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Ušaj, Marko
Publications (10 of 15) Show all publications
Kandušer, M., Kokalj Imsirovic, M. & Ušaj, M. (2019). The Effect of Lipid Antioxidant α-Tocopherol on Cell Viability and Electrofusion Yield of B16-F1 Cells In Vitro. The Journal of Membrane Biology, 252(1), 105-114
Open this publication in new window or tab >>The Effect of Lipid Antioxidant α-Tocopherol on Cell Viability and Electrofusion Yield of B16-F1 Cells In Vitro
2019 (English)In: The Journal of Membrane Biology, ISSN 0022-2631, Vol. 252, no 1, p. 105-114Article in journal (Refereed) Published
Abstract [en]

Induced cell fusion is a powerful method for production of hybridoma in biotechnology and cell vaccines in medical applications. Among different alternatives, physical methods have an advantage, as they do not require any additives. Among them electrofusion, an electroporation-based cell fusion method holds a great promise. Electric pulses cause cell membrane permeabilization and due to pore formation bring cell membrane into the fusogenic state. At the same time, however, they compromise cell viability. We used a train of 8 × 100 µs electric pulses, delivered at 1 Hz with strengths ranging from 400 to 1600 V/cm. We evaluated electrofusion efficiency by dual color microscopy. We determined cell viability, because during electroporation reactive oxygen species are generated affecting cell survival. The novelty of our study is evaluation of the effect of lipid antioxidant α-tocopherol on cell fusion yield and cell viability on mouse B16-F1 cells. Pretreatment with α-tocopherol slowed down dynamic of cell fusion shortly after electroporation. Twenty-four hours later, fusion yields between α-tocopherol treated and untreated cells were comparable. The viability of α-tocopherol pretreated cells was drastically improved. Pretreatment of cells with α-tocopherol improved whole electrofusion process by more than 60%. We believe that α-tocopherol holds great promise to become an important agent to improve cell electrofusion method.

Place, publisher, year, edition, pages
Springer, 2019
Keywords
Electrofusion, Vitamin E, In vitro
National Category
Cell Biology Other Medical Engineering Other Electrical Engineering, Electronic Engineering, Information Engineering
Research subject
Natural Science, Biomedical Sciences; Chemistry, Biotechnology; Natural Science, Cell and Organism Biology
Identifiers
urn:nbn:se:lnu:diva-81830 (URN)10.1007/s00232-019-00059-4 (DOI)
Available from: 2019-04-11 Created: 2019-04-11 Last updated: 2019-05-06Bibliographically approved
Rahman, M. A., Ušaj, M., Rassier, D. E. & Månsson, A. (2018). Blebbistatin Effects Expose Hidden Secrets in the Force-Generating Cycle of Actin and Myosin. Paper presented at Biophysical-Society Thematic Meeting on Single-Cell Biophysics - Mearurement, Modulation, and Modeling, JUN, 2017, Natl Taiwan Univ, Acad Sinica, Inst Atom & Mol Sci, Taipei, TAIWAN. Biophysical Journal, 115(2), 386-397
Open this publication in new window or tab >>Blebbistatin Effects Expose Hidden Secrets in the Force-Generating Cycle of Actin and Myosin
2018 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 115, no 2, p. 386-397Article in journal (Refereed) Published
Abstract [en]

Cyclic interactions between myosin II motors and actin filaments driven by ATP turnover underlie muscle contraction and have key roles in the motility of nonmuscle cells. A remaining enigma in the understanding of this interaction is the relationship between the force-generating structural change and the release of the ATP-hydrolysis product, inorganic phosphate (Pi), from the active site of myosin. Here, we use the small molecular compound blebbistatin to probe otherwise hidden states and transitions in this process. Different hypotheses for the Pi release mechanism are tested by interpreting experimental results from in vitro motility assays and isolated muscle fibers in terms of mechanokinetic actomyosin models. The data fit with ideas that actomyosin force generation is preceded by Pi release, which in turn is preceded by two serial transitions after/coincident with cross-bridge attachment. Blebbistatin changes the rate limitation of the cycle from the first to the second of these transitions, uncovering functional roles of an otherwise short-lived pre-power stroke state that has been implicated by structural data.

