Super-resolution structured illumination microscopy: past, present and future.

Structured illumination microscopy (SIM) has emerged as an essential technique for three-dimensional (3D) and live-cell super-resolution imaging. However, to date, there has not been a dedicated workshop or journal issue covering the various aspects of SIM, from bespoke hardware and software development and the use of commercial instruments to biological applications. This special issue aims to recap recent developments as well as outline future trends. In addition to SIM, we cover related topics such as complementary super-resolution microscopy techniques, computational imaging, visualization and image processing methods. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

An Engineered Human Adipose/Collagen Model for In Vitro Breast Cancer Cell Migration Studies.

Adipocytes are one of the major stromal cell components of the human breast. These cells play a key role in the development of the gland and are implicated in breast tumorigenesis. Frequently, directional stromal collagen I fibers are found surrounding aggressive breast tumors. These fibers enhance breast cancer cell migration and are associated with poor patient prognosis. We sought to recapitulate these stromal components in vitro to provide a three-dimensional (3D) model comprising human adipose tissue and anisotropic collagen fibers. We developed a human mesenchymal stem cell (hMSC) cell line capable of undergoing differentiation into mature adipocytes by immortalizing hMSCs, isolated from breast reduction mammoplasties, through retroviral transduction. These immortalized hMSCs were seeded in engineered collagen I scaffolds with directional internal architecture, and adipogenesis was chemically induced, resulting in human adipose tissue being synthesized in vitro in an architectural structure associated with breast tumorigenesis. Subsequently, fluorescently labeled cells from an established breast cancer cell line were seeded into this model, cocultured for 7 days and imaged using multiphoton microscopy. Enhanced breast cancer cell migration was observed in the adipose-containing model over empty scaffold controls, demonstrating an adipocyte-mediated influence on breast cancer cell migration. Thus, this 3D in vitro model recapitulates the migratory effects of adipocytes observed on breast cancer cells and suggests that it could have utility with fresh breast tumor biopsies as an assay for cancer therapeutic efficacy in personalized medicine strategies.

Mathematical imaging methods for mitosis analysis in live-cell phase contrast microscopy.

In this paper we propose a workflow to detect and track mitotic cells in time-lapse microscopy image sequences. In order to avoid the requirement for cell lines expressing fluorescent markers and the associated phototoxicity, phase contrast microscopy is often preferred over fluorescence microscopy in live-cell imaging. However, common specific image characteristics complicate image processing and impede use of standard methods. Nevertheless, automated analysis is desirable due to manual analysis being subjective, biased and extremely time-consuming for large data sets. Here, we present the following workflow based on mathematical imaging methods. In the first step, mitosis detection is performed by means of the circular Hough transform. The obtained circular contour subsequently serves as an initialisation for the tracking algorithm based on variational methods. It is sub-divided into two parts: in order to determine the beginning of the whole mitosis cycle, a backwards tracking procedure is performed. After that, the cell is tracked forwards in time until the end of mitosis. As a result, the average of mitosis duration and ratios of different cell fates (cell death, no division, division into two or more daughter cells) can be measured and statistics on cell morphologies can be obtained. All of the tools are featured in the user-friendly MATLAB®Graphical User Interface MitosisAnalyser.

CARS based label-free assay for assessment of drugs by monitoring lipid droplets in tumour cells.

Coherent anti-Stokes Raman scattering (CARS) is becoming an established tool for label-free multi-photon imaging based on molecule specific vibrations in the sample. The technique has proven to be particularly useful for imaging lipids, which are abundant in cells and tissues, including cytoplasmic lipid droplets (LD), which are recognized as dynamic organelles involved in many cellular functions. The increase in the number of lipid droplets in cells undergoing cell proliferation is a common feature in many neoplastic processes [1] and an increase in LD number also appears to be an early marker of drug-induced cell stress and subsequent apoptosis [3]. In this paper, a CARS-based label-free method is presented to monitor the increase in LD content in HCT116 colon tumour cells treated with the chemotherapeutic drugs Etoposide, Camptothecin and the protein kinase inhibitor Staurosporine. Using CARS, LDs can easily be distinguished from other cell components without the application of fluorescent dyes and provides a label-free non-invasive drug screening assay that could be used not only with cells and tissues ex vivo but potentially also in vivo.

A primary microcephaly protein complex forms a ring around parental centrioles.

Autosomal recessive primary microcephaly (MCPH) is characterized by a substantial reduction in prenatal human brain growth without alteration of the cerebral architecture and is caused by biallelic mutations in genes coding for a subset of centrosomal proteins. Although at least three of these proteins have been implicated in centrosome duplication, the nature of the centrosome dysfunction that underlies the neurodevelopmental defect in MCPH is unclear. Here we report a homozygous MCPH-causing mutation in human CEP63. CEP63 forms a complex with another MCPH protein, CEP152, a conserved centrosome duplication factor. Together, these two proteins are essential for maintaining normal centrosome numbers in cells. Using super-resolution microscopy, we found that CEP63 and CEP152 co-localize in a discrete ring around the proximal end of the parental centriole, a pattern specifically disrupted in CEP63-deficient cells derived from patients with MCPH. This work suggests that the CEP152-CEP63 ring-like structure ensures normal neurodevelopment and that its impairment particularly affects human cerebral cortex growth.

Spatial coupling of mTOR and autophagy augments secretory phenotypes.

Protein synthesis and autophagic degradation are regulated in an opposite manner by mammalian target of rapamycin (mTOR), whereas under certain conditions it would be beneficial if they occurred in unison to handle rapid protein turnover. We observed a distinct cellular compartment at the trans side of the Golgi apparatus, the TOR-autophagy spatial coupling compartment (TASCC), where (auto)lysosomes and mTOR accumulated during Ras-induced senescence. mTOR recruitment to the TASCC was amino acid- and Rag guanosine triphosphatase-dependent, and disruption of mTOR localization to the TASCC suppressed interleukin-6/8 synthesis. TASCC formation was observed during macrophage differentiation and in glomerular podocytes; both displayed increased protein secretion. The spatial coupling of cells' catabolic and anabolic machinery could augment their respective functions and facilitate the mass synthesis of secretory proteins.

Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.

Computed tomography-navigated transthoracic core biopsy of pulmonary lesions: which factors affect diagnostic yield and complication rates?

RATIONALE AND OBJECTIVES: Only a few studies have systematically evaluated risk factors for pneumothorax and pulmonary hemorrhage in computed tomographically (CT)-guided transthoracic lung biopsy (TLB). We evaluated the diagnostic yield of CT-guided TLB and determined risk factors for pneumothorax and hemorrhage. METHODS: One hundred seventy-two CT-guided TLBs were performed on 159 patients (mean age 66 +/- 11 years; 72% male) using a 16-gauge core biopsy needle. Lesion and patient characteristics, lung function analysis, CT signs of emphysema, histopathologic diagnoses, and complications were recorded. Statistical analysis was performed with multivariate regression analysis. RESULTS: Histopathologic diagnosis was established in 153 cases (89%). Although lesion size was higher (47 +/- 29 vs. 43 +/- 35 mm, P = .191) and depth was lower (22 +/- 23 vs. 6 +/- 23 mm, P = .350) in procedures with histopathologic diagnosis, no parameter showed significant impact on diagnostic yield. Sensitivity and specificity for detection of malignancy were 93% and 100%, respectively, whereas positive and negative predictive values were 100% and 88%. Overall accuracy was 95%. Pneumothorax occurred in 45 procedures (26%). Hemorrhage was recorded in 17 procedures (10%). There was higher frequency of pneumothorax in smaller lesions (35 +/- 23 vs. 50 +/- 31 mm, P = .003; odds ratio = .96) and greater depth (29 +/- 29 vs. 20 +/- 19 mm, P = .05; odds ratio = 1.03). CT signs of emphysema revealed higher incidence of hemorrhage (35% vs. 23%; P = .04; odds ratio=41.03). Other parameters were nonsignificant. CONCLUSIONS: The high diagnostic yield of CT-guided TLB was not affected by lesion characteristics or emphysema. Pneumothorax rate was influenced by lesion size and depth. Hemorrhage was associated with CT signs of emphysema.

The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization.

Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids.

A demonstration of pattern formation without positional information in Dictyostelium.

Although positional information, conveyed by morphogen gradients, is a widely accepted way of forming patterns during development, an alternative method is conceivable, based on the intermingled differentiation of cells with different fates, followed by their sorting into discrete pattern elements. It has been proposed that Dictyostelium prestalk and prespore cells behave in this way at the mound stage of development. However, it has been difficult to conclusively demonstrate that they initially differentiate intermingled, because rapid cell movement within the mound makes it impossible to be sure where prestalk and prespore cells originate. We have taken a novel approach to address this problem by blocking cell movement at different stages in development, using the actin-depolymerizing drug, latrunculin-A. Prestalk and prespore cells differentiate with essentially normal efficiency and timing in such paralyzed structures. When movement is blocked sufficiently early, the major cell types all subsequently differentiate at scattered positions throughout the aggregate, and even in the streams leading into it. Our work strongly supports the idea that the prestalk/prespore pattern in Dictyostelium forms without positional information and demonstrate that latrunculin-A may provide a useful tool for the investigation of patterning in other organisms.

A structure-activity study of the inhibition of HIV-1 Tat-dependent trans-activation by mixmer 2'-O-methyl oligoribonucleotides containing locked nucleic acid (LNA), alpha-L-LNA, or 2'-thio-LNA residues.

The HIV-1 trans-activation responsive element (TAR) RNA stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibitors of these interactions block full-length transcription and, hence, would potentially inhibit HIV replication. We have studied structure-activity relationships in inhibition of trans-activation by steric block 2'-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. Inhibition was measured both in Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract and in a robust HeLa cell reporter assay that involves use of stably integrated plasmids to express firefly luciferase Tat dependently and Renilla luciferase Tat-independently. OMe oligonucleotides with optimally 40%-50% LNA units and a minimum of 12 residues in length were active in the cellular assay when delivered with cationic gemini surfactant GS11 at 50% inhibitory concentrations of 230 +/- 40 nM, whereas activity in the in vitro transcription assay was observed down to 9 residues. No cellular activity was observed for OMe oligonucleotides of 12 or 16 residues, which was shown to be due to poor cellular uptake. Both 12-mer mixmers containing alpha -L-LNA or 2'-thio-LNA (S-LNA) were also active in in vitro transcription and the former in cellular reporter inhibition assays, demonstrating that the property of promotion of cellular uptake by LNA is not due to specific sugar conformational effects. Covalent conjugates of OMe/LNA chimeras with Kaposi-fibroblast growth factor (K-FGF) or Transportan peptides failed to enter HeLa cells without a delivery agent but were fully active when delivered by cationic gemini surfactant, showing that in principle, peptide conjugation does not interfere with cellular activity. Thus, OMe/LNA mixmers are powerful reagents for use as steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.

Regulation of the pollen-specific actin-depolymerizing factor LlADF1.

Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by approximately 60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy.