scholarly journals Nuclear envelope dynamics during plant cell division suggest common mechanisms between kingdoms

2011 ◽  
Vol 435 (3) ◽  
pp. 661-667 ◽  
Author(s):  
Katja Graumann ◽  
David E. Evans
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Plate ◽  
Lamin B ◽  
Nuclear Envelope Breakdown ◽  
Plant Cell Division ◽  
Lamin B Receptor ◽  
Close Proximity ◽  
Green Fluorescent ◽  
Membrane Components

Behaviour of the NE (nuclear envelope) during open mitosis has been explored extensively in metazoans, but lack of native markers has limited similar investigations in plants. In the present study, carried out using living synchronized tobacco BY-2 suspension cultures, the non-functional NE marker LBR (lamin B receptor)–GFP (green fluorescent protein) and two native, functional NE proteins, AtSUN1 [Arapidopsis thaliana SUN (Sad1/UNC84) 1] and AtSUN2, we provide evidence that the ER (endoplasmic reticulum)-retention theory for NE membranes is applicable in plants. We also observe two apparently unique plant features: location of the NE-membrane components in close proximity to chromatin throughout division, and spatially distinct reformation of the NE commencing at the chromatin surface facing the spindle poles and concluding at the surface facing the cell plate. Mobility of the proteins was investigated in the interphase NE, during NE breakdown and reformation, in the spindle membranes and the cell plate. A role for AtSUN2 in nuclear envelope breakdown is suggested.

scholarly journals A New Model for Nuclear Envelope Breakdown

2001 ◽  
Vol 12 (2) ◽  
pp. 503-510 ◽  
Author(s):  
Mark Terasaki ◽  
Paul Campagnola ◽  
Melissa M. Rolls ◽  
Pascal A. Stein ◽  
Jan Ellenberg ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Second Phase ◽  
New Model ◽  
Nuclear Envelope Breakdown ◽  
Two Phases ◽  
Green Fluorescent

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

scholarly journals Dynamics of the Endoplasmic Reticulum and Golgi Apparatus during Early Sea Urchin Development

2000 ◽  
Vol 11 (3) ◽  
pp. 897-914 ◽  
Author(s):  
Mark Terasaki
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Permeability Barrier ◽  
Cell Cycles ◽  
Nuclear Envelope Breakdown ◽  
Quantitative Measurements ◽  
Green Fluorescent

The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2–3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is ∼30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-μm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.

scholarly journals Investigations on protein arginine methyltransferases in differentiation and cancer

2017 ◽  
Author(s):  
Οδυσσέας Πατούνας
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Nuclear Scaffold ◽  
Protein Arginine Methyltransferases ◽  
Lamin B ◽  
Lamin B Receptor ◽  
Green Fluorescent

Η μεθυλίωση των καταλοίπων αργινίνης από την οικογένεια των μεθυλοτρανσφερασών της αργινίνης (Protein Arginine Methyltransferases, PRMTs) είναι ένας σημαντικός ρυθμιστής της λειτουργίας των πρωτεϊνών και εμπλέκεται σε διαδικασίες όπως: η επιγενετική ρύθμιση της γονιδιακής έκφρασης, οι αποκρίσεις σε βλάβες του γενετικού υλικού, η ωρίμανση του RNA και η κυτταρική σηματοδότηση. Ο κυριότερος εκπρόσωπος της οικογένειας των ενζύμων αυτών είναι η PRMT1, από το γονίδιο της οποίας παράγονται εφτά διαφορετικές ισομορφές μετά από εναλλακτικό μάτισμα του αρχικού μεταγράφου στο 5’ άκρο. Οι ισομορφές αυτές εκφράζονται σε διαφορετικά επίπεδα ανάλογα με τον κυτταρικό τύπο, επιδεικνύουν διακριτή ειδικότητα για συγκεκριμένα υποστρώματα καθώς και διαφορετικό υποκυττάριο εντοπισμό. Στην παρούσα διδακτορική διατριβή, ανακαλύψαμε μια νέα ισομορφή της PRMT1 που δε σχετίζεται με το αμινοτελικό άκρο της πρωτεΐνης, όπως οι εφτά γνωστές έως τώρα ισομορφές. Η νέα αυτή ισομορφή δεν εμπεριέχει τα εξόνια 8 και 9 τα οποία είναι υπεύθυνα για την κωδικοποίηση του βραχίονα διμερισμού του ενζύμου. Εξαιτίας αυτού, το ένζυμο δεν μπορεί να δημιουργήσει καταλυτικά ενεργά ολιγομερή με τις υπόλοιπες ισομορφές του. Πειράματα FRAP (Fluorescent Recovery After Photobleaching) έδειξαν την ύπαρξη ενός ακινητοποιημένου κλάσματος της πρωτεΐνης στον πυρήνα. Τα αποτελέσματα αυτά είναι σύμφωνα με παλιότερα ευρήματα του εργαστηρίου μας, που έδειξαν ότι υπάρχει ισχυρή πρόσδεση της ανενεργής ή ανεσταλμένης PRMT1 στη χρωματίνη και στο πυρηνικό ικρίωμα (nuclear scaffold). Η νέα ισομορφή μπορεί να προσδεθεί στα ίδια υποστρώματα με την ενζυματικά ενεργή PRMT1. Η έκφραση της ανιχνεύθηκε σε διαφορετικές κυτταρικές σειρές και ήταν αυξημένη σε αυτές με τα πιο έντονα καρκινικά χαρακτηριστικά ή μετά από την υπερέκραφαση του μεταγραφικού παράγοντα Snail που επάγει τη μετάπτωση από επιθήλιο σε μεσέγχυμα (ΕΜΤ). Θεωρούμε ότι η νέα ισομορφή μπορεί να λειτουργήσει ως ρυθμιστής της δράσης της PRMT1 στα καρκινικά κύτταρα, δρώντας ως ανταγωνιστικός αναστολέας που δεν επιτρέπει την πρόσβαση των ενεργών ολιγομερών της PRMT1 στα υποστρώματά τους. Επίσης, παρουσιάζουμε νέα δεδομένα που καταδεικνύουν ότι ο υποδοχέας της λαμίνης Β (Lamin B Receptor, LBR) αποτελεί υπόστρωμα της PRMT1, κάτι που δεν ήταν γνωστό μέχρι σήμερα. Η μεθυλίωση του LBR από την PRMT1, πιθανώς να αποτελεί μέρος της επιγενετικής ρύθμισης των γονιδίων μέσω υποστρωμάτων που δεν ανήκουν στην οικογένεια των ιστονών. Στο δεύτερο τμήμα της διδακτορικής διατριβής, μελετήσαμε την PRMT8, το όγδοο μέλος της οικογένειας των PRMTs. Η αλληλουχία της PRMT8 είναι κατά ένα μεγάλο ποσοστό ομόλογη με αυτήν της PRMT1, αλλά η έκφραση της είναι περιορισμένη στο κεντρικό νευρικό σύστημα. Μέχρι σήμερα, η PRMT8 δεν είχε μελετηθεί ενδελεχώς, εξαιτίας της έλλειψης κατάλληλων εργαλείων και κυτταρικών συστημάτων. Στα πλαίσια της παρούσας διατριβής αναπτύξαμε νέα εργαλεία και διεξαγάγαμε μία σειρά πειραμάτων με σκοπό να διερευνήσουμε τον φυσιολογικό ρόλο της PRMT8 στη διαφοροποίηση και τη διατήρηση του νευρικού ιστού. Από τα πειράματα μας, προκύπτει ότι η PRMT8 εκφράζεται ενδογενώς στο κυτταρικό μοντέλο νευρικής διαφοροποίησης, LUHMES (LUnd Human MESencephalon). Η υπερέκφραση της PRMT8, συντηγμένης με την πράσινη φθορίζουσα πρωτεΐνη (Green Fluorescent Protein, GFP), κατέδειξε ότι η PRMT8 συσσωρεύεται στον πυρήνα καθώς τα LUHMES διαφοροποιούνται. Βάσει των αποτελεσμάτων μας φαίνεται ότι τα επίπεδα της πρωτεΐνης στον πυρήνα δεν μπορούν να ξεπεράσουν ένα όριο ανεξάρτητα από τη συνολική ποσότητά της στο κύτταρο. Τέλος, κατασκευάσαμε και αξιολογήσαμε νέους φορείς λέντι-ιών για την αποσιώπηση της έκφρασης της PRMT8 και την ανίχνευση μορίων με τα οποία αλληλεπιδρά μέσω της μεθόδου που ονομάζεται BioID. Συνοπτικά, τα αποτελέσματα της διδακτορικής διατριβής μου αποδεικνύουν ότι η PRMT1 και η PRMT8 επιτελούν σημαντικό ρόλο στον καρκίνο και στη νευρική διαφοροποίηση, αντίστοιχα. Τα αποτελέσματα αυτά θα έχουν μεγάλη χρησιμότητα σε επόμενες μελέτες όπου θα διερευνηθεί η πιθανότητα χρήσης τους ως διαγνωστικά εργαλεία ή φαρμακευτικοί στόχοι στον καρκίνο και σε νευροεκφυλιστικές ασθένειες.

scholarly journals An in vitro nuclear disassembly system reveals a role for the RanGTPase system and microtubule-dependent steps in nuclear envelope breakdown

2007 ◽  
Vol 178 (4) ◽  
pp. 595-610 ◽  
Author(s):  
Petra Mühlhäusser ◽  
Ulrike Kutay
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Dynamic Rupture ◽  
Vitro Assay ◽  
Nuclear Envelope Breakdown ◽  
Egg Extracts ◽  
Guanosine Triphosphatase ◽  
Green Fluorescent

