole release from the apical cortex. Overall, this work not only reveals a role for Plk4 in regulating centrosome function but also links the centrosome biogenesis machinery with the MSO apparatus.
Asunto(s)
Proteínas Cdh1/metabolismo , Centriolos/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células-Madre Neurales/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/fisiología , Animales , Proteínas Cdh1/genética , Ciclo Celular , Células Cultivadas , Centrosoma/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Masculino , Células-Madre Neurales/citología , Fosforilación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Centrosome abnormalities are emerging hallmarks of cancer. The overproduction of centrosomes (known as centrosome amplification) has been reported in a variety of cancers and is currently being explored as a promising target for therapy. However, to understand different types of centrosome abnormalities and their impact on centrosome function during tumor progression, as well as to identify tumor subtypes that would respond to the targeting of a centrosome abnormality, a reliable method for accurately quantifying centrosomes in human tissue samples is needed. Here, we established a method of quantifying centrosomes at a single-cell level in different types of human tissue samples. We tested multiple anti-centriole and pericentriolar-material antibodies to identify bona fide centrosomes and multiplexed these with cell border markers to identify individual cells within the tissue. High-resolution microscopy was used to generate multiple Z-section images, allowing us to acquire whole cell volumes in which to scan for centrosomes. The normal cells within the tissue serve as internal positive controls. Our method provides a simple, accurate way to distinguish alterations in centrosome numbers at the level of single cells.
Asunto(s)
Centrosoma/patología , Centrosoma/fisiología , Análisis de la Célula Individual/métodos , Proteínas Portadoras/análisis , Centriolos/patología , Centrosoma/metabolismo , Humanos , Neoplasias/metabolismo , Tubulina (Proteína)/análisisRESUMEN
We genetically characterized the synaptic role of the Drosophila homologue of human DCAF12, a putative cofactor of Cullin4 (Cul4) ubiquitin ligase complexes. Deletion of Drosophila DCAF12 impairs larval locomotion and arrests development. At larval neuromuscular junctions (NMJs), DCAF12 is expressed presynaptically in synaptic boutons, axons, and nuclei of motor neurons. Postsynaptically, DCAF12 is expressed in muscle nuclei and facilitates Cul4-dependent ubiquitination. Genetic experiments identified several mechanistically independent functions of DCAF12 at larval NMJs. First, presynaptic DCAF12 promotes evoked neurotransmitter release. Second, postsynaptic DCAF12 negatively controls the synaptic levels of the glutamate receptor subunits GluRIIA, GluRIIC, and GluRIID. The down-regulation of synaptic GluRIIA subunits by nuclear DCAF12 requires Cul4. Third, presynaptic DCAF12 is required for the expression of synaptic homeostatic potentiation. We suggest that DCAF12 and Cul4 are critical for normal synaptic function and plasticity at larval NMJs.
Asunto(s)
Proteínas Cullin/metabolismo , Proteínas de Drosophila/metabolismo , Homeostasis , Unión Neuromuscular/metabolismo , Plasticidad Neuronal , Neurotransmisores/metabolismo , Animales , Proteínas Cullin/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Larva/genética , Larva/metabolismo , Unión Neuromuscular/genética , Neurotransmisores/genética , Receptores Ionotrópicos de Glutamato/genética , Receptores Ionotrópicos de Glutamato/metabolismo , UbiquitinaciónRESUMEN
Centrosome numerical abnormalities have been reported in a variety of tumors. Centrosome numbers in cancer cells display both inter-tumor and intra-tumor heterogeneity. The over production of centrosomes (centrosome amplification) is unique in cancer cells and is a promising target for therapy. Thus, a method to quantify centrosome numbers on a single cell level is needed. Here, we describe a protocol to quantify centrosome numbers in formalin fixed paraffin embedded (FFPE) tissue samples by multiplexing antibodies to define bona fide centrosomes and cell borders. Centrosomes in single cells are identified using high resolution immunofluorescent microscopy with Z-sectioning. This protocol is easy to perform and has been used to quantify centrosome numbers on a single cell level in a variety of human tissue samples.
RESUMEN
Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole "targeting-factor." In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phospho-residues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4's kinase domain. Thus, Asl-A's phosphorylation state determines which of Asl-A's two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Centriolos/metabolismo , Drosophila , Fosforilación , Unión ProteicaRESUMEN
Centrosomes are the major microtubule-nucleating and microtubule-organizing centers of cells and play crucial roles in microtubule anchoring, organelle positioning, and ciliogenesis. At the centrosome core lies a tightly associated or "engaged" mother-daughter centriole pair. During mitotic exit, removal of centrosomal proteins pericentrin and Cep215 promotes "disengagement" by the dissolution of intercentriolar linkers, ensuring a single centriole duplication event per cell cycle. Herein, we explore a new mechanism involving vesicular trafficking for the removal of centrosomal Cep215. Using small interfering RNA and CRISPR/Cas9 gene-edited cells, we show that the endocytic protein EHD1 regulates Cep215 transport from centrosomes to the spindle midbody, thus facilitating disengagement and duplication. We demonstrate that EHD1 and Cep215 interact and show that Cep215 displays increased localization to vesicles containing EHD1 during mitosis. Moreover, Cep215-containing vesicles are positive for internalized transferrin, demonstrating their endocytic origin. Thus, we describe a novel relationship between endocytic trafficking and the centrosome cycle, whereby vesicles of endocytic origin are used to remove key regulatory proteins from centrosomes to control centriole duplication.
