BMC Cell Biology - Most viewed articles

Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis

2008-06-05

Background: Cholesterol-rich membrane microdomains known as lipid rafts have been implicated in diverse physiologic processes including lipid transport and signal transduction. Lipid rafts were originally defined as detergent-resistant membranes (DRMs) due to their relative insolubility in cold non-ionic detergents. Recent findings suggest that, although DRMs are not equivalent to lipid rafts, the presence of a given protein within DRMs strongly suggests its potential for raft association in vivo. Therefore, isolation of DRMs represents a useful starting point for biochemical analysis of lipid rafts. The physicochemical properties of DRMs present unique challenges to analysis of their protein composition. Existing methods of isolating DRM-enriched fractions involve flotation of cell extracts in a sucrose density gradient, which, although successful, can be labor intensive, time consuming and results in dilute sucrose-containing fractions with limited utility for direct proteomic analysis. In addition, several studies describing the proteomic characterization of DRMs using this and other approaches have reported the presence of nuclear proteins in such fractions. It is unclear whether these results reflect trafficking of nuclear proteins to DRMs or whether they arise from nuclear contamination during isolation. To address these issues, we have modified a published differential detergent extraction method to enable rapid DRM isolation that minimizes nuclear contamination and yields fractions compatible with mass spectrometry. Results: DRM-enriched fractions isolated using the conventional or modified extraction methods displayed comparable profiles of known DRM-associated proteins, including flotillins, GPI-anchored proteins and heterotrimeric G-protein subunits. Thus, the modified procedure yielded fractions consistent with those isolated by existing methods. However, we observed a marked reduction in the percentage of nuclear proteins identified in DRM fractions isolated with the modified method (15%) compared to DRMs isolated by conventional means (36%). Furthermore, of the 21 nuclear proteins identified exclusively in modified DRM fractions, 16 have been reported to exist in other subcellular sites, with evidence to suggest shuttling of these species between the nucleus and other organelles. Conclusion: We describe a modified DRM isolation procedure that generates DRMs that are largely free of nuclear contamination and that is compatible with downstream proteomic analyses with minimal additional processing. Our findings also imply that identification of nuclear proteins in DRMs is likely to reflect legitimate movement of proteins between compartments, and is not a result of contamination during extraction.

The NIMA-family kinase, Nek1 affects the stability of centrosomes and ciliogenesis

Mark C White and Lynne M Quarmby — 2008-06-04

Background: Mutations in Nek1 (NIMA-Related Kinase 1) are causal in the murine models of polycystic kidney disease kat and kat2J. The Neks are known as cell cycle kinases, but recent work in protists has revealed that in addition to roles in the regulation of cell cycle progression, some Neks also regulate cilia. In most cells, cilia are disassembled prior to mitosis and are regenerated after cytokinesis. We propose that Neks participate in the coordination of ciliogenesis with cell cycle progression. Mammalian Nek1 is a candidate for this activity because renal cysts form in response to dysfunctional ciliary signalling. Results: Here we report that over-expression of full-length mNek1 inhibited ciliogenesis without disrupting centrosomes in the murine renal epithelial cell line IMCD3. In contrast, over-expression of the kinase domain with its associated basic region, but without the acidic domain, caused loss of centrosomes. As expected, these cells also failed to grow cilia. Both defective ciliogenesis in response to too much mNek1 and disassembly of centrosomes in response to expression of the kinase lacking the presumptive regulatory domain was abrogated by kinase-inactivating mutations or by removal of the coiled-coil domain. We observed that kinase-inactive, C-terminal truncations of mNek1 retaining the coiled-coil domain localized to the cilium, and we define a ciliary targeting region within the coiled-coil domain. Conclusion: Based on our data, we propose that Nek1 plays a role in centrosome integrity, affecting both ciliogenesis and centrosome stability.

Mechanism of nuclear factor of activated T-cells mediated FasL expression in corticosterone -treated mouse Leydig tumor cells

Wei-Ran Chai, Yong Chen, Qian Wang and Hui-Bao Gao — 2008-06-12

Background: Fas and FasL is important mediators of apoptosis. We have previously reported that the stress levels of corticosterone (CORT, glucocorticoid in rat) increase expression of Fas/FasL and activate Fas/FasL signal pathway in rat Leydig cells, which consequently leads to apoptosis. Moreover, our another study showed that nuclear factor of activated T-cells (NFAT) may play a potential role in up-regulation of FasL during CORT-treated rat Leydig cell. It is not clear yet how NFAT is involved in CORT-induced up-regulation of FasL. The aim of the present study is to investigate the molecular mechanisms of NFAT-mediated FasL expression in CORT-treated Leydig cells. Results: Western blot analysis showed that NFAT2 expression is present in mouse Leydig tumor cell (mLTC-1). CORT-induced increase in FasL expression in mLTC-1 was ascertained by Western Blot analysis and CORT-induced increase in apoptotic frequency of mLTC-1 cells was detected by FACS with annexin-V labeling. Confocal imaging of NFAT2-GFP in mLTC-1 showed that high level of CORT stimulated NFAT translocation from the cytoplasm to the nucleus. RNA interference-mediated knockdown of NFAT2 significantly attenuated CORT-induced up-regulation of FasL expression in mLTC. These results corroborated our previous finding that NFAT2 is involved in CORT-induced FasL expression in rat Leydig cells and showed that mLTC-1 is a suitable model for investigating the mechanism of CORT-induced FasL expression. The analysis of reporter constructs revealed that the sequence between -201 and +71 of mouse FasL gene is essential for CORT-induced FasL expression. The mutation analysis demonstrated that CORT-induced FasL expression is mediated via an NFAT binding element located in the -201 to +71 region. Co-transfection studies with an NFAT2 expression vector and reporter construct containing -201 to +71 region of FasL gene showed that NFAT2 confer a strong inducible activity to the FasL promoter at its regulatory region. In addition, chromatin immunoprecipitation assay further confirmed the results of reporter gene studies by showing the specific binding of NFAT2 to the -201 to +71 region. Conclusion: In the present study, we demonstrated that NFAT2 directly stimulates transcription of FasL in high level CORT-treated mLTC-1. In conclusion, the present study provides further evidence for our finding that CORT-induced FasL expression in Leydig cells is mediated by NFAT.

Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue

David Robertson, Kay Savage, Jorge S Reis-Filho and Clare M Isacke — 2008-03-19

Background: Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues. Results: We present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4°C or in the longer term at -20°C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously. Conclusion: This method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples.

Comparative study of mesenchymal stem cells from C57BL/10 and mdx mice

2008-05-19

Background: Human mesenchymal stem cells (MSCs) have been studied and applied extensively because of their ability to self-renew and differentiate into various cell types. Since most human diseases models are murine, mouse MSCs should have been studied in detail. The mdx mouse – a Duchenne muscular dystrophy model – was produced by introducing a point mutation in the dystrophin gene. To understand the role of dystrophin in MSCs, we compared MSCs from mdx and C57BL/10 mice, focusing particularly on the aspects of light and electron microscopic morphology, immunophenotyping, and differentiation potential. Results: Our study showed that at passage 10, mdx-MSCs exhibited increased heterochromatin, larger vacuoles, and more lysosomes under electron microscopy compared to C57BL/10-MSCs. C57BL/10-MSCs formed a few myotubes, while mdx-MSCs did not at the same passages. By passage 21, mdx-MSCs but not C57BL/10-MSCs had gradually lost their proliferative ability. In addition, a significant difference in the expression of CD34, not Sca-1 and CD11b, was observed between the MSCs from the 2 mice. Conclusion: Our current study reveals that the MSCs from the 2 mice, namely, C57BL/10 and mdx, exhibit differences in proliferative and myogenic abilities. The results suggest that the changes in mouse MSC behavior may be influenced by lack of dystrophin protein in mdx mouse.

