Molecular Basis of Organelle Transport
Kevin T. Vaughan
Ph.D., Cornell University Medical College
Postdoctoral, UMASS Medical Center,
Worcester Foundation for Biomedical Research
One of the most fundamental questions in cell biology is how cells move, organize and compartmentalize internal structures such as organelles and chromosomes (DNA). Normally, this intracellular transport is fast, efficient and tightly regulated. A growing number of diseases are now thought to result from disrupted transport of organelles in specific organs. Loss of coordinated chromosome movement has been implicated as the basis of developmental defects and loss of tumor suppressor genes in cancer.
Regulation of IC-Organelle Binding Through p150 Phosphorylation
here are several mechanisms of transport and each system is composed of two essential elements. Cytoskeletal filaments function as railroads for transport, and “molecular motors” function as the engines. In the last decade, a large number of these “motor proteins” have been identified and analyzed. Members of the myosin superfamily move along actin filaments and are specialized in muscle, whereas members of the kinesin and dynein superfamilies move along microtubules and are specialized in neurons.
The Vaughan lab is focusing on questions in the microtubule motor field, and uses one motor called cytoplasmic dynein as an example. Cytoplasmic dynein moves towards the minus or slow-growing ends of microtubules, and is responsible for centripetal organelle movement, retrograde axonal transport, and several aspects of chromosome segregation during mitosis. Genetic defects in dynein-mediated transport play a role in developmental defects of the nervous system, cancers in epithelial cells, and infertility. Because of its role in axonal transport, dynein activity is also important in recovery from spinal injury. Finally, a number of viruses such as herpes, adenovirus and rabies appear to hijack dynein for transport to the nucleus for replication.
Proposed Role for p150-Glued Microtubule Interaction During Membrane Transport
Although we recognize that cytoplasmic dynein mediates these aspects of organelle and chromosome movement, we do not understand how this motor identifies or binds to its cargo. The primary focus of our lab is to determine how and where cytoplasmic dynein interacts with its cargo, and how this process is regulated. Our recent studies suggest strongly that a related complex called dynactin serves as the dynein receptor on the surface of organelles and chromosomes. We are using a combination of cDNA cloning, mammalian cell culture, immunofluorescence microscopy, DNA transfection and biochemistry to study the interaction of cytoplasmic dynein with dynactin at these sites. Ongoing studies focus on a novel population of dynactin-containing vesicles that accumulate at microtubule plus ends prior to binding cytoplasmic dynein and initiating transport. Using live-cell imaging, we are studying this vesicle population as a new model system for cargo-motor interactions.
One approach we have capitalized on is the analysis of dynein regulation. If we can identify what interactions are regulated, this could provide evidence for which interactions are essential. We have identified two phosphorylated subunits in this pathway and determined the functional impact of this phosphorylation. Phosphorylation of the cytoplasmic dynein intermediate chains regulates binding of the dynein motor to the candidate receptor dynactin (Fig. 1). This finding supports the hypothesis that dynactin functions as an essential cofactor. We have also mapped the first regulatory phosphorylation site in the p150Glued subunit of dynactin. This modification regulates the microtubule binding activity of dynactin, and appears to be coordinated with the recruitment of the dynein motor for transport (Fig. 2). Ongoing work is focused on the respective kinases for these subunits, the signaling pathways that impinge on dynein, and cell cycle specific phosphorylation of microtubule-based motors.Deacon, S.W. , A.S. Serpinskaya, P.S. Vaughan, M. L. Fanarraga, I.Vernos, K.T. Vaughan, and V. I. Gelfand. (2002). Dynactin Serves as a Receptor for Kinesin II on Xenopus laevis Melanosomes. J. Cell Biol. In Revision.
Askham, J.M., K.T. Vaughan, H.V. Goodson, and E.E. Morrison (2002). Evidence That An Interaction Between EB1 and p150Glued is Required for the Formation and Maintenance of a Radial Microtubule Array Anchored at the Centrosome. Mol. Biol. Cell, 13:3627-3645.
Vaughan, PS., P. Miura, M. Henderson, B. Byrne and K.T. Vaughan (2002). A role for regulated binding of p150Glued to microtubule plus ends in organelle transport. J Cell Biol, 158: 305-319.
Susalka, S.J., K. Nikulina, M.W. Salata, PS. Vaughan, S.M. King, K.T. Vaughan and K.K. Pfister (2002). The roadblock light chain binds a novel region of the cytoplasmic dynein intermediate chain. J Biol Chem, 277: 32939-46.
Vaughan, PS., J.D. Leszyk and K.T. Vaughan (2001). Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin. J Biol Chem, 276: 26171-26179.
Ye, G-J., K.T. Vaughan, R.B. Vallee and B. Roizman (2000). The herpes simplex virus 1 U(L)34 protein interacts with a cytoplasmic dynein intermediate chain and targets nuclear membrane.. J Virol, 74: 1355-1363.
Fossella, J., S.A. Samant, L.M. Silver, S.M. King, K.T. Vaughan, P.Olds-Clarke, K.A. Johnson, A. Mikami, R.B. Vallee and S.H. Pilder (2000).An axonemal dynein at the Hybrid Sterility 6 locus: implications for t haplotype-specific male sterility and the evolution of species barriers. Mamm Genome, 3: 8-15.
Vaughan, K.T., S.H. Tynan, N.E. Faulkner, C.J. Echeverri and R.B. Vallee (1999). Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends. J Cell Sci, 112: 1437-1447.
Steffen, W., S. Karki, K.T. Vaughan, R.B. Vallee, E.L.F. Holzbaur, D.G. Weiss and S.A. Kuznetsov (1997). The involvement of the intermediate chain of cytoplasmic dynein in binding the motor complex to membranous organelles of Xenopus oocytes. Mol Biol Cell, 8: 2077-2088.
Block-Galarza, J., K.O. Chase, E. App, K.T. Vaughan, R.B. Vallee, M. DiFiglia and N. Aronin (1997). Fast transport and retrograde movement of huntingtin and HAP 1 in axons. Neuroreport, 8: 2247-2251.
Vaughan, K.T., A. Mikami, B.M. Paschal, E.L.F. Holzbaur, S.M. Hughes, C.J. Echeverri, K.J. Moore, D.J. Gilbert, N.G. Copeland, N.A. Jenkins and R.B. Vallee (1996). Multiple mouse chromosomal loci for dynein-based motility. Genomics, 36: 29-38.
Echeverri, C.J., B.M. Paschal, K.T. Vaughan and R.B. Vallee (1996). Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J Cell Biol, 132: 617-633.
Pfister, K.K, .W. Salata, J.F. Dillman III, K.T. Vaughan, R.B. Vallee, E. Torre and R.J. Lye (1996). Differential expression and phosphorylation of the 74-kDa intermediate chains of cytoplasmic dynein in cultured neurons and glia. J Biol Chem, 271: 1687-1694.
Vaughan, K.T. and R.B. Vallee (1995). Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued. J Cell Biol, 131: 1507-1516.
Vaughan, K.T., E.L.F. Holzbaur and R.B. Vallee (1995). Subcellular targeting of the retrograde motor cytoplasmic dynein. Biochem Soc Trans,23: 50-54.
Vallee, R.B., K.T. Vaughan and C.J. Echeverri (1995). Targeting of Cytoplasmic Dynein to Membranous Organelles and Kinetochores via Dynactin. Cold Spring Harb Symp Quant Biol, 60: 803-811.
Hughes, S.M., K.T. Vaughan, J.S. Herskovits and R.B. Vallee (1995). Molecular analysis of a cytoplasmic dynein light intermediate chain reveals homology to a family of ATPases. J Cell Sci, 108: 17-24.