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Usaj, Marko, Research EngineerORCID iD iconorcid.org/0000-0001-6662-8886
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Publications (10 of 27) Show all publications
Berg, A., Velayuthan, L. P., Månsson, A. & Usaj, M. (2024). Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent. International Journal of Molecular Sciences, 25(12), Article ID 6747.
Open this publication in new window or tab >>Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent
2024 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 25, no 12, article id 6747Article in journal (Refereed) Published
Abstract [en]

Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac beta-myosin heavy chain (beta-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current similar to 4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

Place, publisher, year, edition, pages
MDPI, 2024
Keywords
virus-free gene delivery, C2C12, cell transfection, protein expression, protein purification, human cardiac myosin II, in vitro motility assay, single-molecule assays
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-131772 (URN)10.3390/ijms25126747 (DOI)001257630300001 ()38928453 (PubMedID)2-s2.0-85197301116 (Scopus ID)
Available from: 2024-08-15 Created: 2024-08-15 Last updated: 2024-08-15
Usaj, M., Pavlin, M. & Kanduser, M. (2024). Feasibility Study for the Use of Gene Electrotransfer and Cell Electrofusion as a Single-Step Technique for the Generation of Activated Cancer Cell Vaccines. Journal of Membrane Biology
Open this publication in new window or tab >>Feasibility Study for the Use of Gene Electrotransfer and Cell Electrofusion as a Single-Step Technique for the Generation of Activated Cancer Cell Vaccines
2024 (English)In: Journal of Membrane Biology, ISSN 0022-2631, E-ISSN 1432-1424Article in journal (Refereed) Epub ahead of print
Abstract [en]

Cell-based therapies hold great potential for cancer immunotherapy. This approach is based on manipulation of dendritic cells to activate immune system against specific cancer antigens. For the development of an effective cell vaccine platform, gene transfer, and cell fusion have been used for modification of dendritic or tumor cells to express immune (co)stimulatory signals and to load dendritic cells with tumor antigens. Both, gene transfer and cell fusion can be achieved by single technique, a cell membrane electroporation. The cell membrane exposed to external electric field becomes temporarily permeable, enabling introduction of genetic material, and also fusogenic, enabling the fusion of cells in the close contact. We tested the feasability of combining gene electrotransfer and electrofusion into a single-step technique and evaluated the effects of electroporation buffer, pulse parameters, and cell membrane fluidity for single or combined method of gene delivery or cell fusdion. We determined the percentage of fused cells expressing green fluorescence protein (GFP) in a murine cell model of melanoma B16F1, cell line used in our previous studies. Our results suggest that gene electrotransfer and cell electrofusion can be applied in a single step. The percentage of viable hybrid cells expressing GFP depends on electric pulse parameters and the composition of the electroporation buffer. Furthermore, our results suggest that cell membrane fluidity is not related to the efficiency of the gene electrotransfer and electrofusion. The protocol is compatible with microfluidic devices, however further optimization of electric pulse parameters and buffers is still needed.

Place, publisher, year, edition, pages
Springer, 2024
Keywords
Electrofusion, Gene electrotransfer, Cancer cell vaccines, Cell membrane fluidity, Electroporation
National Category
Medical Biotechnology
Research subject
Chemistry, Biotechnology
Identifiers
urn:nbn:se:lnu:diva-132148 (URN)10.1007/s00232-024-00320-5 (DOI)001289372800002 ()39133276 (PubMedID)2-s2.0-85200997842 (Scopus ID)
Available from: 2024-08-29 Created: 2024-08-29 Last updated: 2024-10-23
Melbacke, A., Salhotra, A., Usaj, M. & Månsson, A. (2024). Improved longevity of actomyosin in vitro motility assays for sustainable lab-on-a-chip applications. Scientific Reports, 14(1), Article ID 22768.
Open this publication in new window or tab >>Improved longevity of actomyosin in vitro motility assays for sustainable lab-on-a-chip applications
2024 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 14, no 1, article id 22768Article in journal (Refereed) Published
Abstract [en]

In the in vitro motility assay (IVMA), actin filaments are observed while propelled by surface-adsorbed myosin motor fragments such as heavy meromyosin (HMM). In addition to fundamental studies, the IVMA is the basis for a range of lab-on-a-chip applications, e.g. transport of cargoes in nanofabricated channels in nanoseparation/biosensing or the solution of combinatorial mathematical problems in network-based biocomputation. In these applications, prolonged myosin function is critical as is the potential to repeatedly exchange experimental solutions without functional deterioration. We here elucidate key factors of importance in these regards. Our findings support a hypothesis that early deterioration in the IVMA is primarily due to oxygen entrance into in vitro motility assay flow cells. In the presence of a typically used oxygen scavenger mixture (glucose oxidase, glucose, and catalase), this leads to pH reduction by a glucose oxidase-catalyzed reaction between glucose and oxygen but also contributes to functional deterioration by other mechanisms. Our studies further demonstrate challenges associated with evaporation and loss of actin filaments with time. However, over 8 h at 21-26 degrees C, there is no significant surface desorption or denaturation of HMM if solutions are exchanged manually every 30 min. We arrive at an optimized protocol with repeated exchange of carefully degassed assay solution of 45 mM ionic strength, at 30 min intervals. This is sufficient to maintain the high-quality function in an IVMA over 8 h at 21-26 degrees C, provided that fresh actin filaments are re-supplied in connection with each assay solution exchange. Finally, we demonstrate adaptation to a microfluidic platform and identify challenges that remain to be solved for real lab-on-a-chip applications.

Place, publisher, year, edition, pages
Nature Publishing Group, 2024
Keywords
In vitro motility assay, Actomyosin, Lab-on-a-chip, Biosensing, Network based biocomputation, Microfluidics
National Category
Biophysics
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-133044 (URN)10.1038/s41598-024-73457-x (DOI)001328801300065 ()39354041 (PubMedID)2-s2.0-85205527204 (Scopus ID)
Available from: 2024-10-28 Created: 2024-10-28 Last updated: 2024-10-28
Berg, A., Velayuthan, L. P., Tågerud, S., Ušaj, M. & Månsson, A. (2024). Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence. Cytoskeleton
Open this publication in new window or tab >>Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence
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2024 (English)In: Cytoskeleton, ISSN 1949-3584Article in journal (Refereed) Epub ahead of print
Abstract [en]

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (k(cat)). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

Place, publisher, year, edition, pages
John Wiley & Sons, 2024
Keywords
actin, actin-activated ATPase, Alexa 647 ATP, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, single molecule fluorescence, beta-Cardiac myosin II
National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-129124 (URN)10.1002/cm.21858 (DOI)001203081500001 ()38623952 (PubMedID)2-s2.0-85191016814 (Scopus ID)
Available from: 2024-05-02 Created: 2024-05-02 Last updated: 2024-08-22
Salhotra, A., Rahman, M. A., Ruijgrok, P. V., Meinecke, C. R., Ušaj, M., Zemsky, S., . . . Månsson, A. (2023). Exploitation of Engineered Light-Switchable Myosin XI for Nanotechnological Applications. ACS Nano, 17(17), 17233-17244
Open this publication in new window or tab >>Exploitation of Engineered Light-Switchable Myosin XI for Nanotechnological Applications
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2023 (English)In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 17, no 17, p. 17233-17244Article in journal (Refereed) Published
Abstract [en]

For certain nanotechnological applications of the contractile proteins actin and myosin, e.g., in biosensing and network-based biocomputation, it would be desirable to temporarily switch on/off motile function in parts of nanostructured devices, e.g., for sorting or programming. Myosin XI motor constructs, engineered with a light-switchable domain for switching actin motility between high and low velocities (light-sensitive motors (LSMs) below), are promising in this regard. However, they were not designed for use in nanotechnology, where longevity of operation, long shelf life, and selectivity of function in specific regions of a nanofabricated network are important. Here, we tested if these criteria can be fulfilled using existing LSM constructs or if additional developments will be required. We demonstrated extended shelf life as well as longevity of the actin-propelling function compared to those in previous studies. We also evaluated several approaches for selective immobilization with a maintained actin propelling function in dedicated nanochannels only. Whereas selectivity was feasible using certain nanopatterning combinations, the reproducibility was not satisfactory. In summary, the study demonstrates the feasibility of using engineered light-controlled myosin XI motors for myosin-driven actin transport in nanotechnological applications. Before use for, e.g., sorting or programming, additional work is however needed to achieve reproducibility of the nanofabrication and, further, optimize the motor properties.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2023
Keywords
engineered myosin XI, actin, light-switchablemotor, nanofabrication, surface chemistry, spatiotemporal motility control
National Category
Biophysics
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-125040 (URN)10.1021/acsnano.3c05137 (DOI)001063827500001 ()37639711 (PubMedID)2-s2.0-85171203426 (Scopus ID)
Available from: 2023-10-05 Created: 2023-10-05 Last updated: 2023-11-07Bibliographically approved
Månsson, A., Ušaj, M., Moretto, L., Matusovsky, O., Velayuthan, L. P., Friedman, R. & Rassier, D. E. (2023). New paradigms in actomyosin energy transduction: Critical evaluation of non-traditional models for orthophosphate release. Bioessays, 45(9), Article ID 2300040.
Open this publication in new window or tab >>New paradigms in actomyosin energy transduction: Critical evaluation of non-traditional models for orthophosphate release
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2023 (English)In: Bioessays, ISSN 0265-9247, E-ISSN 1521-1878, Vol. 45, no 9, article id 2300040Article in journal (Refereed) Published
Abstract [en]

