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Publications (6 of 6) Show all publications
Pulkkinen, L. I., Barrass, S. V., Lindgren, M., Pace, H., Överby, A. K., Anastasina, M., . . . Butcher, S. J. (2023). Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein. PLoS Pathogens, 19(2), Article ID e1011125.
Open this publication in new window or tab >>Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein
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2023 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 19, no 2, article id e1011125Article in journal (Refereed) Published
Abstract [en]

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate the interactions of the wild-type and truncated capsid proteins with membranes with biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids, which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

Place, publisher, year, edition, pages
Public Library of Science, 2023
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-205497 (URN)10.1371/journal.ppat.1011125 (DOI)000966733300001 ()36787339 (PubMedID)2-s2.0-85149054055 (Scopus ID)
Available from: 2023-03-14 Created: 2023-03-14 Last updated: 2023-09-05Bibliographically approved
Liu, K.-C., Pace, H., Larsson, E., Hossain, S., Kabedev, A., Shukla, A., . . . Lundmark, R. (2022). Membrane insertion mechanism of the caveola coat protein Cavin1. Proceedings of the National Academy of Sciences of the United States of America, 119(25), Article ID 2202295119.
Open this publication in new window or tab >>Membrane insertion mechanism of the caveola coat protein Cavin1
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2022 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 119, no 25, article id 2202295119Article in journal (Refereed) Published
Abstract [en]

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Place, publisher, year, edition, pages
Proceedings of the National Academy of Sciences, 2022
Keywords
caveolae, Cavin1, membrane curvature, membrane-shaping protein, protein-lipid interactions
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-203198 (URN)10.1073/pnas.2202295119 (DOI)000838706900008 ()2-s2.0-85133725056 (Scopus ID)
Funder
Swedish Research Council, 2018-05973European CommissionThe Kempe FoundationsSwedish Cancer SocietyWallenberg Foundations
Available from: 2023-01-18 Created: 2023-01-18 Last updated: 2023-01-18Bibliographically approved
Nadeem, A., Berg, A., Pace, H., Alam, A., Toh, E., Ådén, J., . . . Wai, S. N. (2022). Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae. eLIFE, 11, Article ID e73439.
Open this publication in new window or tab >>Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae
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2022 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 11, article id e73439Article in journal (Refereed) Published
Abstract [en]

The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.

Place, publisher, year, edition, pages
eLife Sciences Publications, Ltd, 2022
Keywords
Vibrio cholerae, MakA, lipid
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-192300 (URN)10.7554/eLife.73439 (DOI)2-s2.0-85124321786 (Scopus ID)
Funder
Swedish Research Council, 2018–02914Swedish Research Council, 2016–05009Swedish Research Council, 2019–01720Swedish Research Council, 2016–06963Swedish Research Council, 2019–02011Swedish Cancer Society, 2017–419Swedish Cancer Society, 2020–711The Kempe Foundations, JCK-1728The Kempe Foundations, SMK-1756.2The Kempe Foundations, SMK-1553The Kempe Foundations, JCK-1724The Kempe Foundations, SMK-1961Knut and Alice Wallenberg FoundationFamiljen Erling-Perssons Stiftelse
Available from: 2022-02-08 Created: 2022-02-08 Last updated: 2024-01-12Bibliographically approved
Nadeem, A., Nagampalli, R., Toh, E., Alam, A., Myint, S. L., Heidler, T., . . . Persson, K. (2021). A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion. Proceedings of the National Academy of Sciences of the United States of America, 118(47), Article ID e2111418118.
Open this publication in new window or tab >>A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion
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2021 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 118, no 47, article id e2111418118Article in journal (Refereed) Published
Abstract [en]

Vibrio cholerae, responsible for outbreaks of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe how the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto-unrecognized genomic island. The proteins MakA/B/E can form a tripartite toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the cytolytic activity of the Mak proteins was obtained by X-ray crystallography. Flagellum-facilitated secretion ensuring spatially coordinated delivery of Mak proteins revealed a role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of diagnostics and therapeutic strategies against pathogenic Vibrionaceae.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-191257 (URN)10.1073/pnas.2111418118 (DOI)000727697700014 ()34799450 (PubMedID)2-s2.0-85121209218 (Scopus ID)
Funder
Swedish Research Council, 2016-05009Swedish Research Council, 2018-02914Swedish Research Council, 2019-01720Swedish Research Council, 2007-08673The Kempe Foundations, SMK-1756.2The Kempe Foundations, SMK-1553The Kempe Foundations, JCK-1728Swedish Cancer Society, 2017-419The Kempe Foundations, SMK-1961Swedish Research Council
Available from: 2022-01-12 Created: 2022-01-12 Last updated: 2023-05-11Bibliographically approved
Thorsteinsson, K., Olsén, E., Schmidt, E., Pace, H. & Bally, M. (2020). FRET-Based assay for the quantification of extracellular vesicles and other vesicles of complex composition. Analytical Chemistry, 92(23), 15336-15343
Open this publication in new window or tab >>FRET-Based assay for the quantification of extracellular vesicles and other vesicles of complex composition
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2020 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 92, no 23, p. 15336-15343Article in journal (Refereed) Published
Abstract [en]

