Temperature Dependence of Force, Velocity, and Processivity of Single Kinesin Molecules

Kawaguchi, Kenji; Ishiwata, Shin’ichi

doi:10.1006/bbrc.2000.2856

Cited by 119 publications

(122 citation statements)

References 25 publications

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“…Third, endogenous vesicles in the shear zone move dorsally from the vegetal pole with a velocity and saltatory behavior suggestive of kinesin-mediated transport . Fourth, Dsh-GFP, GBP-GFP and XKLC4-GFP (Miller et al, 1999; this work) move with velocities that are in the range in which kinesin and kinesinlike proteins have been measured to move in vitro and in vivo (Howard et al, 1989;Kawaguchi and Ishiwata, 2000;Zhou et al, 2001;Yajima et al, 2002). Together, these observations support our evidence that a motor protein in the kinesin family is involved in translocating dorsal determinants along the subcortical microtubule array.…”

Section: Discussionsupporting

confidence: 81%

GBP binds kinesin light chain and translocates during cortical rotation inXenopuseggs

et al. 2003

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Section: Discussionsupporting

confidence: 81%

GBP binds kinesin light chain and translocates during cortical rotation inXenopuseggs

et al. 2003

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“…At stall (near equilibrium), the energy available to power an elementary step does not depend on temperature; however, when cells are moving, the stepping rate does depend on temperature. The same thing is true for ATP-driven motor proteins (16,17).…”

Section: Discussionmentioning

confidence: 77%

Force and Velocity of Mycoplasma mobile Gliding

2002

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The effects of temperature and force on the gliding speed of Mycoplasma mobile were examined. Gliding speed increased linearly as a function of temperature from 0.46 m/s at 11.5°C to 4.0 m/s at 36.5°C. A polystyrene bead was attached to the tail of M. mobile using a polyclonal antibody raised against whole M. mobile cells. Cells attached to beads glided at the same speed as cells without beads. When liquid flow was applied in a flow chamber, cells reoriented and moved upstream with reduced speeds. Forces generated by cells at various gliding speeds were calculated by multiplying their estimated frictional drag coefficients with their velocities relative to the liquid. The gliding speed decreased linearly with force. At zero speed, the force measurements extrapolated to 26 pN at 22.5 and 27.5°C. At zero force, the speed extrapolated to 2.3 and 3.3 m/s at 22.5 and 27.5°C, respectively-the same speeds as those observed for free gliding cells. Cells attached to beads were also trapped by an optical tweezer, and the stall force was measured to be 26 to 28 pN (17.5 to 27.5°C). The gliding speed depended on temperature, but the maximum force did not, suggesting that the mechanism is composed of at least two steps, one that generates force and another that allows displacement. Other implications of these results are discussed.Mycoplasmas are parasitic bacteria with a small genome and no peptidoglycan layer (27). Several mycoplasma species have a distinct cell polarity characterized by a protruding membrane extension, the attachment organelle (27). They are able to attach to and glide on glass, plastic, and eukaryotic cell surfaces, always moving in the direction of the organelle (19). The gliding mechanism is unknown. Mycoplasmas do not have any appendages such as flagella or pili (19) or any genes obviously related to motility, including motor proteins such as myosin or kinesin (7, 10, 13). However, a transmembrane protein associated with a cytoskeleton-like structure has been shown to be necessary for glass binding in Mycoplasma pneumoniae (22).Mycoplasma mobile, isolated from a fish gill organ, has a conical cell structure, with the attachment organelle at the apex of the cone. It glides up to four cell lengths per second (2.5 m/s) in the direction of the apex, which is designated as a head-like structure (30). However, little is known about force generation (28,29). In this study, to characterize its gliding motility, we attached a bead to the tail end of M. mobile and measured the gliding force as a function of speed using viscous flow and an optical tweezer. MATERIALS AND METHODSCultivation. M. mobile strain 163K (ATCC 43663) was grown as described previously (25).Measurements of gliding speed. M. mobile cells in a culture at an optical density at 600 nm of 0.03 to 0.1 were collected by centrifugation at 10,000 ϫ g for 4 min at room temperature and resuspended in a fivefold-smaller volume of medium. A 5-l aliquot was sealed between a coverslip and a slide within a thin ring of Apiezon M grease (Apiezon Products, L...