Place, publisher, year, edition, pages
Rockville, MD: Biophysical Society, 2018
National Category
Biophysics
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-77397 (URN)10.1016/j.bpj.2018.05.037 (DOI)000438958800024 ()30021113 (PubMedID)2-s2.0-85048935566 (Scopus ID)
Conference
Biophysical-Society Thematic Meeting on Single-Cell Biophysics - Mearurement, Modulation, and Modeling, JUN, 2017, Natl Taiwan Univ, Acad Sinica, Inst Atom & Mol Sci, Taipei, TAIWAN
Available from: 2018-08-29 Created: 2018-08-29 Last updated: 2019-08-29Bibliographically approved
Månsson, A. & Ušaj, M. (2018). Challenges in TIRF-Microscopy Based Single Molecule ATPase and Binding Assays for Myosin and Actin. In: Biophysical Journal Supplement 1: . Paper presented at Biophysical Society 62nd Annual Meeting, February 17-21, 2018, San Francisco. (pp. 211a-211a). , 114(3), Article ID 1062-Plat.
Open this publication in new window or tab >>Challenges in TIRF-Microscopy Based Single Molecule ATPase and Binding Assays for Myosin and Actin
2018 (English)In: Biophysical Journal Supplement 1, 2018, Vol. 114, no 3, p. 211a-211a, article id 1062-PlatConference paper, Oral presentation with published abstract (Refereed)
Abstract [en]

Total internal reflection fluorescence (TIRF) microscopy has permitted visualization of myosin and actin mechanochemistry on a single-molecule level. It has, for instance, been possible to study complex phenomena such as temporal relationship between ATP turnover and the force producing transition. However, despite quite large number of studies using fluorescent ATP analogs, several challenges remain in the interpretation of TIRF data in terms of ATP binding to the active site of myosin. We here present improvements in assay conditions for more reliable detection of ATP on-time (i.e. dwell time) at the active site(s) of myosin using approaches to suppress fluorophore blinking events related to the photophysics of the fluorescent probe. We also suggest assay designs for analysis of myosin-binding to actin filaments on a surface in the presence of nucleotide. The experiments were performed using a custom built TIRF microscopy platform and fluorescent Alexa Fluor 647 ATP (Alexa-ATP) whose turnover to Alexa Fluor 647 ADP (Alexa-ADP) and inorganic phosphate (Pi) by myosin and actomyosin occurs by a similar mechanism as for unlabeled ATP. Using this system we present solutions for improved single molecule assays for actin and myosin with emphasis on the use of reducing and oxidizing agents (trolox/trolox-quinone; TTQ) in assay buffers to suppress photo-blinking while maintaining long-lasting fluorescence signals. The cumulative frequency distribution of dwell-time events exhibited more than one exponential phase both in the presence and absence of TTQ. The relative amplitude of the slowest phase increased appreciably in the presence of TTQ as well as the overall half-time.

National Category
Biophysics
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-75776 (URN)10.1016/j.bpj.2017.11.1180 (DOI)
Conference
Biophysical Society 62nd Annual Meeting, February 17-21, 2018, San Francisco.
Available from: 2018-06-14 Created: 2018-06-14 Last updated: 2018-06-15Bibliographically approved
Månsson, A., Ušaj, M., Moretto, L. & Rassier, D. E. (2018). Do Actomyosin Single-Molecule Mechanics Data Predict Mechanics of Contracting Muscle?. International Journal of Molecular Sciences, 19(7), Article ID 1863.
Open this publication in new window or tab >>Do Actomyosin Single-Molecule Mechanics Data Predict Mechanics of Contracting Muscle?
2018 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 19, no 7, article id 1863Article, review/survey (Refereed) Published
Abstract [en]

In muscle, but not in single-molecule mechanics studies, actin, myosin and accessory proteins are incorporated into a highly ordered myofilament lattice. In view of this difference we compare results from single-molecule studies and muscle mechanics and analyze to what degree data from the two types of studies agree with each other. There is reasonable correspondence in estimates of the cross-bridge power-stroke distance (7-13 nm), cross-bridge stiffness (similar to 2 pN/nm) and average isometric force per cross-bridge (6-9 pN). Furthermore, models defined on the basis of single-molecule mechanics and solution biochemistry give good fits to experimental data from muscle. This suggests that the ordered myofilament lattice, accessory proteins and emergent effects of the sarcomere organization have only minor modulatory roles. However, such factors may be of greater importance under e.g., disease conditions. We also identify areas where single-molecule and muscle data are conflicting: (1) whether force generation is an Eyring or Kramers process with just one major power-stroke or several sub-strokes; (2) whether the myofilaments and the cross-bridges have Hookean or non-linear elasticity; (3) if individual myosin heads slip between actin sites under certain conditions, e.g.,in lengthening; or (4) if the two heads of myosin cooperate.

Place, publisher, year, edition, pages
MDPI, 2018
Keywords
optical tweezers, optical traps, muscle fiber, myofibril, myosin, actin, cross-bridge, mechanochemical model
National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-77732 (URN)10.3390/ijms19071863 (DOI)000442807400042 ()29941816 (PubMedID)2-s2.0-85049149996 (Scopus ID)
Available from: 2018-09-13 Created: 2018-09-13 Last updated: 2019-08-29Bibliographically approved
Ušaj, M., Zattelman, L., Regev, R., Shneyer, B. I., Wiesel-Motiuk, N. & Henn, A. (2018). Overexpression and purification of human myosins from transiently and stably transfected suspension adapted HEK293SF-3F6 cells. Analytical Biochemistry, 558(1), 19-27
Open this publication in new window or tab >>Overexpression and purification of human myosins from transiently and stably transfected suspension adapted HEK293SF-3F6 cells
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2018 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 558, no 1, p. 19-27Article in journal (Refereed) Published
Abstract [en]

The myosin family of motor proteins is an attractive target of therapeutic small-molecule protein inhibitors and modulators. Milligrams of protein quantities are required to conduct proper biophysical and biochemical studies to understand myosin functions. Myosin protein expression and purification represent a critical starting point towards this goal. Established utilization of Dictyostelium discoideum, Drosophila melanogaster, insect and mouse cells for myosin expression and purification is limited, cost, labor and time inefficient particularly for (full-length) human myosins. Here we are presenting detailed protocols for production of several difficult-to-purify recombinant human myosins in efficient quantities up to 1 mg of protein per liter of cell culture. This is the first time that myosins have been purified in large scales from suspension adapted transiently and stably expressing human cells. The method is also useful for expressing other human proteins in quantities sufficient to perform extensive biochemical and biophysical characterization.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Recombinant myosins, Human cell expression system, HEK293SF-3F6, Suspension cell culture, Gene expression, Polyethyleneimine
National Category
Biochemistry and Molecular Biology Biophysics Cell Biology
Research subject
Chemistry, Biotechnology; Chemistry, Biochemistry; Natural Science, Cell and Organism Biology
Identifiers
urn:nbn:se:lnu:diva-81832 (URN)10.1016/j.ab.2018.07.026 (DOI)
Available from: 2019-04-11 Created: 2019-04-11 Last updated: 2019-05-03Bibliographically approved
Ušaj, M. & Henn, A. (2017). Kinetic adaptation of human Myo19 for active mitochondrial transport to growing filopodia tips. Scientific Reports, 7(1), Article ID 11596.
Open this publication in new window or tab >>Kinetic adaptation of human Myo19 for active mitochondrial transport to growing filopodia tips
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, no 1, article id 11596Article in journal (Refereed) Published
Abstract [en]

Myosins are actin-based molecular motors which are enzymatically adapted for their cellular functions such as transportation and membrane tethering. Human Myo19 affects mitochondrial motility, and promotes their localization to stress-induced filopodia. Therefore, studying Myo19 enzymology is essential to understand how this motor may facilitate mitochondrial motility. Towards this goal, we have purified Myo19 motor domain (Myo19-3IQ) from a human-cell expression system and utilized transient kinetics to study the Myo19-3IQ ATPase cycle. We found that Myo19-3IQ exhibits noticeable conformational changes (isomerization steps) preceding both ATP and ADP binding, which may contribute to nucleotide binding regulation. Notably, the ADP isomerization step and subsequent ADP release contribute significantly to the rate-limiting step of the Myo19-3IQ ATPase cycle. Both the slow ADP isomerization and ADP release prolong the time Myo19-3IQ spend in the strong actin binding state and hence contribute to its relatively high duty ratio. However, the predicted duty ratio is lower than required to support motility as a monomer. Therefore, it may be that several Myo19 motors are required to propel mitochondria movement on actin filaments efficiently. Finally, we provide a model explaining how Myo19 translocation may be regulated by the local ATP/ADP ratio, coupled to the mitochondria presence in the filopodia.