The HIV Tat protein affects processing of ribosomal RNA precursor

2008-06-17

Background: Inside the cell, the HIV Tat protein is mainly found in the nucleus and nucleolus. The nucleolus, the site of ribosome biogenesis, is a highly organized, non-membrane-bound sub-compartment where proteins with a high affinity for nucleolar components are found. While it is well known that Tat accumulates in the nucleolus via a specific nucleolar targeting sequence, its function in this compartment it still unknown. Results: To clarify the significance of the Tat nucleolar localization, we induced the expression of the protein during oogenesis in Drosophila melanogaster strain transgenic for HIV-tat gene. Here we show that Tat localizes in the nucleoli of Drosophila oocyte nurse cells, where it specifically co-localizes with fibrillarin. Tat expression is accompanied by a significant decrease of cytoplasmic ribosomes, which is apparently related to an impairment of ribosomal rRNA precursor processing. Such an event is accounted for by the interaction of Tat with fibrillarin and U3 snoRNA, which are both required for pre-rRNA maturation. Conclusion: Our data contribute to understanding the function of Tat in the nucleolus, where ribosomal RNA synthesis and cell cycle control take place. The impairment of nucleolar pre-rRNA maturation through the interaction of Tat with fibrillarin-U3snoRNA complex suggests a process by which the virus modulates host response, thus contributing to apoptosis and protein shut-off in HIV-uninfected cells.

The influence of N-acetyl-L-cysteine on oxidative stress and nitric oxide synthesis in stimulated macrophages treated with a mustard gas analogue

Victor Paromov, Min Qui, Hongsong Yang, Miltorn Smith and William L Stone — 2008-06-20

Background: Sulphur mustard gas, 2, 2a-dichlorodiethyl sulphide (HD), is a chemical warfare agent. Both mustard gas and its monofunctional analogue, 2-chloroethyl ethyl sulphide (CEES), are alkylating agents that react with and diminish cellular thiols and are highly toxic. Previously, we reported that lipopolysaccharide (LPS) significantly enhances the cytotoxicity of CEES in murine RAW 264.7 macrophages and that CEES transiently inhibits nitric oxide (NO) production via suppression of inducible NO synthase (iNOS) protein expression. NO generation is an important factor in wound healing. In this paper, we explored the hypotheses that LPS increases CEES toxicity by increasing oxidative stress and that treatment with N-acetyl-L-cysteine (NAC) would block LPS induced oxidative stress and protect against loss of NO production. NAC stimulates glutathione (GSH) synthesis and also acts directly as a free radical scavenger. The potential therapeutic use of the antibiotic, polymyxin B, was also evaluated since it binds to LPS and could thereby block the enhancement of CEES toxicity by LPS and also inhibit the secondary infections characteristic of HD/CEES wounds. Results: We found that 10 mM NAC, when administered simultaneously or prior to treatment with 0.5 mM CEES, increased the viability of LPS stimulated macrophages. Surprisingly, NAC failed to protect LPS stimulated macrophages from CEES induced loss of NO production. Macrophages treated with both LPS and CEES show increased oxidative stress parameters (cellular thiol depletion and increased protein carbonyl levels). NAC effectively protected RAW 264.7 cells simultaneously treated with CEES and LPS from GSH loss and oxidative stress. Polymyxin B was found to partially block nitric oxide production and diminish CEES toxicity in LPS-treated macrophages. Conclusion: The present study shows that oxidative stress is an important mechanism contributing to CEES toxicity in LPS stimulated macrophages and supports the notion that antioxidants could play a therapeutic role in preventing mustard gas toxicity. Although NAC reduced oxidative stress in LPS stimulated macrophages treated with CEES, it did not reverse CEES-induced loss of NO production. NAC and polymyxin B were found to help prevent CEES toxicity in LPS-treated macrophages.

Straw blood cell count, growth, inhibition and comparison to apoptotic bodies

Yonnie Wu, David C Henry, Kyle Heim, Jeffrey P Tomkins and Cheng-Yi Kuan — 2008-05-20