Asunto(s)
Centriolos/metabolismo , Vesículas Citoplasmáticas/metabolismo , Antígenos/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Citocinesis , Endocitosis , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transporte de Proteínas , Transferrina/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 is then phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STil/ANa2 (STAN) domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N terminus of Ana2, which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Centriolos/enzimología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Centriolos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Transducción de SeñalRESUMEN
Basal cells in a simple secretory epithelium adhere to the extracellular matrix (ECM), providing contextual cues for ordered repopulation of the luminal cell layer. Early high-grade prostatic intraepithelial neoplasia (HG-PIN) tissue has enlarged nuclei and nucleoli, luminal layer expansion and genomic instability. Additional HG-PIN markers include loss of α6ß4 integrin or its ligand laminin-332, and budding of tumor clusters into laminin-511-rich stroma. We modeled the invasive budding phenotype by reducing expression of α6ß4 integrin in spheroids formed from two normal human stable isogenic prostate epithelial cell lines (RWPE-1 and PrEC 11220). These normal cells continuously spun in culture, forming multicellular spheroids containing an outer laminin-332 layer, basal cells (expressing α6ß4 integrin, high-molecular-weight cytokeratin and p63, also known as TP63) and luminal cells that secrete PSA (also known as KLK3). Basal cells were optimally positioned relative to the laminin-332 layer as determined by spindle orientation. ß4-integrin-defective spheroids contained a discontinuous laminin-332 layer corresponding to regions of abnormal budding. This 3D model can be readily used to study mechanisms that disrupt laminin-332 continuity, for example, defects in the essential adhesion receptor (ß4 integrin), laminin-332 or abnormal luminal expansion during HG-PIN progression.
Asunto(s)
Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/patología , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Humanos , Integrina alfa6beta4/metabolismo , Masculino , Modelos Biológicos , Morfogénesis , Clasificación del Tumor , Invasividad Neoplásica , Fenotipo , Próstata/metabolismo , Próstata/patología , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , KalininaRESUMEN
The centrosome is the major microtubule-organizing centre of many cells, best known for its role in mitotic spindle organization. How the proteins of the centrosome are accurately assembled to carry out its many functions remains poorly understood. The non-membrane-bound nature of the centrosome dictates that protein-protein interactions drive its assembly and functions. To investigate this massive macromolecular organelle, we generated a 'domain-level' centrosome interactome using direct protein-protein interaction data from a focused yeast two-hybrid screen. We then used biochemistry, cell biology and the model organism Drosophila to provide insight into the protein organization and kinase regulatory machinery required for centrosome assembly. Finally, we identified a novel role for Plk4, the master regulator of centriole duplication. We show that Plk4 phosphorylates Cep135 to properly position the essential centriole component Asterless. This interaction landscape affords a critical framework for research of normal and aberrant centrosomes.
Asunto(s)
Centrosoma/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Duplicación de Gen , Orgánulos/metabolismo , Mapas de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Fosforilación , Unión Proteica , Multimerización de Proteína , Especificidad por SustratoRESUMEN
The spatial organization of the genome within the eukaryotic nucleus is a dynamic process that plays a central role in cellular processes such as gene expression, DNA replication, and chromosome segregation. Condensins are conserved multi-subunit protein complexes that contribute to chromosome organization by regulating chromosome compaction and homolog pairing. Previous work in our laboratory has shown that the Cap-H2 subunit of condensin II physically and genetically interacts with the Drosophila homolog of human MORF4-related gene on chromosome 15 (MRG15). Like Cap-H2, Mrg15 is required for interphase chromosome compaction and homolog pairing. However, the mechanism by which Mrg15 and Cap-H2 cooperate to maintain interphase chromatin organization remains unclear. Here, we show that Cap-H2 localizes to interband regions on polytene chromosomes and co-localizes with Mrg15 at regions of active transcription across the genome. We show that co-localization of Cap-H2 on polytene chromosomes is partially dependent on Mrg15. We have identified a binding motif within Cap-H2 that is essential for its interaction with Mrg15, and have found that mutation of this motif results in loss of localization of Cap-H2 on polytene chromosomes and results in partial suppression of Cap-H2-mediated compaction and homolog unpairing. Our data are consistent with a model in which Mrg15 acts as a loading factor to facilitate Cap-H2 binding to chromatin and mediate changes in chromatin organization.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Interfase/genética , Complejos Multiproteicos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Adenosina Trifosfatasas/química , Secuencias de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/química , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Expresión Génica , Genes Reporteros , Genoma , Complejos Multiproteicos/química , Mutación , Cromosomas Politénicos , Unión Proteica , Transporte de Proteínas , Transcripción Genética , Activación TranscripcionalRESUMEN
Polo-like kinase 4 (Plk4) is a master regulator of centriole duplication, and its hyperactivity induces centriole amplification. Homodimeric Plk4 has been shown to be ubiquitinated as a result of autophosphorylation, thus promoting its own degradation and preventing centriole amplification. Unlike other Plks, Plk4 contains three rather than two Polo box domains, and the function of its third Polo box (PB3) is unclear. Here, we performed a functional analysis of Plk4's structural domains. Like other Plks, Plk4 possesses a previously unidentified autoinhibitory mechanism mediated by a linker (L1) near the kinase domain. Thus, autoinhibition is a conserved feature of Plks. In the case of Plk4, autoinhibition is relieved after homodimerization and is accomplished by PB3 and by autophosphorylation of L1. In contrast, autophosphorylation of the second linker promotes separation of the Plk4 homodimer. Therefore, autoinhibition delays the multiple consequences of activation until Plk4 dimerizes. These findings reveal a complex mechanism of Plk4 regulation and activation which govern the process of centriole duplication.