RESUMO
The nucleolus, a membrane-less organelle, is responsible for ribosomal RNA transcription, ribosomal RNA processing, and ribosome assembly. Nucleolar size and number are indicative of a cell's protein synthesis rate and proliferative capacity, and abnormalities in the nucleolus have been linked to neurodegenerative diseases and cancer. In this study, we demonstrated that the nucleolar protein ZNF692 directly interacts with nucleophosmin 1 (NPM1). Knocking down ZNF692 resulted in the nucleolar redistribution of NPM1 in ring-like structures and reduced protein synthesis. Purified NPM1 forms spherical condensates in vitro but mixing it with ZNF692 produces irregular condensates more closely resembling living cell nucleoli. Our findings indicate that ZNF692, by interacting with NPM1, plays a critical role in regulating nucleolar architecture and function in living cells.
Assuntos
Nucléolo Celular , Proteínas de Ligação a DNA , Nucleofosmina , Fatores de Transcrição , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Ribossômico/metabolismo , Humanos , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.
Assuntos
Proteínas de Ligação a DNA , Biossíntese de Proteínas , Proteínas Ribossômicas , Subunidades Ribossômicas Menores de Eucariotos , Fatores de Transcrição , Nucléolo Celular/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Humanos , Animais , Ratos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The primary cilium is a nexus for cell signaling and relies on specific protein trafficking for function. The tubby family protein TULP3 transports integral membrane proteins into cilia through interactions with the intraflagellar transport complex-A (IFT-A) and phosphoinositides. It was previously shown that short motifs called ciliary localization sequences (CLSs) are necessary and sufficient for TULP3-dependent ciliary trafficking of transmembrane cargoes. However, the mechanisms by which TULP3 regulates ciliary compartmentalization of nonintegral, membrane-associated proteins and whether such trafficking requires TULP3-dependent CLSs is unknown. Here we show that TULP3 is required for ciliary transport of the Joubert syndrome-linked palmitoylated GTPase ARL13B through a CLS. An N-terminal amphipathic helix, preceding the GTPase domain of ARL13B, couples with the TULP3 tubby domain for ciliary trafficking, irrespective of palmitoylation. ARL13B transport requires TULP3 binding to IFT-A but not to phosphoinositides, indicating strong membrane-proximate interactions, unlike transmembrane cargo transport requiring both properties of TULP3. TULP3-mediated trafficking of ARL13B also regulates ciliary enrichment of farnesylated and myristoylated downstream effectors of ARL13B. The lipidated cargoes show distinctive depletion kinetics from kidney epithelial cilia with relation to Tulp3 deletion-induced renal cystogenesis. Overall, these findings indicate an expanded role of the tubby domain in capturing analogous helical secondary structural motifs from diverse cargoes.
Assuntos
Cílios , Proteínas de Membrana , Cílios/metabolismo , Transporte Proteico , Proteínas de Membrana/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Fosfatidilinositóis/metabolismoRESUMO
The spindle matrix has been proposed to facilitate mitotic spindle assembly. In this issue, Huang et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201706103) show that the spindle matrix protein BuGZ is sufficient to form micron-scale compartments that recruit and activate Aurora A, a critical kinase for spindle assembly.
Assuntos
Aurora Quinase A/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Mitose/fisiologia , Fuso Acromático/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismoRESUMO
Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the pericentriolar material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Ciclo Celular/química , Centríolos/química , Animais , Proteínas de Caenorhabditis elegans/isolamento & purificação , Proteínas de Ciclo Celular/isolamento & purificação , Cromatografia de Afinidade , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Proteínas de Fluorescência Verde/química , Microscopia de Fluorescência , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/isolamento & purificação , Células Sf9 , Quinase 1 Polo-LikeRESUMO
The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Centrossomo/diagnóstico por imagem , Redes e Vias Metabólicas , Fosforilação , Polimerização , Ligação Proteica , Estrutura Terciária de Proteína , Ultrassonografia , Quinase 1 Polo-LikeRESUMO
Dynein is a minus-end-directed microtubule motor important for mitotic spindle positioning. In budding yeast, dynein activity is restricted to anaphase when the nucleus enters the bud neck, yet the nature of the underlying regulatory mechanism is not known. Here, the microtubule-associated protein She1p is identified as a novel regulator of dynein activity. In she1 Delta cells, dynein is activated throughout the cell cycle, resulting in aberrant spindle movements that misposition the spindle. We also found that dynactin, a cofactor essential for dynein motor function, is a dynamic complex whose recruitment to astral microtubules (aMTs) increases dramatically during anaphase. Interestingly, loss of She1p eliminates the cell-cycle regulation of dynactin recruitment and permits enhanced dynactin accumulation on aMTs throughout the cell cycle. Furthermore, localization of the dynactin complex to aMTs requires dynein, suggesting that dynactin is recruited to aMTs via interaction with dynein and not the microtubule itself. Lastly, we present evidence supporting the existence of an incomplete dynactin subcomplex localized at the SPB, and a complete complex that is loaded onto aMTs from the cytoplasm. We propose that She1p restricts dynein-dependent spindle positioning to anaphase by inhibiting the association of dynein with the complete dynactin complex.