Release of the ATP hydrolysis product ortophosphate (Pi) from the active site of myosin is central in chemo-mechanical energy transduction and closely associated with the main force-generating structural change, the power-stroke. Despite intense investigations, the relative timing between Pi-release and the power-stroke remains poorly understood. This hampers in depth understanding of force production by myosin in health and disease and our understanding of myosin-active drugs. Since the 1990s and up to today, models that incorporate the Pi-release either distinctly before or after the power-stroke, in unbranched kinetic schemes, have dominated the literature. However, in recent years, alternative models have emerged to explain apparently contradictory findings. Here, we first compare and critically analyze three influential alternative models proposed previously. These are either characterized by a branched kinetic scheme or by partial uncoupling of Pi-release and the power-stroke. Finally, we suggest critical tests of the models aiming for a unified picture.

Place, publisher, year, edition, pages
John Wiley & Sons, 2023
Keywords
actin, actomyosin, ATP turnover, ATPase, inorganic phosphate, myosin, ortophosphate
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-123551 (URN)10.1002/bies.202300040 (DOI)001013725400001 ()37366639 (PubMedID)2-s2.0-85163000556 (Scopus ID)
Available from: 2023-08-09 Created: 2023-08-09 Last updated: 2023-08-31Bibliographically approved
Velayuthan, L. P., Moretto, L., Tågerud, S., Ušaj, M. & Månsson, A. (2023). Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays. Scientific Reports, 13(1), Article ID 4101.
Open this publication in new window or tab >>Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays
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2023 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 13, no 1, article id 4101Article in journal (Refereed) Published
Abstract [en]

Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesting conditions, we functionally characterized the expressed protein, co-purified with murine essential and regulatory light chains. The gliding velocity (1.5-1.7 mu m/s; 25 degrees C) in the in vitro motility assay as well as maximum actin activated catalytic activity (k(cat); 8-9 s(-1)) and actin concentration for half maximal activity (K-ATPase; 70-80 mu M) were similar to those found previously using virus based infection. The results should allow new types of studies, e.g., screening of a wide range of mutations to be selected for further characterization.

Place, publisher, year, edition, pages
Nature Publishing Group, 2023
National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-120565 (URN)10.1038/s41598-023-30576-1 (DOI)000949110500003 ()36907906 (PubMedID)2-s2.0-85150205206 (Scopus ID)
Available from: 2023-05-05 Created: 2023-05-05 Last updated: 2023-05-31Bibliographically approved
Ušaj, M., Moretto, L. & Månsson, A. (2022). Critical Evaluation of Current Hypotheses for the Pathogenesis of Hypertrophic Cardiomyopathy. International Journal of Molecular Sciences, 23(4), Article ID 2195.
Open this publication in new window or tab >>Critical Evaluation of Current Hypotheses for the Pathogenesis of Hypertrophic Cardiomyopathy
2022 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 4, article id 2195Article in journal (Refereed) Published
Abstract [en]

