kth.sePublications
Change search
Link to record
Permanent link

Direct link
Publications (10 of 37) Show all publications
Nordahl, L., Akkuratov, E. E., Heimgärtner, J., Schach, K., Meineke, B., Elsässer, S., . . . Brismar, H. (2024). Detection and quantification of Na,K-ATPase dimers in the plasma membrane of living cells by FRET-FCS. Biochimica et Biophysica Acta - General Subjects, 1868(7), Article ID 130619.
Open this publication in new window or tab >>Detection and quantification of Na,K-ATPase dimers in the plasma membrane of living cells by FRET-FCS
Show others...
2024 (English)In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1868, no 7, article id 130619Article in journal (Refereed) Published
Abstract [en]

The sodium potassium pump, Na,K-ATPase (NKA), is an integral plasma membrane protein, expressed in all eukaryotic cells. It is responsible for maintaining the transmembrane Na+ gradient and is the major determinant of the membrane potential. Self-interaction and oligomerization of NKA in cell membranes has been proposed and discussed but is still an open question. Here, we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET-FCS, to analyze NKA in the plasma membrane of living cells. Click chemistry was used to conjugate the fluorescent labels Alexa 488 and Alexa 647 to non-canonical amino acids introduced in the NKA α1 and β1 subunits. We demonstrate that FRET-FCS can detect an order of magnitude lower concentration of green-red labeled protein pairs in a single-labeled red and green background than what is possible with cross-correlation (FCCS). We show that a significant fraction of NKA is expressed as a dimer in the plasma membrane. We also introduce a method to estimate not only the number of single and double labeled NKA, but the number of unlabeled, endogenous NKA and estimate the density of endogenous NKA at the plasma membrane to 1400 ± 800 enzymes/μm2.

Place, publisher, year, edition, pages
Elsevier BV, 2024
Keywords
FRET-FCS, NaK-ATPase, Non-canonical amino acids
National Category
Biochemistry Molecular Biology
Identifiers
urn:nbn:se:kth:diva-346157 (URN)10.1016/j.bbagen.2024.130619 (DOI)001235008700001 ()38643888 (PubMedID)2-s2.0-85191150723 (Scopus ID)
Note

QC 20240626

Available from: 2024-05-03 Created: 2024-05-03 Last updated: 2025-02-20Bibliographically approved
Carrante, N. F., Dubackic, M., Makasewicz, K., Wennmalm, S., Liu, X., Hermodsson, T., . . . Sparr, E. (2023). alpha-Synuclein Cooperativity in Lipid Membranes Binding. European Biophysics Journal, 52(SUPPL 1), S130-S130
Open this publication in new window or tab >>alpha-Synuclein Cooperativity in Lipid Membranes Binding
Show others...
2023 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 52, no SUPPL 1, p. S130-S130Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2023
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-336017 (URN)001029235400416 ()
Note

QC 20230911

Available from: 2023-09-11 Created: 2023-09-11 Last updated: 2025-02-20Bibliographically approved
Fornasier, M., Wennmalm, S., Andersson, A., Sparr, E. & Jonsson, P. (2023). Effect of the negative lipid fraction on alpha-synuclein cooperativity and binding. European Biophysics Journal, 52(SUPPL 1), S81-S81
Open this publication in new window or tab >>Effect of the negative lipid fraction on alpha-synuclein cooperativity and binding
Show others...
2023 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 52, no SUPPL 1, p. S81-S81Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2023
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-335969 (URN)001029235400225 ()
Note

QC 20230911

Available from: 2023-09-11 Created: 2023-09-11 Last updated: 2025-02-20Bibliographically approved
Nordahl, L., Wennmalm, S., Jönsson, J., Elsässer, S., Akkuratov, E. E. & Brismar, H. (2022). Direct Observation of Na,K‐ATPase Oligomers in The Plasma Membrane of Living Cells by FRET‐FCS. The FASEB Journal, 36(S1)
Open this publication in new window or tab >>Direct Observation of Na,K‐ATPase Oligomers in The Plasma Membrane of Living Cells by FRET‐FCS
Show others...
2022 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 36, no S1Article in journal (Refereed) Published
Abstract [en]

The sodium pump, Na,K-ATPase, is an integral plasma membrane protein, expressed in all eukaryotic cells. Na,K-ATPase transforms chemical energy from ATP into a gradient of Na+ and K+ over the plasma membrane by actively exporting three Na+ ions and importing two K+ ions for each hydrolyzed ATP. It is responsible for maintenance of the transmembrane Na+ gradient and is the major determinant of the membrane potential. It provides the driving force for all Na+-coupled transport processes, thereby controlling essential functions in the cell. Na,K-ATPase is formed by three subunits alpha/beta/FXYD, where alpha is the catalytic ion-transporting subunit, beta is a regulatory subunit and FXYD is accessory.

Self-interaction and oligomerization of the Na,K-ATPase alpha/beta heterodimer in cell membranes has been proposed and discussed for a long time but is still an open question.

Here we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET-FCS, in order to detect oligomers of Na,K-ATPase. Compared to conventional cross-correlation FCCS, FRET-FCS is one to two orders of magnitude more sensitive when detecting oligomers. Moreover, FRET-FCS is inherently insensitive to unbalanced labeling, which is a great advantage during live cell measurements.

We hypothesized that Na,K-ATPase can exist in a higher order oligomeric state and demonstrate the use of FRET-FCS to test this hypothesis

We have introduced fluorescent labels by using expression of non-canonical amino acid modified beta subunits. The FRET pair Alexa488 and Alexa647 was directly conjugated to the beta subunits using selective click chemistry. Conventional FCS measurements of labeled cells revealed the absolute density of labeled and unlabeled Na,K-ATPase. With FRET-FCS we could observe FRET signals and FCS curves demonstrating the existence of oligomers. Positive controls for the FRET-FCS measurements were constructed by labeling alpha subunits with Alexa488 and beta subunits with Alexa647.

Furthermore, we performed Monte Carlo simulations of Na,K-ATPase, as monomer and as oligomer of increasing order, together with its ligands in a picket and fence diffusion model of the plasma membrane. The simulations suggest that oligomerization can have an impact on the net efficiency of the Na,K-ATPase measured as ATP turnover.

In conclusion we find that Na,K-ATPase can be found in the plasma membrane as oligomers. Further we discuss the consequences of oligomerization and propose that it can have a regulatory effect for the Na,K-ATPase net efficiency.

Place, publisher, year, edition, pages
Wiley, 2022
National Category
Biochemistry Molecular Biology Biophysics
Identifiers
urn:nbn:se:kth:diva-312484 (URN)10.1096/fasebj.2022.36.s1.r4657 (DOI)000878430605101 ()35553550 (PubMedID)
Note

QC 20220520

Available from: 2022-05-19 Created: 2022-05-19 Last updated: 2025-02-20Bibliographically approved
Nordenström, M., Benselfelt, T., Hollertz, R., Wennmalm, S., Larsson, P. A., Mehandzhiyski, A., . . . Wågberg, L. (2022). The structure of cellulose nanofibril networks at low concentrations and their stabilizing action on colloidal particles. Carbohydrate Polymers, 297, 120046, Article ID 120046.
Open this publication in new window or tab >>The structure of cellulose nanofibril networks at low concentrations and their stabilizing action on colloidal particles
Show others...
2022 (English)In: Carbohydrate Polymers, ISSN 0144-8617, E-ISSN 1879-1344, Vol. 297, p. 120046-, article id 120046Article in journal (Refereed) Published
Abstract [en]

The structure and dynamics of networks formed by rod-shaped particles can be indirectly investigated by measuring the diffusion of spherical tracer particles. This method was used to characterize cellulose nanofibril (CNF) networks in both dispersed and arrested states, the results of which were compared with coarse-grained Brownian dynamics simulations. At a CNF concentration of 0.2 wt% a transition was observed where, below this concentration tracer diffusion is governed by the increasing macroscopic viscosity of the dispersion. Above 0.2 wt%, the diffusion of small particles (20-40 nm) remains viscosity controlled, while particles (100-500 nm) become trapped in the CNF network. Sedimentation of silica microparticles (1-5 mu m) in CNF dispersions was also determined, showing that sedimentation of larger particles is significantly affected by the presence of CNF. At concentrations of 0.2 wt%, the sedimentation velocity of 5 mu m particles was reduced by 99 % compared to pure water.

Place, publisher, year, edition, pages
Elsevier BV, 2022
Keywords
Cellulose nanofibrils, Colloid stability, Simulations, Diffusion, Sedimentation, Models
National Category
Polymer Chemistry
Identifiers
urn:nbn:se:kth:diva-319717 (URN)10.1016/j.carbpol.2022.120046 (DOI)000860482300005 ()36184183 (PubMedID)2-s2.0-85137161224 (Scopus ID)
Note

QC 20221017

Available from: 2022-10-17 Created: 2022-10-17 Last updated: 2022-12-12Bibliographically approved
Makasewicz, K., Wennmalm, S., Linse, S. & Sparr, E. (2022). α-Synuclein-induced deformation of small unilamellar vesicles. QRB Discovery, 3, Article ID e10.
Open this publication in new window or tab >>α-Synuclein-induced deformation of small unilamellar vesicles
2022 (English)In: QRB Discovery, ISSN 2633-2892, Vol. 3, article id e10Article in journal (Refereed) Published
Abstract [en]

Abstract α-Synuclein is a small neuronal protein that reversibly associates with lipid membranes. The membrane interactions are believed to be central to the healthy function of this protein involved in synaptic plasticity and neurotransmitter release. α-Synuclein has been speculated to induce vesicle fusion as well as fission, processes which are analogous to each other but proceed in different directions and involve different driving forces. In the current work, we analyse α-synuclein-induced small unilamellar vesicle deformation from a thermodynamics point of view. We show that the structures interpreted in the literature as fusion intermediates are in fact a stable deformed state and neither fusion nor vesicle clustering occurs. We speculate on the driving force for the observed deformation and put forward a hypothesis that α-synuclein self-assembly on the lipid membrane precedes and induces membrane remodelling.

Place, publisher, year, edition, pages
Cambridge University Press (CUP), 2022
Keywords
fission, lipid membrane, membrane remodelling, α-Synuclein
National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-326464 (URN)10.1017/qrd.2022.9 (DOI)2-s2.0-85135595871 (Scopus ID)
Note

QC 20230522

Available from: 2023-05-22 Created: 2023-05-22 Last updated: 2023-05-22Bibliographically approved
Makasewicz, K., Wennmalm, S., Stenqvist, B., Fornasier, M., Andersson, A., Jonsson, P., . . . Sparr, E. (2021). Cooperativity of alpha-Synuclein Binding to Lipid Membranes. ACS Chemical Neuroscience, 12(12), 2099-2109
Open this publication in new window or tab >>Cooperativity of alpha-Synuclein Binding to Lipid Membranes
Show others...
2021 (English)In: ACS Chemical Neuroscience, E-ISSN 1948-7193, Vol. 12, no 12, p. 2099-2109Article in journal (Refereed) Published
Abstract [en]

Cooperative binding is a key feature of metabolic pathways, signaling, and transport processes. It provides tight regulation over a narrow concentration interval of a ligand, thus enabling switching to be triggered by small concentration variations. The data presented in this work reveal strong positive cooperativity of alpha-synuclein binding to phospholipid membranes. Fluorescence cross-correlation spectroscopy, confocal microscopy, and cryo-TEM results show that in excess of vesicles alpha-synuclein does not distribute randomly but binds only to a fraction of all available vesicles. Furthermore, alpha-synuclein binding to a supported lipid bilayer observed with total internal reflection fluorescence microscopy displays a much steeper dependence of bound protein on total protein concentration than expected for independent binding. The same phenomenon was observed in the case of alpha-synuclein binding to unilamellar vesicles of sizes in the nm and mu m range as well as to flat supported lipid bilayers, ruling out that nonuniform binding of the protein is governed by differences in membrane curvature. Positive cooperativity of alpha-synuclein binding to lipid membranes means that the affinity of the protein to a membrane is higher where there is already protein bound compared to a bare membrane. The phenomenon described in this work may have implications for alpha-synuclein function in synaptic transmission and other membrane remodeling events.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2021
Keywords
Cooperative binding, homotropic allostery, alpha-synuclein, lipid membrane, Adair equation, fluorescence correlation spectroscopy
National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-298998 (URN)10.1021/acschemneuro.1c00006 (DOI)000664290200004 ()34076426 (PubMedID)2-s2.0-85108386476 (Scopus ID)
Note