Research in the field of extracellular vesicles is rapidly expanding and finding footholds in many areas of medical science. However, the availability of methodologies to quantify the concentration of membrane material present in a sample remains limited. Herein, we present a novel approach for the quantification of vesicle material, specifically the quantification of the total lipid membrane surface area, found in a sample using Förster resonance energy transfer (FRET). In this assay, sonication is used to drive the fusion between vesicles in the sample to be quantified and liposomes containing a pair of FRET fluorophores. The change in emission spectrum upon vesicle fusion is directly related to the total membrane surface area of the sample added, and a calibration curve allows for the quantification of a variety of vesicle species, including enveloped viruses, bacterial outer membrane vesicles, and mammalian extracellular vesicles. Without extensive optimization of experimental parameters, we were able to quantify down to ∼109 vesicles/mL, using as little as 60 μL of the sample. The assay precision was comparable to that of a commercial nanoparticle tracking analysis system. While its limit of detection was slightly higher, the FRET assay is superior for the detection of small vesicles, as its performance is vesicle-size-independent. Taken together, the FRET assay is a simple, robust, and versatile method for the quantification of a variety of purified vesicle samples.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2020
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:umu:diva-176977 (URN)10.1021/acs.analchem.0c02271 (DOI)000596727600012 ()33179908 (PubMedID)2-s2.0-85096617026 (Scopus ID)
Funder
Swedish Research Council, 2017-04029Knut and Alice Wallenberg Foundation
Available from: 2020-11-23 Created: 2020-11-23 Last updated: 2023-09-05Bibliographically approved
Lubart, Q., Hannestad, J. K., Pace, H., Fjällborg, D., Westerlund, F., Esbjörner, E. K. & Bally, M. (2020). Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO. Physical Chemistry, Chemical Physics - PCCP, 22(16), 8781-8790
Open this publication in new window or tab >>Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO
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2020 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 22, no 16, p. 8781-8790Article in journal (Refereed) Published
Abstract [en]

Lipophilic carbocyanine dyes are widely used as fluorescent cell membrane probes in studies ranging from biophysics to cell biology. While they are extremely useful for qualitative observation of lipid structures, a major problem impairing quantitative studies is that the chemical environment of the lipid bilayer affects both the dye's insertion efficiency and photophysical properties. We present a systematic investigation of the sulphonated carbocyanine dye 3,3 '-dioctadecyl-5,5 '-di(4-sulfophenyl) (SP-DiO) and demonstrate how its insertion efficiency into pre-formed lipid bilayers and its photophysical properties therein determine its apparent fluorescence intensity in different lipid environments. For this purpose, we use large unilamellar vesicles (LUVs) made of lipids with distinct chain unsaturation, acyl chain length, head group charge, and with variation in membrane cholesterol content as models. Using a combination of absorbance, fluorescence emission, and fluorescence lifetime measurements we reveal that SP-DiO incorporates more efficiently into liquid disordered phases compared to gel phases. Moreover, incorporation into the latter phase is most efficient when the mismatch between the length of the lipid and dye hydrocarbon chains is small. Furthermore, SP-DiO incorporation is less efficient in LUVs composed of negatively charged lipids. Lastly, when cholesterol was included in the LUV membranes, we observed significant spectral shifts, consistent with dye aggregation. Taken together, our study highlights the complex interplay between membrane composition and labeling efficiency with lipophilic dyes and advocates for careful assessment of fluorescence data when attempting a quantitative analysis of fluorescence data with such molecules.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2020
National Category
Physical Chemistry Biophysics
Identifiers
urn:nbn:se:umu:diva-172504 (URN)10.1039/c9cp04158c (DOI)000537175100050 ()32285050 (PubMedID)2-s2.0-85084167302 (Scopus ID)
Funder
Swedish Research Council, 2017-04029Swedish Research Council, 2016-03902Wallenberg FoundationsWenner-Gren Foundations
Available from: 2020-07-02 Created: 2020-07-02 Last updated: 2020-07-02Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0001-5116-2577

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