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  • 1.
    Adler, J
    et al.
    Stockholms universitet, Wenner-Grens institut för experimentell biologi.
    Pagakis, S N
    Parmryd, I
    Stockholms universitet, Wenner-Grens institut för experimentell biologi.
    Replicate-based noise corrected correlation for accurate measurements of colocalization.2008In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 230, no Pt 1, p. 121-33Article in journal (Refereed)
  • 2. Adler, Jeremy
    et al.
    Bergholm, Fredrik
    Pagakis, Stamatis
    Parmryd, Ingela
    Noise and colocalization in fluorescence microscopy: solving a problem2008In: Microscopy and Analysis, Vol. 22, no 5, p. 7-10Article, review/survey (Other academic)
    Abstract [en]

    The Pearson correlation coefficient (PCC) is regularly used in colocalization measurements, but it is sensitive to image noise. Images of fluorophores are usually degraded by Poisson and background noise and we have found that, even with apparently high quality images, the measured PCC is substantially understated, to the extent that the numbers become misleading. This means that ostensibly significant differences in the PCC between two populations may just reflect differing image quality while dissimilar levels of noise may mask significant differences. A new correction, based on measurements of image quality, derived from a pair of images for each fluorophore, aligns the measured PCC with the true PCC. Our method, the Replicate Based Noise Corrected Correlation (RBNCC), generates an accurate PCC even from poor images. It is highly photon efficient and therefore well suited for use in live cell fluorescence imaging or with rapidly bleaching fluorophores.

  • 3.
    Adler, Jeremy
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Colocalization Analysis in Fluorescence Microscopy2012In: Cell Imaging Techniques: Methods and Protocols / [ed] Taatjes, Douglas J. & Roth, Jürgen, New York: Humana Press, 2012, p. 97-109Chapter in book (Refereed)
    Abstract [en]

    The measurement of colocalization requires images of two fluorophores that are aligned, with no cross talk, and that the intensities remain within the response range of the microscope. Quantitation depends upon differentiating between the presence and absence of fluorescence, and measurements should be made within biologically relevant regions of interest. Co-occurrence can be measured simply by area or with the M1 and M2 coefficients, and should be compared to random distributions. Correlation analysis should use the Pearson and Spearman coefficients, which need to be measured by replicate based noise corrected correlation to eliminate errors arising from differences in image quality. Ideally, both co-occurrence and correlation should be reported.

  • 4.
    Adler, Jeremy
    et al.
    Stockholms universitet, Wenner-Grens institut för experimentell biologi.
    Parmryd, Ingela
    Stockholms universitet, Wenner-Grens institut för experimentell biologi.
    In support of the Pearson correlation coefficient.2007In: Journal of Microscopy, Vol. 227, no Pt 1, p. 83; author reply 84-5Article in journal (Other academic)
  • 5.
    Adler, Jeremy
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Quantification of Colocalisation; Co-Occurrence, Correlation, Empty Voxels, Regions of Interest and Thresholding2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 602A-602AArticle in journal (Other academic)
    Abstract [en]

    Measuring colocalisation is not straightforward with a plethora of coefficients that encapsulate different definitions. Measurements may also be implemented differently. Not only do measurements differ; interconversion is impossible making comparisons challenging. There is a need to cull coefficients and for clear definitions of what precisely is meant by colocalisation in individual studies. Colocalisation can be considered to have two components; co-occurrence which reports whether the fluorophores are found together and correlation which reports on the similarity in their patterns of intensity.

  • 6.
    Adler, Jeremy
    et al.
    Stockholms universitet, Wenner-Grens institut.
    Parmryd, Ingela
    Stockholms universitet, Wenner-Grens institut.
    Quantifying Colocalization by Correlation: The Pearson Correlation Coefficient is Superior to the Mander's Overlap Coefficient2010In: CYTOMETRY PART A, ISSN 1552-4922, Vol. 77A, no 8, p. 733-742Article in journal (Refereed)
    Abstract [en]

    The Pearson correlation coefficient (PCC) and the Mander's overlap coefficient (MOC) are used to quantify the degree of colocalization between fluorophores. The MOC was introduced to overcome perceived problems with the PCC. The two coefficients are mathematically similar, differing in the use of either the absolute intensities (MOC) or of the deviation from the mean (PCC). A range of correlated datasets, which extend to the limits of the PCC, only evoked a limited response from the MOC. The PCC is unaffected by changes to the offset while the MOC increases when the offset is positive. Both coefficients are independent of gain. The MOC is a confusing hybrid measurement, that combines correlation with a heavily weighted form of co-occurrence, favors high intensity combinations, downplays combinations in which either or both intensities are low and ignores blank pixels. The PCC only measures correlation. A surprising finding was that the addition of a second uncorrelated population can substantially increase the measured correlation, demonstrating the importance of excluding background pixels. Overall, since the MOC is unresponsive to substantial changes in the data and is hard to interpret, it is neither an alternative to nor a useful substitute for the PCC. The MOC is not suitable for making measurements of colocalization either by correlation or co-occurrence.

  • 7.
    Adler, Jeremy
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Quantifying colocalization: thresholding, void voxels and the H-coef2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 11, p. e111983-Article in journal (Refereed)
    Abstract [en]

    A critical step in the analysis of images is identifying the area of interest e.g. nuclei. When the nuclei are brighter than the remainder of the image an intensity can be chosen to identify the nuclei. Intensity thresholding is complicated by variations in the intensity of individual nuclei and their intensity relative to their surroundings. To compensate thresholds can be based on local rather than global intensities. By testing local thresholding methods we found that the local mean performed poorly while the Phansalkar method and a new method based on identifying the local background were superior. A new colocalization coefficient, the Hcoef, highlights a number of controversial issues. (i) Are molecular interactions measurable (ii) whether to include voxels without fluorophores in calculations, and (iii) the meaning of negative correlations. Negative correlations can arise biologically (a) because the two fluorophores are in different places or (b) when high intensities of one fluorophore coincide with low intensities of a second. The cases are distinct and we argue that it is only relevant to measure correlation using pixels that contain both fluorophores and, when the fluorophores are in different places, to just report the lack of co-occurrence and omit these uninformative negative correlation. The Hcoef could report molecular interactions in a homogenous medium. But biology is not homogenous and distributions also reflect physico-chemical properties, targeted delivery and retention. The Hcoef actually measures a mix of correlation and co-occurrence, which makes its interpretation problematic and in the absence of a convincing demonstration we advise caution, favouring separate measurements of correlation and of co-occurrence.

