Skip to main content
SpringerLink
Log in
Book cover

The Cytoskeleton pp 19–39Cite as

Visualizing In Vitro Trafficking

  • Protocol
  • First Online:

  • 1136 Accesses

Part of the book series: Neuromethods ((NM,volume 79))

Abstract

Here we present a detailed guide for performing in vitro trafficking assays. These are high-resolution light microscopy assays designed to look at the cytoskeletal filament-based trafficking of cellular organelles. The strategy is to partially purify organelles from lysed mammalian cells and freeze them as single-use aliquots. The organelles are then thawed and allowed to bind microtubule and actin filaments that have been coated onto handmade optical microchambers. Time lapse multichannel fluorescence microscopy is then performed to identify specific vesicles and associated proteins and to observe and quantify how the material is transported. These protocols were initially developed to study rodent liver endosomes but are adapted here for the study of cultured cells and include commentary on their use with other types of organelles.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Vale RD, Reese TS, Sheetz MP (1985) Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42(1):39–50

    Article  PubMed  CAS  Google Scholar 

  2. Lye RJ, Porter ME, Scholey JM, McIntosh JR (1987) Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans. Cell 51(2):309–318. doi:0092-8674(87)90157-7[pii]

    Article  PubMed  CAS  Google Scholar 

  3. Waterman-Storer CM (2001) Microtubule/organelle motility assays. Curr Protoc Cell Biol 11 doi:10.1002/0471143030.cb1301s00 (Chapter 13:Unit 13)

    Google Scholar 

  4. Hendricks AG, Perlson E, Ross JL, Schroeder HW 3rd, Tokito M, Holzbaur EL (2010) Motor coordination via a tug-of-war mechanism drives bidirectional vesicle transport. Curr Biol 20(8):697–702. doi:10.1016/j.cub.2010.02.058, S0960-9822(10)00331-3[pii]

    Article  PubMed  CAS  Google Scholar 

  5. Murray JW, Bananis E, Wolkoff AW (2000) Reconstitution of ATP-dependent movement of endocytic vesicles along microtubules in vitro: an oscillatory bidirectional process. Mol Biol Cell 11(2):419–433

    PubMed  CAS  Google Scholar 

  6. Hyman A, Drechsel D, Kellogg D, Salser S, Sawin K, Steffen P, Wordeman L, Mitchison T (1991) Preparation of modified tubulins. Methods Enzymol 196:478–485

    Article  PubMed  CAS  Google Scholar 

  7. Pollock N, Koonce MP, de Hostos EL, Vale RD (1998) In vitro microtubule-based organelle transport in wild-type Dictyostelium and cells overexpressing a truncated dynein heavy chain. Cell Motil Cytoskeleton 40(3):304–314. doi:10.1002/(SICI)1097-0169(1998) 40:3<304::AID-CM8>3.0.CO;2-C[pii]

    Article  PubMed  CAS  Google Scholar 

  8. Hyman AA, Salser S, Drechsel DN, Unwin N, Mitchison TJ (1992) Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP. Mol Biol Cell 3(10):1155–1167

    PubMed  CAS  Google Scholar 

  9. Soppina V, Rai A, Mallik R (2009) Simple non-fluorescent polarity labeling of microtubules for molecular motor assays. Biotechniques 46(7):543–549. doi:000113124[pii]10.2144/000113124

    Article  PubMed  CAS  Google Scholar 

  10. Kim T, Kao MT, Hasselbrink EF, Meyhofer E (2007) Active alignment of microtubules with electric fields. Nano Lett 7(1):211–217. doi:10.1021/nl061474k

    Article  PubMed  CAS  Google Scholar 

  11. Yokokawa R, Tarhan MC, Kon T, Fujita H (2008) Simultaneous and bidirectional transport of kinesin-coated microspheres and dynein-coated microspheres on polarity-oriented microtubules. Biotechnol Bioeng 101(1):1–8. doi:10.1002/bit.21874

    Article  PubMed  CAS  Google Scholar 

  12. Yanagida T, Nakase M, Nishiyama K, Oosawa F (1984) Direct observation of motion of single F-actin filaments in the presence of myosin. Nature 307(5946):58–60

