Live cell imaging of heavy-ion-induced radiation responses by beamline microscopy

To study the dynamics of protein recruitment to DNA lesions, ion beams can be used to generate extremely localized DNA damage within restricted regions of the nuclei. This inhomogeneous spatial distribution of lesions can be visualized indirectly and rapidly in the form of radiation-induced foci using immunocytochemical detection or GFP-tagged DNA repair proteins. To analyze faster protein translocations and a possible contribution of radiation-induced chromatin movement in DNA damage recognition in live cells, we developed a remote-controlled system to obtain high-resolution fluorescence images of living cells during ion irradiation with a frame rate of the order of seconds. Using scratch replication labeling, only minor chromatin movement at sites of ion traversal was observed within the first few minutes of impact. Furthermore, time-lapse images of the GFP-coupled DNA repair protein aprataxin revealed accumulations within seconds at sites of ion hits, indicating a very fast recruitment to damaged sites. Repositioning of the irradiated cells after fixation allowed the comparison of live cell observation with immunocytochemical staining and retrospective etching of ion tracks. These results demonstrate that heavy-ion radiation-induced changes in sub-nuclear structures can be used to determine the kinetics of early protein recruitment in living cells and that the changes are not dependent on large-scale chromatin movement at short times postirradiation. © 2005 by Radiation Research Society.

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by Golgin-97

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.

The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation

The receptor protein tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1) has been implicated in aberrant cancer cell growth and immune cell function, however, its function within cells has yet to be properly elucidated. To investigate the cellular function of DEP-1, stable cell lines inducibly expressing DEP-1 were generated. Induction of DEP-1 expression was found to decrease PDGF-stimulated tyrosine phosphorylation of a number of cellular proteins including the PDGF receptor, and to inhibit growth factor-stimulated phosphorylation of components of the MAPK pathway, indicating that DEP-1 antagonised PDGF receptor signalling. This was supported by data showing that DEP-1 expression resulted in a reduction in cell proliferation. DEP-1-expressing cells had fewer actin-containing microfilament bundles, reduced vinculin and paxillin-containing adhesion plaques, and were defective in interactions with fibronectin. Defective cell-substratum adhesion correlated with lack of activation of FAK in DEP-1-expressing cells. Time-lapse interference reflection microscopy of live cells revealed that although small focal contacts at the leading edge were generated in DEP-1-expressing cells, they failed to mature into stable focal adhesions, as found in control cells. Further motility analysis revealed that DEP-1-expressing cells retained limited random motility, but showed no chemotaxis towards a gradient of PDGF. In addition, cell-cell contacts were disrupted, with a change in the localisation of cadherin from discrete areas of cell-cell contact to large areas of membrane interaction, and there was a parallel redistribution of beta-catenin. These results demonstrate that DEP-1 is a negative regulator of cell proliferation, cell-substratum contacts, motility and chemotaxis in fibroblasts.

A comparison between capillary and imaging techniques for sizing bubbles in flotation systems

This communication reports a laboratory and plant comparison between the University of Cape Town (UCT) device (capillary) and the McGill University bubble sizing method (imaging). The laboratory work was conducted on single bubbles to establish the accuracy of the techniques by comparing with a reference method (capture in a burette). Single bubble measurements with the McGill University technique showed a tendency to slightly underestimate (4% for a 1.3 mm bubble) and the UCT technique to slightly overestimate (1% for the 1.3 man bubble). Both trends are anticipated from fundamental considerations. In the UCT technique bubble breakup was observed when measuring a 2.7 mm bubble using a 0.5 mm ID capillary tube. A discrepancy of 11% was determined when comparing the techniques in an industrial-scale mechanical flotation cell. The possible sources of bias are discussed. (C) 2003 Elsevier Ltd. All rights reserved.

Cell death mechanisms associated with G(2) radiosensitivity in patients with prostate cancer and benign prostatic hyperplasia

Cells respond to genotoxic insults such as ionizing radiation by halting in the G(2) phase of the cell cycle. Delayed cell death (mitotic death) can occur when the cell is released from G(2), and specific spindle defects form endopolyploid cells (endoreduplication/tetraploidy). Enhanced G(2) chromosomal radiosensitivity has been observed in many cancers and genomic instability syndromes, and it is manifested by radiation-induced chromatid aberrations observed in lymphocytes of patients. Here we compare the G(2) chromosomal radiosensitivity in prostate patients with benign prostatic hyperplasia (BPH) or prostate cancer with disease-free controls. We also investigated whether there is a correlation between G(2) chromosomal radiosensitivity and aneuploidy (tetraploidy and endoreduplication), which are indicative of mitotic cell death. The G(2) assay was carried out on all human blood samples. Metaphase analysis was conducted on the harvested chromosomes by counting the number of aberrations and the mitotic errors (endoreduplication/tetraploidy) separately per 100 metaphases. A total of 1/14 of the controls were radiosensitive in G(2) compared to 6/15 of the BPH patients and 15/17 of the prostate cancer patients. Radiation-induced mitotic inhibition was assessed to determine the efficacy of G(2) checkpoint control in the prostate patients. There was no significant correlation of G(2) radiosensitivity scores and mitotic inhibition in BPH patients (P = 0.057), in contrast to prostate cancer patients, who showed a small but significant positive correlation (P = 0.029). Furthermore, there was no significant correlation between G(2) radiosensitivity scores of BPH patients and endoreduplication/ tetraploidy (P = 0.136), which contrasted with an extremely significant correlation observed in prostate cancer patients (P < 0.0001). In conclusion, cells from prostate cancer patients show increased sensitivity to the induction of G(2) aberrations from ionizing radiation exposure but paradoxically show reduced mitotic indices and aneuploidy as a function of aberration frequency.