During prophase, vertebrate cells disassemble their nuclear envelope (NE) in the process of NE breakdown (NEBD). We have established an in vitro assay that uses mitotic Xenopus laevis egg extracts and semipermeabilized somatic cells bearing a green fluorescent protein–tagged NE marker to study the molecular requirements underlying the dynamic changes of the NE during NEBD by live microscopy. We applied our in vitro system to analyze the role of the Ran guanosine triphosphatase (GTPase) system in NEBD. Our study shows that high levels of RanGTP affect the dynamics of late steps of NEBD in vitro. Also, inhibition of RanGTP production by RanT24N blocks the dynamic rupture of nuclei, suggesting that the local generation of RanGTP around chromatin may serve as a spatial cue in NEBD. Furthermore, the microtubule-depolymerizing drug nocodazole interferes with late steps of nuclear disassembly in vitro. High resolution live cell imaging reveals that microtubules are involved in the completion of NEBD in vivo by facilitating the efficient removal of membranes from chromatin.

scholarly journals Organization and Dynamics of Growing Microtubule Plus Ends during Early Mitosis

2003 ◽  
Vol 14 (3) ◽  
pp. 916-925 ◽  
Author(s):  
Michelle Piehl ◽  
Lynne Cassimeris
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Stable Cell Line ◽  
Cell Periphery ◽  
Late Prophase ◽  
Nuclear Envelope Breakdown ◽  
Cycle Dependent ◽  
Green Fluorescent ◽  
Early Mitosis

A stable cell line expressing EB1-green fluorescent protein was used to image growing microtubule plus ends at the G2/M transition. By late prophase growing ends no longer extend to the cell periphery and were not uniformly distributed around each centrosome. Growing ends were much more abundant in the area surrounding the nuclear envelope, and microtubules growing around the nucleus were 1.5 fold longer than those growing in the opposite direction. The growth of longer ends toward the nucleus did not result from a localized faster growth rate, because this rate was ∼11 μm/min in all directions from the centrosome. Rather, microtubule ends growing toward the nucleus seemed stabilized by dynein/dynactin associated with the nuclear envelope. Injection of p50 into late prophase cells removed dynein from the nuclear envelope, reduced the density of growing ends near the nuclear envelope and resulted in a uniform distribution of growing ends from each centrosome. We suggest that the cell cycle-dependent binding of dynein/dynactin to the nuclear envelope locally stabilizes growing microtubules. Both dynein and microtubules would then be in a position to participate in nuclear envelope breakdown, as described in recent studies.

scholarly journals A Visual Screen of a Gfp-Fusion Library Identifies a New Type of Nuclear Envelope Membrane Protein

1999 ◽  
Vol 146 (1) ◽  
pp. 29-44 ◽  
Author(s):  
Melissa M. Rolls ◽  
Pascal A. Stein ◽  
Stephen S. Taylor ◽  
Edward Ha ◽  
Frank McKeon ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Nuclear Envelope ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Physical Separation ◽  
Culture Cells ◽  
New Type ◽  
Green Fluorescent ◽  
Membrane Components

The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

Alterations of nuclear envelope and chromatin organization in mandibuloacral dysplasia, a rare form of laminopathy

2005 ◽  
Vol 23 (2) ◽  
pp. 150-158 ◽  
Author(s):  
Ilaria Filesi ◽  
Francesca Gullotta ◽  
Giovanna Lattanzi ◽  
Maria Rosaria D'Apice ◽  
Cristina Capanni ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Protein A ◽  
Nuclear Architecture ◽  
Mendelian Inheritance ◽  
Premature Aging ◽  
Lamin A ◽  
Lamin B ◽  
Lamin B Receptor ◽  
Epigenetic Events ◽  
Mandibuloacral Dysplasia

Autosomal recessive mandibuloacral dysplasia [mandibuloacral dysplasia type A (MADA); Online Mendelian Inheritance in Man (OMIM) no. 248370 ] is caused by a mutation in LMNA encoding lamin A/C. Here we show that this mutation causes accumulation of the lamin A precursor protein, a marked alteration of the nuclear architecture and, hence, chromatin disorganization. Heterochromatin domains are altered or completely lost in MADA nuclei, consistent with the finding that heterochromatin-associated protein HP1β and histone H3 methylated at lysine 9 and their nuclear envelope partner protein lamin B receptor (LBR) are delocalized and solubilized. Both accumulation of lamin A precursor and chromatin defects become more severe in older patients. These results strongly suggest that altered chromatin remodeling is a key event in the cascade of epigenetic events causing MADA and could be related to the premature-aging phenotype.

scholarly journals The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

2001 ◽  
Vol 152 (2) ◽  
pp. 385-400 ◽  
Author(s):  
Patrick Heun ◽  
Thierry Laroche ◽  
M.K. Raghuraman ◽  
Susan M. Gasser
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Yeast Cells ◽  
G1 Phase ◽  
Nuclear Pore ◽  
Origin Firing ◽  
Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

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