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Figueroa, Ricardo A.ORCID iD iconorcid.org/0000-0003-1476-6675
Alternative names
Publications (10 of 24) Show all publications
Bergqvist, C., Niss, F., Figueroa, R. A., Beckman, M., Maksel, D., Jafferali, M. H., . . . Hallberg, E. (2019). Monitoring of chromatin organization in live cells by FRIC. Effects of the inner nuclear membrane protein Samp1. Nucleic Acids Research, 47(9), Article ID e49.
Open this publication in new window or tab >>Monitoring of chromatin organization in live cells by FRIC. Effects of the inner nuclear membrane protein Samp1
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2019 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 9, article id e49Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:su:diva-168660 (URN)10.1093/nar/gkz123 (DOI)000473756300001 ()
Funder
Swedish Research Council, 621-2010-448Swedish Cancer Society, 110590Stiftelsen Olle Engkvist Byggmästare
Available from: 2019-05-02 Created: 2019-05-02 Last updated: 2022-04-29Bibliographically approved
Larsson, V. J., Jafferali, M. H., Vijayaraghavan, B., Figueroa, R. A. & Hallberg, E. (2018). Mitotic spindle assembly and γ-tubulin localisation depend on the integral nuclear membrane protein, Samp1. Journal of Cell Science, 131(8), Article ID jcs211664.
Open this publication in new window or tab >>Mitotic spindle assembly and γ-tubulin localisation depend on the integral nuclear membrane protein, Samp1
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2018 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 131, no 8, article id jcs211664Article in journal (Refereed) Published
Abstract [en]

We have investigated a possible role of the inner nuclear membrane protein, Samp1, in the mitotic machinery. Live cell imaging showed that Samp1aYFP distributed as filamentous structures in the mitotic spindle, partially co-localising with ß-tubulin. Samp1 depletion resulted in an increased frequency of cells with signs of chromosomal mis-segregation and prolonged metaphase, indicating problems with spindle assembly and/or chromosomal alignment. Consistently, mitotic spindles in Samp1 depleted cells contained significantly lower levels of ß-tubulin and γ-tubulin, phenotypes which were rescued by overexpression of Samp1aYFP. We found that Samp1 can bind directly to γ-tubulin and that Samp1 co-precipitated with γ-tubulin and HAUS6 of the Augmin complex in live cells. The levels of Haus6, in the mitotic spindle also decreased after Samp1 depletion. We show that Samp1 is involved in the recruitment of Haus6 and γ-tubulin to the mitotic spindle. Samp1 is the first inner nuclear membrane protein shown to have a function in mitotic spindle assembly.

Keywords
Samp1, nuclear membrane, mitotic spindle, gamma tubulin, Augmin, Cancer
National Category
Cell Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-154091 (URN)10.1242/jcs.211664 (DOI)000440536400010 ()
Funder
Swedish Research Council, 621-2010-448Swedish Cancer Society, 110590Stiftelsen Olle Engkvist Byggmästare
Available from: 2018-03-14 Created: 2018-03-14 Last updated: 2022-03-23Bibliographically approved
Vijayaraghavan, B., Figueroa, R. A., Bergqvist, C., Gupta, A. J., Sousa, P. & Hallberg, E. (2018). RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin. Biochimica et Biophysica Acta - Biomembranes, 1860(6), 1326-1334
Open this publication in new window or tab >>RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin
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2018 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 6, p. 1326-1334Article in journal (Refereed) Published
Abstract [en]

Samp1, spindle associated membrane protein 1, is a type II integral membrane protein localized in the inner nuclear membrane. Recent studies have shown that the inner nuclear membrane protein, Emerin and the small monomeric GTPase, Ran are direct binding partners of Samp1. Here we addressed the question whether Ran could regulate the interaction between Samp1 and Emerin in the inner nuclear membrane. To investigate the interaction between Samp1 and Emerin in live cells, we performed FRAP experiments in cells overexpressing YFP-Emerin. We compared the mobility of YFP-Emerin in Samp1 knock out cells and cells overexpressing Samp1. The results showed that the mobility of YFP-Emerin was higher in Samp1 knock out cells and lower in cells overexpressing Samp1, suggesting that Samp1 significantly attenuates the mobility of Emerin in the nuclear envelope. FRAP experiments using tsBN2 cells showed that the mobility of Emerin depends on RanGTP. Consistently, in vitro binding experiments showed that the affinity between Samp1 and Emerin is decreased in the presence of Ran, suggesting that Ran attenuates the interaction between Samp1 and Emerin. This is the first demonstration that Ran can regulate the interaction between two proteins in the nuclear envelope.

Keywords
Muscular dystrophy, Nuclear membrane, Samp1, Emerin, Ran, FRAP
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-157708 (URN)10.1016/j.bbamem.2018.03.001 (DOI)000432758400009 ()29510091 (PubMedID)
Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2022-03-23Bibliographically approved
Shimoji, M., Figueroa, R. A., Neve, E., Maksel, D., Imreh, G., Morgenstern, R. & Hallberg, E. (2017). Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1. Biochimica et Biophysica Acta - Biomembranes, 1859(2), 238-244
Open this publication in new window or tab >>Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1
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2017 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1859, no 2, p. 238-244Article in journal (Refereed) Published
Abstract [en]

Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress.

Keywords
Subcellular targeting, Dual distribution, Glutathione transferase
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-140296 (URN)10.1016/j.bbamem.2016.11.014 (DOI)000392772100011 ()27913278 (PubMedID)
Available from: 2017-03-13 Created: 2017-03-13 Last updated: 2022-02-28Bibliographically approved
Lehto, T., Vasconcelos, L., Margus, H., Figueroa, R., Pooga, M., Hällbrink, M. & Langel, Ü. (2017). Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides. Bioconjugate chemistry, 28(3), 782-792
Open this publication in new window or tab >>Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides
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2017 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 28, no 3, p. 782-792Article in journal (Refereed) Published
Abstract [en]

Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well studied CPP, PepFectl4, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.

Keywords
peptide, delivery, oligonucleotide
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-140331 (URN)10.1021/acs.bioconjchem.6b00680 (DOI)000396801500012 ()
Available from: 2017-03-06 Created: 2017-03-06 Last updated: 2022-02-28Bibliographically approved
Jafferali, M. H., Figueroa, R. A., Hasan, M. & Hallberg, E. (2017). Spindle associated membrane protein 1 (Samp1) is required for the differentiation of muscle cells. Scientific Reports, 7, Article ID 16655.
Open this publication in new window or tab >>Spindle associated membrane protein 1 (Samp1) is required for the differentiation of muscle cells
2017 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 7, article id 16655Article in journal (Refereed) Published
Abstract [en]

Muscles are developed and regenerated in a differentiation process called myogenesis, which involves components of the nuclear envelope. We have investigated Samp1 (Spindle Associated Membrane Protein 1), a transmembrane nuclear envelope protein, which interacts with emerin and lamin A, both of which are linked to Emery-Dreifuss muscular dystrophy (EDMD). We found that the levels of Samp1 increased seven-fold during differentiation of mouse C2C12 muscle progenitor cells. To test if Samp1 could have a role in myogenesis we developed stable C2C12 knockdown cell lines expressing short hairpin RNA targeting Samp1 expression. The Samp1 depleted C2C12 cells displayed normal mobility and normal distribution of emerin and lamin A. However, Samp1 depletion increased ERK signaling and completely blocked differentiation of C2C12 cells, which failed to express myogenic marker proteins and failed to form myotubes. The block in myogenesis in Samp1 depleted cells was completely rescued by ectopic expression of RNAi resistant human Samp1, showing that Samp1 is required for muscle differentiation.

National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-150882 (URN)10.1038/s41598-017-16746-y (DOI)000416891400057 ()29192166 (PubMedID)
Available from: 2018-01-10 Created: 2018-01-10 Last updated: 2022-09-15Bibliographically approved
Ivanova, E. V., Figueroa, R. A., Gatsinzi, T., Hallberg, E. & Iverfeldt, K. (2016). Anchoring of FRET Sensors-A Requirement for Spatiotemporal Resolution. Sensors, 16(5), Article ID 703.
Open this publication in new window or tab >>Anchoring of FRET Sensors-A Requirement for Spatiotemporal Resolution
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2016 (English)In: Sensors, E-ISSN 1424-8220, Vol. 16, no 5, article id 703Article in journal (Refereed) Published
Abstract [en]

FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling.

Keywords
apoptosis, caspase, FRET sensor, live cell imaging, neurodegeneration, signal transduction
National Category
Biological Sciences Biomedical Laboratory Science/Technology Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-132518 (URN)10.3390/s16050703 (DOI)000378510400074 ()
Available from: 2016-08-15 Created: 2016-08-15 Last updated: 2022-03-23Bibliographically approved
Jafferali, M. H., Figueroa, R. A. & Hallberg, E. (2016). MCLIP Detection of Novel Protein-Protein Interactions at the Nuclear Envelope. In: Katherine L. Wilson, Arnoud Sonnenberg (Ed.), Intermediate Filament Associated Proteins: (pp. 503-515). Elsevier, 569
Open this publication in new window or tab >>MCLIP Detection of Novel Protein-Protein Interactions at the Nuclear Envelope
2016 (English)In: Intermediate Filament Associated Proteins / [ed] Katherine L. Wilson, Arnoud Sonnenberg, Elsevier, 2016, Vol. 569, p. 503-515Chapter in book (Refereed)
Abstract [en]

The organization and function of the nuclear envelope (NE) involves hundreds of nuclear membrane proteins and myriad protein-protein interactions, most of which are still uncharacterized. Many NE proteins interact stably or dynamically with the nuclear lamina or chromosomes. This can make them difficult to extract under non-denaturing conditions, and greatly limits our ability to explore and identify functional protein interactions at the NE. This knowledge is needed to understand nuclear envelope structure and the mechanisms of human laminopathy diseases. This chapter provides detailed protocols for MCLIP (membrane cross-linking immunoprecipitation) identification of novel protein-protein interactions in mammalian cells.

Place, publisher, year, edition, pages
Elsevier, 2016
Series
Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988 ; 569
Keywords
Nucleus, Nuclear envelope, Nuclear lamina, Nuclear membrane, Protein–protein interactions, Proteomics, MCLIP
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-148114 (URN)10.1016/bs.mie.2015.08.022 (DOI)000410546200025 ()26778573 (PubMedID)978-0-12-803469-9 (ISBN)
Available from: 2017-10-19 Created: 2017-10-19 Last updated: 2022-02-28Bibliographically approved
Vijayaraghavan, B., Jafferali, M. H., Figueroa, R. A. & Hallberg, E. (2016). Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane. Nucleus, 7(4), 415-423
Open this publication in new window or tab >>Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane
2016 (English)In: Nucleus, ISSN 1949-1034, E-ISSN 1949-1042, Vol. 7, no 4, p. 415-423Article in journal (Refereed) Published
Abstract [en]

Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and during mitosis, it localizes to the mitotic spindle. Samp1 was recently found to coprecipitate a protein complex containing Ran, a GTPase with fundamental regulatory functions both in interphase and in mitosis. To investigate the interaction between Samp1 and Ran in further detail, we have designed and expressed recombinant fusion proteins of the Chaetomium thermophilum homolog of Samp1 (Ct. Samp1) and human Ran. Pulldown experiments show that Samp1 binds directly to Ran and that Samp1 binds better to RanGTP compared to RanGDP. Samp1 also preferred RanGTP over RanGDP in living tsBN2 cells. We also show that the Ran binding domain is located between amino acids 75-135 in the nucleoplasmically exposed N-terminal tail of Samp1. This domain is unique for Samp1, without homology in any other proteins in fungi or metazoa. Samp1 is the first known transmembrane protein that binds to Ran and could provide a unique local binding site for RanGTP in the INM. Samp1 overexpression resulted in increased Ran concentrations in the nuclear periphery supporting this idea.

Keywords
EDMD, laminopathies, LINC complex, nucleus, nuclear membrane, Ran
National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-135087 (URN)10.1080/19491034.2016.1220465 (DOI)000384442800010 ()27541860 (PubMedID)
Available from: 2016-11-23 Created: 2016-10-31 Last updated: 2022-03-23Bibliographically approved
Lindberg, S., Regberg, J., Eriksson, J., Helmfors, H., Muñoz-Alarcón, A., Srimanee, A., . . . Langel, Ü. (2015). A convergent uptake route for peptide- and polymer-based nucleotide delivery systems. Journal of Controlled Release, 206, 58-66
Open this publication in new window or tab >>A convergent uptake route for peptide- and polymer-based nucleotide delivery systems
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2015 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 206, p. 58-66Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) have been used as vehicles to deliver various cargos into cells and are promising as tools to deliver therapeutic biomolecules such as oligonucleotides both in vitro and in vivo. CPPs are positively charged and it is believed that CPPs deliver their cargo in a receptor-independent manner by interactingwith the negatively charged plasmamembrane and thereby inducing endocytosis. In this study we examine the mechanism of uptake of several different, well known, CPPs that form complexes with oligonucleotides.We show that these CPP:oligonucleotide complexes are negatively charged in transfection-media and their uptake is mediated by class A scavenger receptors (SCARA). These receptors are known to promiscuously bind to, and mediate uptake of poly-anionic macromolecules. Uptake of CPP:oligonucleotide complexes was abolished using pharmacological SCARA inhibitors as well as siRNA-mediated knockdown of SCARA. Additionally, uptake of CPP:oligonucleotide was significantly increased by transiently overexpressing SCARA. Furthermore, SCARA inhibitors also blocked internalization of cationic polymer:oligonucleotide complexes.Our results demonstrate that the previous held belief that CPPs act receptor independently does not hold true for CPP:oligonucleotide complexes, as scavenger receptor class A (SCARA) mediates the uptake of all the examined CPP:oligonucleotide complexes in this study.

Keywords
Cell-penetrating peptides, Oligonucleotide delivery, CPP, Scavenger receptor class A, SCARA, Receptor-mediated endocytosis
National Category
Chemical Sciences Biological Sciences Pharmacology and Toxicology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-108343 (URN)10.1016/j.jconrel.2015.03.009 (DOI)000353361400006 ()
Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2022-02-23Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-1476-6675

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