RESUMO
Dendritic filopodia are dynamic structures thought to be the precursors of spines during synapse development. Morphological maturation to spines is associated with the stabilization and strengthening of synapses, and can be altered in various neurological disorders. Telencephalin (TLN/intercellular adhesion molecule-5 (ICAM5)) localizes to dendritic filopodia, where it facilitates their formation/maintenance, thereby slowing spine morphogenesis. As spines are largely devoid of TLN, its exclusion from the filopodia surface appears to be required in this maturation process. Using HeLa cells and primary hippocampal neurons, we demonstrate that surface removal of TLN involves internalization events mediated by the small GTPase ADP-ribosylation factor 6 (ARF6), and its activator EFA6A. This endocytosis of TLN affects filopodia-to-spine transition, and requires Rac1-mediated dephosphorylation/release of actin-binding ERM proteins from TLN. At the somato-dendritic surface, TLN and EFA6A are confined to distinct, flotillin-positive membrane subdomains. The co-distribution of TLN with this lipid raft marker also persists during its endosomal targeting to CD63-positive late endosomes. This suggests a specific microenvironment facilitating ARF6-mediated mobilization of TLN that contributes to promotion of dendritic spine development.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Moléculas de Adesão Celular/metabolismo , Dendritos/fisiologia , Espinhas Dendríticas/metabolismo , Endossomos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Pseudópodes/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Microambiente Celular/genética , Microambiente Celular/fisiologia , Dendritos/genética , Dendritos/metabolismo , Espinhas Dendríticas/genética , Espinhas Dendríticas/fisiologia , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Cultura Primária de Células , Transporte Proteico/genética , Pseudópodes/genética , Pseudópodes/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Amyloid ß (Aß) peptides, the primary constituents of senile plaques and a hallmark in Alzheimer's disease pathology, are generated through the sequential cleavage of amyloid precursor protein (APP) by ß-site APP cleaving enzyme 1 (BACE1) and γ-secretase. The early endosome is thought to represent a major compartment for APP processing; however, the mechanisms of how BACE1 encounters APP are largely unknown. In contrast to APP internalization, which is clathrin-dependent, we demonstrate that BACE1 is sorted to early endosomes via a route controlled by the small GTPase ADP ribosylation factor 6 (ARF6). Altering ARF6 levels or its activity affects endosomal sorting of BACE1, and consequently results in altered APP processing and Aß production. Furthermore, sorting of newly internalized BACE1 from ARF6-positive towards RAB GTPase 5 (RAB5)-positive early endosomes depends on its carboxyterminal short acidic cluster-dileucine motif. This ARF6-mediated sorting of BACE1 is confined to the somatodendritic compartment of polarized neurons in agreement with Aß peptides being primarily secreted from here. These results demonstrate a spatial separation between APP and BACE1 during surface-to-endosome transport, suggesting subcellular trafficking as a regulatory mechanism for this proteolytic processing step. It thereby provides a novel avenue to interfere with Aß production through a selective modulation of the distinct endosomal transport routes used by BACE1 or APP.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endossomos/enzimologia , Processamento de Proteína Pós-Traducional , Fator 6 de Ribosilação do ADP , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Animais , Antígenos CD59/metabolismo , Compartimento Celular , Polaridade Celular , Dendritos/metabolismo , Endocitose , Células HeLa , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Leucina/metabolismo , Camundongos , Modelos Biológicos , Transporte Proteico , Ratos , Receptores da Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
In recent years, Drosophila melanogaster has emerged as a powerful model for neuronal circuit development, pathology, and function. A major impediment to these studies has been the lack of a genetically encoded, specific, universal, and phenotypically neutral marker of the somatodendritic compartment. We have developed such a marker and show that it is effective and specific in all neuronal populations tested in the peripheral and central nervous system. The marker, which we name DenMark (Dendritic Marker), is a hybrid protein of the mouse protein ICAM5/Telencephalin and the red fluorescent protein mCherry. We show that DenMark is a powerful tool for revealing novel aspects of the neuroanatomy of developing dendrites, identifying previously unknown dendritic arbors, and elucidating neuronal connectivity.
Assuntos
Dendritos/genética , Drosophila melanogaster/genética , Marcadores Genéticos/genética , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Proteínas Recombinantes de Fusão/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Eletrorretinografia , Hipocampo/citologia , Imuno-Histoquímica , Proteínas Luminescentes/genética , Glicoproteínas de Membrana/genética , Camundongos , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Proteínas Recombinantes de Fusão/genética , Proteína Vermelha FluorescenteRESUMO
Mitotic spindle assembly is mediated by two processes: a centrosomal and a chromosomal pathway. RanGTP regulates the latter process by releasing microtubule-associated proteins from inhibitory complexes. NuSAP, a microtubule- and DNA-binding protein, is a target of RanGTP and promotes the formation of microtubules near chromosomes. However, the contribution of NuSAP to cell proliferation in vivo is unknown. Here, we demonstrate that the expression of NuSAP highly correlates with cell proliferation during embryogenesis and adult life, making it a reliable marker of proliferating cells. Additionally, we show that NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. As a result of sustained spindle checkpoint activity, the cells are unable to progress through mitosis, eventually leading to caspase activation and apoptotic cell death. Together, our findings demonstrate that NuSAP is essential for proliferation of embryonic cells and, simultaneously, they underscore the importance of chromatin-induced spindle assembly.
Assuntos
Biomarcadores/metabolismo , Cromatina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Osso e Ossos , Proliferação de Células , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/genética , Fuso Acromático/genéticaRESUMO
The gamma-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this "proteasome of the membrane" are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of gamma-secretase subcomplexes and, concomitantly, total cellular gamma-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates gamma-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular gamma-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/química , Animais , Ligação Competitiva , Regulação para Baixo , Endopeptidases , Retículo Endoplasmático/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Peptídeo Hidrolases , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The spindle apparatus is a microtubule (MT)-based machinery that attaches to and segregates the chromosomes during mitosis and meiosis. Self-organization of the spindle around chromatin involves the assembly of MTs, their attachment to the chromosomes, and their organization into a bipolar array. One regulator of spindle self-organization is RanGTP. RanGTP is generated at chromatin and activates a set of soluble, Ran-regulated spindle factors such as TPX2, NuMA, and NuSAP . How the spindle factors direct and attach MTs to the chromosomes are key open questions. Nucleolar and Spindle-Associated Protein (NuSAP) was recently identified as an essential MT-stabilizing and bundling protein that is enriched at the central part of the spindle . Here, we show by biochemical reconstitution that NuSAP efficiently adsorbs to isolated chromatin and DNA and that it can directly produce and retain high concentrations of MTs in the immediate vicinity of chromatin or DNA. Moreover, our data reveal that NuSAP-chromatin interaction is subject to Ran regulation and can be suppressed by Importin alpha (Impalpha) and Imp7. We propose that the presence of MT binding agents such as NuSAP, which can be directly immobilized on chromatin, are critical for targeting MT production to vertebrate chromosomes during spindle self-organization.
Assuntos
Cromossomos de Mamíferos/metabolismo , Cromossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Linhagem Celular , DNA/metabolismo , Células HeLa , Humanos , Carioferinas/metabolismo , Camundongos , Mitose , Oócitos/citologia , Oócitos/metabolismo , Proteínas Recombinantes/metabolismo , Xenopus laevis/metabolismo , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Here, we report on the identification of nucleolar spindle-associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/genética , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Linhagem Celular , Nucléolo Celular/genética , Núcleo Celular/genética , Núcleo Celular/patologia , Segregação de Cromossomos/genética , DNA Complementar/análise , Células Eucarióticas/metabolismo , Fase G2/genética , Camundongos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Fuso Acromático/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Neuron. 32:579-589). Here, we demonstrate that TLN is not a substrate for gamma-secretase cleavage, but displays a prolonged half-life in PS1(-/-) hippocampal neurons. TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3. Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from gamma-secretase activity. Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles. TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations. Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation. Collectively, PS1 deficiency affects in a gamma-secretase-independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes.
Assuntos
Autofagia/fisiologia , Hipocampo/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Actinas/genética , Actinas/metabolismo , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases , Proteína 12 Relacionada à Autofagia , Catepsina D/genética , Moléculas de Adesão Celular , Endopeptidases/genética , Endopeptidases/metabolismo , Endossomos/genética , Endossomos/metabolismo , Células HeLa , Hipocampo/ultraestrutura , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neurônios/ultraestrutura , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Presenilina-1 , Proteínas/genética , Proteínas/metabolismo , Transdução de Sinais/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) alpha, Impbeta, and Imp7. While Impalpha and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impbeta specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.