    Article  PubMed  CAS  Google Scholar 

  13. Kron SJ, Spudich JA (1986) Fluorescent actin filaments move on myosin fixed to a glass surface. Proc Natl Acad Sci U S A 83(17):6272–6276

    Article  PubMed  CAS  Google Scholar 

  14. Geuze HJ, Slot JW, Strous GJ, Lodish HF, Schwartz AL (1983) Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell 32(1):277–287

    Article  PubMed  CAS  Google Scholar 

  15. Oka JA, Weigel PH (1983) Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. Dissociation of internalized ligand from receptor occurs in two kinetically and thermally distinguishable compartments. J Biol Chem 258(17):10253–10262

    PubMed  CAS  Google Scholar 

  16. Steer CJ, Ashwell G (1980) Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes. J Biol Chem 255(7):3008–3013

    PubMed  CAS  Google Scholar 

  17. Hubbard AL, Wilson G, Ashwell G, Stukenbrok H (1979) An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. I. Distribution of 125I-ligands among the liver cell types. J Cell Biol 83(1):47–64

    Article  PubMed  CAS  Google Scholar 

  18. Weibel ER, Staubli W, Gnagi HR, Hess FA (1969) Correlated morphometric and biochemical studies on the liver cell. I. Morphometric model, stereologic methods, and normal morphometric data for rat liver. J Cell Biol 42(1):68–91

    Article  PubMed  CAS  Google Scholar 

  19. Hagiwara H, Yorifuji H, Sato-Yoshitake R, Hirokawa N (1994) Competition between motor molecules (kinesin and cytoplasmic dynein) and fibrous microtubule-associated proteins in binding to microtubules. J Biol Chem 269(5):3581–3589

    PubMed  CAS  Google Scholar 

  20. Loubery S, Wilhelm C, Hurbain I, Neveu S, Louvard D, Coudrier E (2008) Different microtubule motors move early and late endocytic compartments. Traffic 9(4):492–509. doi:TRA704[pii]10.1111/j.1600-0854.2008.00704.x

    Article  PubMed  CAS  Google Scholar 

  21. Soppina V, Rai AK, Ramaiya AJ, Barak P, Mallik R (2009) Tug-of-war between dissimilar teams of microtubule motors regulates transport and fission of endosomes. Proc Natl Acad Sci U S A 106(46):19381–19386. doi:0906524106[pii]10.1073/pnas.0906524106

    Article  PubMed  CAS  Google Scholar 

  22. Ghosh RN, Gelman DL, Maxfield FR (1994) Quantification of low density lipoprotein and transferrin endocytic sorting HEp2 cells using confocal microscopy. J Cell Sci 107(Pt 8):2177–2189

    PubMed  CAS  Google Scholar 

  23. Schroer TA, Schnapp BJ, Reese TS, Sheetz MP (1988) The role of kinesin and other soluble factors in organelle movement along microtubules. J Cell Biol 107(5):1785–1792

    Article  PubMed  CAS  Google Scholar 

  24. Kishino A, Yanagida T (1988) Force measurements by micromanipulation of a single actin filament by glass needles. Nature 334(6177):74–76. doi:10.1038/334074a0

    Article  PubMed  CAS  Google Scholar 

  25. Kapoor TM, Mitchison TJ (1999) Allele-specific activators and inhibitors for kinesin. Proc Natl Acad Sci U S A 96(16):9106–9111

    Article  PubMed  CAS  Google Scholar 

  26. Bananis E, Murray JW, Stockert RJ, Satir P, Wolkoff AW (2000) Microtubule and motor-dependent endocytic vesicle sorting in vitro. J Cell Biol 151(1):179–186

    Article  PubMed  CAS  Google Scholar 

  27. Fort AG, Murray JW, Dandachi N, Davidson MW, Dermietzel R, Wolkoff AW, Spray DC (2011) In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors. J Biol Chem 286(26):22875–22885. doi:M111.219709[pii]10.1074/jbc.M111.219709

    Article  PubMed  CAS  Google Scholar 

  28. Murray JW, Bananis E, Wolkoff AW (2002) Immunofluorescence microchamber technique for characterizing isolated organelles. Anal Biochem 305(1):55–67

    Article  PubMed  CAS  Google Scholar 

  29. Ross JL, Wallace K, Shuman H, Goldman YE, Holzbaur EL (2006) Processive bidirectional motion of dynein-dynactin complexes in vitro. Nat Cell Biol 8(6):562–570. doi:ncb1421[pii]10.1038/ncb1421

    Article  PubMed  CAS  Google Scholar 

  30. Pucadyil TJ, Schmid SL (2008) Real-time visualization of dynamin-catalyzed membrane fission and vesicle release. Cell 135(7):1263–1275. doi:S0092-8674(08)01495-5[pii]10.1016/j.cell.2008.11.020

    Article  PubMed  CAS  Google Scholar 

  31. Murray JW, Sarkar S, Wolkoff AW (2008) Single vesicle analysis of endocytic fission on microtubules in vitro. Traffic 9(5):833–847. doi:TRA725[pii]10.1111/j.1600-0854.2008.00725.x

    Article  PubMed  CAS  Google Scholar 

  32. Hoffman MT, Sheung J, Selvin PR (2011) Fluorescence imaging with one nanometer accuracy: in vitro and in vivo studies of molecular motors. Methods Mol Biol 778:33–56. doi:10.1007/978-1-61779-261-8_4

    Article  PubMed  CAS  Google Scholar 

  33. Patterson G, Davidson M, Manley S, Lippincott-Schwartz J (2010) Superresolution imaging using single-molecule localization. Annu Rev Phys Chem 61:345–367. doi:10.1146/annurev.physchem.012809.103444

    Article  PubMed  CAS  Google Scholar 

  34. Nath S, Bananis E, Sarkar S, Stockert RJ, Sperry AO, Murray JW, Wolkoff AW (2007) Kif5B and Kifc1 interact and are required for motility and fission of early endocytic vesicles in mouse liver. Mol Biol Cell 18(5):1839–1849. doi:E06-06-0524[pii]10.1091/mbc.E06-06-0524

    Article  PubMed  CAS  Google Scholar 

  35. Sarkar S, Bananis E, Nath S, Anwer MS, Wolkoff AW, Murray JW (2006) PKCzeta is required for microtubule-based motility of vesicles containing the ntcp transporter. Traffic 7(8):1078–1091. doi:TRA447[pii]10.1111/j.1600-0854.2006.00447.x

    Article  PubMed  CAS  Google Scholar 

  36. Murray JW, Wolkoff AW (2003) Roles of the cytoskeleton and motor proteins in endocytic sorting. Adv Drug Deliv Rev 55(11):1385–1403

    Article  PubMed  CAS  Google Scholar 

  37. Mukhopadhyay A, Nieves E, Che FY, Wang J, Jin L, Murray JW, Gordon K, Angeletti RH, Wolkoff AW (2011) Proteomic analysis of endocytic vesicles: Rab1a regulates motility of early endocytic vesicles. J Cell Sci 124(Pt 5):765–775. doi:jcs.079020[pii]10.1242/jcs.079020

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Anatomy and Structural Biology, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY, USA

    John W. Murray

  2. Division of Gastroenterology and Liver Disease, Albert Einstein College of Medicine, Bronx, NY, USA

    John W. Murray

Authors
  1. John W. Murray
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John W. Murray .

Editor information

Editors and Affiliations

  1. Faculty of Medicine, Neuroanatomy & Molecular Brain Research, Ruhr-Universität Bochum, Universitätsstrasse 150, Bochum, 44801, Germany

    Rolf Dermietzel

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Murray, J.W. (2013). Visualizing In Vitro Trafficking. In: Dermietzel, R. (eds) The Cytoskeleton. Neuromethods, vol 79. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-266-7_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-266-7_2

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-265-0

  • Online ISBN: 978-1-62703-266-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation