RESUMO
Proper connection between the sperm head and tail is critical for sperm motility and fertilization. Head-tail linkage is mediated by the Head-Tail Coupling Apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is poorly understood. Here, we use Drosophila to investigate formation and remodeling of the HTCA throughout spermiogenesis by visualizing key components of this complex. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a 'Centriole Cap' that surrounds the centriole (or Basal Body) as it invaginates into the surface of the nucleus. As development progresses, the centriole is laterally displaced to the side of the nucleus while the HTCA expands under the nucleus, forming what we term the 'Nuclear Shelf.' We next show that the proximal centriole-like (PCL) structure is positioned under the Nuclear Shelf, functioning as a critical stabilizer of centriole-nuclear attachment. Together, our data indicate that the HTCA is a complex, multi-point attachment site that simultaneously engages the PCL, the centriole, and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis.
RESUMO
Proper connection between the sperm head and tail is critical for sperm motility and fertilization. The link between the head and tail is mediated by the Head-Tail Coupling Apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is not well understood. Here, we use Drosophila to create a high-resolution map of proteins and structures at the HTCA throughout spermiogenesis. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a 'Centriole Cap' that surrounds the centriole (or Basal Body) as it is inserted, or embedded into the surface of the nucleus. As development progresses, the centriole is laterally displaces to the side of the nucleus, during which time the HTCA expands under the nucleus, forming what we term the 'Nuclear Shelf.' We next show that the proximal centriole-like (PCL) structure is positioned under the Nuclear Shelf and functions as a critical stabilizer of the centriole-nuclear attachment. Together, our data indicate that the HTCA is complex, multi-point attachment site that simultaneously engages the PCL, the centriole, and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis.
RESUMO
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.
Assuntos
Centríolos , Proteínas Associadas aos Microtúbulos , Masculino , Animais , Centríolos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Drosophila/metabolismo , Centrossomo/metabolismo , Espermatogênese , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Centrosomes are essential parts of diverse cellular processes, and precise regulation of the levels of their constituent proteins is critical for their function. One such protein is Pericentrin (PCNT) in humans and Pericentrin-like protein (PLP) in Drosophila. Increased PCNT expression and its protein accumulation are linked to clinical conditions including cancer, mental disorders, and ciliopathies. However, the mechanisms by which PCNT levels are regulated remain underexplored. Our previous study demonstrated that PLP levels are sharply down-regulated during early spermatogenesis and this regulation is essential to spatially position PLP on the proximal end of centrioles. We hypothesized that the sharp drop in PLP protein was a result of rapid protein degradation during the male germ line premeiotic G2 phase. Here, we show that PLP is subject to ubiquitin-mediated degradation and identify multiple proteins that promote the reduction of PLP levels in spermatocytes, including the UBR box containing E3 ligase Poe (UBR4), which we show binds to PLP. Although protein sequences governing posttranslational regulation of PLP are not restricted to a single region of the protein, we identify a region that is required for Poe-mediated degradation. Experimentally stabilizing PLP, via internal PLP deletions or loss of Poe, leads to PLP accumulation in spermatocytes, its mispositioning along centrioles, and defects in centriole docking in spermatids.
Assuntos
Centríolos , Ubiquitina-Proteína Ligases , Masculino , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Antígenos/metabolismoRESUMO
Centrosomes are multi-protein organelles that function as microtubule (MT) organizing centers (MTOCs), ensuring spindle formation and chromosome segregation during cell division.1,2,3 Centrosome structure includes core centrioles that recruit pericentriolar material (PCM) that anchors γ-tubulin to nucleate MTs.1,2 In Drosophila melanogaster, PCM organization depends on proper regulation of proteins like Spd-2, which dynamically localizes to centrosomes and is required for PCM, γ-tubulin, and MTOC activity in brain neuroblast (NB) mitosis and male spermatocyte (SC) meiosis.4,5,6,7,8 Some cells have distinct requirements for MTOC activity due to differences in characteristics like cell size9,10 or whether they are mitotic or meiotic.11,12 How centrosome proteins achieve cell-type-specific functional differences is poorly understood. Previous work identified alternative splicing13 and binding partners14 as contributors to cell-type-specific differences in centrosome function. Gene duplication, which can generate paralogs with specialized functions,15,16 is also implicated in centrosome gene evolution,17 including cell-type-specific centrosome genes.18,19 To gain insight into cell-type-specific differences in centrosome protein function and regulation, we investigated a duplication of Spd-2 in Drosophila willistoni, which has Spd-2A (ancestral) and Spd-2B (derived). We find that Spd-2A functions in NB mitosis, whereas Spd-2B functions in SC meiosis. Ectopically expressed Spd-2B accumulates and functions in mitotic NBs, but ectopically expressed Spd-2A failed to accumulate in meiotic SCs, suggesting cell-type-specific differences in translation or protein stability. We mapped this failure to accumulate and function in meiosis to the C-terminal tail domain of Spd-2A, revealing a novel regulatory mechanism that can potentially achieve differences in PCM function across cell types.
