Subcellular fractionation of intra-axonally transport polypeptides in the rabbit visual system.

Abstract

We analyzed the subcellular distributions of proteins that are transported down the axons of rabbit retinal ganglion cells and compared these distributions to those of enzyme markers for endoplasmic reticulum, plasma membrane, and mitochondria. The proteins of each of five previously identified transport groups were uniquely distributed through the subcellular fractions, suggesting that each transport group is associated with different subcellular organelles. In particular, all of the observed group I polypeptides (the most rapidly moving group, maximum velocity > 240 mm/day) were associated with material of hydrodynamic properties similar to those of the plasma membrane. The proteins of group II (maximum velocity = 34-68 mm/day) were heterogeneous in their subcellular distributions but included mitochondrial proteins. Groups III and IV (maximum velocity = 4-8 and 2-4 mm/day, respectively) included materials that may be involved in motile processes; group V (maximum velocity = 0.7-1.1 mm/day) contained material of very high density which may be associated with neurofilaments. Many of the proteins required by the axons and synaptic terminals of neurons are synthesized in the neuronal cell bodies and conveyed to their destinations by the process of intra-axonal transport. The identities and organization of the transported proteins are of interest not only because of the importance of these proteins in axonal and synaptic functions but also because their transport may represent a modification of transport processes used to convey proteins over less dramatic distances in other cell types.In the retinal ganglion cells of the rabbit, the intra-axonal transport of proteins appears to be a complex but highly organized process. Proteins synthesized in the cell bodies of these neurons can be labeled with radioactive amino acids injected into the vitreous of the eye, and the labeled transported proteins can be subsequently monitored as they move down the axons.[The axons of the retinal ganglion cells pass through the optic nerve (ON) and contralateral optic tract (OT), a total distance of about 3 cm, to form synapses in the lateral geniculate nucleus (LGN) and superior colliculus (SC)]. We previously used sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography of the gels to resolve more than 40 of the transported polypeptides; these polypeptides can be assigned to at least five groups, according to the time at which they first appear in labeled form in segments of the ON and OT-i.e., according to their maximum transport velocities (1, 2). The significance of this organization is not understood; however, the principles governing this grouping could be reflected in the subcellular associations of the polypeptides within each group.We have therefore begun to analyze the subcellular distributions of the transported polypeptides in homogenates of the ON and OT (which contain the axons of the retinal ganglion cells) as well as of the LGN and SC (which contain nerve endings in addition to t...

The Anterograde Transport of Rabies Virus in Rat Sensory Dorsal Root Ganglia Neurons

“…We presume that the virus is transported across the nerve cell without replicating in the cell soma. Similar fast anterograde transport mechanisms have been widely reported for other cellular components (Couraud & DiGiamberardino, 1980;Gilbert et al, 1985;Lorenz & Willard, 1978;Lubinska & Niemierko, 1971 ;Ochs, 1972). The mean velocity of rabies virus fast transport was estimated to be in the range of 100 to 400 mm per day which is in accordance with the velocities (360 mm/day) reported for rat sensory neurons (Aquino et al, 1987).…”

“…The anterograde transport of virus is probably a complex phenomenon; perhaps it is the mode of viral entry that determines which mechanism will be used. Indeed different modes and velocities of transport have been described (Lorenz & Willard, 1978;Tytell et al, 1981). For example, different molecular forms of acetylcholinesterase are transported at various velocities ranging from 3 to 400 mm/day (Couraud & DiGiamberardino, 1980).…”

Cytoskeletal Requirements in Axonal Transport of Slow Component-b

“…First, radioisotopic labeling studies have provided data suggesting that many of the SCb proteins move together as if they comprised discrete structures that are transported in axons (Willard et al, 1974(Willard et al, , 1979Black and Lasek, 1980;Tytell et al, 1981;Lasek, 1981, 1982). This possibility was reinforced by studies in which large subsets of SCb proteins were isolated by subcellular fractionation (Lorenz and Willard, 1978) or nondenaturing immunoprecipitation (Black et al, 1991). Finally, a review of the literature reveals that many other SCb proteins interact with the three proteins that are cotransported in our system.…”

Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms.

“…One plausible explanation for these results is that KHC-A and KHC-B were associated with different subclasses of membrane bounded organelles having different average rates. Both radiolabel studies (Lorenz and Willard, 1978) and video microscopy (Brady et al, , 1985 demonstrated that small tubulovesicular structures moved in axonal transport at a rate severalfold over that of mitochondria. If KHC-A and KHC-B represented kinesin associated with small tubulovesicular organelles and mitochondria respectively, protein markers for these structures should display transport kinetics similar to the corresponding kinesin variant.…”