[Show abstract][Hide abstract]ABSTRACT: Lamin A (LaA) is a component of the nuclear lamina, an intermediate filament meshwork that underlies the inner nuclear membrane (INM) of the nuclear envelope (NE). Newly synthesized prelamin A (PreA) undergoes extensive processing involving C-terminal farnesylation followed by proteolysis yielding non-farnesylated mature lamin A. Different inhibitors of these processing events are currently used therapeutically. Hutchinson-Gilford Progeria Syndrome (HGPS) is most commonly caused by mutations leading to an accumulation of a farnesylated LaA isoform, prompting a clinical trial using farnesyltransferase inhibitors (FTI) to reduce this modification. At therapeutic levels, HIV protease inhibitors (PI) can unexpectedly inhibit the final processing step in PreA maturation. We have examined the dynamics of LaA processing and associated cellular effects during PI or FTI treatment and following inhibitor washout. While PI reversibility was rapid, with respect to both LaA maturation and associated cellular phenotype, recovery from FTI treatment was more gradual. FTI reversibility is influenced by both cell type and rate of proliferation. These results suggest a less static lamin network than has previously been observed.
[Show abstract][Hide abstract]ABSTRACT: Sun-2 and Nesprin-3 are present in abnormal cytoplasmic aggregates during mitosis and early G1 during Lop treatment. A 48hr treatment of Saos-2 cells with Lop led to the accumulation of Sun 2 and Nesprin-3 at LaA/C immunoreactive cytoplasmic aggregates in metaphase (upper panels) and early G1 (lower panels). DNA is labeled by Hoechst dye in blue. Bar, 10µm.
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[Show abstract][Hide abstract]ABSTRACT: Inhibition of protein synthesis does not impair PreA processing following PI washout. Saos-2 cells were treated with cyclohexamide at the time of Lop washout. PreA levels dramatically disappear by 3 hrs following washout. Percent of mature LaA listed below.
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[Show abstract][Hide abstract]ABSTRACT: In HGPS cells, HDJ-2 exhibits prolonged maturation following FTI washout. (A) An anti-HA immunoblot of extracts from WT human fibroblasts expressing exogenous HA-progerin were either treated with DMSO (control) or FTI-277 for 48 hrs immediately following transfection. The FTI-277 treated progerin migrates more slowly. (B) In HGPS cells treated with FTI-277 for 96hrs, HDJ-2 is incompletely processed by 24hrs following FTI-277. Percent of mature HDJ-2 listed above.
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[Show abstract][Hide abstract]ABSTRACT: Multiple FTIs failed to permit rapid processivity of PreA following washout. As detected by anti-LaA immunoblots of Saos-2 cell lysates, PreA was refractory to processing following washout of 10µm L-744, 832 or 1µm BMS-214662. Percent of mature LaA listed below.
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[Show abstract][Hide abstract]ABSTRACT: Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin-Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1-3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning.
Full-text · Article · Feb 2009 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract]ABSTRACT: Sun1 and 2 are A-type lamin-binding proteins that, in association with nesprins, form a link between the inner nuclear membranes (INMs) and outer nuclear membranes of mammalian nuclear envelopes. Both immunofluorescence and immunoelectron microscopy reveal that Sun1 but not Sun2 is intimately associated with nuclear pore complexes (NPCs). Topological analyses indicate that Sun1 is a type II integral protein of the INM. Localization of Sun1 to the INM is defined by at least two discrete regions within its nucleoplasmic domain. However, association with NPCs is dependent on the synergy of both nucleoplasmic and lumenal domains. Cells that are either depleted of Sun1 by RNA interference or that overexpress dominant-negative Sun1 fragments exhibit clustering of NPCs. The implication is that Sun1 represents an important determinant of NPC distribution across the nuclear surface.
Full-text · Article · Sep 2007 · The Journal of Cell Biology
[Show abstract][Hide abstract]ABSTRACT: The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH(2)-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419-3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).
Full-text · Article · Feb 2006 · The Journal of Cell Biology