  • 8. Adler, Jeremy
    et al.
    Shevchuk, Andrew I
    Novak, Pavel
    Korchev, Yuri E
    Parmryd, Ingela
    Plasma membrane topography and interpretation of single-particle tracks.2010In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 7, no 3, p. 170-1Article in journal (Refereed)
  • 9. Appelkvist, E L
    et al.
    Aberg, F
    Guan, Z
    Parmryd, Ingela
    Dallner, G
    Regulation of coenzyme Q biosynthesis1994In: Molecular Aspects of Medicine, ISSN 0098-2997, E-ISSN 1872-9452, Vol. 15, no Suppl., p. 37-46Article, review/survey (Refereed)
    Abstract [en]

    The side-chain moiety of coenzyme Q is synthesized by a trans-prenyltransferase present in microsomes. Condensation of this moiety with the precursor ring takes place in the Golgi system. The enzymes involved, as well as the cytosolic geranylgeranyl-PP synthase, are regulated in an independent fashion. When the size of the farnesyl-PP pool is decreased or increased by employing appropriate inhibitors, the rate of CoQ synthesis is modified accordingly, indicating the dependence of trans-prenyltransferase activity on the level of intracellular substrate concentrations. Administration of peroxisome proliferators elevates CoQ concentrations not only in blood, but also in various tissues. Thus, it may be possible in the future to selectively increase CoQ concentrations in certain organs, without increasing the level of cholesterol.

  • 10. Appelkvist, E L
    et al.
    Venizelos, N
    Zhang, Y
    Parmryd, I
    Hagenfeldt, L
    Dallner, G
    Synthesis of mevalonate pathway lipids in fibroblasts from Zellweger and X-linked ALD patients.1999In: Pediatric Research, ISSN 0031-3998, E-ISSN 1530-0447, Vol. 46, no 3, p. 345-50Article in journal (Refereed)
    Abstract [en]

    Fibroblasts were cultured to determine the involvement of peroxisomes in cholesterol and dolichol synthesis. For this purpose, the behavior of cells from patients with Zellweger syndrome, with X-linked adrenoleukodystrophy, and from nondiseased control subjects was studied. Cells both after pretreatment with mevinolin and without pretreatment were incubated in a medium containing [3H]-mevalonate. In fibroblasts from patients with peroxisomal defects, the cholesterol content and mevalonate incorporation into cholesterol were decreased by 10-20% in comparison with control cells. Mevinolin pretreatment decreased the incorporation rate of [3H]-mevalonate into cholesterol but increased the labeling of ubiquinone and dolichol both in diseased and control cells. Squalene synthase activity was unchanged, whereas the activity of farnesyl-pyrophosphate synthase was increased in the diseased states. The results show that in patients with peroxisomal deficiency neither the amount nor the rate of synthesis of cholesterol and dolichol is reduced to any greater extent.

  • 11.
    Ashrafzadeh, Parham
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Dinic, Jelena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Actin Filaments Attachment to the Plasma Membrane Cause the Formation of Ordered Lipid Domains in Live Cells2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 706A-706AArticle in journal (Other academic)
    Abstract [en]

    The aim of this study was to investigate the relationship between ordered plasma membrane nanodomains and actin filaments using di-4-ANEPPDHQ and laurdan together with the reagents that affect actin filament dynamics in live Jurkat and primary T cells. The degree of lipid packing can be quantified using polarity sensitive membrane dyes such as laurdan and di-4-ANEPPDHQ. These two dyes display a red shift in their emission peaks for membranes in ld phase relative to lo phase. Laurdan is uncharged and can easily flip between two leaflets of the plasma membrane and we demonstrate that it reports equally on the two leaflets of the plasma membrane.

  • 12.
    Ashrafzadeh, Parham
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Methods applicable to membrane nanodomain studies?2015In: Essays in Biochemistry, ISSN 0071-1365, E-ISSN 1744-1358, Vol. 57, p. 57-68Article, review/survey (Refereed)
    Abstract [en]

    Membrane nanodomains are dynamic liquid entities surrounded by another type of dynamic liquid. Diffusion can take place inside, around and in and out of the domains, and membrane components therefore continuously shift between domains and their surroundings. In the plasma membrane, there is the further complexity of links between membrane lipids and proteins both to the extracellular matrix and to intracellular proteins such as actin filaments. In addition, new membrane components are continuously delivered and old ones removed. On top of this, cells move. Taking all of this into account imposes great methodological challenges, and in the present chapter we discuss some methods that are currently used for membrane nanodomain studies, what information they can provide and their weaknesses.

  • 13. Bergholm, Fredrik
    et al.
    Adler, Jeremy
    Stockholms universitet, Wenner-Grens institut.
    Parmryd, Ingela
    Stockholms universitet, Wenner-Grens institut.
    Analysis of Bias in the Apparent Correlation Coefficient Between Image Pairs Corrupted by Severe Noise2010In: Journal of Mathematical Imaging and Vision, ISSN 0924-9907, E-ISSN 1573-7683, Vol. 37, no 3, p. 204-219Article in journal (Refereed)
    Abstract [en]

    The correlation coefficient r is a measure of similarity used to compare regions of interest in image pairs. In fluorescence microscopy there is a basic tradeoff between the degree of image noise and the frequency with which images can be acquired and therefore the ability to follow dynamic events. The correlation coefficient r is commonly used in fluorescence microscopy for colocalization measurements, when the relative distributions of two fluorophores are of interest. Unfortunately, r is known to be biased understating the true correlation when noise is present. A better measure of correlation is needed. This article analyses the expected value of r and comes up with a procedure for evaluating the bias of r, expected value formulas. A Taylor series of so-called invariant factors is analyzed in detail. These formulas indicate ways to correct r and thereby obtain a corrected value free from the influence of noise that is on average accurate (unbiased). One possible correction is the attenuated corrected correlation coefficient R, introduced heuristically by Spearman (in Am. J. Psychol. 15:72-101, 1904). An ideal correction formula in terms of expected values is derived. For large samples R tends towards the ideal correction formula and the true noise-free correlation. Correlation measurements using simulation based on the types of noise found in fluorescence microscopy images illustrate both the power of the method and the variance of R. We conclude that the correction formula is valid and is particularly useful for making correct analyses from very noisy datasets.

  • 14. Daly, Craig J
    et al.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    McGrath, John C
    Visualization and analysis of vascular receptors using confocal laser scanning microscopy and fluorescent ligands2012In: Receptor Binding Techniques / [ed] Anthony P. Davenport, New York: Humana Press, 2012, Vol. 897, p. 95-107Chapter in book (Refereed)
    Abstract [en]

    The use of fluorescent ligands to analyze receptor distribution is increasing in popularity. This is due to the ever growing number of fluorescent ligands and the increased sensitivity of microscope-based technologies. Image-analysis methods have advanced to a stage where quantification of fluorescent signals is relatively simple (if used appropriately). In this chapter we describe a method of analyzing the 2D and 3D distribution of fluorescent ligands in segments of blood vessels. In addition, we introduce the issues surrounding the accurate analysis of colocalization of two different fluorescent ligands.

  • 15.
    Dinic, Jelena
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Ashrafzadeh, Parham
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid domains2013In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 3, p. 1102-1111Article in journal (Refereed)
    Abstract [en]

    The relationship between ordered plasma membrane nanodomains, known as lipid rafts, and actin filaments is the focus of this study. Plasma membrane order was followed in live cells at 37°C using laurdan and di-4-ANEPPDHQ to report on lipid packing. Disrupting actin polymerisation decreased the fraction of ordered domains, which strongly argue that unstimulated cells have a basal level of ordered domains. Stabilising actin filaments had the opposite effect and increased the proportion of ordered domains. Decreasing the plasma membrane level of 4-phosphate-inositides lowers the number of attachment points for actin filaments and reduced the proportion of ordered domains. Aggregation of plasma membrane molecules, both lipid raft and non-lipid raft markers, lead to the formation of ordered domains. The increase in ordered domains was correlated with an increase in actin filaments just beneath the plasma membrane. In live cell plasma membrane blebs, which are detached from the underlying actin filaments, the fraction of ordered domains was low and GM1 could not be patched to form ordered domains. We conclude that ordered domains form when actin filaments attach to the plasma membrane. This downplays lipid-lipid interactions as the main driving force behind the formation of ordered membrane domains in vivo, giving greater prominence to membrane-intracellular filament interactions.

  • 16.
    Dinic, Jelena
    et al.
    Stockholms universitet, Wenner-Grens institut.
    Biverståhl, Henrik
    Stockholms universitet, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Institutionen för biokemi och biofysik.
    Parmryd, Ingela
    Stockholms universitet, Wenner-Grens institut.
    Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing2011In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1808, no 1, p. 298-306Article in journal (Refereed)
    Abstract [en]

    Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.

  • 17.
    Dinic, Jelena
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Riehl, Astrid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Adler, Jeremy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The T cell receptor resides in ordered plasma membrane nanodomains that aggregate upon patching of the receptor2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 10082Article in journal (Refereed)
    Abstract [en]

    Two related models for T cell signalling initiation suggest either that T cell receptor (TCR) engagement leads to its recruitment to ordered membrane domains, often referred to as lipid rafts, where signalling molecules are enriched or that ordered TCR-containing membrane nanodomains coalesce upon TCR engagement. That ordered domains form upon TCR engagement, as they do upon lipid raft marker patching, has not been considered. The target of this study was to differentiate between those three options. Plasma membrane order was followed in live T cells at 37 °C using laurdan to report on lipid packing. Patching of the TCR that elicits a signalling response resulted in aggregation, not formation, of ordered plasma membrane domains in both Jurkat and primary T cells. The TCR colocalised with actin filaments at the plasma membrane in unstimulated Jurkat T cells, consistent with it being localised to ordered membrane domains. The colocalisation was most prominent in cells in G1 phase when the cells are ready to commit to proliferation. At other cell cycle phases the TCR was mainly found at perinuclear membranes. Our study suggests that the TCR resides in ordered plasma membrane domains that are linked to actin filaments and aggregate upon TCR engagement.

  • 18. Fotin-Mleczek, Mariola
    et al.
    Henkler, Frank
    Samel, Dierk
    Reichwein, Monica
    Hausser, Angelika
    Parmryd, Ingela
    Scheurich, Peter
    Schmid, Johannes A
    Wajant, Harald
    Apoptotic crosstalk of TNF receptors: TNF-R2-induces depletion of TRAF2 and IAP proteins and accelerates TNF-R1-dependent activation of caspase-82002In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 115, no Pt 13, p. 2757-70Article in journal (Refereed)
    Abstract [en]

    We have recently shown that stimulation of TNF-R2 selectively enhances apoptosis induction by the death receptor TNF-R1. Here, we demonstrate that stimulation of CD30 or CD40 also leads to selective enhancement of TNF-R1-induced cell death. Enhancement of apoptosis was correlated with the depletion of endogenous TRAF2 within 1 to 6 hours. Selective prestimulation of TNF-R2 for several hours inhibited TNF-R2-induced activation of the anti-apoptotic NF-kappaB pathway up to 90% and dramatically enhanced apoptosis induction by this receptor. When both TNF-receptors were stimulated simultaneously, TNF-R1-induced NF-kappaB activation remained unaffected but TNF-R1-induced apoptosis was still significantly enhanced. Compared with FasL-induced cell death TNF-R1-induced activation of caspase-8 was significantly weaker and delayed. Costimulation or prestimulation of TNF-R2 enhanced caspase-8 processing. Life cell imaging and confocal microscopy revealed that both TNF-R1 and TNF-R2 recruited the anti-apoptotic factor cIAP1 in a TRAF2-dependent manner. Thus, TNF-R2 may compete with TNF-R1 for the recruitment of newly synthesized TRAF2-bound anti-apoptotic factors, thereby promoting the formation of a caspase-8-activating TNF-R1 complex. Hence, TNF-R2 triggering can interfere with TNF-R1-induced apoptosis by inhibition of NF-kappaB-dependent production of anti-apoptotic factors and by blocking the action of anti-apoptotic factors at the post-transcriptional level.

  • 19. Fujimoto, Toyoshi
    et al.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation2016In: Frontiers in Cell and Developmental Biology, ISSN 2296-634X, Vol. 4, article id 155Article, review/survey (Refereed)
    Abstract [en]

    The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence.

  • 20.
    Grünler, J
    et al.
    Department of Biochemistry, Stockholm University, Sweden.
    Parmryd, Ingela
    Department of Biochemistry, Stockholm University, Sweden.
    Subcellular distribution of farnesyl protein transferase in rat liver1999In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 455, no 3, p. 233-237Article in journal (Refereed)
    Abstract [en]

    Farnesyl protein transferase (FPT) activity was measured in rat liver subcellular fractions by using an unspecific acceptor for the farnesyl groups. The highest specific activity was found in mitochondria and it exceeded that of the microsomes three-fold. Considerably lower specific activities were found in the nuclei and cytosol. Further subfractionation revealed that the mitochondrial FPT activity is located in the matrix. The beta-subunit of the mitochondrial enzyme has an apparent molecular mass of 46 kDa, which is similar to its cytosolic counterpart. The results suggest that protein farnesylation can take place in a number of subcellular organelles.

  • 21.
    Huang, Fang
    et al.
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
    Parmryd, Ingela
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
    Nilsson, Fredrik
    AstraZeneca R&D Mölndal, SE-43183 Mölndal, Sweden .
    Persson, Annika L.
    Department of Zoological Cell Biology, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden.
    Pakrasi, Himadri B.
    Department of Biology, Washington University, St. Louis, Missouri 63130.
    Andersson, Bertil
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden;Division of Cell Biology, Linköping University, SE-58185 Linköping, Sweden.
    Norling, Birgitta
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
    Proteomics of Synechocystis sp. strain PCC 6803: identification of plasma membrane proteins2002In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 1, no 12, p. 956-966Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F(0) membrane part of the ATP synthase.

  • 22. Lindberg, Bo G
    et al.
    Merritt, Eleanor A
    Rayl, Melanie
    Liu, Chenxiao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Olofsson, Berit
    Faye, Ingrid
    Immunogenic and Antioxidant Effects of a Pathogen-Associated Prenyl Pyrophosphate in Anopheles gambiae2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 8, p. e73868-Article in journal (Refereed)
    Abstract [en]

    Despite efficient vector transmission, Plasmodium parasites suffer great bottlenecks during their developmental stages within Anopheles mosquitoes. The outcome depends on a complex three-way interaction between host, parasite and gut bacteria. Although considerable progress has been made recently in deciphering Anopheles effector responses, little is currently known regarding the underlying microbial immune elicitors. An interesting candidate in this sense is the pathogen-derived prenyl pyrophosphate and designated phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), found in Plasmodium and most eubacteria but not in higher eukaryotes. HMBPP is the most potent stimulant known of human Vγ9Vδ2 T cells, a unique lymphocyte subset that expands during several infections including malaria. In this study, we show that Vγ9Vδ2 T cells proliferate when stimulated with supernatants from intraerythrocytic stages of Plasmodium falciparum cultures, suggesting that biologically relevant doses of phosphoantigens are excreted by the parasite. Next, we used Anopheles gambiae to investigate the immune- and redox- stimulating effects of HMBPP. We demonstrate a potent activation in vitro of all but one of the signaling pathways earlier implicated in the human Vγ9Vδ2 T cell response, as p38, JNK and PI3K/Akt but not ERK were activated in the A. gambiae 4a3B cell line. Additionally, both HMBPP and the downstream endogenous metabolite isopentenyl pyrophosphate displayed antioxidant effects by promoting cellular tolerance to hydrogen peroxide challenge. When provided in the mosquito blood meal, HMBPP induced temporal changes in the expression of several immune genes. In contrast to meso-diaminopimelic acid containing peptidoglycan, HMBPP induced expression of dual oxidase and nitric oxide synthase, two key determinants of Plasmodium infection. Furthermore, temporal fluctuations in midgut bacterial numbers were observed. The multifaceted effects observed in this study indicates that HMBPP is an important elicitor in common for both Plasmodium and gut bacteria in the mosquito.

  • 23. Lisowska, Halina
    et al.
    Deperas-Kaminska, Marta
    Haghdoost, Siamak
    Stockholms universitet, Institutionen för genetik, mikrobiologi och toxikologi.
    Parmryd, Ingela
    Stockholms universitet, Avdelningen för cellbiologi.
    Wojcik, Andrzej
    Stockholms universitet, Institutionen för genetik, mikrobiologi och toxikologi.
    Radiation-induced DNA damage and repair in human gammadelta and alphabeta T-lymphocytes analysed by the alkaline comet assay2010In: Genome integrity, ISSN 2041-9414, Vol. 1, no 1, p. 8-Article in journal (Refereed)
    Abstract [en]

    It has been shown by a number of authors that the radiosensitivity of peripheral blood mononuclear cells (PBMC) is higher in cancer patients compared to healthy donors, which is interpreted as a sign of genomic instability. PBMC are composed of different cell subpopulations which are differently radiosensitive and the difference between cancer patients and healthy donors could also be due to different composition of their PBMC pools. Gamma-delta T-lymphocytes play an important role in immunosurveillance and are promising cells for immunotherapy. Their abundance is frequently reduced in cancer patients so should their sensitivity to radiation be lower than that of other T-lymphocytes, this could, at least partly explain the low radiosensitivity of PBMC from healthy individuals compared to cancer patients. The present investigation was carried out to test this. Using the alkaline comet assay we analysed the level of DNA damage and repair in isolated gammadelta T-lymphocytes, pan T-lymphocytes and in total PBMC exposed in vitro to gamma radiation. We found no difference in the level of DNA damage and the capacity of DNA repair between the T cell populations. This is the first study that addresses the question of sensitivity to radiation of gamma-delta T-cells.

  • 24. Magee, Anthony I
    et al.
    Adler, Jeremy
    Parmryd, Ingela
    Cold-induced coalescence of T-cell plasma membrane microdomains activates signalling pathways2005In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 118, no Pt 14, p. 3141-51Article in journal (Refereed)
    Abstract [en]

    The plasma membranes of eukaryotic cells are hypothesised to contain microdomains with distinct lipid and protein composition known as lipid rafts. In T cells, cross-linking of lipid raft components triggers signalling cascades. We show that the T-cell antigen receptor (TCR) and a protein tyrosine kinase, Lck, have a patchy plasma membrane distribution in Jurkat T cells at reduced temperatures, although they have a continuous distribution at physiological temperature (37 degrees C). GM1 displays a patchy distribution at reduced temperature after Triton X-100 extraction. The archetypal non-lipid raft marker, the transferrin receptor, displays a more continuous plasma membrane distribution uncorrelated with that of Lck at 0 degrees C. Cold-induced aggregation of the lipid raft-partitioning proteins is accompanied by increased tyrosine phosphorylation and ERK activation, peaking at 10-20 degrees C. Tyrosine phosphorylation is further greatly increased by ligating the TCR with anti-CD3 at 10-20 degrees C. The tyrosine phosphorylation mainly occurred at the plasma membrane, was dependent on Lck and on the surface expression of the TCR. The activation of tyrosine phosphorylation and ERK by TCR ligation at reduced temperature also occurred in human primary T cells. These results support the concept that lipid rafts can form in membranes of live cells and that their coalescence stimulates signalling.

  • 25. Magee, Anthony I
    et al.
    Parmryd, Ingela
    Detergent-resistant membranes and the protein composition of lipid rafts2003In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 4, no 11, p. 234-Article, review/survey (Refereed)
    Abstract [en]

    The plasma membrane of eukaryotic cells contains lipid rafts with protein and lipid compositions differing from the bulk plasma membrane. Several recent proteomic studies have addressed the composition of lipid rafts, but the different definitions used for lipid rafts need scrutinizing before results can be evaluated.

  • 26. Magee, Tony
    et al.
    Pirinen, Niina
    Adler, Jeremy
    Pagakis, Stamatis N
    Parmryd, Ingela
    Lipid rafts: cell surface platforms for T cell signaling2002In: Biological Research (Print), ISSN 0716-9760, E-ISSN 0717-6287, Vol. 35, no 2, p. 127-31Article, review/survey (Refereed)
    Abstract [en]

    The Src family tyrosine kinase Lck is essential for T cell development and T cell receptor (TCR) signaling. Lck is post-translationally fatty acylated at its N-terminus conferring membrane targeting and concentration in plasma membrane lipid rafts, which are lipid-based organisational platforms. Confocal fluorescence microscopy shows that Lck colocalizes in rafts with GPI-linked proteins, the adaptor protein LAT and Ras, but not with non-raft membrane proteins including the protein tyrosine phosphatase CD45. The TCR also associates with lipid rafts and its cross-linking causes coaggregation of raft-associated proteins including Lck, but not of CD45. Cross-linking of either the TCR or rafts strongly induces specific tyrosine phosphorylation of the TCR in the rafts. Remarkably, raft patching alone induces signalling events analogous to TCR stimulation, with the same dependence on expression of key TCR signalling molecules. Our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of signaling proteins including Lck, LAT, and the TCR, while excluding CD45, thereby potentiating protein tyrosine phosphorylation and downstream signaling. We are currently testing this hypothesis as well as using imaging techniques such as fluorescence resonance energy transfer (FRET) microscopy to study the dynamics of proteins and lipids in lipid rafts in living cells undergoing signaling events. Recent data show that the key phosphoinositide PI(4,5)P2 is concentrated in T cell lipid rafts and that on stimulation of the cells it is rapidly converted to PI(3,4,5)P3 and diacylglycerol within rafts. Thus rafts are hotspots for both protein and lipid signalling pathways.

  • 27.
    Mahammad, Saleemulla
    Stockholms universitet, Wenner-Grens institut.
    Cholesterol in T cells: homeostasis, plasma membrane organization and signaling2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The plasma membrane of eukaryotic cells contains cholesterol and glycosphingolipids enriched nanodomains known as lipid rafts; which are believed to exist in a liquid ordered (lo) state. Methyl-beta-cyclodextrin (MBCD) is used to deplete cellular cholesterol and a widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts. To analyze this in T cells a progressive cholesterol extraction protocols was established. At 37ºC, MBCD treatment does not lead to the preferential loss of cholesterol from TX-DRMs. At 0ºC only 35% of total cholesterol could be extracted demonstrating that less than 35% of the cell’s cholesterol is found in the plasma membrane. Moreover, incubation of cells at 0ºC causes loss of plasma membrane cholesterol and an increase in cholesteryl esters. The increase in cholesterol esters upon cold exposure is linked to the cholesterol concentration induced activation of ACAT enzyme which converts cholesterol to cholesteryl esters. Cholesterol concentration specific activation of ACAT and conversion of cholesterol to cholesteryl esters during the loading of cholesterol onto T cells by MBCD was also observed. By using MBCD for progressive cholesterol depletion from T cells at 37ºC, the effect of cholesterol depletion on T cell signaling was addressed. At 10-20% cholesterol depletion levels, tyrosine phosphorylation is increased and ERK is activated. Peripheral actin polymerization, cell spreading and membrane protrusions are also triggered by limited cholesterol depletion. Upon limited cholesterol depletion aggregation of lipid rafts in the plasma membrane was observed. The aggregation of lipid rafts upon cholesterol depletion does not dependent on the signaling proteins such as Src-kinases. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains.

  • 28.
    Mahammad, Saleemulla
    et al.
    Stockholms universitet, Wenner-Grens institut.
    Dinic, Jelena
    Stockholms universitet, Wenner-Grens institut.
    Adler, Jeremy
    Stockholms universitet, Wenner-Grens institut.
    Parmryd, Ingela
    Stockholms universitet, Wenner-Grens institut.
    Limited cholesterol depletion causes aggregation of plasma membrane lipid raftsinducing T cell activation2010In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1801, no 6, p. 625-634Article in journal (Refereed)
    Abstract [en]

    Acute cholesterol depletion is generally associated with decreased or abolished T cell signalling but it can also cause T cell activation. This anomaly has been addressed in Jurkat T cells using progressive cholesterol depletion with methyl-beta-cyclodextrin (MBCD). At depletion levels higher than 50% there is substantial cell death, which explains reports of signalling inhibition. At 10–20% depletion levels, tyrosine phosphorylation is increased, ERK is activated and there is a small increase in cytoplasmic Ca2+. Peripheral actin polymerisation is also triggered by limited cholesterol depletion. Strikingly, the lipid raft marker GM1 aggregates upon cholesterol depletion and these aggregated domains concentrate the signalling proteins Lck and LAT, whereas the opposite is true for the non lipid raft marker the transferrin receptor. Using PP2, an inhibitor of Src family kinase activation, it is demonstrated that the lipid raft aggregation occurs independently of and thus upstream of the signalling response. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. That cholesterol depletion and not unspecific effects of MBCD was behind the reported results was confirmed by performing all experiments with MBCD–cholesterol, when no net cholesterol extraction took place. We conclude that non-lethal cholesterol depletion causes the aggregation of lipid rafts which then induces T cell signalling.

  • 29.
    Mahammad, Saleemulla
    et al.
    Department of Cell and Molecular Biology, Northwestern University, Feinberg School of Medicine, Chicago, IL, United States.
    Parmryd, Ingela
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Cholesterol Depletion Using Methyl-β-cyclodextrin2015In: Methods in Membrane Lipids / [ed] Dylan M. Owen, Springer, 2015, Vol. 1232, p. 91-102Chapter in book (Refereed)
    Abstract [en]

    Cholesterol is an essential component of mammalian cells. It is the major lipid constituent of the plasma membrane and is also abundant in most other organelle membranes. In the plasma membrane cholesterol plays critical physical roles in the maintenance of membrane fluidity and membrane permeability. It is also important for membrane trafficking, cell signalling, and lipid as well as protein sorting. Cholesterol is essential for the formation of liquid ordered domains in model membranes, which in cells are known as lipid nanodomains or lipid rafts. Cholesterol depletion is widely used to study the role of cholesterol in cellular processes and can be performed over days using inhibitors of its synthesis or acutely over minutes using chemical reagents. Acute cholesterol depletion by methyl-β-cyclodextrin (MBCD) is the most widely used method and here we describe how it should be performed to avoid the common side-effect cell death.

  • 30.
    Mahammad, Saleemulla
    et al.
    Stockholms universitet, Wenner-Grens institut.
    Parmryd, Ingela
    Stockholms universitet, Wenner-Grens institut.
    Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions.2008In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1778, no 5, p. 1251-8Article in journal (Refereed)
    Abstract [en]

    Methyl-beta-cycloclextrin (MBCD) is frequently used to acutely deplete cells of cholesterol. A widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts and that sensitivity to MBCD is proof of lipid raft involvement in a cellular process. To analyse any MBCD preference systematically, progressive cholesterol depletion of Jurkat T cells was performed using MBCD and [H-3]-cholesterol. It was found that at 37 degrees C, MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions and that the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, cells restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, mere incubation at 0 degrees C caused a loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. Moreover, only 35% of total cholesterol could be extracted by MBCD at 0 degrees C and was accompanied by a complete loss of plasma membrane and endocytotic recycling centre filipin staining. This study clearly shows that MBCD does not specifically extract cholesterol from any cellular fraction, that cholesterol redistributes upon temperature changes and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol.

  • 31. Parmryd, I
    et al.
    Dallner, G
    In vivo prenylation of rat proteins: modification of proteins with penta- and hexaprenyl groups.1999In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 364, no 2, p. 153-60Article in journal (Refereed)
    Abstract [en]

    In vivo protein prenylation was studied in newborn rats by repeated injections of [3H]mevalonate. The highest level of protein-bound mevalonate metabolites was found in the kidney, but incorporation was observed in all organs studied. After fluorography of SDS-polyacrylamide gel electrophoresis-separated polypeptides, labeling was found in the 21- to 28-kDa molecular mass region and, after prolonged exposure of the film, additional bands at both higher and lower molecular masses could be detected. Protein prenylation in the kidney increased during the first 5 days after birth, whereas that in the liver reached a maximum on the fourth day. After methyliodide treatment of the prenylated proteins, farnesol, geranylgeraniol, and two larger isoprenoids, pentaprenol and hexaprenol, were released. In the liver, the ratio of protein-bound geranylgeraniol to farnesol increased from 2 to 4.5 during the first 5 days after birth. Upon subfractionation of the kidney, the highest level of labeling was found in mitochondria and microsomes. When the mitochondria were subfractionated into outer membranes, intermembrane space and an inner membrane/matrix fraction, the labeling pattern of prenylated polypeptides differed in all fractions. The results demonstrate that in vivo labeling of rats can be performed to study the extent, type, and distribution of protein prenylation.

  • 32. Parmryd, I
    et al.
    Shipton, C A
    Andersson, B
    Dallner, G
    Protein prenylation in spinach--tissue specificity and greening-induced changes.1997In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 339, no 1, p. 73-8Article in journal (Refereed)
    Abstract [en]

    Etiolated spinach seedlings, as well as petioles and blades of leaves of green seedlings, were labeled with [3H]mevalonate to study protein prenylation in several plant developmental stages. The polypeptide prenylation pattern of the leaf petiole and the leaf blade differed considerably, although some prenylated proteins were present in both tissues. During greening several prenylated polypeptides in the 30- to 46-kDa molecular mass region and two at 15 kDa became more abundant, while others in the 21.5- to 30-kDa region and one at 62 kDa showed a relative decrease. However, the relative amount of several of the prenylated polypeptides did not appear to be altered during the greening process. In etiolated seedlings, more thioether-linked farnesol than geranylgeraniol was found, whereas in seedlings grown under normal light conditions the converse situation prevailed.

  • 33.
    Parmryd, Ingela
    Stockholms universitet.
    Protein Prenylation in Higher Eukaryotes1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Protein prenylation is a post-translational process where proteins become covalently modified by an isoprenoid group. This modification adds hydrophobicity to the proteins. For all prenylated proteins identified to date, the prenyl group is an absolute requirement for their biological functions. To date, this process has been thoroughly characterised in mammals and yeast with the majority of the studies being performed either in vitro or in tissue-cultured cells. This thesis presents evidence that protein prenylation is a widespread process that is also present in plants. It is focused mainly on in vivo protein prenylation in plants and mammals. In vitro studies of the enzyme farnesyl protein transferase are also discussed.

    By using spinach seedlings, whose protein synthesis rates are high, an in vivo method for studying protein prenylation was developed. Spinach, the first plant to be analysed for isoprenoid-modified proteins, was found to contain numerous proteins that were prenylated with metabolites of the isoprenoid precursor mevalonate. The majority of the prenylated proteins originated from cellular organelles. The nature of the covalently-bound isoprenoids was assessed by hydrolysis of lipid-extracted proteins in combination with high performance liquid chromatography (HPLC) analysis. Both thioether-linked farnesyl and geranylgeranyl groups, as well as longer chain isoprenoids, were released.

    The soluble fraction of spinach contained an enzyme that could farnesylate the sulphydryl reagent dithiothreitol (DTT). This enzyme was immunoprecipitated with antisera raised against mammalian farnesyl protein transferase (FPT), which strongly suggesting it being a plant FPT. A new assay for measuring prenyl protein transferases was developed. The spinach FPT depended on zinc ions for its activity and functioned preferentially at pH 7.0. DTT inhibited in vivo prenylation of a subset of the prenylated spinach proteins in a dose dependent manner.

    Labelling of prenylated proteins in spinach stems was much more intense than that in spinach leaves. In addition, the relative labelling intensity of the prenylated proteins differed markedly in stem and leaf tissue. During greening, the pattern of labelled prenylated proteins was altered. Several chloroplastic proteins displayed an increase in intensity when etiolated seedlings were subjected to light. Etiolated plants contained a higher proportion of thioether-linked farnesyl groups than did plants grown under normal light conditions.

    Chloroplasts were shown to contain at least 20 prenylated polypeptides in the first study addressing protein prenylation in this organelle. These were all found in the thylakoid and envelope membrane fractions. A large proportion of the prenylated polypeptides originated from the outer envelope membranes, as assessed by proteolytic treatment of intact chloroplasts. The majority of the chloroplastic prenylated polypeptides were part of multimeric protein complexes, as shown by sucrose density centrifugation of detergent-treated membrane preparations. The thylakoid membranes contained an FPT activity. This is unique as all other prenyl protein transferases characterised to date are soluble enzymes.

    Chloroplasts were demonstrated to contain a hitherto undescribed type of prenylated polypeptides. The prenylation of this new type of prenylated polypeptides depended upon expression of chloroplast genes. Furthermore, these polypeptides were modified by isoprenoids via a bond of non-thioether character. The polypeptides encompassing the novel type of modification were readily prenylated when labelling was performed with either mevalonate or farnesol, but not with geranylgeraniol.

    Protein prenylation in rats, when studied by mevalonate labelling, was most prominent in the kidney. The majority of the prenylated polypeptides had molecular masses between 21-28 kDa. A substantial proportion of the prenylated proteins in kidney originated from the mitoplast subfraction of mitochondria. In addition to farnesyl and geranylgeranyl groups, which are both well-known prenylating moieties, pentaprenyl and hexaprenyl groups were also bound to rat proteins via thioether linkages, as demonstrated by methyl iodide hydrolysis in combination with HPLC analysis.

  • 34.
    Parmryd, Ingela
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Adler, Jeremy
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Colocalisation - the Tale of Co-Occurrence and Correlation2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 294A-294AArticle in journal (Other academic)
  • 35.
    Parmryd, Ingela
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Adler, Jeremy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Bernhem, Kristoffer
    Royal Inst Technol, Dept Appl Phys, Stockholm, Sweden..
    Membrane Topography can Cause Apparent Clustering - Identification and Differentiation from Genuine Clustering2018In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 114, no 3, p. 165A-165AArticle in journal (Other academic)
  • 36. Parmryd, Ingela
    et al.
    Adler, Jeremy
    Patel, Roopal
    Magee, Anthony I
    Imaging metabolism of phosphatidylinositol 4,5-bisphosphate in T-cell GM1-enriched domains containing Ras proteins.2003In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 285, no 1, p. 27-38Article in journal (Refereed)
    Abstract [en]

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and Ras proteins are involved in signalling pathways originating at the plasma membrane. The localisation and metabolism of PI(4,5)P(2) was studied in Jurkat T cells using fluorescence microscopic imaging with EGFP-tagged and antibody probes. Software was developed to objectively quantitate colocalisation and was used to show that plasma membrane PI(4,5)P(2) was enriched in lipid raft-containing patches of GM1 ganglioside, formed by crosslinking cholera toxin B-subunit (CT-B). The PI(4,5)P(2) metabolites phosphatidylinositol 3,4,5-trisphosphate and diacylglycerol appeared in plasma membrane CT-B-GM1 patches upon induction of signalling. Transferrin receptor and the CD45 tyrosine phosphatase did not colocalise with CT-B-GM1 patches, whereas the tyrosine kinase Lck, the scaffolding protein LAT, and endogenous Ras proteins did partially colocalise with CT-B-GM1 patches as did transfected EGFP-K-Ras(4B) and EGFP-H-Ras. The results demonstrate that T-cell PI(4,5)P(2) metabolism is occurring in GM1-enriched domains and that Ras proteins are present in these domains in vivo.

  • 37.
    Parmryd, Ingela
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Adler, Jeremy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Sintorn, Ida-Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Strand, Robin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Movement on Uneven Surfaces Displays Characteristic Features of Hop Diffusion2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 524A-524AArticle in journal (Other academic)
  • 38.
    Parmryd, Ingela
    et al.
    Stockholm University.
    Andersson, B
    Stockholm University.
    Dallner, G
    Stockholm University.
    Protein prenylation in spinach chloroplasts.1999In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 96, no 18, p. 10074-10079Article in journal (Refereed)
    Abstract [en]

    Protein prenylation in plants was studied by in vivo metabolic (3)H-mevalonate labeling in combination with a range of protein synthesis inhibitors. In spinach cotyledons, this posttranslational protein modification was found to be divided into two categories, one representing the conventional prenylation involving farnesyl and geranylgeranyl groups bound to cysteine residues via thioether linkages. This category revealed a similar pattern of prenylated proteins to that observed in mammalian cells and depends on nuclear gene expression. The other category was shown to represent a type of prenylation confined to chloroplasts. It depends on plastid gene expression and does not involve a thioether bond. The modifying isoprenoid could be released from the chloroplastic polypeptides by alkaline treatment and was identified as phytol upon GC-MS analysis. The phytol could readily be derived from all-trans-[(3)H]farnesol, which, like all-trans-[(3)H]geranylgeraniol, was taken up by the cotyledons, resulting in incorporation of radiolabel into proteins.

  • 39. Parmryd, Ingela
    et al.
    Dallner, G
    Organization of isoprenoid biosynthesis1996In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 24, no 3, p. 677-682Article, review/survey (Refereed)
  • 40.
    Parmryd, Ingela
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Riehl, Astrid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Dinic, Jelena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Adler, Jeremy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    The T Cell Receptor Resides in Ordered Plasma Membrane Nanodomains that Aggregate Upon T Cell Activation2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2, p. 98A-98AArticle in journal (Other academic)
  • 41.
    Parmryd, Ingela
    et al.
    Arrhenius Laboratories for Natural Sciences, Biochemistry Department, Stockholm University, S-106 91 Stockholm, Sweden.
    Shipton, C. A.
    Swiezewska, E.
    Andersson, B.
    Dallner, G.
    Identification of spinach farnesyl protein transferase: Dithiothreitol as an acceptor in vitro1995In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 234, no 3, p. 723-731Article in journal (Refereed)
    Abstract [en]

    Spinach seedlings were found to contain farnesyl protein transferase. The enzyme is activated by Zn2+, but not by Mg2+. The pH optimum is approximately 7.0 and maximal activity is obtained at 40-45 degrees C. The apparent Km for the farnesyl diphosphate substrate is 7 microM. Western blotting of soluble proteins with an antiserum raised against mammalian farnesyl protein transferase demonstrated a specific cross-reactivity with the spinach enzyme. The antiserum preferentially recognises the beta-subunit of the heterodimeric farnesyl protein transferase, and the corresponding spinach polypeptide has a molecular mass of 42 kDa on SDS/PAGE. The enzyme can employ dithiothreitol as an acceptor for the farnesyl moiety and catalyses the formation of a thioether linkage between these substrates. On the basis of this discovery, a new method was developed utilising the hydrophobicity of the reaction product, and its interaction with poly(propylene). During in vivo labelling, the plants took up dithiothreitol, which inhibited the incorporation of [3H]mevalonate metabolites into proteins, indicating that dithiothreitol might be isoprenylated in vivo as well as in vitro. However, isoprenylation of some proteins remains unaffected by dithiothreitol suggesting the existence of different isoprenylation mechanisms. Thus, it is demonstrated that plants possess farnesyl protein transferase, which resembles its mammalian and yeast homologues.

