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  • 1.
    Henn, Arnon
    et al.
    Technion, Israel.
    Shneyer, Boris
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Myosin 19 is an Outer Mitochondrial Membrane Motor and Effector of Starvation Induced Filopodia with Unique Kinetic Features2016In: Biophysical Journal Supplement 1, 2016, Vol. 110, p. 615a-616a, article id 3040-PosConference paper (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.

  • 2.
    Kandušer, Maša
    et al.
    University of Ljubljana, Slovenia.
    Kokalj Imsirovic, Mojca
    University of Ljubljana, Slovenia.
    Ušaj, Marko
    University of Ljubljana, Slovenia.
    The Effect of Lipid Antioxidant α-Tocopherol on Cell Viability and Electrofusion Yield of B16-F1 Cells In Vitro2019In: The Journal of Membrane Biology, ISSN 0022-2631, Vol. 252, no 1, p. 105-114Article in journal (Refereed)
    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.

  • 3.
    Kandušer, Maša
    et al.
    University of Ljubljana, Slovenia.
    Ušaj, Marko
    University of Ljubljana, Slovenia.
    Cell electrofusion: past and future perspectives for antibody production and cancer cell vaccines2014In: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 11, no 12, p. 1885-1898Article in journal (Refereed)
    Abstract [en]

    Introduction: In the past few decades, new methods for drug and gene delivery have been developed, among which electroporation and electrofusion have gained noticeable attention. Lately, advances in the field of immunotherapy have enabled new cancer therapies based on immune response, including monoclonal antibodies and cell vaccines. Efficient cell fusion is needed for both hybridoma production and cell vaccine preparation, and electrofusion is a promising method to achieve this goal.Areas covered: In the present review, we cover new strategies of cancer treatment related to antibody production and cell vaccines. In more detail, cell electroporation and electrofusion are addressed. We briefly describe principles of cell electroporation and focus on electrofusion and its influential factors, with special attention on the fusogenic state of the cell membrane, contact formation, the effect of electrofusion media and cell viability. We end the review with an overview of the very promising field of microfluidic devices for electrofusion.Expert opinion: In our opinion, electrofusion can be a very efficient method for hybridoma and cell vaccine production. Advances in the development of microfluidic devices and a better understanding of the underlying (biological) mechanisms will overcome the current limitations.

  • 4.
    Moretto, Luisa
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ušaj, Marko
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Matusovsky, Oleg
    McGill Univ, Canada.
    Rassier, Dilson E.
    McGill Univ, Canada.
    Friedman, Ran
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Linnaeus University, Linnaeus Knowledge Environments, Advanced Materials.
    Multistep orthophosphate release tunes actomyosin energy transduction2022In: Nature Communications, E-ISSN 2041-1723, Vol. 13, no 1, article id 4575Article in journal (Refereed)
    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.

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  • 5.
    Månsson, Alf
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ušaj, Marko
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Challenges in TIRF-Microscopy Based Single Molecule ATPase and Binding Assays for Myosin and Actin2018In: Biophysical Journal Supplement 1, 2018, Vol. 114, no 3, p. 211a-211a, article id 1062-PlatConference paper (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.

  • 6.
    Månsson, Alf
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ušaj, Marko
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Moretto, Luisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Matusovsky, Oleg
    McGill Univ, Canada.
    Velayuthan, Lok Priya
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Friedman, Ran
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Rassier, Dilson E.
    McGill Univ, Canada.
    New paradigms in actomyosin energy transduction: Critical evaluation of non-traditional models for orthophosphate release2023In: Bioessays, ISSN 0265-9247, E-ISSN 1521-1878, Vol. 45, no 9, article id 2300040Article in journal (Refereed)
    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.

  • 7.
    Månsson, Alf
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ušaj, Marko
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Moretto, Luisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Rassier, Dilson E.
    McGill Univ, Canada.
    Do Actomyosin Single-Molecule Mechanics Data Predict Mechanics of Contracting Muscle?2018In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 19, no 7, article id 1863Article, review/survey (Refereed)
    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.

  • 8.
    Rahman, Mohammad A.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ušaj, Marko
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Rassier, Dilson E.
    McGill Univ, Canada.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Blebbistatin Effects Expose Hidden Secrets in the Force-Generating Cycle of Actin and Myosin2018In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 115, no 2, p. 386-397Article in journal (Refereed)
    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.

  • 9.
    Rems, Lea
    et al.
    University of Ljubljana, Slovenia.
    Ušaj, Marko
    University of Ljubljana, Slovenia.
    Kandušer, Maša
    University of Ljubljana, Slovenia.
    Reberšek, Matej
    University of Ljubljana, Slovenia.
    Miklavčič, Damijan
    University of Ljubljana, Slovenia.
    Pucihar, Gorazd
    University of Ljubljana, Slovenia.
    Cell electrofusion using nanosecond electric pulses2013In: Scientific Reports, E-ISSN 2045-2322, Vol. 3, article id 3382Article in journal (Refereed)
    Abstract [en]

    Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.

  • 10.
    Salhotra, Aseem
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Lund University, Sweden.
    Rahman, Mohammad A.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ruijgrok, Paul, V
    Stanford Univ, USA.
    Meinecke, Christoph R.
    Tech Univ Chemnitz, Germany.
    Ušaj, Marko
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Lund University, Sweden.
    Zemsky, Sasha
    Stanford Univ, USA.
    Lindberg, Frida W.
    Lund University, Sweden.
    Surendiran, Pradheebha
    Lund University, Sweden.
    Lyttleton, Roman W.
    Lund University, Sweden.
    Linke, Heiner
    Lund University, Sweden.
    Korten, Till
    Tech Univ Dresden, Germany.
    Bryant, Zev
    Stanford Univ, USA.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Lund University, Sweden.
    Exploitation of Engineered Light-Switchable Myosin XI for Nanotechnological Applications2023In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 17, no 17, p. 17233-17244Article in journal (Refereed)
    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.

  • 11.
    Salhotra, Aseem
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Zhu, Jingyuan
    Lund University, Sweden.
    Surendiran, Pradheebha
    Lund University, Sweden.
    Meinecke, Christoph Robert
    Technische Universität Chemnitz, Germany;Fraunhofer Institute for Electronic Nanosystems (ENAS), Germany.
    Lyttleton, Roman
    Lund University, Sweden.
    Ušaj, Marko
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Lindberg, Frida
    Lund University, Sweden.
    Norrby, Marlene
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Linke, Heiner
    Lund University, Sweden.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Linnaeus University, Linnaeus Knowledge Environments, Advanced Materials.
    Prolonged function and optimization of actomyosin motility for up scaled network-based biocomputation2021In: New Journal of Physics, E-ISSN 1367-2630, Vol. 23, article id 085005Article in journal (Refereed)
    Abstract [en]

    Significant advancements have been made towards exploitation of naturally available molecular motors and their associated cytoskeletal filaments in nanotechnological applications. For instance, myosin motors and actin filaments from muscle have been used with the aims to establish new approaches in biosensing and network-based biocomputation. The basis for these developments is a version of the in vitro motility assay (IVMA) where surface-adsorbed myosin motors propel the actin filaments along suitably derivatized nano-scale channels on nanostructured chips. These chips are generally assembled into custom-made microfluidic flow cells. For effective applications, particularly in biocomputation, it is important to appreciably prolong function of the biological system. Here, we systematically investigated potentially critical factors necessary to achieve this, such as biocompatibility of different components of the flow cell, the degree of air exposure, assay solution composition and nanofabrication methods. After optimizing these factors we prolonged the function of actin and myosin in nanodevices for biocomputation from <20 min to >60 min. In addition, we demonstrated that further optimizations could increase motility run times to >20 h. Of great importance for the latter development was a switch of glucose oxidase in the chemical oxygen scavenger system (glucose oxidase–glucose–catalase) to pyranose oxidase, combined with the use of blocking actin (non-fluorescent filaments that block dead motors). To allow effective testing of these approaches we adapted commercially available microfluidic channel slides, for the first time demonstrating their usefulness in the IVMA. As part of our study, we also demonstrate that myosin motor fragments can be stored at −80 °C for more than 10 years before use for nanotechnological purposes. This extended shelf-life is important for the sustainability of network-based biocomputation.

  • 12.
    Shneyer, Boris I.
    et al.
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Henn, Arnon
    Technion, Israel.
    Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia2016In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 129, no 3, p. 543-556Article in journal (Refereed)
    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.

  • 13.
    Shneyer, Boris I.
    et al.
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Wiesel-Motiuk, Naama
    Technion, Israel.
    Regev, Ronit
    Technion, Israel.
    Henn, Arnon
    Technion, Israel.
    ROS induced distribution of mitochondria to filopodia by Myo19 depends on a class specific tryptophan in the motor domain2017In: Scientific Reports, E-ISSN 2045-2322, Vol. 7, no 1, article id 11577Article in journal (Refereed)
    Abstract [en]

    The role of the actin cytoskeleton in relation to mitochondria function and dynamics is only recently beginning to be recognized. Myo19 is an actin-based motor that is bound to the outer mitochondrial membrane and promotes the localization of mitochondria to filopodia in response to glucose starvation. However, how glucose starvation induces mitochondria localization to filopodia, what are the dynamics of this process and which enzymatic adaptation allows the translocation of mitochondria to filopodia are not known. Here we show that reactive oxygen species (ROS) mimic and mediate the glucose starvation induced phenotype. In addition, time-lapse fluorescent microscopy reveals that ROS-induced Myo19 motility is a highly dynamic process which is coupled to filopodia elongation and retraction. Interestingly, Myo19 motility is inhibited by back-to-consensus-mutation of a unique residue of class XIX myosins in the motor domain. Kinetic analysis of the purified mutant Myo19 motor domain reveals that the duty ratio (time spent strongly bound to actin) is highly compromised in comparison to that of the WT motor domain, indicating that Myo19 unique motor properties are necessary to propel mitochondria to filopodia tips. In summary, our study demonstrates the contribution of actin-based motility to the mitochondrial localization to filopodia by specific cellular cues.

  • 14.
    Shneyer, Boris
    et al.
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Henn, Arnon
    Technion, Israel.
    Myosin 19 is Anchored to the Mitochondria, Affecting its Localization and Morphology2015In: Biophysical Journal supplement 1, 2015, Vol. 108, p. 303a-, article id 1517-PosConference paper (Refereed)
    Abstract [en]

    Mitochondria undergo continuous cycles of fission and fusion creating a highly dynamic network, which is essential for its proper functions in apoptosis, ATP generation and calcium homeostasis. Mitochondria long-range motility relies on the microtubule motors kinesin and dynein. Recently, actin and myosin 19 have been implicated in mitochondrial motility in vertebrates. However, the interaction of endogenous myosin 19 with the mitochondria remains unknown. Here, we show using multiple complementary approaches that endogenous myosin 19 is anchored directly to the outer mitochondrial membrane (OMM) in a monotopic fashion. We have identified a region of 30 residues at the tail domain of myosin 19, which is both essential and sufficient for myosin 19-OMM interaction. Furthermore, we have purified to near homogeneity a 45 long peptide comprised of this region to study its biochemical and biophysical properties. We performed in-vitro binding assay by fluorescence anisotropy of this specific purified peptide to vesicles with different phospholipid compositions. Our results revealed that that this peptide binds to vesicles mimicking the OMM with the highest affinity. To relate this tight binding to the mitochondria to myosin 19 ATPase activity, we have purified myosin 19-3IQ construct and measured its actin-dependent steady state ATPase activity. Interestingly, we found that it is completely inhibited by very low calcium concentration, suggesting that myosin 19 activity may be regulated by local calcium concentration. The interaction between a motor protein and an organelle, and the calcium dependence implicates that myosin 19 plays a role in mitochondria network dynamics.

  • 15.
    Ušaj, Marko
    et al.
    University of Ljubljana, Slovenia.
    Flisar, Karel
    University of Ljubljana, Slovenia.
    Miklavcic, Damijan
    University of Ljubljana, Slovenia.
    Kanduser, Masa
    University of Ljubljana, Slovenia.
    Electrofusion of B16-F1 and CHO cells: the comparison of the pulse first and contact first protocols2013In: Bioelectrochemistry, ISSN 1567-5394, E-ISSN 1878-562X, Vol. 89, p. 34-41Article in journal (Refereed)
    Abstract [en]

    High voltage electric pulses induce permeabilisation (i.e. electroporation) of cell membranes. Electric pulses also induce fusion of cells which are in contact. Contacts between cells can be established before electroporation, in so-called contact first or after electroporation in pulse first protocol. The lowest fusion yield was obtained by pulse first protocol (0.8%±0.3%) and it was only detected by phase contrast microscopy. Higher fusion yield detected by fluorescence microscopy was obtained by contact first protocol. The highest fusion yield (15%) was obtained by modified adherence method whereas fusion yield obtained by dielectrophoresis was lower (4%). The results are in agreement with current understanding of electrofusion process and with existing electrochemical models. Our data indicate that probability of stalk formation leading to fusion pores and cytoplasmic mixing is higher in contact first protocol where cells in contact are exposed to electric pulses. Another contribution of present study is the comparison of two detection methods. Although fusion yield can be more precisely determined with fluorescence microscopy we should note that by using this detection method single coloured fused cells cannot be detected. Therefore low fusion yields are more reliably detected by phase contrast microscopy.

