Vesicle Transport Assay

Torsten Wöllert, Syracuse University, Syracuse, New York, USA
George M Langford, Syracuse University, Syracuse, New York, USA

Published online: December 2009

DOI: 10.1002/9780470015902.a0002611.pub2

Eukaryotic cells contain actin filaments and microtubules that function as cytoplasmic filaments for vesicle transport. The study of vesicle transport on these filaments in vitro advanced rapidly when vesicle transport assays were developed. High-resolution multimode video-enhanced microscopy is the method used for such assays to detect vesicles as small as 100 nm in diameter and microtubules 25 nm in diameter. Vesicle transport is recorded in real time on high-capacity digital storage media. Motion analysis is performed to quantify different parameters of vesicle movement along cytoplasmic filaments including velocity, distances travelled and straightness of path. In vitro vesicle transport assays proved highly successful and enabled the identification of important factors that are essential for regulation of intracellular transport.

Key concepts

  • The cytoskeleton contains actin filaments and microtubules, both of which serve as tracks for vesicle transport.
  • Vesicle transport is the directed movement of membrane vesicles on filaments of the cytoskeleton.
  • Microtubules function as intracellular tracks for transport of vesicles by two classes of motor proteins, kinesins and dyneins.
  • Actin filaments serve as tracks for vesicle transport by myosin motors.
  • Motor proteins are mechanochemical nanomachines that use the energy of adenosine triphosphate (ATP) for muscle contraction, cell motility and transport of cargo along the cytoskeleton.
  • The superfamily of myosin motor proteins found in eukaryotic cells is known to contain over 20 different classes.
  • The kinesin superfamily is subdivided into 14 classes and the family of dyneins is grouped into axonemal and cytoplasmic dyneins.
  • Motor protein defects are associated with diseases including Griscelli syndrome, Usher syndrome, myopathies and deafness.
  • Differential interference microscopy, also known as Nomarski microscopy, uses polarized light to produce contrast in unstained biological specimens.
  • Cell-free extracts are important tools for cell biologists and have a variety of applications including cell cycle studies, intracellular transport mechanisms, signal transduction events and maintenance of cell architecture.

Keywords: video microscopy; vesicle transport; motility assays; actin; myosin; microtubules

Figure 1. Schematic diagram of a multimode high-resolution light microscope for epifluorescence and video-enhanced contrast microscopy. The microscope is placed on a vibration isolation table and is equipped with light sources for bright-field (100 W halogen lamp) and epifluorescence (100 W HBO mercury lamp or 120 W mercury vapour short arc) imaging. A high-resolution video camera with a framing rate of 30 frames/s (e.g. Hamamatsu Newvicon tube camera) and low-light cooled CCD camera (e.g. Hamamatsu ORCAII) are attached for real-time imaging. A digital image processor (e.g. Hamamatsu Argus-20 real-time image processor) is used for contrast enhancement (e.g. frame averaging, background subtraction and contrast enhancement) of the specimen image. The differential interference contrast (DIC) optics includes polarizer, beam splitter (Wollaston prism), condenser (1.4NA), beam combiner (Wollaston prism) and analyser that are located above the 1.4NA objectives.
Figure 2. A comparison of images acquired by the analogue video – (a), (b), (c) – and the digital – (d), (e), (f) – cameras. The contrast of the video images (a) and (d) is improved by subtraction of an out-of-focus background image to remove electronic noise and background. The contrast in the final images (c) and (f) is digitally adjusted with an image processor operating at video rates by setting the high and low grey values to black and white (0 and 256 in an 8-bit digital image), respectively and expanding the range of levels in the image to 256. Scale bar, 5 m.
Figure 3. Actin-based in vitro vesicle transport assay reconstituted in the axoplasm of the squid giant axon. (a) Four sequential frames from a video clip (9 s total) show vesicles moving on actin filaments. The vesicles are detected by VEC-DIC microscopy but the actin filaments are invisible. Actin filaments are not visible by VEC-DIC microscopy. The numbers indicate quantity and position of moving vesicles in each micrograph whereas the asterisk marks a microtubule. Time of image acquisition is given in seconds at the higher right corner. (a¢) Diagram of the tracks of the five vesicles (numbers 1–5) shown in (a). (b) Corresponding actin filament network visualized by fluorescence microscopy after staining with 0.5 M rhodamine/phalloidin. (b¢) Overlay of actin filaments (b) and tracks shown in (a¢). The MetaMorph software was used to create the track diagram. Scale bar, 5 m.
Figure 4. Reconstituted in vitro vesicle transport on actin filaments in extracts of clam oocytes. (a) and (a¢) Networks of actin filaments and actin bundles assembled spontaneously in these extracts and were visualized by fluorescence microscopy after staining with 0.5 M rhodamine/phalloidin. The actin bundles were detectable by VEC-DIC microscopy. (b) and (b¢) Networks of tubular vesicles that are highly reminiscent of endoplasmic reticulum formed in the presence of actin filaments and actin bundles. (b¢), (c), (c¢) and (c¢¢) Sequences of DIC images monitored by VEC-DIC microscopy for a period of 4 s and diagram of the tracks (c¢¢) of the two vesicles (numbers 1–2) moving on actin filaments (c¢¢). (c¢¢¢) Overlay of actin filament network (c¢¢) and tracks shown in (c¢). The MetaMorph software was used to create the track diagram. Scale bars 5 m and 2 m.
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    Langford GM (2001) Video-enhanced microscopy for analysis of cytoskeleton structure and function. Methods in Molecular Biology 161: 31–43.
    Paul AS and Pollard TD (2009) Review of the mechanism of processive actin filament elongation by formins. Cell Motility and the Cytoskeleton 66: 606–617.
    book Schliwa M (ed.) (2004) Molecular Motors. Hoboken, NJ: Wiley-VCH Verlag GMbH.
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    book Weiss DG, Maile W, Wick RA and Steffen W (1999) "Video microscopy". In: Lacey AJ (ed.) Electronic Light Microscopy in Biology – A Practical Approach, pp. 221–278. Oxford: IRL Press.

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Cellular Transport | Techniques in Cell Biology
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Article Title: Vesicle Transport Assay
 
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Wöllert, Torsten, and Langford, George M(Dec 2009) Vesicle Transport Assay. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002611.pub2]