  • 42.
    Parmryd, Ingela
    et al.
    UNIV STOCKHOLM,DEPT BIOCHEM,ARRHENIUS LABS NAT SCI,S-10691 STOCKHOLM,SWEDEN.
    Shipton, C A
    Swiezewska, E.
    Dallner, G.
    Andersson, B.
    Chloroplastic prenylated proteins1997In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 414, no 3, p. 527-531Article in journal (Refereed)
    Abstract [en]

    By in vivo [3H]mevalonate labelling of spinach combined with biochemical analysis, evidence is provided for the existence of protein prenylation in chloroplasts. Approximately 20 prenylated polypeptides were resolved by SDS-PAGE followed by autoradiography. Thermolysin treatment of intact chloroplasts revealed that about 40% of the prenylated polypeptides were associated with the cytoplasmic surface of the outer envelope membrane. The remaining portion was present in thylakoids and/or the inner envelope membrane. The majority of the prenylated polypeptides were associated with larger membrane protein complexes. A farnesyl protein transferase activity was found to be associated with the thylakoid membrane.

  • 43.
    Parmryd, Ingela
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Önfelt, Björn
    Consequences of Membrane Topography2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 12, p. 2775-2784Article, review/survey (Refereed)
    Abstract [en]

    The surface of mammalian cells is neither smooth nor flat and cells have several times more plasma membrane than the minimum area required to accommodate their shape. We discuss the biological function of this apparent excess membrane that allows the cells to migrate, undergo shape changes and probably plays a role in signal transduction. Methods for studying membrane folding and topography; atomic force microscopy, scanning ion conductance microscopy, fluorescence polarisation microscopy and linear dichroism, are described and evaluated. Membrane folding and topography is frequently ignored when interpreting microscopy data. This has resulted in several misconceptions for instance regarding colocalisation, membrane organisation and molecular clustering. We suggest simple ways to avoid these pitfalls and invoke Occam's razor - that simple explanations are preferable to complex ones. Topography, i. e. deviations from a smooth surface, should always be ruled out as the cause of anomalous data before other explanations are presented.

  • 44. Runquist, M
    et al.
    Parmryd, I
    Thelin, A
    Chojnacki, T
    Dallner, G
    Distribution of branch point prenyltransferases in regions of bovine brain.1995In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 65, no 5, p. 2299-306Article in journal (Refereed)
    Abstract [en]

    Bovine brains contain large amounts of isoprenoid compounds and the enzymes involved in their biosynthesis were investigated. Ten different regions were dissected from fresh bovine brains and, in addition, fractions from cerebellum, spinal cord, and hypophysis were obtained. The cholesterol concentration was found to be approximately 8 mg/g in the cortex regions and three times higher in the pons, medulla oblongata, and white matter. Dolichol concentration varied between 8 and 40 micrograms/g in the different tissues, and ubiquinone was found at a lower level, which varied between 3 and 25 micrograms/g. Farnesylpyrophosphate synthase activity in cytosolic fractions from various regions exhibited only a twofold variation, whereas geranylgeranyl pyrophosphate synthase displayed larger differences, being particularly rich in the pons, medulla oblongata, white matter, and spinal cord. Squalene synthase activity was lowest in the thalamus and threefold higher in the pons. Determination of specific activity based on cholesterol content revealed that enzyme activities in various regions are not related to the actual lipid amount present. Both cis- and trans-prenyltransferases exhibited similarities in their regional distribution showing up to 20-fold differences in activity. Thus, it appears that the mevalonate pathway lipids and the various branch point enzymes involved in their syntheses vary greatly in different brain regions and are subjected to separate regulation.

  • 45.
    Shipton, C. A.
    et al.
    Arrhenius Laboratories for Natural Sciences, Biochemistry Department, University of Stockholm, Stockholm S-106 91 Sweden.
    Parmryd, Ingela
    Arrhenius Laboratories for Natural Sciences, Biochemistry Department, University of Stockholm, Stockholm S-106 91 Sweden.
    Swiezewska, E.
    Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-532 Warsaw, Poland.
    Andersson, B.
    Arrhenius Laboratories for Natural Sciences, Biochemistry Department, University of Stockholm, Stockholm S-106 91 Sweden.
    Dallner, G.
    Arrhenius Laboratories for Natural Sciences, Biochemistry Department, University of Stockholm, Stockholm S-106 91 Sweden;Clinical Research Center at Novum, Karolinska Institutet, S-141 86 Huddinge, Sweden.
    Isoprenylation of plant proteins in vivo: Isoprenylated proteins are abundant in the mitochondria and nuclei of spinach1995In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 270, no 2, p. 566-572Article in journal (Refereed)
    Abstract [en]

    Protein isoprenylation in vivo is demonstrated using spinach seedlings labeled with [3H]mevalonate. This report provides evidence for the occurrence of a large number of isoprenylated proteins in plants. Seedlings, without roots, were labeled quantitatively through the cut stem. Mevinolin treatment of the seedlings resulted in increased incorporation of radiolabel into proteins. Approximately 30 labeled bands could be detected after autoradiography of SDS-polyacrylamide gel electrophoresis-separated polypeptides, ranging in molecular mass from 6 to 200 kDa. Methyl iodide hydrolysis resulted in the release of covalently bound farnesol, geranylgeraniol, phytol, and some unidentified isoprenoid compounds from mevalonate-labeled proteins. It was found that all cellular fractions contained some isoprenylated proteins, although most were located in the mitochondria and nuclei. Subfractionation of the nucleus revealed that the majority of isoprenylated proteins in this compartment were components of the nuclear matrix. The results demonstrate that in vivo labeling of a complex organism can be performed using a plant system in order to study protein isoprenylation and distribution of modified proteins in different cellular compartments.

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