  • 16.
    Ušaj, Marko
    et al.
    Technion, Israel.
    Henn, Arnon
    Technion, Israel.
    Kinetic adaptation of human Myo19 for active mitochondrial transport to growing filopodia tips2017In: Scientific Reports, E-ISSN 2045-2322, Vol. 7, no 1, article id 11596Article in journal (Refereed)
    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.

  • 17.
    Ušaj, Marko
    et al.
    University of Ljubljana, Slovenia.
    Kandušer, Maša
    University of Ljubljana, Slovenia.
    Modified Adherence Method (MAM) for Electrofusion of Anchorage-Dependent Cells2015In: 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.

  • 18.
    Ušaj, Marko
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Moretto, Luisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Linnaeus University, Linnaeus Knowledge Environments, Advanced Materials.
    Critical Evaluation of Current Hypotheses for the Pathogenesis of Hypertrophic Cardiomyopathy2022In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 4, article id 2195Article in journal (Refereed)
    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.

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  • 19.
    Ušaj, Marko
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Moretto, Luisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Vemula, Venukumar
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Salhotra, Aseem
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Linnaeus University, Linnaeus Knowledge Environments, Advanced Materials.
    Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms2021In: Communications Biology, E-ISSN 2399-3642, Vol. 4, no 1, p. 1-12, article id 64Article in journal (Refereed)
    Abstract [en]

    Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2-0.5 s(-1)) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction.

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  • 20.
    Ušaj, Marko
    et al.
    Technion - Israel Institute of Technology, Israel.
    Zattelman, Lilach
    Technion - Israel Institute of Technology, Israel.
    Regev, Ronit
    Technion - Israel Institute of Technology, Israel.
    Shneyer, Boris I.
    Technion - Israel Institute of Technology, Israel.
    Wiesel-Motiuk, Naama
    Technion - Israel Institute of Technology, Israel.
    Henn, Arnon
    Technion - Israel Institute of Technology, Israel.
    Overexpression and purification of human myosins from transiently and stably transfected suspension adapted HEK293SF-3F6 cells2018In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 558, no 1, p. 19-27Article in journal (Refereed)
    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.

  • 21.
    Velayuthan, Lok Priya
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Moretto, Luisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Tågerud, Sven
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ušaj, Marko
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays2023In: Scientific Reports, E-ISSN 2045-2322, Vol. 13, no 1, article id 4101Article in journal (Refereed)
    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.

  • 22.
    Vemula, Venukumar
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Huber, Tamás
    University of Pécs, Hungary.
    Ušaj, Marko
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Bugyi, Beáta
    University of Pécs, Hungary.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Linnaeus University, Linnaeus Knowledge Environments, Advanced Materials.
    Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity2021In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 296, article id 100181Article in journal (Refereed)
    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.

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  • 23.
    Zattelman, Lilach
    et al.
    Technion, Israel.
    Regev, Ronit
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Reinke, Patrick Y. A.
    Institute for Biophysical Chemistry, Germany.
    Giese, Sven
    Institute for Biophysical Chemistry, Germany.
    Samson, Abraham O.
    Bar-Ilan University, Israel.
    Taft, Manuel H.
    Hannover Medical School, Germany.
    Manstein, Dietmar J.
    Hannover Medical School, Germany.
    Henn, Arnon
    Technion, Israel.
    N-terminal splicing extensions of the human MYO1C gene fine-tune the kinetics of the three full-length myosin IC isoforms2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 43, p. 17804-17818Article in journal (Refereed)
    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.

  • 24.
    Zoabi, Muhammad
    et al.
    Technion, Israel.
    Nadar-Ponniah, Prathamesh T.
    Technion, Israel.
    Khoury-Haddad, Hanan
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Budowski-Tal, Inbal
    Technion, Israel.
    Haran, Tali
    Technion, Israel.
    Henn, Arnon
    Technion, Israel.
    Mandel-Gutfreund, Yael
    Technion, Israel.
    Ayoub, Nabieh
    Technion, Israel.
    RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 21, p. 13026-13038Article in journal (Refereed)
    Abstract [en]

    The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.

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