| 1. |
- Beal, Jacob, et al.
(author)
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Robust estimation of bacterial cell count from optical density
- 2020
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Journal article (peer-reviewed)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Boyero, Luz, et al.
(author)
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Riparian plant litter quality increases with latitude
- 2017
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In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Journal article (peer-reviewed)abstract
- Plant litter represents a major basal resource in streams, where its decomposition is partly regulated by litter traits. Litter-trait variation may determine the latitudinal gradient in decomposition in streams, which is mainly microbial in the tropics and detritivore-mediated at high latitudes. However, this hypothesis remains untested, as we lack information on large-scale trait variation for riparian litter. Variation cannot easily be inferred from existing leaf-trait databases, since nutrient resorption can cause traits of litter and green leaves to diverge. Here we present the first global-scale assessment of riparian litter quality by determining latitudinal variation (spanning 107 degrees) in litter traits (nutrient concentrations; physical and chemical defences) of 151 species from 24 regions and their relationships with environmental factors and phylogeny. We hypothesized that litter quality would increase with latitude (despite variation within regions) and traits would be correlated to produce 'syndromes' resulting from phylogeny and environmental variation. We found lower litter quality and higher nitrogen: phosphorus ratios in the tropics. Traits were linked but showed no phylogenetic signal, suggesting that syndromes were environmentally determined. Poorer litter quality and greater phosphorus limitation towards the equator may restrict detritivore-mediated decomposition, contributing to the predominance of microbial decomposers in tropical streams.
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| 3. |
- Bergqvist, Cecilia, et al.
(author)
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Monitoring of chromatin organization in live cells by FRIC. Effects of the inner nuclear membrane protein Samp1
- 2019
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 47:9
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Journal article (peer-reviewed)abstract
- In most cells, transcriptionally inactive heterochromatin is preferentially localized in the nuclear periphery and transcriptionally active euchromatin is localized in the nuclear interior. Different cell types display characteristic chromatin distribution patterns, which change dramatically during cell differentiation, proliferation, senescence and different pathological conditions. Chromatin organization has been extensively studied on a cell population level, but there is a need to understand dynamic reorganization of chromatin at the single cell level, especially in live cells. We have developed a novel image analysis tool that we term Fluorescence Ratiometric Imaging of Chromatin (FRIC) to quantitatively monitor dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation. We also found that peripheral heterochromatin depended on the levels of the inner nuclear membrane protein Samp1, suggesting an important role in promoting peripheral heterochromatin. Taken together, FRIC is a powerful and robust new tool to study dynamic chromatin redistribution in live cells.
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| 4. |
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| 5. |
- Buch, Charlotta, et al.
(author)
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An integral protein of the inner nuclear membrane localizes to the mitotic spindle in mammalian cells
- 2009
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 122:12, s. 2100-2107
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Journal article (peer-reviewed)abstract
- Here, we characterize a transmembrane protein of the nuclear envelope that we name spindle-associated membrane protein 1 (Samp1). The protein is conserved in metazoa and fission yeast and is homologous to Net5 in rat and Ima1 in Schizosaccharomyces pombe. We show that, in human cells, the protein is a membrane-spanning polypeptide with an apparent molecular mass of 43 kDa. This is consistent with a predicted polypeptide of 392 amino acids that has five transmembrane segments and its C-terminus exposed to the nucleoplasm. During interphase, Samp1 was specifically distributed in the inner nuclear membrane. Post-transcriptional silencing of Samp1 expression resulted in separation of centrosomes from the nuclear envelope, indicating that it is functionally connected to the cytoskeleton. At the onset of mitosis, most of the protein dispersed out into the ER, as expected. However, during mitosis, a significant fraction of the protein specifically localized to the polar regions of the mitotic spindle. We demonstrate for the first time, in human cells, the existence of a membranous structure overlapping with the mitotic spindle. Interestingly, another integral inner nuclear membrane protein, emerin, was absent from the spindle-associated membranes. Thus, Samp1 defines a specific membrane domain associated with the mitotic spindle.
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| 6. |
- Figueroa, Ricardo A., et al.
(author)
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Anchored FRET sensors detect local caspase activation prior to neuronal degeneration
- 2011
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In: Molecular Neurodegeneration. - : Springer Science and Business Media LLC. - 1750-1326. ; 6, s. 35-
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Journal article (peer-reviewed)abstract
- BACKGROUND: Recent studies indicate local caspase activation in dendrites or axons during development and in neurodegenerative disorders such as Alzheimer's disease (AD). Emerging evidences point to soluble oligomeric amyloid-beta peptide as a causative agent in AD.RESULTS: Here we describe the design of fluorescence resonance energy transfer (FRET)-based caspase sensors, fused to the microtubule associated protein tau. Specific caspase sensors preferentially cleaved by caspase-3, -6 or -9 were expressed in differentiated human neuroblastoma SH-SY5Y cells. The anchoring of the sensors resulted in high FRET signals both in extended neurites and soma and made analysis of spatiotemporal signal propagation possible. Caspase activation was detected as loss of FRET after exposure to different stimuli. Interestingly, after staurosporine treatment caspase-6 activation was significantly delayed in neurites compared to cell bodies. In addition, we show that exposure to oligomer-enriched amyloid-beta peptide resulted in loss of FRET in cells expressing sensors for caspase-3 and -6, but not -9, in both soma and neurites before neurite degeneration was observed.CONCLUSIONS: Taken together, the results show that by using anchored FRET sensors it is possible to detect stimuli-dependent differential activation of caspases and to distinguish local from global caspase activation in live neuronal cells. Furthermore, in these cells oligomer-enriched amyloid-beta peptide induces a global, rather than local activation of caspase-3 and -6, which subsequently leads to neuronal cell death.
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| 7. |
- Figueroa, Ricardo A., et al.
(author)
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Microtubule-associated nuclear envelope proteins in interphase and mitosis
- 2011
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In: Biochemical Society Transactions. - 0300-5127 .- 1470-8752. ; 39, s. 1786-1789
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Journal article (peer-reviewed)abstract
- The LINC (linker of nucleoskeleton and cytoskeleton) complex forms a transcisternal bridge across the NE (nuclear envelope) that connects the cytoskeleton with the nuclear interior. This enables some proteins of the NE to communicate with the centrosome and the microtubule cytoskeleton. The position of the centrosome relative to the NE is of vital importance for many cell functions, such as cell migration and division, and centrosomal dislocation is a frequent phenotype in laminopathic disorders. Also in mitosis, a small group of transmembrane NE proteins associate with microtubules when they concentrate in a specific membrane domain associated with the mitotic spindle. The present review discusses structural and functional aspects of microtubule association with NE proteins and how this association may be maintained over the cell cycle.
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| 8. |
- Figueroa, Ricardo A., 1979-
(author)
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The functional organization of nuclear membrane proteins and development of new technology for studies of cell signaling
- 2011
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Doctoral thesis (other academic/artistic)abstract
- The eukaryotic cell is defined by the nucleus, which is delimited by a double membrane structure termed the nuclear envelope (NE). The NE is implicated in a multitude of different processes, for example chromatin organization. During mitosis in higher eukaryotes the nucleus is disassembled to allow the formation of the mitotic spindle, which segregates the duplicated chromosomes between daughter cells. We have characterized a novel transmembrane protein of the inner nuclear membrane. Because of its distribution along spindle microtubule during mitosis, we termed the protein Samp1 (Spindle associated membrane protein 1). Samp1 is the founding member of transmembrane proteins that define a novel membrane domain that we have termed the SE (spindle endomembrane). Furthermore, we have shown that in interphase Samp1 specifically interacts with the centrosome and A-type lamina network proteins. Moreover, Samp1 contains an evolutionary highly conserved N-terminal tail containing two putative zinc fingers. Recent studies indicate local caspase activity in dendrites or axons during development and in neurodegenerative disorders. Here I present the development of a novel and unique system to monitor protease activity at sub-cellular resolution in live cells. This system relies on a cleavable FRET sensor that is anchored to the cytoskeleton. Using this system we demonstrate local caspase activation of the soma in neuronaly differentiated cells. We also used the anchored FRET sensors to monitor caspase activation after treatment with the Alzheimer’s decease related amyloid-β peptide. Moreover we have improved a NF-ĸB decoy delivery system. The system consists of a cell penetrating peptide, transportan-10, covalently linked to a peptide nucleic acid sequence that hybridizes with a nonanucleotide sequence in the decoy. We show that this system effectively delivered the decoy and inhibited an inflammatory response in primary rat glial cells.
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| 9. |
- Figueroa, Ricardo, et al.
(author)
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A transmembrane inner nuclear membrane protein in the mitotic spindle
- 2010
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In: Nucleus (Austin). - 1949-1042. ; 1:3, s. 249-253
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Journal article (peer-reviewed)abstract
- We have recently characterized a novel transmembrane protein of the inner nuclear membrane of mammalian cells. The protein has two very interesting features. First, despite being an integral membrane protein it is able to concentrate in the membranes colocalizing with the mitotic spindle in metaphase and anaphase. Hence, the protein was named Samp1, Spindle associated membrane protein 1. Secondly, it displays a functional connection to centrosomes. This article discusses various aspects of Samp1 in relation to possible cellular function(s).
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| 10. |
- Fisher, Linda, et al.
(author)
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Targeting cytokine expression in glial cells by cellular delivery of an NFκB decoy
- 2007
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In: Journal of Molecular Neuroscience. - 0895-8696 .- 1559-1166. ; 31:3, s. 209-219
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Journal article (peer-reviewed)abstract
- Inhibition of nuclear factor (NF)-κB has emerged as an important strategy for design of anti-inflammatory therapies. In neurodegenerative disorders like Alzheimer’s disease, inflammatory reactions mediated by glial cells are believed to promote disease progression. Here, we report that uptake of a double-stranded oligonucleotide NF-κB decoy in rat primary glial cells is clearly facilitated by noncovalent binding to a cell-penetrating peptide, transportan 10, via a complementary peptide nucleic acid (PNA) sequence. Fluorescently labeled oligonucleotide decoy was detected in the cells within 1 h only when cells were incubated with the decoy in the presence of cell-penetrating peptide. Cellular delivery of the decoy also inhibited effects induced by a neurotoxic fragment of the Alzheimer β amyloid peptide in the presence of the inflammatory cytokine interleukin (IL) 1β. Pretreatment of the cells with the complex formed by the decoy and the cell-penetrating peptide-PNA resulted in 80% and 50% inhibition of the NF-κB binding activity and IL-6 mRNA expression, respectively.
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