RESUMO
The Golgi complex processes secretory proteins and lipids, carries out protein sorting and signaling, and supports growth and composition of the plasma membrane. Golgi complex size likely is regulated to meet the demands of each function, and this may involve differential changes of its distinct subdomains. Nevertheless, the primary size change is elongation of the Golgi ribbon-like network as occurs during Golgi complex doubling for mitosis and during differentiation involving upregulated secretion. One hypothesis states that Golgi complex size is set by the abundance of secretory cargo and Golgi complex components that, through binding vesicle coat complexes, drive vesicle coat formation to alter Golgi complex influx and efflux. Regulation of transport factors controlling Golgi membrane traffic is also observed and may control Golgi complex size, but more work is needed to directly link these events to Golgi complex size regulation, especially during differentiation of specialized cell types.
Assuntos
Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Tamanho das Organelas , Transporte Biológico/fisiologia , Ciclo Celular , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Via SecretóriaRESUMO
The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.
Assuntos
Benzamidas/metabolismo , Proteínas Qa-SNARE/metabolismo , Tiofenos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Benzamidas/farmacologia , Transporte Biológico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Transporte Proteico , Ricina/metabolismo , Toxina Shiga/metabolismo , Toxinas Shiga/metabolismo , Tiofenos/farmacologia , Proteínas de Transporte Vesicular/fisiologiaRESUMO
The surface glycoprotein (GP) of Ebola virus causes many of the virus's pathogenic effects, including a dramatic loss of endothelial cell adhesion associated with widespread hemorrhaging during infection. Although the GP-mediated deadhesion depends on its extracellular mucin-like domain, it is unknown whether any, or all, of this domain's densely clustered O-glycosylation sites are required. It is also unknown whether any of the 20 distinct polypeptide GalNAc-transferases (ppGalNAc-Ts) that initiate mucin-type O-glycosylation in human cells are functionally required. Here, using HEK293 cell lines lacking specific glycosylation enzymes, we demonstrate that GP requires extended O-glycans to exert its deadhesion effect. We also identified ppGalNAc-T1 as largely required for the GP-mediated adhesion defects. Despite its profound effect on GP function, the absence of ppGalNAc-T1 only modestly reduced the O-glycan mass of GP, indicating that even small changes in the bulky glycodomain can cause loss of GP function. Indeed, protein-mapping studies identified a small segment of the mucin-like domain critical for function and revealed that mutation of five glycan acceptor sites within this segment are sufficient to abrogate GP function. Together, these results argue against a mechanism of Ebola GP-induced cell detachment that depends solely on ectodomain bulkiness and identify a single host-derived glycosylation enzyme, ppGalNAc-T1, as a potential target for therapeutic intervention against Ebola virus disease.
Assuntos
Adesão Celular , Ebolavirus/fisiologia , N-Acetilgalactosaminiltransferases/metabolismo , Proteínas Virais de Fusão/metabolismo , Sítios de Ligação , Ebolavirus/metabolismo , Glicosilação , Células HEK293 , Humanos , Ligação Proteica , Especificidade por Substrato , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
The GalNAc-type O-glycoproteome is orchestrated by a large family of polypeptide GalNAc-transferase isoenzymes (GalNAc-Ts) with partially overlapping contributions to the O-glycoproteome besides distinct nonredundant functions. Increasing evidence indicates that individual GalNAc-Ts co-regulate and fine-tune specific protein functions in health and disease, and deficiencies in individual GALNT genes underlie congenital diseases with distinct phenotypes. Studies of GalNAc-T specificities have mainly been performed with in vitro enzyme assays using short peptide substrates, but recently quantitative differential O-glycoproteomics of isogenic cells with and without GALNT genes has enabled a more unbiased exploration of the nonredundant contributions of individual GalNAc-Ts. Both approaches suggest that fairly small subsets of O-glycosites are nonredundantly regulated by specific GalNAc-Ts, but how these isoenzymes orchestrate regulation among competing redundant substrates is unclear. To explore this, here we developed isogenic cell model systems with Tet-On inducible expression of two GalNAc-T genes, GALNT2 and GALNT11, in a knockout background in HEK293 cells. Using quantitative O-glycoproteomics with tandem-mass-tag (TMT) labeling, we found that isoform-specific glycosites are glycosylated in a dose-dependent manner and that induction of GalNAc-T2 or -T11 produces discrete glycosylation effects without affecting the major part of the O-glycoproteome. These results support previous findings indicating that individual GalNAc-T isoenzymes can serve in fine-tuned regulation of distinct protein functions.
Assuntos
N-Acetilgalactosaminiltransferases/metabolismo , Proteoma/metabolismo , Sequência de Aminoácidos , Glicosilação , Células HEK293 , Humanos , Isoenzimas/metabolismo , Proteômica/métodos , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Protein O-glycosylation is important in numerous processes including the regulation of proteolytic processing sites by O-glycan masking in select newly synthesized proteins. To investigate O-glycan-mediated masking using an assay amenable to large-scale screens, we generated a fluorescent biosensor with an O-glycosylation site situated to mask a furin cleavage site. The sensor is activated when O-glycosylation fails to occur because furin cleavage releases a blocking domain allowing dye binding to a fluorogen activating protein. Thus, by design, glycosylation should block furin from activating the sensor only if it occurs first, which is predicted by the conventional view of Golgi organization. Indeed, and in contrast to the recently proposed rapid partitioning model, the sensor was non-fluorescent under normal conditions but became fluorescent when the Golgi complex was decompartmentalized. To test the utility of the sensor as a screening tool, cells expressing the sensor were exposed to a known inhibitor of O-glycosylation extension or siRNAs targeting factors known to alter glycosylation efficiency. These conditions activated the sensor substantiating its potential in identifying new inhibitors and cellular factors related to protein O-glycosylation. In summary, these findings confirm sequential processing in the Golgi, establish a new tool for studying the regulation of proteolytic processing by O-glycosylation, and demonstrate the sensor's potential usefulness for future screening projects.
Assuntos
Furina/metabolismo , Complexo de Golgi/metabolismo , Processamento de Proteína Pós-Traducional , Furina/genética , Glicosilação , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , ProteóliseRESUMO
Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzymes deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.
Assuntos
Corantes Fluorescentes/química , N-Acetilgalactosaminiltransferases/fisiologia , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/química , Técnicas Biossensoriais , Inibidores Enzimáticos/farmacologia , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/química , Corantes Fluorescentes/metabolismo , Glicosilação , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/fisiologia , Microscopia de Fluorescência , N-Acetilgalactosaminiltransferases/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismoRESUMO
The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni(2+)-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering.
Assuntos
Proteínas de Transporte/química , Bicamadas Lipídicas/química , Lipoilação , Proteínas de Membrana/química , Membranas Artificiais , Ácido Mirístico/química , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ácido Mirístico/metabolismo , Estrutura Terciária de ProteínaRESUMO
Acute inhibition is a powerful technique to test proteins for direct roles and order their activities in a pathway, but as a general gene-based strategy, it is mostly unavailable in mammalian systems. As a consequence, the precise roles of proteins in membrane trafficking have been difficult to assess in vivo. Here we used a strategy based on a genetically encoded fluorescent protein that generates highly localized and damaging reactive oxygen species to rapidly inactivate exit from the endoplasmic reticulum (ER) during live-cell imaging and address the long-standing question of whether the integrity of the Golgi complex depends on constant input from the ER. Light-induced blockade of ER exit immediately perturbed Golgi membranes, and surprisingly, revealed that cis-Golgi-resident proteins continuously cycle to peripheral ER-Golgi intermediate compartment (ERGIC) membranes and depend on ER exit for their return to the Golgi. These experiments demonstrate that ER exit and extensive cycling of cis-Golgi components to the cell periphery sustain the mammalian Golgi complex.
Assuntos
Complexo de Golgi/enzimologia , Proteínas/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos da radiação , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Cinética , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
P-type ATPases transport a wide array of ions, regulate diverse cellular processes, and are implicated in a number of human diseases. However, mechanisms that increase ion transport by these ubiquitous proteins are not known. SPCA1 is a P-type pump that transports Mn(2+) from the cytosol into the Golgi. We developed an intra-Golgi Mn(2+) sensor and used it to screen for mutations introduced in SPCA1, on the basis of its predicted structure, which could increase its Mn(2+) pumping activity. Remarkably, a point mutation (Q747A) predicted to increase the size of its ion permeation cavity enhanced the sensor response and a compensatory mutation restoring the cavity to its original size abolished this effect. In vivo and in vitro Mn(2+) transport assays confirmed the hyperactivity of SPCA1-Q747A. Furthermore, increasing Golgi Mn(2+) transport by expression of SPCA1-Q747A increased cell viability upon Mn(2+) exposure, supporting the therapeutic potential of increased Mn(2+) uptake by the Golgi in the management of Mn(2+)-induced neurotoxicity.
Assuntos
Adenosina Trifosfatases/química , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/fisiologia , Complexo de Golgi/metabolismo , Manganês/química , Mutação , ATPases Translocadoras de Prótons/química , Alanina/química , ATPases Transportadoras de Cálcio/química , Citosol/metabolismo , Complexo de Golgi/química , Células HeLa , Humanos , Modelos Moleculares , Conformação Molecular , Fosforilação , Mutação Puntual , Proteínas de Transporte Vesicular/químicaRESUMO
Mitotic phosphorylation of the conserved GRASP domain of GRASP65 disrupts its self-association, leading to a loss of Golgi membrane tethering, cisternal unlinking, and Golgi breakdown. Recently, the structural basis of the GRASP self-interaction was determined, yet the mechanism by which phosphorylation disrupts this activity is unknown. Here, we present the crystal structure of a GRASP phosphomimic containing an aspartic acid substitution for a serine residue (Ser-189) that in GRASP65 is phosphorylated by PLK1, causing a block in membrane tethering and Golgi ribbon formation. The structure revealed a conformational change in the GRASP internal ligand that prevented its insertion into the PDZ binding pocket, and gel filtration assays showed that this phosphomimic mutant exhibited a significant reduction in dimer formation. Interestingly, the structure also revealed an apparent propagation of conformational change from the site of phosphorylation to the shifted ligand, and alanine substitution of two residues (Glu-145 and Ser-146) at penultimate positions in this chain rescued dimer formation by the phosphomimic. These data reveal the structural basis of the phosphoinhibition of GRASP-mediated membrane tethering and provide a mechanism for its allosteric regulation.
Assuntos
Proteínas de Transporte/química , Complexo de Golgi/química , Membranas Intracelulares/química , Proteínas de Membrana/química , Mitose/fisiologia , Regulação Alostérica/fisiologia , Substituição de Aminoácidos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Relação Estrutura-Atividade , Quinase 1 Polo-LikeRESUMO
The mammalian Golgi apparatus exists as stacks of cisternae that are laterally linked to form a continuous membrane ribbon, but neither the molecular requirements for, nor the purpose of, Golgi ribbon formation are known. Here, we demonstrate that ribbon formation is mediated by specific membrane-fusion events that occur during Golgi assembly, and require the Golgi proteins GM130 and GRASP65. Furthermore, these GM130 and GRASP65-dependent lateral cisternal-fusion reactions are necessary to achieve uniform distribution of enzymes in the Golgi ribbon. The membrane continuity created by ribbon formation facilitates optimal processing conditions in the biosynthetic pathway.
Assuntos
Complexo de Golgi/enzimologia , Complexo de Golgi/fisiologia , Membranas Intracelulares/fisiologia , Fusão de Membrana , Proteínas de Membrana/fisiologia , Autoantígenos , Glicosilação , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Membranas Intracelulares/ultraestrutura , Proteínas de Membrana/genética , Microscopia Eletrônica de Transmissão , Transporte Proteico , RNA Interferente Pequeno/genéticaRESUMO
Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.
Assuntos
Complexo de Golgi/química , Complexo de Golgi/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dobramento de Proteína , Cristalografia por Raios X , Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Domínios PDZRESUMO
The subcompartmentalized structure of the Golgi apparatus contributes to efficient glycosylation in the secretory pathway. Subcompartmentalization driven by maturation relies primarily on constant and accurate vesicle-mediated local recycling of Golgi residents. The precision of this vesicle transport is dependent on the interplay between the key factors that mediate vesicle budding and fusion--the coat proteins and the SNARE fusion machinery. These alone, however, may not be sufficient to ensure establishment of compartments de novo, and additional regulatory mechanisms operate to modify their activity.
Assuntos
Compartimento Celular , Complexo de Golgi/metabolismo , Transporte Biológico , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Transporte Vesicular/metabolismoRESUMO
GRASP65 links cis-Golgi cisternae via a homotypic, N-terminal PDZ interaction, and its mitotic phosphorylation disrupts this activity. Neither the identity of the PDZ ligand involved in the GRASP65 self-interaction nor the mechanism by which phosphorylation inhibits its interaction is known. Phospho-mimetic mutation of known cyclin-dependent kinase 1/cyclin B sites, all of which are in the C-terminal "regulatory domain" of the molecule, failed to block organelle tethering. However, we identified a site phosphorylated by Polo-like kinase 1 (PLK1) in the GRASP65 N-terminal domain for which mutation to aspartic acid blocked tethering and alanine substitution prevented mitotic Golgi unlinking. Further, using interaction assays, we discovered an internal PDZ ligand adjacent to the PLK phosphorylation site that was required for tethering. These results reveal the mechanism of phosphoinhibition as direct inhibition by PLK1 of the PDZ ligand underlying the GRASP65 self-interaction.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Substituição de Aminoácidos , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , Ciclina B1/metabolismo , Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Domínios PDZ/fisiologia , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Quinase 1 Polo-LikeRESUMO
GRASP proteins share an N-terminal GRASP domain and mediate homotypic tethering of Golgi cisternae to form extended Golgi ribbons. The golgin GM130 is thought to bind the C-terminal side of the GRASP domain to recruit GRASP65 onto the Golgi whereas stable membrane association appears to also depend on anchoring of the N terminus by myristoylation. Here, we examine the nature of the GM130/GRASP65 interaction and test whether the dual membrane contacts of the GRASP domain have a role in tethering beyond membrane recruitment. GM130 was found to contain a C-terminal PDZ ligand that binds the putative groove of the second PDZ-like domain in GRASP65. To test tethering activity independent of targeting, we took advantage of a tethering assay carried out on the mitochondrial membrane in which the GRASP membrane attachment points were individually or simultaneously substituted with mitochondrially targeted transmembrane sequences. N-terminally anchored constructs tethered only if the C terminus was also anchored; and likewise, C-terminally anchored constructs tethered only if the N terminus was anchored. One explanation for the role of this dual anchoring is that it orients the GRASP domain to prevent cis interactions within the same membrane thereby favoring trans interactions between adjacent membranes. Indeed, singly anchored GRASP constructs, although nonfunctional in tethering, interacted with one another and also bound and inhibited dually anchored constructs. This work thus elucidates the GM130/GRASP65 interaction and supports a novel orientation-based model of membrane tether regulation in which dual membrane contact orients the tethering interaction interface to favor trans over cis interactions.
Assuntos
Autoantígenos/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Autoantígenos/genética , Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Proteínas de Membrana/genética , Estrutura Terciária de ProteínaRESUMO
Under experimental conditions, the Golgi apparatus can undergo de novo biogenesis from the endoplasmic reticulum (ER), involving a rapid phase of growth followed by a return to steady state, but the mechanisms that control growth are unknown. Quantification of coat protein complex (COP) II assembly revealed a dramatic up-regulation at exit sites driven by increased levels of Golgi proteins in the ER. Analysis in a permeabilized cell assay indicated that up-regulation of COPII assembly occurred in the absence GTP hydrolysis and any cytosolic factors other than the COPII prebudding complex Sar1p-Sec23p-Sec24p. Remarkably, acting via a direct interaction with Sar1p, increased expression of the Golgi enzyme N-acetylgalactosaminyl transferase-2 induced increased COPII assembly on the ER and an overall increase in the size of the Golgi apparatus. These results suggest that direct interactions between Golgi proteins exiting the ER and COPII components regulate ER exit, providing a variable exit rate mechanism that ensures homeostasis of the Golgi apparatus.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Complexo de Golgi/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Retículo Endoplasmático/metabolismo , Células HeLa , Homeostase , Humanos , Modelos Biológicos , N-Acetilgalactosaminiltransferases/metabolismo , RatosRESUMO
Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G(2)/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle "checkpoint." A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G(2) to control G(2)/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G(2) Golgi unlinking and the G(2)/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.
Assuntos
Complexo de Golgi/enzimologia , MAP Quinase Quinase 1/fisiologia , Mitose , Divisão Celular/genética , Fase G2/genética , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , Proteínas de Membrana/metabolismo , Mitose/genética , Fosforilação , Interferência de RNARESUMO
We propose a new active mask algorithm for the segmentation of fluorescence microscope images of punctate patterns. It combines the (a) flexibility offered by active-contour methods, (b) speed offered by multiresolution methods, (c) smoothing offered by multiscale methods, and (d) statistical modeling offered by region-growing methods into a fast and accurate segmentation tool. The framework moves from the idea of the "contour" to that of "inside and outside," or masks, allowing for easy multidimensional segmentation. It adapts to the topology of the image through the use of multiple masks. The algorithm is almost invariant under initialization, allowing for random initialization, and uses a few easily tunable parameters. Experiments show that the active mask algorithm matches the ground truth well and outperforms the algorithm widely used in fluorescence microscopy, seeded watershed, both qualitatively, as well as quantitatively.
Assuntos
Algoritmos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Reconhecimento Automatizado de Padrão/métodos , Técnicas Citológicas/métodos , Células HeLa , HumanosRESUMO
The Golgi apparatus is comprised of stacked cisternal membranes forming subcompartments specialized for posttranslational processing of newly synthesized secretory cargo. Recent experimental evidence indicates that the Golgi apparatus can undergo de novo biogenesis from the endoplasmic reticulum, but the mechanism by which the membranes self assemble into compartmentalized structures remains unknown. We developed a discrete-event computer simulation model to test whether two fundamental mechanisms-vesicle-coat-mediated selective concentration of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins during vesicle formation, and SNARE-mediated selective fusion of vesicles-suffice to generate and maintain compartments. Simulations verified that this minimal model is adequate for homeostasis of preestablished compartments, even in response to small perturbations, and for de novo formation of stable compartments. The model led to a novel prediction that Golgi size is in part dependent on target SNARE expression level. This prediction was supported by a demonstration that exogenous expression of the Golgi target SNARE syntaxin-5 alters Golgi size in living cells.
Assuntos
Complexo de Golgi/química , Complexo de Golgi/fisiologia , Fusão de Membrana/fisiologia , Modelos Biológicos , Vesículas Transportadoras/química , Vesículas Transportadoras/fisiologia , Simulação por Computador , Modelos Químicos , Proteínas SNARE/química , Proteínas SNARE/fisiologiaRESUMO
It is unclear whether the mammalian Golgi apparatus can form de novo from the ER or whether it requires a preassembled Golgi matrix. As a test, we assayed Golgi reassembly after forced redistribution of Golgi matrix proteins into the ER. Two conditions were used. In one, ER redistribution was achieved using a combination of brefeldin A (BFA) to cause Golgi collapse and H89 to block ER export. Unlike brefeldin A alone, which leaves matrix proteins in relatively large remnant structures outside the ER, the addition of H89 to BFA-treated cells caused ER accumulation of all Golgi markers tested. In the other, clofibrate treatment induced ER redistribution of matrix and nonmatrix proteins. Significantly, Golgi reassembly after either treatment was robust, implying that the Golgi has the capacity to form de novo from the ER. Furthermore, matrix proteins reemerged from the ER with faster ER exit rates. This, together with the sensitivity of BFA remnants to ER export blockade, suggests that presence of matrix proteins in BFA remnants is due to cycling via the ER and preferential ER export rather than their stable assembly in a matrix outside the ER. In summary, the Golgi apparatus appears capable of efficient self-assembly.