Motor Property of Mammalian Myosin 10: A Dissertation

Myosin 10 is a vertebrate specific actin-based motor protein that is expressed in a variety of cell types. Cell biological evidences suggest that myosin 10 plays a role in cargo transport and filopodia extension. In order to fully appreciate these physiological processes, it is crucial to understand the motor property of myosin 10. However, little is known about its mechanoenzymatic characteristics. In vitro biochemical characterization of myosin 10 has been hindered by the low expression level of the protein in most tissues. In this study, we succeeded in obtaining sufficient amount of recombinant mammalian myosin 10 using the baculovirus expression system. The movement directionality of the heterologously expressed myosin 10 was determined to be plus end-directed by the in vitro motility assay with polarity-marked actin filament we developed. The result is consistent with the proposed physiological function of myosin 10 as a plus end-directed transporter inside filopodia. The duty ratio of myosin 10 was determined to be 0.6~0.7 by the enzyme kinetic analysis, suggesting that myosin 10 is a processive motor. Unexpectedly, we were unable to confirm the processive movement of dimeric myosin 10 along actin filaments in a single molecule study. The result does not support the proposed function of myosin 10 as a transporter. One possible explanation for this discrepancy is that the apparent nonprocessive nature of myosin 10 is important for generating sufficient force required for the intrafilopodial transport by working in concert with numbers of other myosin 10 molecules while not interfering with each other. Altogether, the present study provided qualitative and quantitative biochemical evidences for the better understanding of the motor property of myosin 10 and of the biological processes in which it is involved. Finally, a general molecular mechanism of myosin motors behind the movement directionality and the processivity is discussed based on our results together with the currently available experimental evidences. The validity of the widely accepted ‘leverarm hypothesis’ is reexamined

The Origin of Minus-end Directionality and Mechanochemistry of Ncd Motors

Adaptation of molecular structure to the ligand chemistry and interaction with the cytoskeletal filament are key to understanding the mechanochemistry of molecular motors. Despite the striking structural similarity with kinesin-1, which moves towards plus-end, Ncd motors exhibit minus-end directionality on microtubules (MTs). Here, by employing a structure-based model of protein folding, we show that a simple repositioning of the neck-helix makes the dynamics of Ncd non-processive and minus-end directed as opposed to kinesin-1. Our computational model shows that Ncd in solution can have both symmetric and asymmetric conformations with disparate ADP binding affinity, also revealing that there is a strong correlation between distortion of motor head and decrease in ADP binding affinity in the asymmetric state. The nucleotide (NT) free-ADP (?-ADP) state bound to MTs favors the symmetric conformation whose coiled-coil stalk points to the plus-end. Upon ATP binding, an enhanced flexibility near the head-neck junction region, which we have identified as the important structural element for directional motility, leads to reorienting the coiled-coil stalk towards the minus-end by stabilizing the asymmetric conformation. The minus-end directionality of the Ncd motor is a remarkable example that demonstrates how motor proteins in the kinesin superfamily diversify their functions by simply rearranging the structural elements peripheral to the catalytic motor head domain

Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

Cytoskeleton and Cell Motility

The present article is an invited contribution to the Encyclopedia of Complexity and System Science, Robert A. Meyers Ed., Springer New York (2009). It is a review of the biophysical mechanisms that underly cell motility. It mainly focuses on the eukaryotic cytoskeleton and cell-motility mechanisms. Bacterial motility as well as the composition of the prokaryotic cytoskeleton is only briefly mentioned. The article is organized as follows. In Section III, I first present an overview of the diversity of cellular motility mechanisms, which might at first glance be categorized into two different types of behaviors, namely "swimming" and "crawling". Intracellular transport, mitosis - or cell division - as well as other extensions of cell motility that rely on the same essential machinery are briefly sketched. In Section IV, I introduce the molecular machinery that underlies cell motility - the cytoskeleton - as well as its interactions with the external environment of the cell and its main regulatory pathways. Sections IV D to IV F are more detailed in their biochemical presentations; readers primarily interested in the theoretical modeling of cell motility might want to skip these sections in a first reading. I then describe the motility mechanisms that rely essentially on polymerization-depolymerization dynamics of cytoskeleton filaments in Section V, and the ones that rely essentially on the activity of motor proteins in Section VI. Finally, Section VII is devoted to the description of the integrated approaches that have been developed recently to try to understand the cooperative phenomena that underly self-organization of the cell cytoskeleton as a whole.Comment: 31 pages, 16 figures, 295 reference

Anaphase B.

Anaphase B spindle elongation is characterized by the sliding apart of overlapping antiparallel interpolar (ip) microtubules (MTs) as the two opposite spindle poles separate, pulling along disjoined sister chromatids, thereby contributing to chromosome segregation and the propagation of all cellular life. The major biochemical "modules" that cooperate to mediate pole-pole separation include: (i) midzone pushing or (ii) braking by MT crosslinkers, such as kinesin-5 motors, which facilitate or restrict the outward sliding of antiparallel interpolar MTs (ipMTs); (iii) cortical pulling by disassembling astral MTs (aMTs) and/or dynein motors that pull aMTs outwards; (iv) ipMT plus end dynamics, notably net polymerization; and (v) ipMT minus end depolymerization manifest as poleward flux. The differential combination of these modules in different cell types produces diversity in the anaphase B mechanism. Combinations of antagonist modules can create a force balance that maintains the dynamic pre-anaphase B spindle at constant length. Tipping such a force balance at anaphase B onset can initiate and control the rate of spindle elongation. The activities of the basic motor filament components of the anaphase B machinery are controlled by a network of non-motor MT-associated proteins (MAPs), for example the key MT cross-linker, Ase1p/PRC1, and various cell-cycle kinases, phosphatases, and proteases. This review focuses on the molecular mechanisms of anaphase B spindle elongation in eukaryotic cells and briefly mentions bacterial DNA segregation systems that operate by spindle elongation

Myosin IX: A Single-Headed Processive Motor

The class IX myosin is a member of the myosin superfamily and found in variety of tissues. Myosin IX is quite unique among the myosin superfamily in that the tail region contains a GTPase activating protein (GAP) domain for the small GTP-binding protein, Rho. Recently it was reported that myosin IX shows processive movement that travels on an actin filament for a long distance. This was an intriguing discovery, because myosin IX is a “single-headed†myosin unlike other processive myosins which have “double-headed†structure. It has been thought that “processive†motors walk on their track with their two heads, thus traveling for a long distance. Therefore, it is reasonable to expect that the processive movement of single headed myosin IX is based on the unique feature of myosin IX motor function. In this study, I investigated the mechanism of processive movement of single-headed myosins by analyzing the mechanism of ATPase cycle of myosin IX that is closely correlated with the cross-bridge cycle (the mechanical cycle of actomyosin). In the first part, I performed the transient enzyme kinetic analysis of myosin IX using the motor domain construct to avoid the complexity raised by the presence of the tail domain. It was revealed that the kinetical characteristics of myosin IX ATPase is quite different from other processive myosins. It was particularly notable that the affinity of the weak actin binding state of Myosin IX was extremely high comparing with known myosins. It is thought that the high affinity for actin throughout the ATPase cycle is a major component to explain the processive movement of myosin IX. In the second part of this study, I cloned full length human myosin IX construct to further investigate the regulation of motor activity of myosin IX. It was revealed that the basal ATPase activity but not the actin dependent ATPase activity of myosin IX is inhibited by its tail region. Furthermore full-length myosin IX is regulated by calcium, presumably due to the calcium binding to the CaM light chain. These result suggest that the tail domain serves as a regulatory component of myosin IX

Kinesin Is an Evolutionarily Fine-Tuned Molecular Ratchet-and-Pawl Device of Decisively Locked Direction

Conventional kinesin is a dimeric motor protein that transports membranous organelles toward the plus-end of microtubules (MTs). Individual kinesin dimers show steadfast directionality and hundreds of consecutive steps, yetthe detailed physical mechanism remains unclear. Here we compute free energies for the entire dimer-MT system for all possible interacting configurations by taking full account of molecular details. Employing merely first principles and several measured binding and barrier energies, the system-level analysis reveals insurmountable energy gaps between configurations, asymmetric ground state caused by mechanically lifted configurational degeneracy, and forbidden transitions ensuring coordination between both motor domains for alternating catalysis. This wealth of physical effects converts a kinesin dimer into a molecular ratchet-and-pawl device, which determinedly locks the dimer's movement into the MT plus-end and ensures consecutive steps in hand-over-hand gait.Under a certain range of extreme loads, however, the ratchet-and-pawl device becomes defective but not entirely abolished to allow consecutive back-steps. This study yielded quantitative evidence that kinesin's multiple molecular properties have been evolutionarily adapted to fine-tune the ratchet-and-pawl device so as to ensure the motor's distinguished performance.Comment: 10 printed page

Functional characterization of Leishmania donovani myosin-XXI in transfection and lipid binding studies

Myosins establish a superfamily of cytoskeleton-associated motor proteins, which are encoded in the genome of virtually all eukaryotic organisms. As molecular motors powered by the hydrolysis of ATP, myosins are capable of translocating along filamentous actin. The transduction of chemical into mechanical energy enables myosins to fulfill a plethora of different functions, as for example muscle contraction, cell-cell adhesion, protein transport, or membrane trafficking. In this context, myosin-XXI plays an extraordinary role. The motor protein, which has recently been described in the literature, is the only myosin the expression of which could thus far be verified in the parasite Leishmania donovani. For the parasite, myosin-XXI is a vital motor protein mediating the important cellular processes of endocytosis, exocytosis, vesicular trafficking, and flagellum formation. In the present work, the molecular bases underlying the function of this essential multifunctional motor protein were aimed to be further elucidated. For this purpose, the full-length myosin as well as individual myosin-XXI domains were characterized using in vivo and in vitro methods. In mammalian cell transfection experiments, functionality and cellular distribution of myosin-XXI were examined in a physiological environment devoid of endogenously expressed myosin-XXI. With the help of gliding filament assays and lipid bilayer binding experiments, directionality of myosin-XXI translocation along F-actin was determined and membrane attachment of the molecular motor was investigated. The transfection studies indicated that myosin-XXI is capable of performing processive movement in mammalian cells, and that the motor protein most probably promotes actin polymerization within the filopodia of transfected cells. These processes depended on the presence of the N-terminal portion of myosin-XXI (aa 1 to 800) comprising an SH3-like domain, the complete motor domain, and part of the adjacent neck and tail region. Gliding filament assays verified for the first time that myosin-XXI, like the vast majority of myosins known to date, is a plus end-directed molecular motor. The analysis of myosin-XXI´s association with membranes demonstrated that the motor protein´s attachment to lipid bilayers requires positive bilayer curvature. In addition, it was shown that myosin-XXI exhibits at least two separate curvature sensitive lipid binding sites. The results obtained in the present study give evidence that the N-terminal domains of myosin-XXI are of great importance for the motor protein´s functionality, though they are known not to be involved in the localization of myosin-XXI within Leishmania donovani. A potential function of the N-terminal parts of myosin-XXI might include the regulation of actin polymerization in membrane protrusions, a process that could possibly play a role in the myosin-XXI-mediated formation of the Leishmania donovani flagellum. From the results of the membrane binding studies, it can be concluded that membrane curvature has a regulatory effect on the spatial distribution of myosin-XXI. Accordingly, myosin-XXI is the first myosin described to bind to membranes depending on membrane geometry. The existence of several separate curvature sensitive lipid binding motifs within the motor protein might in fact guarantee maintenance of the myosin-XXI-membrane association during the conformational changes occurring when myosin-XXI passes through its catalytic cycle.Myosine bilden eine Superfamilie Cytoskelett-assoziierter Motorproteine, die im Genom nahezu aller eukaryotischen Organismen kodiert sind. Angetrieben durch die Hydrolyse von ATP können sich Myosine als molekulare Motoren entlang von filamentösem Aktin fortbewegen. Die Umwandlung chemischer in mechanische Energie ermöglicht es Myosinen, eine Vielzahl verschiedener Funktionen, wie zum Beispiel Muskelkontraktion, Zell-Zell-Adhäsion, Proteintransport oder membran-verlagernden Transport zu erfüllen. Myosin-XXI nimmt hierbei eine besondere Stellung ein. Dieses in der Literatur beschriebene Motorprotein ist das bislang einzige Myosin, dessen Expression in dem Parasiten Leishmania donovani nachgewiesen werden konnte. Für den Parasiten stellt Myosin-XXI ein lebensnotwendiges Motorprotein dar, welches die wichtigen zellulären Prozesse Endozytose, Exozytose, Vesikeltransport und Flagellenbildung vermittelt. Ziel der vorliegenden Arbeit war es, die molekularen Grundlagen der Funktion dieses essenziellen multifunktionalen Motorproteins weiter aufzuklären. Hierzu wurden sowohl das vollständige Myosin als auch einzelne seiner Domänen mit Hilfe von in-vivo- und in-vitro-Methoden charakterisiert. In Transfektionsexperimenten an Säugetierzellen wurden die Funktionalität und die zelluläre Verteilung von Myosin-XXI in einer physiologischen Umgebung untersucht, in der kein endogen exprimiertes Myosin-XXI vorhanden war. Mittels Gleitfilamentversuchen und Experimenten zur Bindung an Lipiddoppelschichten wurde die Richtung der Myosinbewegung entlang von F-Aktin bestimmt sowie das Membranbindeverhalten des molekularen Motors genauer erforscht. Die Transfektionsversuche zeigten, dass Myosin-XXI in Säugetierzellen in der Lage ist, sich prozessiv zu bewegen und dass es sehr wahrscheinlich die Aktinpolymerisation in den Filopodien der transfizierten Zellen verstärkt. Für diese Prozesse ist der N-terminale Abschnitt des Myosin-XXI-Moleküls (Aminosäuren 1 bis 800) erforderlich, welcher eine SH3-ähnliche Domäne, die vollständige Motordomäne und einen Teil der anschließenden Hals- und Schwanz-Region umfasst. In Gleitfilamentversuchen wurde erstmalig nachgewiesen, dass sich Myosin-XXI wie die überwiegende Mehrheit der bisher bekannten Myosine in Richtung des Plus-Endes von F-Aktin bewegt. Die Untersuchungen zum Membranbindeverhalten von Myosin-XXI verdeutlichten, dass die Anlagerung des Motorproteins an Lipiddoppelschichten eine positive Oberflächenkrümmung der Doppelschichten voraussetzt. Ferner konnte gezeigt werden, dass das Motorprotein mindestens zwei eigenständige krümmungssensitive Lipidbindungsstellen aufweist. Die Ergebnisse der vorliegenden Arbeit belegen, dass die N-terminalen Domänen von Myosin-XXI von hoher Wichtigkeit für die Funktionalität des Motorproteins sind, auch wenn sie bekanntermaßen bei der Lokalisation von Myosin-XXI in Leishmania donovani keine Rolle spielen. Eine mögliche Funktion des N-terminalen Abschnitts von Myosin-XXI könnte die Regulation der Aktinpolymerisation in Membranausstülpungen sein, ein Prozess, der möglicherweise bei der Myosin-XXI-vermittelten Bildung des Leishmania-donovani-Flagellums von Bedeutung ist. Aus den Ergebnissen der Membranbindungsstudien lässt sich schlussfolgern, dass die Oberflächenkrümmung von Membranen regulatorisch auf die räumliche Verteilung von Myosin-XXI wirkt. Hierbei ist Myosin-XXI das erste Myosin, für das eine Abhängigkeit der Membranbindung von der Membrangeometrie beschrieben wird. Das Vorhandensein mehrerer eigenständiger krümmungssensitiver Lipidbindemotive innerhalb des Motorproteins könnte dabei gewährleisten, dass die Membranbindung von Myosin-XXI aufrechterhalten werden kann, während das Motorprotein seinen katalytischen Zyklus und die damit einhergehenden Konformationsänderungen durchläuft