QC 20210728

Available from: 2021-07-28 Created: 2021-07-28 Last updated: 2023-08-28Bibliographically approved
Gao, Y., Wennmalm, S., Winblad, B., Schedin-Weiss, S. & Tjernberg, L. O. (2021). Live Cell FRET Imaging Reveals Amyloid beta-Peptide Oligomerization in Hippocampal Neurons. International Journal of Molecular Sciences, 22(9), Article ID 4530.
Open this publication in new window or tab >>Live Cell FRET Imaging Reveals Amyloid beta-Peptide Oligomerization in Hippocampal Neurons
Show others...
2021 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 22, no 9, article id 4530Article in journal (Refereed) Published
Abstract [en]

Amyloid beta-peptide (A beta) oligomerization is believed to contribute to the neuronal dysfunction in Alzheimer disease (AD). Despite decades of research, many details of A beta oligomerization in neurons still need to be revealed. Forster resonance energy transfer (FRET) is a simple but effective way to study molecular interactions. Here, we used a confocal microscope with a sensitive Airyscan detector for FRET detection. By live cell FRET imaging, we detected A beta 42 oligomerization in primary neurons. The neurons were incubated with fluorescently labeled A beta 42 in the cell culture medium for 24 h. A beta 42 were internalized and oligomerized in the lysosomes/late endosomes in a concentration-dependent manner. Both the cellular uptake and intracellular oligomerization of A beta 42 were significantly higher than for A beta 40. These findings provide a better understanding of A beta 42 oligomerization in neurons.

Place, publisher, year, edition, pages
MDPI AG, 2021
Keywords
amyloid &#946, -peptide, oligomerization, aggregation, FRET
National Category
Neurosciences
Identifiers
urn:nbn:se:kth:diva-296194 (URN)10.3390/ijms22094530 (DOI)000650423800001 ()33926107 (PubMedID)2-s2.0-85104672943 (Scopus ID)
Note

QC 20210609

Available from: 2021-06-09 Created: 2021-06-09 Last updated: 2022-06-25Bibliographically approved
Pettersson, P., Patrick, J., Jakob, M., Jacobs, M., Kloesgen, R. B., Wennmalm, S. & Maler, L. (2021). Soluble TatA forms oligomers that interact with membranes: Structure and insertion studies of a versatile protein transporter. Biochimica et Biophysica Acta - Biomembranes, 1863(2), Article ID 183529.
Open this publication in new window or tab >>Soluble TatA forms oligomers that interact with membranes: Structure and insertion studies of a versatile protein transporter
Show others...
2021 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1863, no 2, article id 183529Article in journal (Refereed) Published
Abstract [en]

The twin-arginine translocase (Tat) mediates the transport of already-folded proteins across membranes in bacteria, plants and archaea. TatA is a small, dynamic subunit of the Tat-system that is believed to be the active component during target protein translocation. TatA is foremost characterized as a bitopic membrane protein, but has also been found to partition into a soluble, oligomeric structure of yet unknown function. To elucidate the interplay between the membrane-bound and soluble forms we have investigated the oligomers formed by Arabidopsis thaliana TatA. We used several biophysical techniques to study the oligomeric structure in solution, the conversion that takes place upon interaction with membrane models of different compositions, and the effect on bilayer integrity upon insertion. Our results demonstrate that in solution TatA oligomerizes into large objects with a high degree of ordered structure. Upon interaction with lipids, conformational changes take place and TatA disintegrates into lower order oligomers. The insertion of TatA into lipid bilayers causes a temporary leakage of small molecules across the bilayer. The disruptive effect on the membrane is dependent on the liposome's negative surface charge density, with more leakage observed for purely zwitterionic bilayers. Overall, our findings indicate that A. thaliana TatA forms oligomers in solution that insert into bilayers, a process that involves reorganization of the protein oligomer.

Place, publisher, year, edition, pages
Elsevier BV, 2021
Keywords
Twin-arginine translocase, Oligomer, Vesicles, Membrane insertion, Membrane leakage, Electron microscopy, Light scattering, Chemical crosslinking, Circular dichroism, Fluorescence Correlation Spectroscopy
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-289288 (URN)10.1016/j.bbamem.2020.183529 (DOI)000603419900021 ()33279512 (PubMedID)2-s2.0-85097774344 (Scopus ID)
Note

QC 20210126

Available from: 2021-01-26 Created: 2021-01-26 Last updated: 2022-06-25Bibliographically approved
Villamil Giraldo, A.-M. -., Eriksson, I., Wennmalm, S., Fyrner, T., Ederth, T. & Öllinger, K. (2020). Interactions of the lysosomotropic detergent omethyl-serine dodecylamide hydrochloride (Msdh) with lipid bilayer membranes-implications for cell toxicity. International Journal of Molecular Sciences, 21(9), Article ID 3136.
Open this publication in new window or tab >>Interactions of the lysosomotropic detergent omethyl-serine dodecylamide hydrochloride (Msdh) with lipid bilayer membranes-implications for cell toxicity
Show others...
2020 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 21, no 9, article id 3136Article in journal (Refereed) Published
Abstract [en]

O-methyl-serine dodecylamine hydrochloride (MSDH) is a detergent that accumulates selectively in lysosomes, a so-called lysosomotropic detergent, with unexpected chemical properties. At physiological pH, it spontaneously forms vesicles, which disassemble into small aggregates (probably micelles) below pH 6.4. In this study, we characterize the interaction between MSDH and liposomes at different pH and correlate the findings to toxicity in human fibroblasts. We find that the effect of MSDH on lipid membranes is highly pH-dependent. At neutral pH, the partitioning of MSDH into the liposome membrane is immediate and causes the leakage of small fluorophores, unless the ratio between MSDH and lipids is kept low. At pH 5, the partitioning of MSDH into the membrane is kinetically impeded since MSDH is charged and a high ratio between MSDH and the lipids is required to permeabilize the membrane. When transferred to cell culture conditions, the ratio between MSDH and plasma membrane lipids must therefore be low, at physiological pH, to maintain plasma membrane integrity. Transmission electron microscopy suggests that MSDH vesicles are taken up by endocytosis. As the pH of the endosomal compartment progressively drops, MSDH vesicles disassemble, leading to a high concentration of increasingly charged MSDH in small aggregates inside the lysosomes. At sufficiently high MSDH concentrations, the lysosome is permeabilized, the proteolytic content released to the cytosol and apoptotic cell death is induced.

Place, publisher, year, edition, pages
MDPI AG, 2020
Keywords
Liposome, Lysosomal membrane permeabilization, Lysosome, Lysosomotropic detergent, MSDH, caspase 3, detergent, lactate dehydrogenase, methyl serine dodecylamide hydrochloride, n acetyl beta glucosaminidase, unclassified drug, AG1518 cell line, apoptosis, Article, cell damage, cell permeabilization, cell viability, confocal laser scanning microscopy, controlled study, cytotoxicity, cytotoxicity assay, endocytosis, enzyme activity, fibroblast cell line, fluorescence correlation spectroscopy, human, human cell, immunocytochemistry, incubation time, lipid bilayer, live cell imaging, molecular interaction, molecular stability, MTT assay, pH, photon correlation spectroscopy, spectrofluorometry, transmission electron microscopy, Western blotting
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-277140 (URN)10.3390/ijms21093136 (DOI)000535581700107 ()32365555 (PubMedID)2-s2.0-85084276477 (Scopus ID)
Note

QC 20200716

Available from: 2020-07-16 Created: 2020-07-16 Last updated: 2025-02-20Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-1850-5440

Search in DiVA

Show all publications