Assuntos
Proteínas do Citoesqueleto , Proteínas de Drosophila , Drosophila , Duplicação Gênica , Tubulina (Proteína) , Animais , Masculino , Centríolos/genética , Centríolos/metabolismo , Centrossomo/metabolismo , Drosophila/genética , Drosophila/metabolismo , Meiose , Mitose , Tubulina (Proteína)/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas de Drosophila/genéticaRESUMO
Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosomal microtubules (MTs) with motor proteins anchored at the cortex or the nuclear surface. However, it remains unknown how centrioles migrate in cellular contexts in which they do not nucleate MTs. Here, we demonstrate that during interphase, inactive centrioles move directly along the interphase MT network as Kinesin-1 cargo. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. In vitro assays show that PLP directly interacts with the cargo binding domain of Kinesin-1, allowing PLP to migrate on MTs. Binding assays using purified proteins revealed that relief of Kinesin-1 autoinhibition is critical for its interaction with PLP. Finally, our studies of neural stem cell asymmetric divisions in the Drosophila brain show that the PLP-Kinesin-1 interaction is essential for the timely separation of centrioles, the asymmetry of centrosome activity, and the age-dependent centrosome inheritance.
Assuntos
Antígenos , Centríolos , Cinesinas , Animais , Antígenos/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Células-Tronco Neurais , Transporte ProteicoRESUMO
Given their copy number differences and unique modes of inheritance, the evolved gene content and expression of sex chromosomes is unusual. In many organisms the X and Y chromosomes are inactivated in spermatocytes, possibly as a defense mechanism against insertions into unpaired chromatin. In addition to current sex chromosomes, Drosophila has a small gene-poor X-chromosome relic (4th) that re-acquired autosomal status. Here we use single cell RNA-Seq on fly larvae to demonstrate that the single X and pair of 4th chromosomes are specifically inactivated in primary spermatocytes, based on measuring all genes or a set of broadly expressed genes in testis we identified. In contrast, genes on the single Y chromosome become maximally active in primary spermatocytes. Reduced X transcript levels are due to failed activation of RNA-Polymerase-II by phosphorylation of Serine 2 and 5.
Assuntos
Drosophila/genética , Cromossomos Sexuais/genética , Espermatócitos/metabolismo , Animais , Drosophila/crescimento & desenvolvimento , Regulação da Expressão Gênica , Genes Ligados ao Cromossomo X/genética , Genes Ligados ao Cromossomo Y/genética , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Especificidade de Órgãos , RNA Polimerase II/metabolismo , Cromossomos Sexuais/metabolismo , Espermatogênese/genética , Testículo/citologia , Testículo/metabolismo , Transcrição GênicaRESUMO
The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, we identify a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of pericentrin-like protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage.
Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Microtúbulos/metabolismo , Cabeça do Espermatozoide/metabolismo , Cauda do Espermatozoide/metabolismo , Animais , Corpos Basais/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , MasculinoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/genética , Centríolos/genética , Proteínas de Drosophila/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Ciclo Celular/genética , Linhagem Celular , Drosophila melanogaster/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Associadas aos Microtúbulos/genética , Fosforilação/genética , Ligação Proteica/genéticaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Centríolos/enzimologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Centríolos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Transdução de SinaisRESUMO
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.
Assuntos
Centrossomo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Duplicação Gênica , Organelas/metabolismo , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Fosforilação , Ligação Proteica , Multimerização Proteica , Especificidade por SubstratoRESUMO
Centrioles are the foundation of two organelles, centrosomes and cilia. Centriole numbers and functions are tightly controlled, and mutations in centriole proteins are linked to a variety of diseases, including microcephaly. Loss of the centriole protein Asterless (Asl), the Drosophila melanogaster orthologue of Cep152, prevents centriole duplication, which has limited the study of its nonduplication functions. Here, we identify populations of cells with Asl-free centrioles in developing Drosophila tissues, allowing us to assess its duplication-independent function. We show a role for Asl in controlling centriole length in germline and somatic tissue, functioning via the centriole protein Cep97. We also find that Asl is not essential for pericentriolar material recruitment or centrosome function in organizing mitotic spindles. Lastly, we show that Asl is required for proper basal body function and spermatid axoneme formation. Insights into the role of Asl/Cep152 beyond centriole duplication could help shed light on how Cep152 mutations lead to the development of microcephaly.
Assuntos
Centríolos/metabolismo , Centríolos/fisiologia , Proteínas de Drosophila/metabolismo , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/metabolismo , Animais , Axonema/metabolismo , Axonema/fisiologia , Corpos Basais/metabolismo , Corpos Basais/fisiologia , Proteínas de Ciclo Celular/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Masculino , Mitose/fisiologia , Espermatozoides/fisiologiaRESUMO
Endocytosis mediated by clathrin, a cellular process by which cells internalize membrane receptors and their extracellular ligands, is an important component of cell signaling regulation. Actin polymerization is involved in endocytosis in varying degrees depending on the cellular context. In yeast, clathrin-mediated endocytosis requires a pulse of polymerized actin and its regulators, which recruit and activate the Arp2/3 complex. In this article, we seek to identify the main protein-protein interactions that 1) cause actin and its regulators to appear in pulses, and 2) determine the effects of key mutations and drug treatments on actin and regulator assembly. We perform a joint modeling/experimental study of actin and regulator dynamics during endocytosis in the budding yeast Saccharomyces cerevisiae. We treat both a stochastic model that grows an explicit three-dimensional actin network, and a simpler two-variable Fitzhugh-Nagumo type model. The models include a negative-feedback interaction of F-actin onto the Arp2/3 regulators. Both models explain the pulse time courses and the effects of interventions on actin polymerization: the surprising increase in the peak F-actin count caused by reduced regulator branching activity, the increase in F-actin resulting from slowing of actin disassembly, and the increased Arp2/3 regulator lifetime resulting from latrunculin treatment. In addition, they predict that decreases in the regulator branching activity lead to increases in accumulation of regulators, and we confirmed this prediction with experiments on yeast harboring mutations in the Arp2/3 regulators, using quantitative fluorescence microscopy. Our experimental measurements suggest that the regulators act quasi-independently, in the sense that accumulation of a particular regulator is most strongly affected by mutations of that regulator, as opposed to the others.
Assuntos
Actinas/metabolismo , Endocitose , Retroalimentação Fisiológica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Simulação por Computador , Modelos Biológicos , Mutação/genética , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Processos Estocásticos , Fatores de TempoRESUMO
CP190 is a large, multi-domain protein, first identified as a centrosome protein with oscillatory localization over the course of the cell cycle. During interphase it has a well-established role within the nucleus as a chromatin insulator. Upon nuclear envelope breakdown, there is a striking redistribution of CP190 to centrosomes and the mitotic spindle, in addition to the population at chromosomes. Here, we investigate CP190 in detail by performing domain analysis in cultured Drosophila S2 cells combined with protein structure determination by X-ray crystallography, in vitro biochemical characterization, and in vivo fixed and live imaging of cp190 mutant flies. Our analysis of CP190 identifies a novel N-terminal centrosome and microtubule (MT) targeting region, sufficient for spindle localization. This region consists of a highly conserved BTB domain and a linker region that serves as the MT binding domain. We present the 2.5 Å resolution structure of the CP190 N-terminal 126 amino acids, which adopts a canonical BTB domain fold and exists as a stable dimer in solution. The ability of the linker region to robustly localize to MTs requires BTB domain-mediated dimerization. Deletion of the linker region using CRISPR significantly alters spindle morphology and leads to DNA segregation errors in the developing Drosophila brain neuroblasts. Collectively, we highlight a multivalent MT-binding architecture in CP190, which confers distinct subcellular cytoskeletal localization and function during mitosis.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Nucleares/fisiologia , Fuso Acromático/ultraestrutura , Animais , Núcleo Celular , Centrossomo , Segregação de Cromossomos , Cromossomos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cristalografia por Raios X , DNA/metabolismo , Fuso Acromático/metabolismo , Células-Tronco/metabolismoRESUMO
Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle-dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability.
