Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro.

Eukaryotic cells tether the nucleoskeleton to the cytoskeleton via a conserved molecular bridge, called the LINC complex. The core of the LINC complex comprises SUN-domain and KASH-domain proteins that directly associate within the nuclear envelope lumen. Intra- and inter-chain disulphide bonds, along with KASH-domain protein interactions, both contribute to the tertiary and quaternary structure of vertebrate SUN-domain proteins. The significance of these bonds and the role of PDIs (protein disulphide isomerases) in LINC complex biology remains unclear. Reducing and non-reducing SDS-PAGE analyses revealed a prevalence of SUN2 homodimers in non-tumorigenic breast epithelia MCF10A cells, but not in the invasive triple-negative breast cancer MDA-MB-231 cell line. Furthermore, super-resolution microscopy revealed SUN2 staining alterations in MCF10A, but not in MDA-MB-231 nuclei, upon reducing agent exposure. While PDIA1 levels were similar in both cell lines, pharmacological inhibition of PDI activity in MDA-MB-231 cells led to SUN-domain protein down-regulation, as well as Nesprin-2 displacement from the nucleus. This inhibition also caused changes in perinuclear cytoskeletal architecture and lamin downregulation, and increased the invasiveness of PDI-inhibited MDA-MB-231 cells in space-restrictive in vitro environments, compared to untreated cells. These results emphasise the key roles of PDIs in regulating LINC complex biology, cellular architecture, biomechanics, and invasion.

BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQP0 water permeability.

BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434-440). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 is not required for membrane association. Biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalized with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various C-terminal domain fragments of BFSP1 generated by caspase cleavage. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca2+. The complete C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments can enhance regulation by elevated calcium or reverse the response, indicative of the regulatory potential of BFSP1 with respect to AQP0. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca2+ concentrations.

Farnesyltransferase inhibitor and rapamycin correct aberrant genome organisation and decrease DNA damage respectively, in Hutchinson-Gilford progeria syndrome fibroblasts.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal premature ageing disease in children. HGPS is one of several progeroid syndromes caused by mutations in the LMNA gene encoding the nuclear structural proteins lamins A and C. In classic HGPS the mutation G608G leads to the formation of a toxic lamin A protein called progerin. During post-translational processing progerin remains farnesylated owing to the mutation interfering with a step whereby the farnesyl moiety is removed by the enzyme ZMPSTE24. Permanent farnesylation of progerin is thought to be responsible for the proteins toxicity. Farnesyl is generated through the mevalonate pathway and three drugs that interfere with this pathway and hence the farnesylation of proteins have been administered to HGPS children in clinical trials. These are a farnesyltransferase inhibitor (FTI), statin and a bisphosphonate. Further experimental studies have revealed that other drugs such as N-acetyl cysteine, rapamycin and IGF-1 may be of use in treating HGPS through other pathways. We have shown previously that FTIs restore chromosome positioning in interphase HGPS nuclei. Mis-localisation of chromosomes could affect the cells ability to regulate proper genome function. Using nine different drug treatments representing drug regimes in the clinic we have shown that combinatorial treatments containing FTIs are most effective in restoring specific chromosome positioning towards the nuclear periphery and in tethering telomeres to the nucleoskeleton. On the other hand, rapamycin was found to be detrimental to telomere tethering, it was, nonetheless, the most effective at inducing DNA damage repair, as revealed by COMET analyses.

Xenopus LAP2ß protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

Xenopus LAP2ß protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2ß fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2ß antibody. Knockdown of the XLAP2ß protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells.

While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells.

A gradient of matrix-bound FGF-2 and perlecan is available to lens epithelial cells.

Fibroblast growth factors play a key role in regulating lens epithelial cell proliferation and differentiation via an anteroposterior gradient that exists between the aqueous and vitreous humours. FGF-2 is the most important for lens epithelial cell proliferation and differentiation. It has been proposed that the presentation of FGF-2 to the lens epithelial cells involves the lens capsule as a source of matrix-bound FGF-2. Here we used immunogold labelling to measure the matrix-bound FGF-2 gradient on the inner surface of the lens capsule in flat-mounted preparations to visualize the FGF-2 available to lens epithelial cells. We also correlated FGF-2 levels with levels of its matrix-binding partner perlecan, a heparan sulphate proteoglycan (HSPG) and found the levels of both to be highest at the lens equator. These also coincided with increased levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in lens epithelial cells that localised to condensed chromosomes of epithelial cells that were Ki-67 positive. The gradient of matrix-bound FGF-2 (anterior pole: 3.7 ± 1.3 particles/µm2; equator: 8.2 ± 1.9 particles/µm2; posterior pole: 4 ± 0.9 particles/µm2) and perlecan (anterior pole: 2.1 ± 0.4 particles/µm2; equator: 5 ± 2 particles/µm2; posterior pole: 1.9 ± 0.7 particles/µm2) available at the inner lens capsule surface was measured for the bovine lens. These data support the anteroposterior gradient hypothesis and provide the first measurement of the gradient for an important morphogen and its HSPG partner, perlecan, at the epithelial cell-lens capsule interface.

