Single-molecule investigation of the interference between kinesin, tau and MAP2c
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Kinesin
Molecular motor
Processivity
Microtubule-associated protein
Organelle
This work focusses on characterizing the effect of SYD-2/kinesin interaction on the motility and mechanochemical properties of UNC-104 motor. The motility properties of molecular motors can be best studied in vitro by using recombinant motors fused to fluorescent proteins. We used fluorescent tagged motor fragment to test the effect of SYD-2 on UNC-104. Microtubule gliding assay and single molecule analysis by TIRF microscopy are well established techniques to study the motile properties of motors like velocity and processivity. In this study we aim to gain a more clear understanding of the importance of SYD-2 and UNC-104 interaction.
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Kinesin
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Significance The kinesin-3 family is one of the largest among the kinesin superfamily and its members play important roles in a variety of cellular functions ranging from intracellular transport to mitosis. Defects in kinesin-3 transport have been implicated in a variety of neurodegenerative, developmental, and cancer diseases, yet the molecular mechanisms of kinesin-3 regulation and cargo transport are largely unknown. We show that kinesin-3 motors undergo cargo-mediated dimerization to transport cellular cargoes. We also show that dimerization results in kinesin-3 motors that are fast and superprocessive. Such high processivity has not been observed for any other motor protein and suggests that kinesin-3 motors are evolutionarily adapted to serve as the marathon runners of the cellular world.
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Coiled coil
Molecular motor
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Kinesin
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Molecular motor
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Kinesin
Molecular motor
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Microtubule-associated protein
Organelle
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Molecular motor
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There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and wide-field illumination microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.
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Fluorescence-lifetime imaging microscopy
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Evanescent light illumination was introduced into a multi-mode microscope to construct a new type of total internal reflection fluorescence microscope (TIRFM). This microscope, capable of TIRFM, high resolution video-enhanced differential interference contrast (DIC), epifluorescence, interference relfection (IR) imaging, was combined with an image acquisition system for time-lapse microscopy. For the understanding the integrin dynamics, human umbilical vein endothelial cells (HUVECs) were stained with FITC labeled anti-CD29 (beta 1 subunit of integrin) and plated on a glass coverslip coated with fibronectin to visualize clustering processes of integrins in the living HUVECs. Dynamics of integrins in HUVECs were observed by total internal reflection fluorescence microscopy (TIRFM).
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Dynactin
Kinesin
Molecular motor
Coiled coil
Magnetic tweezers
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There are several methods for visualizing purified biomolecules near surfaces.Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules.Also, photobleaching and photodamage are concerns.In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM).This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro.In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images.IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy.It is also less susceptible to surface defects and solution impurities than darkfield microscopy.Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and wide-field illumination microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz. Video LinkThe video component of this article can be found at https://www.jove.com/video/59520/ 1. Insert a 50/50 mirror into the filter wheel of the fluorescent microscope using an appropriate filter cube (Figure 1).Handle the 50/50 mirror with care as often they have anti-reflection coating.NOTE: We used a 50/50 mirror in an empty filter cube of the microscope.The 50/50 mirror is inserted where the dichroic mirror is located.2. Use a high magnification/high NA oil objective.
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