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“…Once we observed the movement of Qdot-labeled myoVa on microtubules, a series of experiments and controls were performed in which microtubules were adhered directly to the coverslip surface without any actin present. To observe this movement, microtubules were fixed to the glass surface as described (37). After a 2-min incubation, the flow-cell was washed with AB and then infused with AB with 1 mg/ml BSA.…”

Section: Materials and Methods Proteinmentioning

confidence: 99%

Myosin Va maneuvers through actin intersections and diffuses along microtubules

Ali

Krementsova²,

Kennedy³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

154

158

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Certain types of intracellular organelle transport to the cell periphery are thought to involve long-range movement on microtubules by kinesin with subsequent handoff to vertebrate myosin Va (myoVa) for local delivery on actin tracks. This process may involve direct interactions between these two processive motors. Here we demonstrate using single molecule in vitro techniques that myoVa is flexible enough to effectively maneuver its way through actin filament intersections and Arp2/3 branches. In addition, myoVa surprisingly undergoes a one-dimensional diffusive search along microtubules, which may allow it to scan efficiently for kinesin and/or its cargo. These features of myoVa may help ensure efficient cargo delivery from the cell center to the periphery.cytoskeleton ͉ molecular motor ͉ motility ͉ processivity C oordinated organelle transport along certain secretory pathways starts with kinesin-powered movement on microtubules and finishes with vertebrate myosin Va (myoVa)-based motility on actin (1-4). This trip requires that both motors be present when a microtubule-actin intersection is encountered, but how these two motors find one another or the cargo they share is still unknown. Another challenge is to understand how cargo is delivered to its destination by way of the complex cytoskeletal network. Vertebrate myoVa is a processive motor that can travel long distances along its actin track (5). However, the cytoskeletal intersections that myoVa must encounter along its journey may present a physical barrier to forward motion or an alternate path to its final destination.To investigate this issue from myoVa's point of view, we observed single myoVa molecules labeled with highly photostable quantum dots (Qdots) in an objective-type total internal reflectance microscope (6) as they maneuvered through a model of cytoskeletal intersections created on a microscope coverslip coated with either actin filaments (Fig. 1A) or actin filaments and microtubules (Fig. 1B). In this model system, actin-actin intersections could inhibit or alter myoVa's direction of travel while actin-microtubule intersections should act as a hurdle to myoVa's processive movement. Given myoVa's ability to take 72-nm steps as it walks processively along actin filaments in a handover-hand fashion (6-9) and its inherent flexibility (10, 11), we have observed that myoVa can easily maneuver past actin filament intersections. In contrast, when encountering a microtubule, myoVa cannot step over this physical barrier, but surprisingly can step onto the microtubule and begin a onedimensional diffusive search. Thus, myoVa has evolved to handle the challenges of the cytoskeletal network and through its interaction with the microtubule can effectively scan for its transport partner, kinesin, and/or its cargo. Results and Discussion MyoVa Dynamics at Actin Filament Intersections.To determine what happens when myoVa encounters an overlapping actin filament, we adhered Alexa Fluor 660 phalloidin-labeled actin filaments to the coverslip, followed by TRITC...

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Temperature Dependence of Force, Velocity, and Processivity of Single Kinesin Molecules

Cited by 119 publications

References 25 publications

GBP binds kinesin light chain and translocates during cortical rotation inXenopuseggs

GBP binds kinesin light chain and translocates during cortical rotation inXenopuseggs

Force and Velocity of Mycoplasma mobile Gliding

Myosin Va maneuvers through actin intersections and diffuses along microtubules

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