Background: Mammalian cells transform into individual tubular straw cells naturally in tissues and in response to desiccation related stress in vitro. The transformation event is characterized by a dramatic cellular deformation process which includes: condensation of certain cellular materials into a much smaller tubular structure, synthesis of a tubular wall and growth of filamentous extensions. This study continues the characterization of straw cells in blood, as well as the mechanisms of tubular transformation in response to stress; with specific emphasis placed on investigating whether tubular transformation shares the same signaling pathway as apoptosis. Results: There are approximately 100 billion, unconventional, tubular straw cells in human blood at any given time. The straw blood cell count (SBC) is 45 million/ml, which accounts for 6.9% of the bloods dry weight. Straw cells originating from the lungs, liver and lymphocytes have varying nodules, hairiness and dimensions. Lipid profiling reveals severe disruption of the plasma membrane in CACO cells during transformation. The growth rates for the elongation of filaments and enlargement of rabbit straw cells is 0.6~1.1 (μm/hr) and 3.8 (μm3/hr), respectively. Studies using apoptosis inhibitors and a tubular transformation inhibitor in CACO2 cells and in mice suggested apoptosis produced apoptotic bodies are mediated differently than tubular transformation produced straw cells. A single dose of 0.01 mg/kg/day of p38 MAPK inhibitor in wild type mice results in a 30% reduction in the SBC. In 9 domestic animals SBC appears to correlate inversely with an animal's average lifespan (R2 = 0.7). Conclusion: Straw cells are observed residing in the mammalian blood with large quantities. Production of SBC appears to be constant for a given animal and may involve a stress-inducible protein kinase (P38 MAPK). Tubular transformation is a programmed cell survival process that diverges from apoptosis. SBCs may be an important indicator of intrinsic aging-related stress.

Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis

2008-05-28

Background: The V-ATPase (VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms. Results: The transmembrane topology and stoichiometry of the proteolipid VHA-c" as well as the stoichiometry of the membrane integral subunit VHA-e within the V-ATPase complex were investigated by in vivo fluorescence resonance energy transfer (FRET). VHA-c", VHA-e1 and VHA-e2, VHA-a, VHA-c3, truncated variants of VHA-c3 and a chimeric VHA-c/VHA-c" hybrid were fused to cyan (CFP) and yellow fluorescent protein (YFP), respectively. The constructs were employed for transfection experiments with Arabidopsis thaliana mesophyll protoplasts. Subcellular localization and FRET analysis by confocal laser scanning microscopy (CLSM) demonstrated that (i.) the N- and C-termini of VHA-c" are localised in the vacuolar lumen, (ii.) one copy of VHA-c" is present within the VHA-complex, and (iii.) VHA-c" is localised at the ER and associated Golgi bodies. (iv.) A similar localisation was observed for VHA-e2, whereas (v.) the subcellular localisation of VHA-e1 indicated the trans Golgi network (TGN)-specifity of this subunit. Conclusion: The plant proteolipid ring is a highly flexible protein subcomplex, tolerating the incorporation of truncated and hybrid proteolipid subunits, respectively. Whereas the membrane integral subunit VHA-e is present in two copies within the complex, the proteolipid subunit VHA-c" takes part in complex formation with only one copy. However, neither VHA-c" isoform 1 nor any of the two VHA-e isoforms were identified at the tonoplast. This suggest a function in endomembrane specific VHA-assembly or targeting rather than proton transport.

The influence of nicotine on granulocytic differentiation – Inhibition of the oxidative burst and bacterial killing and increased matrix metalloproteinase-9 release

2008-04-15

Background: Neutrophils leave the bone marrow as terminally differentiated cells, yet little is known of the influence of nicotine or other tobacco smoke components on neutrophil differentiation. Therefore, promyelocytic HL-60 cells were differentiated into neutrophils using dimethylsulfoxide in the presence and absence of nicotine (3-(1-methyl-2-pyrrolidinyl) pyridine). Differentiation was evaluated over 5 days by monitoring terminal differentiation markers (CD11b expression and formazan deposition); cell viability, growth phase, kinetics, and apoptosis; assessing cellular morphology and ultrastructure; and conformational changes to major cellular components. Key neutrophil effector functions (oxidative burst, bacterial killing, matrix metalloproteinase release) were also examined. Results: Nicotine increased the percentage of cells in late differentiation phases (metamyelocytes, banded neutrophils and segmented neutrophils) compared to DMSO alone (p < 0.05), but did not affect any other marker of neutrophil differentiation examined. However, nicotine exposure during differentiation suppressed the oxidative burst in HL-60 cells (p < 0.001); inhibited bacterial killing (p < 0.01); and increased the LPS-induced release of MMP-9, but not MMP-2 (p < 0.05). These phenomena may be α-7-acetylcholine nicotinic receptor-dependent. Furthermore, smokers exhibited an increased MMP-9 burden compared to non-smokers in vivo (p < 0.05). Conclusion: These findings may partially explain the known increase in susceptibility to bacterial infection and neutrophil-associated destructive inflammatory diseases in individuals chronically exposed to nicotine.