Imaging plant nuclei and membrane-associated cytoskeleton by field emission scanning electron microscopy.

Scanning electron microscopy (SEM) is a powerful technique that can image exposed surfaces in 3D. Modern scanning electron microscopes, with field emission electron sources and in-lens specimen chambers, achieve resolutions of better than 0.5 nm and thus offer views of ultrastructural details of subcellular structures or even macromolecular complexes. Obtaining a reliable image is, however, dependent on sample preparation methods that robustly but accurately preserve biological structures. In plants, exposing the object of interest may be difficult due to the existence of a cell wall. This protocol shows how to isolate plant nuclei for SEM imaging of the nuclear envelope and associated structures from both sides of the nuclear envelope in cultured cells as well as in leaf or root cells. Further, it provides a method for uncovering membrane-associated cytoskeletal structures.

System analysis shows distinct mechanisms and common principles of nuclear envelope protein dynamics.

The nuclear envelope contains >100 transmembrane proteins that continuously exchange with the endoplasmic reticulum and move within the nuclear membranes. To better understand the organization and dynamics of this system, we compared the trafficking of 15 integral nuclear envelope proteins using FRAP. A surprising 30-fold range of mobilities was observed. The dynamic behavior of several of these proteins was also analyzed after depletion of ATP and/or Ran, two functions implicated in endoplasmic reticulum-inner nuclear membrane translocation. This revealed that ATP- and Ran-dependent translocation mechanisms are distinct and not used by all inner nuclear membrane proteins. The Ran-dependent mechanism requires the phenylalanine-glycine (FG)-nucleoporin Nup35, which is consistent with use of the nuclear pore complex peripheral channels. Intriguingly, the addition of FGs to membrane proteins reduces FRAP recovery times, and this also depends on Nup35. Modeling of three proteins that were unaffected by either ATP or Ran depletion indicates that the wide range in mobilities could be explained by differences in binding affinities in the inner nuclear membrane.

Embryonic and adult isoforms of XLAP2 form microdomains associated with chromatin and the nuclear envelope.

Laminin-associated polypeptide 2 (LAP2) proteins are alternatively spliced products of a single gene; they belong to the LEM domain family and, in mammals, locate to the nuclear envelope (NE) and nuclear lamina. Isoforms lacking the transmembrane domain also locate to the nucleoplasm. We used new specific antibodies against the N-terminal domain of Xenopus LAP2 to perform immunoprecipitation, identification and localization studies during Xenopus development. By immunoprecipitation and mass spectrometry (LC/MS/MS), we identified the embryonic isoform XLAP2γ, which was downregulated during development similarly to XLAP2ω. Embryonic isoforms XLAP2ω and XLAP2γ were located in close association with chromatin up to the blastula stage. Later in development, both embryonic isoforms and the adult isoform XLAP2ß were localized in a similar way at the NE. All isoforms colocalized with lamin B2/B3 during development, whereas XLAP2ß was colocalized with lamin B2 and apparently with the F/G repeat nucleoporins throughout the cell cycle in adult tissues and culture cells. XLAP2ß was localized in clusters on chromatin, both at the NE and inside the nucleus. Embryonic isoforms were also localized in clusters at the NE of oocytes. Our results suggest that XLAP2 isoforms participate in the maintenance and anchoring of chromatin domains to the NE and in the formation of lamin B microdomains.

Nuclear envelope and nuclear pore complex structure and organization in tobacco BY-2 cells.

The nuclear envelope (NE) is a fundamental structure of eukaryotic cells with a dual role: it separates two distinct compartments, and enables communication between them via nuclear pore complexes (NPCs). Little is known about NPCs and NE structural organization in plants. We investigated the structure of NPCs from both sides of the NE in tobacco BY-2 cells. We detected structural differences between the NPCs of dividing and quiescent nuclei. Importantly, we also traced the organizational pattern of the NPCs, and observed non-random NPC distribution over the nuclear surface. Lastly, we observed an organized filamentous protein structure that underlies the inner nuclear membrane, and interconnects NPCs. The results are discussed within the context of the current understanding of NE structure and function in higher eukaryotes.

FGF-2 release from the lens capsule by MMP-2 maintains lens epithelial cell viability.

The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival.