Howard S. Shpetner

The role of dynein in retrograde axonal transport

Fast axonal transport is manifested at the sub-cellular level as the anterograde or retrograde movement of membrane-bounded organelles along microtubules. Earlier work implicated the protein kinesin as the motor for anterograde axonal transport. More recent work indicates that a brain microtubule-associated protein, MAP 1C, is responsible for retrograde transport. Of additional interest, MAP 1C has been found to be a cytoplasmic form of the ciliary and flagellar ATPase dynein, indicating a much more general functional role for this enzyme in cells than had been suspected.

Purification of brain cytoplasmic dynein and characterization of its in vitro properties

Publisher Summary The force-producing enzyme dynein was first isolated from ciliary and flagellar axonemes. In the axoneme, dynein is situated between outer doublet microtubules, where it converts the energy from ATP hydrolysis into mechanical force. Based on the direction of force production and pharmacology of the enzyme, it was proposed that cytoplasmic dynein was responsible for retrograde axonal transport and a variety of other forms of intracellular motility. This chapter describes the purification and assay of brain cytoplasmic dynein. This approach provides a preparation of microtubules enriched in the entire class of nucleotide-sensitive MAPs. Subsequent extraction of the microtubules with appropriate nucleotide releases at least three force-producing MAPs: kinesin, dynein, and dynamin. Once dynein has been released from microtubules, it can be purified away from trace contaminating polypeptides by sucrose density gradient centrifugation. Its sedimentation coefficient (20S) facilitates its separation from MAP1 and MAP2 (3-5S), as well as from kinesin and dynamin (9-10S).

Dynamin: a microtubule-associated GTP-binding protein.

Summary We recently identified dynamin as a third nucleotide-sensitive microtubule-associated protein in brain tissue, in addition to kinesin and cytoplasmic dynein. Molecular cloning analysis has revealed that dynamin contains the three consensus elements characteristic of GTP-binding proteins, and biochemical results support a role for GTP in dynamin function. Dynamin is also homologous to the Mx proteins, involved in interferon-induced viral resistance, and the product of the yeast VPS1 gene, involved in vacuolar protein sorting. These results identify a novel class of GTP-utilizing proteins, with apparently diverse functions.

Potential roles of microtubule-associated motor molecules in cell division.

It has been established for almost three decades that the mitotic spindle is composed of microtubules. Although much work has been done on the assembly dynamics and organization of mitotic spindle microtubules, relatively little is known about the forces underlying chromosome movement. The mitotic spindle also plays an indirect role in cytokinesis by specifying the position of the cleavage plane. Whether this is also a function of microtubules or of some other spindle element is not known. However, the microtubules are the most obvious and most clearly organized structural elements of the spindle. As such, they are appealing candidates for this role, despite the lack of evidence for a direct interaction of spindle microtubules with the cortical actomyosin network. Recent work has led to the identification of two mechanochemical proteins associated with cytoplasmic microtubules, kinesin’ and dynein These two proteins produce force in opposite directions and may account for many aspects of intracellular motility. They have been most strongly implicated in organelle transport (for review, see ref. 5 ) , but other roles, such as in chromosome separation and other aspects of cell division, have not been extensively explored. Our laboratory has recently identified and purified a third protein that shows many of the mechanochemical characteristics of kinesin and dynein, yet it is clearly a distinct This protein, which we have termed “dynamin,” exhibits a number of characteristics that suggest a role in mitosis. Some evidence already exists to indicate that kinesin is present in the mitotic spindle. Generalized spindle fiber staining was observed in sea urchin eggs by immunocytochemical means; * however, in the mammalian spindle staining was observed only at the p01es.~ Antibodies to axonemal and sea urchin egg dynein have been found to stain the spindle. (For example, see ref. 10.) Such staining has generally been weak and diffuse, with no evidence for a specific localization of dynein within the spindle. Determining the distribution of these molecules within the spindle with precision may prove to be particularly difficult, because it will probably involve discriminating localized staining (as in the kinetochore) from more generalized staining of membranous

Purification and characterization of dynamin.

Publisher Summary Dynamin is a microtubule-associated mechanochemical ATPase recently identified in calf brain white matter cytosol. It exhibits nucleotide-sensitive binding to microtubules, its ATPase activity can be stimulated severalfold by microtubules, and it has shown evidence of force production against microtubules in in vitro assays. Dynamin consists of a 100-kDa polypeptide and an activating factor, which has been only partially purified. The purified 100-kDa species forms cross-bridges between microtubules that appear morphologically identical to those formed in the presence of the activating factor. This chapter describes a method for obtaining both the purified 100-kDa protein and the activating factor in separate fractions from a single preparation of brain microtubules. Extraction of the 100-kDa protein from the microtubules requires elevated salt concentrations as well as nucleotide, apparently due to binding to microtubules via both nucleotide-insensitive and nucleotide-sensitive sites. The extracted 100-kDa protein is subsequently purified by anion-exchange chromatography.

Functional Analysis of Dynamin, a GTPase Mediating Early Endocytosis

Dynamin was initially identified as a nucleotide-dissociable 100 kD microtubule-binding protein in calf brain cytosol, distinct from cytoplasmic dynein and kinesin (Shpetner and Vallee, 1989). Initial biochemical studies indicated a likely nucleotidase activity, and subsequent molecular cloning in rat indicated that the N-terminal 300 amino acids contained three consensus sequence elements for GTP-binding (Obar et al., 1990). No other homology was found with either ras proteins, the α-subunits of G proteins, or any of the GTPases associated with either protein translation or protein translocation into the endoplasmic reticulum. However, extensive homology has been found with three other GTP-binding proteins: the mammalian Mx proteins (Mr 72–80 kD; Horisberger et al., 1990), involved in mediating the interferon-induced reponse to viral infection, the product of the yeast VPS1 gene (Mr 79 kD; Rothman et al, 1990), involved in protein sorting from the Golgi body to the vacuole, and the product of the yeast MGM1 gene (Mr 94 kD; Jones and Fangman, 1992), involved in replication of the mitochondrial genome.