Hereditary hypertrophic cardiomyopathy (HCM), due to mutations in sarcomere proteins, occurs in more than 1/500 individuals and is the leading cause of sudden cardiac death in young people. The clinical course exhibits appreciable variability. However, typically, heart morphology and function are normal at birth, with pathological remodeling developing over years to decades, leading to a phenotype characterized by asymmetric ventricular hypertrophy, scattered fibrosis and myofibrillar/cellular disarray with ultimate mechanical heart failure and/or severe arrhythmias. The identity of the primary mutation-induced changes in sarcomere function and how they trigger debilitating remodeling are poorly understood. Support for the importance of mutation-induced hypercontractility, e.g., increased calcium sensitivity and/or increased power output, has been strengthened in recent years. However, other ideas that mutation-induced hypocontractility or non-uniformities with contractile instabilities, instead, constitute primary triggers cannot yet be discarded. Here, we review evidence for and criticism against the mentioned hypotheses. In this process, we find support for previous ideas that inefficient energy usage and a blunted Frank-Starling mechanism have central roles in pathogenesis, although presumably representing effects secondary to the primary mutation-induced changes. While first trying to reconcile apparently diverging evidence for the different hypotheses in one unified model, we also identify key remaining questions and suggest how experimental systems that are built around isolated primarily expressed proteins could be useful.

Place, publisher, year, edition, pages
MDPI, 2022
Keywords
hypertrophic cardiomyopathy, hypocontractility, hypercontractility, non-uniformity, hierarchical organization
National Category
Cardiac and Cardiovascular Systems Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-110772 (URN)10.3390/ijms23042195 (DOI)000763616800001 ()35216312 (PubMedID)2-s2.0-85124609914 (Scopus ID)2022 (Local ID)2022 (Archive number)2022 (OAI)
Available from: 2022-03-11 Created: 2022-03-11 Last updated: 2023-05-31Bibliographically approved
Moretto, L., Ušaj, M., Matusovsky, O., Rassier, D. E., Friedman, R. & Månsson, A. (2022). Multistep orthophosphate release tunes actomyosin energy transduction. Nature Communications, 13(1), Article ID 4575.
Open this publication in new window or tab >>Multistep orthophosphate release tunes actomyosin energy transduction
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2022 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 13, no 1, article id 4575Article in journal (Refereed) Published
Abstract [en]

Release of the ATP hydrolysis product orthophosphate (Pi) from the myosin active site is central in force generation but is poorly understood. Here, Moretto et al. present evidence for multistep Pi-release reconciling apparently contradictory results. Muscle contraction and a range of critical cellular functions rely on force-producing interactions between myosin motors and actin filaments, powered by turnover of adenosine triphosphate (ATP). The relationship between release of the ATP hydrolysis product ortophosphate (Pi) from the myosin active site and the force-generating structural change, the power-stroke, remains enigmatic despite its central role in energy transduction. Here, we present a model with multistep Pi-release that unifies current conflicting views while also revealing additional complexities of potential functional importance. The model is based on our evidence from kinetics, molecular modelling and single molecule fluorescence studies of Pi binding outside the active site. It is also consistent with high-speed atomic force microscopy movies of single myosin II molecules without Pi at the active site, showing consecutive snapshots of pre- and post-power stroke conformations. In addition to revealing critical features of energy transduction by actomyosin, the results suggest enzymatic mechanisms of potentially general relevance.

Place, publisher, year, edition, pages
Nature Publishing Group, 2022
National Category
Biochemistry and Molecular Biology Biophysics
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-116580 (URN)10.1038/s41467-022-32110-9 (DOI)000836609800023 ()35931685 (PubMedID)2-s2.0-85135471373 (Scopus ID)
Available from: 2022-09-29 Created: 2022-09-29 Last updated: 2023-05-31Bibliographically approved
Vemula, V., Huber, T., Ušaj, M., Bugyi, B. & Månsson, A. (2021). Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity. Journal of Biological Chemistry, 296, Article ID 100181.
Open this publication in new window or tab >>Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity
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2021 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 296, article id 100181Article in journal (Refereed) Published
Abstract [en]

Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements. Its dynamic behavior, such as polymerization and depolymerization of actin filaments in response to intracellular and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor-induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy–based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

Place, publisher, year, edition, pages
ASBMB Publications, 2021
National Category
Biophysics
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-102095 (URN)10.1074/jbc.RA120.015863 (DOI)000672866400159 ()33303625 (PubMedID)2-s2.0-85102939761 (Scopus ID)2021 (Local ID)2021 (Archive number)2021 (OAI)
Funder
Swedish Research Council, 2015-05290;2019-03456EU, Horizon 2020, 732482
Available from: 2021-04-11 Created: 2021-04-11 Last updated: 2023-05-05Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-6662-8886

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