National Category
Biochemistry and Molecular Biology Biophysics
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-75916 (URN)10.1038/s41598-017-11984-6 (DOI)
Available from: 2018-06-14 Created: 2018-06-14 Last updated: 2018-06-15Bibliographically approved
Zattelman, L., Regev, R., Ušaj, M., Reinke, P. Y. A., Giese, S., Samson, A. O., . . . Henn, A. (2017). N-terminal splicing extensions of the human MYO1C gene fine-tune the kinetics of the three full-length myosin IC isoforms. Journal of Biological Chemistry, 292(43), 17804-17818
Open this publication in new window or tab >>N-terminal splicing extensions of the human MYO1C gene fine-tune the kinetics of the three full-length myosin IC isoforms
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2017 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 43, p. 17804-17818Article in journal (Refereed) Published
Abstract [en]

The MYO1C gene produces three alternatively spliced isoforms, differing only in their N-terminal regions (NTRs). These isoforms, which exhibit both specific and overlapping nuclear and cytoplasmic functions, have different expression levels and nuclear–cytoplasmic partitioning. To investigate the effect of NTR extensions on the enzymatic behavior of individual isoforms, we overexpressed and purified the three full-length human isoforms from suspension-adapted HEK cells. MYO1CC favored the actomyosin closed state (AMC), MYO1C16 populated the actomyosin open state (AMO) and AMC equally, and MYO1C35 favored the AMO state. Moreover, the full-length constructs isomerized before ADP release, which has not been observed previously in truncated MYO1CC constructs. Furthermore, global numerical simulation analysis predicted that MYO1C35 populated the actomyosin·ADP closed state (AMDC) 5-fold more than the actomyosin·ADP open state (AMDO) and to a greater degree than MYO1CC and MYO1C16 (4- and 2-fold, respectively). On the basis of a homology model of the 35-amino acid NTR of MYO1C35 (NTR35) docked to the X-ray structure of MYO1CC, we predicted that MYO1C35 NTR residue Arg-21 would engage in a specific interaction with post-relay helix residue Glu-469, which affects the mechanics of the myosin power stroke. In addition, we found that adding the NTR35 peptide to MYO1CC yielded a protein that transiently mimics MYO1C35 kinetic behavior. By contrast, NTR35, which harbors the R21G mutation, was unable to confer MYO1C35-like kinetic behavior. Thus, the NTRs affect the specific nucleotide-binding properties of MYO1C isoforms, adding to their kinetic diversity. We propose that this level of fine-tuning within MYO1C broadens its adaptability within cells.

National Category
Biochemistry and Molecular Biology Biophysics
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-75919 (URN)10.1074/jbc.M117.794008 (DOI)
Available from: 2018-06-14 Created: 2018-06-14 Last updated: 2018-06-15Bibliographically approved
Shneyer, B. I., Ušaj, M. & Henn, A. (2016). Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia. Journal of Cell Science, 129(3), 543-556
Open this publication in new window or tab >>Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia
2016 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 129, no 3, p. 543-556Article in journal (Refereed) Published
Abstract [en]

Mitochondria respond to environmental cues and stress conditions. Additionally, the disruption of the mitochondrial network dynamics and its distribution is implicated in a variety of neurodegenerative diseases. Here, we reveal a new function for Myo19 in mitochondrial dynamics and localization during the cellular response to glucose starvation. Ectopically expressed Myo19 localized with mitochondria to the tips of starvation-induced filopodia. Corollary to this, RNA interference (RNAi)-mediated knockdown of Myo19 diminished filopodia formation without evident effects on the mitochondrial network. We analyzed the Myo19–mitochondria interaction, and demonstrated that Myo19 is uniquely anchored to the outer mitochondrial membrane (OMM) through a 30–45-residue motif, indicating that Myo19 is a stably attached OMM molecular motor. Our work reveals a new function for Myo19 in mitochondrial positioning under stress.

National Category
Cell Biology Biochemistry and Molecular Biology Biophysics
Research subject
Natural Science, Cell and Organism Biology
Identifiers
urn:nbn:se:lnu:diva-75921 (URN)10.1242/jcs.175349 (DOI)
Available from: 2018-06-14 Created: 2018-06-14 Last updated: 2018-06-15Bibliographically approved
Henn, A., Shneyer, B. & Ušaj, M. (2016). Myosin 19 is an Outer Mitochondrial Membrane Motor and Effector of Starvation Induced Filopodia with Unique Kinetic Features. In: Biophysical Journal Supplement 1: . Paper presented at Biophysical Society 60nd Annual Meeting, Feb 27-Mar 02, 2016, Los Angeles (pp. 615a-616a). , 110, Article ID 3040-Pos.
Open this publication in new window or tab >>Myosin 19 is an Outer Mitochondrial Membrane Motor and Effector of Starvation Induced Filopodia with Unique Kinetic Features
2016 (English)In: Biophysical Journal Supplement 1, 2016, Vol. 110, p. 615a-616a, article id 3040-PosConference paper, Poster (with or without abstract) (Refereed)
Abstract [en]