Assuntos
Microtúbulos/fisiologia , Oócitos/fisiologia , Xenopus laevis/fisiologia , Proteína ran de Ligação ao GTP/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Meiose , Microtúbulos/ultraestrutura , Oócitos/citologia , Transporte Proteico , Interferência de RNA , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/metabolismo , Proteína ran de Ligação ao GTP/genéticaRESUMO
Cilia project from the surface of most vertebrate cells and are important for several physiological and developmental processes. Ciliary defects are linked to a variety of human diseases, named ciliopathies, underscoring the importance of understanding signaling pathways involved in cilia formation and maintenance. In this paper, we identified Rer1p as the first endoplasmic reticulum/cis-Golgi-localized membrane protein involved in ciliogenesis. Rer1p, a protein quality control receptor, was highly expressed in zebrafish ciliated organs and regulated ciliary structure and function. Both in zebrafish and mammalian cells, loss of Rer1p resulted in the shortening of cilium and impairment of its motile or sensory function, which was reflected by hearing, vision, and left-right asymmetry defects as well as decreased Hedgehog signaling. We further demonstrate that Rer1p depletion reduced ciliary length and function by increasing γ-secretase complex assembly and activity and, consequently, enhancing Notch signaling as well as reducing Foxj1a expression.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Fatores de Transcrição Forkhead/biossíntese , Regulação da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Secretases da Proteína Precursora do Amiloide/genética , Animais , Linhagem Celular , Cílios/genética , Cílios/metabolismo , Fatores de Transcrição Forkhead/genética , Humanos , Glicoproteínas de Membrana/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Suínos , Peixe-Zebra , Proteínas de Peixe-ZebraAssuntos
Animais Geneticamente Modificados , Raquitismo , Absorção , Animais , Osso e Ossos/patologia , Calcificação Fisiológica , Cálcio/deficiência , Cálcio/metabolismo , Deficiências Nutricionais/complicações , Hipofosfatemia Familiar/genética , Camundongos , Camundongos Knockout , Receptores de Calcitriol/genética , Raquitismo/genética , Raquitismo/metabolismo , Raquitismo/patologiaRESUMO
ADAM10 is involved in the proteolytic processing and shedding of proteins such as the amyloid precursor protein (APP), cadherins, and the Notch receptors, thereby initiating the regulated intramembrane proteolysis (RIP) of these proteins. Here, we demonstrate that the sheddase ADAM10 is also subject to RIP. We identify ADAM9 and -15 as the proteases responsible for releasing the ADAM10 ectodomain, and Presenilin/gamma-Secretase as the protease responsible for the release of the ADAM10 intracellular domain (ICD). This domain then translocates to the nucleus and localizes to nuclear speckles, thought to be involved in gene regulation. Thus, ADAM10 performs a dual role in cells, as a metalloprotease when it is membrane-bound, and as a potential signaling protein once cleaved by ADAM9/15 and the gamma-Secretase.
Assuntos
Proteínas ADAM/metabolismo , Proteínas ADAM/fisiologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Receptores Notch/metabolismo , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/fisiologia , Animais , Núcleo Celular/metabolismo , Camundongos , Microscopia de Fluorescência , Presenilinas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Frações Subcelulares/metabolismo , Distribuição TecidualRESUMO
The conserved oligomeric Golgi (COG) complex is a heterooctameric complex that regulates intraGolgi trafficking and the integrity of the Golgi compartment in eukaryotic cells. Here, we describe a patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex. This patient has a defect in both N- and O-glycosylation. Mass spectrometric analysis of the structures of the N-linked glycans released from glycoproteins from the patient's serum revealed a reduction in sialic acid and galactose residues. Peanut agglutinin (PNA) lectin staining revealed a decrease in sialic acids on core 1 mucin type O-glycans, indicating a combined defect in N- and O-glycosylation. Sequence analysis of the COG1 cDNA and gene identified a homozygous insertion of a single nucleotide (2659-2660insC), which is predicted to lead to a premature translation stop and truncation of the C terminus of the Cog1 protein by 80 amino acids. This mutation destabilizes several other COG subunits and alters their subcellular localization and hence the overall integrity of the COG complex. This results in reduced levels and/or altered Golgi localization of alpha-mannosidase II and beta-1,4 galactosyltransferase I, which links it to the glycosylation deficiency. Transfection of primary fibroblasts of this patient with the full length hemagglutinin-tagged Cog1 indeed restored beta-1,4 galactosyltransferase Golgi localization. We propose naming this disorder CDG-II/Cog1, or CDG-II caused by Cog1 deficiency.