Vesicle Transport Assay

Abstract

Eukaryotic cells contain an extensive cytoplasmic network of actin filaments and microtubules that function as tracks for vesicle transport. The ability to visualise vesicle movement along both sets of filaments advanced rapidly when in vitro vesicle transport (motility) assays were developed. These assays utilise super‐resolution, contrast‐enhanced DIC microscopy to detect structures below the resolution of the light microscope including vesicles as small as 100 nm and microtubules 25 nm in diameter. Images are captured in real time and stored digitally for subsequent analysis. The standard motion analysis parameters include vesicle type, velocity, distances and path length. In vitro motility assays enabled the identification of important factors that are essential for the regulation of intracellular transport including motor proteins and their membrane receptors, the formation of hetero‐motor complexes and transported cargoes.

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 ATP for muscle contraction, cell motility, cell division and transport of different cargoes along the cytoskeleton.
  • The superfamily of myosin motor proteins found in eukaryotic cells is known to contain over 20 different classes and of these, 12 classes are found in vertebrates, including humans.
  • The kinesin superfamily is subdivided into 15 classes and the dynein family is grouped into axonemal and cytoplasmic dyneins.
  • Motor protein defects are associated with diseases including Griscelli syndrome, Usher syndrome, myopathies, deafness, tumour progression and metastasis.
  • Differential interference microscopy, also known as Nomarski microscopy, uses polarised light to enhance gradients in the optical path length and phase shifts in unstained, transparent 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: multimode bright‐field microscopy; vesicle transport; motility assay; actin; myosin; microtubules

Figure 1. Schematic diagram of a multimode high‐resolution light microscope for epifluorescence and DIC 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 (X‐Cite120 light source) imaging. A high‐resolution digital CCD or CMOS camera with a framing rate of 30 frames s−1 (e.g. Hamamatsu Orca 4.0 Flash LT) is attached for real‐time or time‐lapse imaging. A desktop PC with image processing software (e.g. MetaMorph microscope imaging and image analysis software) is used for image acquisition and enhancement (e.g. time‐lapse settings, contrast enhancement and image arithmetic) of the specimen image. The differential interference contrast (DIC) optics includes polariser, beam splitter (Wollaston prism) and condenser (1.4NA) that are located below the microscope stage with specimen holder and beam combiner (Wollaston prism) and analyser that are located above the 1.4NA objectives.
Figure 2. A comparison of images acquired by analogue video (a–c) or digital CCD (d–f) camera. The contrast of the video images (a and d) is improved by a 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 µm.
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 contrast‐enhanced DIC microscopy but the actin filaments are invisible. Actin filaments are not visible by contrast‐enhanced 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 visualised by fluorescence microscopy after staining with 0.5 μM 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: 5μm.
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 visualised by fluorescence microscopy after staining with 0.5 μM rhodamine/phalloidin. The actin bundles were detectable by contrast‐enhanced 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 contrast‐enhanced 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 and 2 µm.
Figure 5. The dynamics of the actin cytoskeleton was observed in vivo in human skin cells (OKF6/TERT‐2). OKF6/TERT‐2 cells were transiently transfected with the pAcGFP1‐actin vector, and the actin cytoskeleton was visualised by total internal reflection fluorescence (TIRF) microscopy. Sequences of TIRF images obtained from a time‐lapse movie of OKF6/TERT‐2 cells monitored for a period of 300 s. Micrographs (0–300s) of the actin‐containing structures and actin dynamics at the cellular edge within the boxed regions of the micrograph. Asterisks indicate regions of filopodia that undergo protrusion and retraction cycles. Scale bars: 10 and 5 µm.
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Wöllert, Torsten, and Langford, George M(Apr 2015) Vesicle Transport Assay. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002611.pub3]