The interaction between the actin cytoskeleton, myosin motors and their function in mitochondria dynamics, morphology and cellular localization is now beginning to emerge. A novel function for actin-based motors as regulators of cellular adaptations to stress, linking actin cytoskeleton remodelling to mitochondria dynamics. We reveal a novel function for myosin 19 in mitochondrial dynamics and localization during cellular response to glucose starvation. Ectopically expressed myosin 19 localizes with mitochondria at the tips of starvation-induced filopodia. Corollary to this, RNAi mediated knockdown of myosin 19 diminished their formation without evident effects on the mitochondrial network. We analyzed myosin 19 mitochondria interaction and demonstrated that it is uniquely anchored to the outer mitochondrial membrane (OMM) via a 30-residue motif, indicating that myosin 19 is a stably attached OMM molecular motor. To this end, we have purified myosin 19-3IQ motor domain construct. Myosin 19-3IQ featured characteristic actin-activated ATPase activity with moderate to slow turnover (kcat) and relatively tight KATPase. Our transient kinetics and steady state equilibrium binding experiments revealed that myosin 19-3IQ binds ATP and ADP with tight affinity that, to the best of our knowledge, have not yet been exhibited by any other myosins. We suspect that this feature allows myosin 19 to operate in a unique cellular environment that may be related to cellular stress conditions as we showed in our previous studies. The detailed knowledge of myosin 19 enzymatic adaptation will provide us with a quantitative working model of myosin 19, and will assist us to understand its cellular function. Our work reveals a novel function for myosin 19 in mitochondrial positioning during homeostasis and under stress conditions and broadens our understanding of the actin cytoskeleton- myosin -mitochondria interplay.

National Category
Biochemistry and Molecular Biology Biophysics Cell Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-75932 (URN)10.1016/j.bpj.2015.11.3302 (DOI)
Conference
Biophysical Society 60nd Annual Meeting, Feb 27-Mar 02, 2016, Los Angeles
Available from: 2018-06-14 Created: 2018-06-14 Last updated: 2018-06-15Bibliographically approved
Ušaj, M. & Kandušer, M. (2015). Modified Adherence Method (MAM) for Electrofusion of Anchorage-Dependent Cells. In: Cell Fusion: Overviews and Methods (pp. 203-216). New York, NY: Humana Press
Open this publication in new window or tab >>Modified Adherence Method (MAM) for Electrofusion of Anchorage-Dependent Cells
2015 (English)In: Cell Fusion: Overviews and Methods, New York, NY: Humana Press, 2015, p. 203-216Chapter in book (Refereed)
Abstract [en]

The artificially induced cell fusion is a useful experimental tool in biology, biotechnology and medicine. The electrofusion is a physical method for cell fusion that applies high-voltage electric pulses. The use of electric pulses causes cell membrane structural changes which bring the cell membrane in the so-called fusogenic state. When such fusogenic membranes are in close contact cell fusion takes place. Physical contact between fusion partners can be achieved by various methods and one of them is modified adherence method (MAM) described in detail here on B16-F1 cell line. The method is based on the fact that living cells form contacts in confluent culture. However, instead of using confluent cell culture, in modified adherence method cells are plated in suitable concentration and allowed to form contacts for only short predetermined period of time. During that time the cells are only slightly attached to the dish surface maintaining the spherical shape. Observed high fusion yields up to 50 % obtained by MAM in situ by dual-color fluorescence microscopy are among the highest in field of electrofusion. The method can be readily adapted to other anchorage-dependent cell lines.

Place, publisher, year, edition, pages
New York, NY: Humana Press, 2015
Series
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029 ; 1313
National Category
Bioprocess Technology Medical Biotechnology Other Medical Engineering
Identifiers
urn:nbn:se:lnu:diva-75931 (URN)10.1007/978-1-4939-2703-6_15 (DOI)978-1-4939-2703-6 (ISBN)978-1-4939-2702-9 (ISBN)
Available from: 2018-06-14 Created: 2018-06-14 Last updated: 2018-06-15Bibliographically approved
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