Chapter 16 - Fast axonal transport in isolated axoplasm from the squid giant axon
Introduction
The number of studies based on live cell imaging has exploded in the last decade as new methodologies were developed for tagging proteins, and high-resolution fluorescence imaging systems became widely available. However, these approaches are not always the only choice for addressing questions associated with intracellular trafficking of cellular proteins and structures.
If the primary question under review is about the properties of the motors moving proteins, then video-enhanced contrast–differential interference contrast (VEC-DIC) microscopy, as introduced by Robert Allen and his colleagues (Allen, 1985, Allen and Allen, 1983, Allen et al., 1981), remains a unique tool for real-time analysis of vesicle trafficking. VEC-DIC is particularly powerful when used in combination with isolated axoplasm from the squid giant axon (Brady et al., 1982, Brady et al., 1985), where limitations in spatial resolution due to diffraction at cell edges are minimized. Suitable squid (Loligo pealeii) are available routinely at the Marine Biological Laboratory in Woods Hole, MA, USA, from April to November (Figure 1). VEC-DIC allows visualization of intracellular organelles moving at rates of up to 2–4 μm/s, which requires acquisition at rates of 30 frames per second (fps) or more. Moreover, it avoids the addition of tags to proteins of interest, which may potentially alter their processing and/or compartmentalization. This is particularly worrisome when such proteins are overexpressed, or whenever those proteins are subunits of larger protein complexes, as the stoichiometry of components is critical for appropriate assembly.
In this chapter, we will first briefly describe the equipment and processing needed for VEC-DIC and then describe the isolation and preparation of the axoplasm. The consideration of technology will serve to illuminate several challenges presented by the need to image trafficking of organelles in cells, particularly neurons. Similarly, our discussion of the axoplasm will provide some insight into why the axon represents a specialized case of intracellular trafficking.
Section snippets
Microscopy and Imaging
The foundation of this method remains real-time (30 fps) VEC-DIC microscopy. Objects below the diffraction limit of resolution (roughly 170 nm for green light) can be detected in live imaging. Objects that are readily imaged under the proper conditions include small vesicles in the 50–100 nm range and individual microtubules. The basis of this approach is conceptually simple: structures smaller than the diffraction limit still interact with photons but project an image that is comparable to the
Preparation of Isolated Axoplasm
Isolated axoplasm is a unique preparation that models in vivo conditions with considerable fidelity, preserving many aspects of the intracellular environment while allowing free access to the intracellular compartment. Perfusions may be limited to very small volumes in the absence of permeability barriers (20–25 μL for a 5 μL axoplasm is typical). Moreover, the concentration of perfusates can be controlled with great precision. Normally, applying proteins and drugs intracellularly faces several
Summary
Although the isolated axoplasm model is not widely available, it has a number of unique characteristics that make it an invaluable model for studying cell biology and molecular biochemistry of the axon (see Chapter Biochemical Analysis of Axon-Specific Phosphorylation Events Using Isolated Squid Axoplasms). The squid giant axon is the only reliable source of pure axoplasm in its native state, allowing unparalleled access to a pure cytoplasmic fraction that is undiluted and retains its
Acknowledgments
The authors would like to express their gratitude to the many students who have spent summers at the Marine Biological Laboratory measuring fast axonal transport in axoplasm for their invaluable work at the MBL. Many of these students have been come from Hunter College, NY, through the HHMI summer research program. This work was mainly supported by grants from the National Institute of Health [NS066942A (to GM), and NS23868, NS23320, NS41170 (to STB)] and from HHMI and the Grass Foundation (YS).
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2016, Experimental NeurologyCitation Excerpt :Motility was analyzed using a Zeiss Axiomat microscope equipped with a 100 × (1.3 numerical aperture) objective and differential interference contrast optics. Images were obtained using a Hammamatsu C2400 CCD with an Argus 20 for image adjustment and the video was further processed using a Hamamatsu Photonics Microscopy C2117 video manipulator for generation of calibrated cursors and scale bars (Song et al., 2016). Anterograde and retrograde FAT were measured by matching calibrated cursor movements to the speed of vesicles moving in the axoplasm preparation.
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2016, Brain Research BulletinCitation Excerpt :Further, cumulative data indicates that phosphorylation represents a major mechanism for the regulation of conventional kinesin-based AT (Gibbs et al., 2015; Hollenbeck, 1993; Lee and Hollenbeck, 1995). Many findings supporting this concept were initially established using isolated squid axoplasm, a model that greatly facilitates the evaluation of axon-autonomous molecular events (Kang et al., 2016; Song et al., 2016). Consistent with the heterogeneity of axonal MBOs and the need for delivering those to specific destinations, multiple kinases were found to regulate both enzymatic (i.e. binding to microtubules and ATPase activity) and non-enzymatic (i.e. attachment to transported cargoes) properties of conventional kinesin.