H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton

Plasma membrane compartmentalization imposes lateral segregation on membrane proteins that is important for regulating signal transduction. We use computational modeling of immunogold spatial point patterns on intact plasma membrane sheets to test different models of inner plasma membrane organization. We find compartmentalization at the nanoscale level but show that a classical raft model of preexisting stable domains into which lipid raft proteins partition is incompatible with the spatial point patterns generated by the immunogold labeling of a palmitoylated raft marker protein. Rather, approximate to 30% of the raft protein exists in cholesterol-dependent nanoclusters, with approximate to 70% distributed as monomers. The cluster/monomer ratio (number of proteins in clusters/number of proteins outside clusters) is independent of expression level. H-rasG12V and K-rasG12V proteins also operate in nanoclusters with fixed cluster/monomer ratios that are independent of expression level. Detailed calibration of the immunogold imaging protocol suggests that radii of raft and RasG12V protein nanoclusters may be as small as 11 and 6 nm, respectively, and shows that the nanoclusters contain small numbers (6.0-7.7) of proteins. Raft nanoclusters do not form if the actin cytoskeleton is disassembled. The formation of K-rasG12V but not H-rasG12V nanoclusters also is actin-dependent. K-rasG12V but not H-rasG12V signaling is abrogated by actin cytoskeleton disassembly, which shows that nanoclustering is critical for Ras function. These findings argue against stable preexisting domains on the inner plasma membrane in favor of dynamic actively regulated nanoclusters similar to those proposed for the outer plasma membrane. RasG12V nanoclusters may facilitate the assembly of essential signal transduction complexes.

Who wants to live forever?

For millennia, human civilization has been fascinated with overcoming death. Immortality, eternal youth or at least the prospect of reaching biblical age have had a strong lure for religion, art and popular beliefs. Life after death, which is, in essence, eternal life, is the one central element of nearly all religions since Ancient Egypt. If we believe the Old Testament, some of the patriarchs lived for several hundreds of years. In the medieval ages, the fountain of youth was a popular myth, often illustrated in paintings, such as Lucas Cranach's The Fountain of Youth (Fig 1). And society today has not lost its fascination with immortality, as seen in Hollywood movies such as the Highlander films (1986–2000), The 6th Day (2000) or Indiana Jones and the Last Crusade (1989), and novels such as H. Rider Haggard's She. But for the first time, modern science may provide the knowledge and tools to interfere with the ageing processes and fulfil this age-old dream. This possibility has triggered an intense debate among scientists and ethicists about the potential of anti-ageing therapies and their ethical and social consequences. Given that anti-ageing therapies could dramatically change the social fabric of modern societies, it is quite astonishing that these debates have neglected the views of the larger public.

Investigation into the ability of Gluconacetobacter sacchari to live as an endophyte in sugarcane

The relatively low numbers and sporadic pattern of incidence of the acetic acid bacterium Gluconacetobacter sacchari with the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homoptera: Pseudococcidae) over time and from different sugarcane-growing regions do not indicate that Glac. sacchari is a significant commensal of the PSMB, as has been previously proposed. This study was conducted to investigate the hypothesis that Glac. sacchari is, like its closest relative Glac. diazotrophicus, an endophyte of sugarcane (Saccharum officinarium L.). In this study, both Glac. sacchari and Glac. diazotrophicus were isolated from internal sugarcane tissue, although the detection of both species was sporadic in all sugarcane-growing regions of Queensland tested. To confirm the ability of Glac. sacchari to live endophytically, an experiment was conducted in which the roots of micropropagated sugarcane plantlets were inoculated with Glac. sacchari, and the plantlets were subsequently examined for the presence of the bacterium in the stem cells. Pure cultures of Glac. sacchari were grown from homogenized surface sterilized sugarcane stems inoculated with Glac. sacchari. Electron microscopy was used to provide further conclusive evidence that Glac. sacchari lives as an endophyte in sugarcane. Scanning electron microscopy of (SEM) sugarcane plantlet stems revealed rod-shaped cells of Glac. sacchari within a transverse section of the plantlet stem cells. The numbers of bacterial cells inside the plant cell indicated a successful infection and colonization of the plant tissue. Using transmission electron microscopy, (TEM) bacterial cells were more difficult to find, due to their spatial separation. In our study, bacteria were mostly found singularly, or in groups of up to four cells inside intercellular spaces, although bacterial cells were occasionally found inside other cells.

Does chemotherapy improve survival in high-risk Stage I and II Merkel cell carcinoma of the skin?

Purpose: The effectiveness of synchronous carboplatin, etoposide, and radiation therapy in improving survival was evaluated by comparison of a matched set of historic control subjects with patients treated in a prospective Phase II study that used synchronous chemotherapy and radiation and adjuvant chemotherapy. Patients and Methods: Patients were included in the analysis if they had disease localized to the primary site and nodes, and they were required to have at least one of the following high-risk features: recurrence after initial therapy, involved nodes, primary size greater than 1 cm, or gross residual disease after surgery. All patients who received chemotherapy were treated in a standardized fashion as part of a Phase II study (Trans-Tasman Radiation Oncology Group TROG 96:07) from 1997 to 2001. Radiation was delivered to the primary site and nodes to a dose of 50 Gy in 25 fractions over 5 weeks, and synchronous carboplatin (AUC 4.5) and etoposide, 80 mg/m(2) i.v. on Days 1 to 3, were given in Weeks 1, 4, 7, and 10. The historic group represents a single institution's experience from 1988 to 1996 and was treated with surgery and radiation alone, and patients were included if they fulfilled the eligibility criteria of TROG 96:07. Patients with occult cutaneous disease were not included for the purpose of this analysis. Because of imbalances in the prognostic variables between the two treatment groups, comparisons were made by application of Cox's proportional hazard modeling. Overall survival, disease-specific survival, locoregional control, and distant control were used as endpoints for the study. Results: Of the 102 patients who had high-risk Stage I and II disease, 40 were treated with chemotherapy (TROG 96:07) and 62 were treated without chemotherapy (historic control subjects). When Cox's proportional hazards modeling was applied, the only significant factors for overall survival were recurrent disease, age, and the presence of residual disease. For disease-specific survival, recurrent disease was the only significant factor. Primary site on the lower limb had an adverse effect on locoregional control. For distant control, the only significant factor was residual disease. Conclusions: The multivariate analysis suggests chemotherapy has no effect on survival, but because of the wide confidence limits, a chemotherapy effect cannot be excluded. A study of this size is inadequately powered to detect small improvements in survival, and a larger randomized study remains the only way to truly confirm whether chemotherapy improves the results in high-risk MCC. (c) 2006 Elsevier Inc.

Imaging, anatomical, and molecular analysis of callosal formation in the developing human fetal brain

A complex set of axonal guidance mechanisms are utilized by axons to locate and innervate their targets. In the developing mouse forebrain, we previously described several midline glial populations as well as various guidance molecules that regulate the formation of the corpus callosum. Since agenesis of the corpus callosum is associated with over 50 different human congenital syndromes, we wanted to investigate whether these same mechanisms also operate during human callosal development. Here we analyze midline glial and commissural development in human fetal brains ranging from 13 to 20 weeks of gestation using both diffusion tensor magnetic resonance imaging and immunohistochemistry. Through our combined radiological and histological studies, we demonstrate the morphological development of multiple forebrain commissures/decussations, including the corpus callosum, anterior commissure, hippocampal commissure, and the optic chiasm. Histological analyses demonstrated that all the midline glial populations previously described in mouse, as well as structures analogous to the subcallosal sling and cingulate pioneering axons, that mediate callosal axon guidance in mouse, are also present during human brain development. Finally, by Northern blot analysis, we have identified that molecules involved in mouse callosal development, including Slit, Robo, Netrin1, DCC, Nfia, Emx1, and GAP-43, are all expressed in human fetal brain. These data suggest that similar mechanisms and molecules required for midline commissure formation operate during both mouse and human brain development. Thus, the mouse is an excellent model system for studying normal and pathological commissural formation in human brain development. (c) 2006 Wiley-Liss, Inc.

Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.

RNA: Networks & imaging

The past few years have brought about a fundamental change in our understanding and definition of the RNA world and its role in the functional and regulatory architecture of the cell. The discovery of small RNAs that regulate many aspects of differentiation and development have joined the already known non-coding RNAs that are involved in chromosome dosage compensation, imprinting, and other functions to become key players in regulating the flow of genetic information. It is also evident that there are tens or even hundreds of thousands of other non-coding RNAs that are transcribed from the mammalian genome, as well as many other yet-to-be-discovered small regulatory RNAs. In the recent symposium RNA: Networks & Imaging held in Heidelberg, the dual roles of RNA as a messenger and a regulator in the flow of genetic information were discussed and new molecular genetic and imaging methods to study RNA presented.