Assuntos
Centrossomo/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Homeodomínio/metabolismo , Interfase , Sequência de Aminoácidos , Animais , Proteínas de Ligação a Calmodulina , Segregação de Cromossomos , Drosophila melanogaster , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Instabilidade Genômica , Microtúbulos/metabolismo , Mitose , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Transporte ProteicoRESUMO
As a large, nonmembrane bound organelle, the centrosome must rely heavily on protein-protein interactions to assemble itself in the cytoplasm and perform its functions as a microtubule-organizing center. Therefore, to understand how this organelle is built and functions, one must understand the protein-protein interactions made by each centrosome protein. Unfortunately, the highly interconnected nature of the centrosome, combined with its predicted unstructured, coil-rich proteins, has made the use of many standard approaches to studying protein-protein interactions very challenging. The yeast-two hybrid (Y2H) system is well suited for studying the centrosome and is an important complement to other biochemical approaches. In this chapter we describe how to carry out a directed Y2H screen to identify the direct interactions between a given centrosome protein and a library of others. Specifically, we detail using a bioinformatics-based approach (structure prediction programs) to subdivide proteins and screen for interactions using an array-based Y2H approach. We also describe how to use the interaction information garnered from this screen to generate mutations to disrupt specific interactions using mutagenic-PCR and a "reverse" Y2H screen. Finally, we discuss how information from such a screen can be integrated into existing models of centrosome assembly and how it can initiate and guide extensive in vitro and in vivo experimentation to test these models.
Assuntos
Centrossomo/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4's tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl-Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.
Assuntos
Centríolos/metabolismo , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Proteínas Serina-Treonina Quinases/química , Animais , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estabilidade Enzimática , Mitose/genética , Fosforilação , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente PequenoRESUMO
Pericentrin is a critical centrosomal protein required for organizing pericentriolar material (PCM) in mitosis. Mutations in pericentrin cause the human genetic disorder Majewski/microcephalic osteodysplastic primordial dwarfism type II, making a detailed understanding of its regulation extremely important. Germaine to pericentrin's function in organizing PCM is its ability to localize to the centrosome through the conserved C-terminal PACT domain. Here we use Drosophila pericentrin-like-protein (PLP) to understand how the PACT domain is regulated. We show that the interaction of PLP with calmodulin (CaM) at two highly conserved CaM-binding sites in the PACT domain controls the proper targeting of PLP to the centrosome. Disrupting the PLP-CaM interaction with single point mutations renders PLP inefficient in localizing to centrioles in cultured S2 cells and Drosophila neuroblasts. Although levels of PCM are unaffected, it is highly disorganized. We also demonstrate that basal body formation in the male testes and the production of functional sperm does not rely on the PLP-CaM interaction, whereas production of functional mechanosensory neurons does.
Assuntos
Corpos Basais/metabolismo , Calmodulina/metabolismo , Proteínas de Drosophila/metabolismo , Neurônios/fisiologia , Espermatozoides/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ligação a Calmodulina , Linhagem Celular , Centríolos/fisiologia , Drosophila melanogaster , Masculino , Mecanotransdução Celular , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Motilidade dos Espermatozoides , EspermatogêneseRESUMO
Many forms of cellular motility are driven by the growth of branched networks of actin filaments, which push against a membrane. In the dendritic nucleation model, Arp2/3 complex is critical, binding to the side of an existing mother filament, nucleating a new daughter filament, and thus creating a branch. Spatial and temporal regulation of Arp2/3 activity is critical for efficient generation of force and movement. A diverse collection of Arp2/3 regulatory proteins has been identified. They bind to and/or activate Arp2/3 complex via an acidic motif with a conserved tryptophan residue. We tested this model for Arp2/3 regulator function in vivo, by examining the roles of multiple Arp2/3 regulators in endocytosis in living yeast cells. We measured the molecular composition of the actin network in cells with mutations that removed the acidic motifs of the four Arp2/3 regulators previously shown to influence the proper function of the actin network. Unexpectedly, we did not find a simple or direct correlation between defects in patch assembly and movement and changes in the composition and dynamics of dendritic nucleation proteins. Taken together our data does not support the simple hypothesis that the primary role for Arp2/3 regulators is to recruit and activate Arp2/3. Rather our data suggests that these regulators may be playing more subtle roles in establishing functional networks in vivo.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Actinas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Modelos Moleculares , Movimento , Proteínas Mutantes/metabolismo , Mutação/genética , Miosinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Endocytosis includes a number of processes by which cells internalize segments of their plasma membrane, enclosing a wide variety of material from outside the cell. Endocytosis can contribute to uptake of nutrients, regulation of signaling molecules, control of osmotic pressure, and function of synapses. The actin cytoskeleton plays an essential role in several of these processes. Actin assembly can create protrusions that encompass extracellular materials. Actin can also support the processes of invagination of a membrane segment into the cytoplasm, elongation of the invagination, scission of the new vesicle from the plasma membrane, and movement of the vesicle away from the membrane. We briefly discuss various types of endocytosis, including phagocytosis, macropinocytosis, and clathrin-independent endocytosis. We focus mainly on new findings on the relative importance of actin in clathrin-mediated endocytosis (CME) in yeast versus mammalian cells.