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Slow axonal protein transport and axoplasmic organization
Journal of the Neurological Sciences, 1986, 72:11-18
[I
Elsevier JNS 2591
Slow Axonal Protein Transport and Axoplasmic Organization David J. G o w e r 1'2 and Michael Tytell 2 ~Section on Neurosurgery, Department of Surgery and 2Department of Anatomy, Wake Forest University Medical Center, Bowman Gray School of Medicine, Winston-Salem, NC (U.S.A.)
(Received 20 May, 1985) (Revised, received 5 August, 1985) (Accepted 7 August, 1985)
SUMMARY A recently formulated structural theory of axonal transport suggests that each group of transported material is a distinct class of functionally related structures and that the proteins making up those structures, with few exceptions, are found in only one rate component, thus maintaining a close noncovalent association during transport. If such a relationship exists, one would postulate that the labelled proteins at the leading edge and trailing edge of a rate component following a pulse of radioactive amino acid would be present in similar proportions. After retinal ganglion cell proteins were labelled by intraocular injection of radioactive amino acid in the guinea pig eye, the optic nerve and tract were analyzed at several post-injection intervals. The data thus derived support the existence of a close relationship among the "soluble" proteins of slow component b and are consistent with the structural concept of axonal transport. If diffusion were the mechanism of transport, the larger proteins would be expected to move slower than the smaller proteins. Such insights into the process of axonal transport should help to identify the variables critical to survival of neurons following acute trauma and degenerative diseases.
Key words: A x o n a l t r a n s p o r t - Guinea p i g - Visual system
Correspondence to: David J. Crower, M.D., Section on Neurosurgery,Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem,NC 27103, U.S.A. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (BiomedicalDivision)
12 INTRODUCTION The orderly anterograde movement of proteins within the axon has been observed by a number of investigators (Hoffman and Lasek 1975; Black and Lasek 1980; Hoffman and Lasek 1980; TyteU et al. 1981; Brady and Lasek 1982; Garner and Lasek 1982; Lasek et al. 1984). This protein transport occurs in three groups, or rate components: "fast component" (FC); "slow component b" (SCb); and "slow component a" (SCa). The family of proteins contained within each rate component move at unique speeds and undergo coordinate transport with little mixing. Two of the rate components contain structural elements that may easily be identified with the electron microscope. The membranous organelles and transmitter vesicles constitute part of the FC, and the cytoskeleton, made up of neurofflaments and microtubules, is included in SCa. However, in the case of SCb, the phenomenon of coordinate transport has been difficult to understand since no easily identifiable structure is contained within SCb, and its proteins traditionally have been thought to be "soluble." Although the idea that cytoplasm behaves like a solution of proteins remains popular, recent studies have suggested that SCb (axoplasm) may correspond, in part, to the structural matrix between the dements of the axonal cytoskeleton, or the "axoplasmic ground substance" (Black and Lasek 1980; Schnapp and Reese 1982; Lasek et al. 1984) and maintain a close non-covalent association with the characteristics of a single structure during axonal transport. This report provides additional evidence that the proteins of SCb behave as an organized aggregate rather than a simple solution. MATERIALS AND METHODS A unilateral intraocular injection of 500 #CI of [aSS]methionine (specific activity > 800 Ci/mmol) in 10 #1 of H 2 0 was administered to eighteen 60-day-old male Hartley guinea pigs under ether anesthesia. The animals were allowed to recover and groups of three were killed at 2, 4, 6, 8, 10 and 25 days following the injection. The appropriate optic nerve with tract was removed and divided into seven 3-ram segments (Fig. 1). Each segment was homogenized in 100 #1 of 1 ~ sodium dodecyl sulfate, 8 M urea, and 2% beta-mercaptoethanol to solubilize the proteins. The three segments from equivalent positions in the optic nerves and tracts from each animal were pooled in order to provide
500#Ci
~
~sS]Methionine~ ~
1JTeetum _~~ , / / ~ "
Fig. 1. Opticsystemofthe guineaNg showingintraoeular injectionfollowedby sectionof the visualsystem.
13 adequate material for repeated analysis, and the trichloroacetic acid-precipitable radioactive protein was measured by liquid scintillation counting. The combined 3-mm segments of optic tracts just posterior to the optic chiasm defined the site of the "axonal window" (Fig. 1). A 30-#1 aliquot of each of these samples was loaded onto a 6-19~/o gradient SDS-polyacrylamide gel and electrophoresis was carried out under a constant power of 8 W for 3 h. The gel was then stained with Serva Blue R ® stain and photographed (Fig. 2). The stained gel was processed for fluorographic detection of radioactive proteins (Bonnet and Laskey 1974; Laskey and Mills 1975), dried and placed in contact with pre-flashed high-speed X-ray film at - 7 0 ° for 2 weeks. The fluorographs were developed as are standard X-ray trims (Fig. 3) and scanned with a laser densitometer to provide a quantitative measure of band density (Fig. 4).
SDS-PA 6-19% Fig. 2. SDS-polyacrylamide gel electrophoresis of 3 mm of optic tract 6-19~ gradient gel concentration: Lane 1 pooled 2 days post-injection, Lane 2 pooled 4 days post-injection, Lane 3 pooled 6 days postinjection, Lane 4 pooled 8 days post-injection, Lane 5 pooled 10 days post-injection, Lane 6 pooled 25 days post-injection.
14
Fig. 3. Fluorograph of gel in Fig. 2.
RESULTS
The graph in Fig. 5 represents the passage of a radioactive wave of labelled proteins from the site ofintraocul~ injection to the superior colliculus over I0 days. The rate of movement of this wave of proteins is consistent with the movement of SCb previously reported (Black and Lasek 1979). A second wave of radioactive proteins may be appreciated at the optic chiasm at 25 days and corresponds to the proteins of SCa. Since scintillation counting gives no information about specific protein species that are undergoing transport, polyacrylamide gel electrophoresis was performed to
15
2 DAY
4 DAY
DAY
8 DAY
10 DAY
25 DAY
Fig. 4. Scanninglaser densitometryoffluorographinFig. 3. These data indicate that the proportion oflarge to small proteins is the same in the leading edge (Day 2) and the trailing edge (Day 10) of slow component b.
Radioactivity 4O.3O
27.57" 1 14.83~
2.1o-1< Retina
,oo
~1~ ~ 1 0 . 0 0 Position in Optic Nerve
Pest.Injecton (Days)
2.00 Superior Colliculus
Fig. 5. Graphic representation of movement of radioactive material associated with slow component b along the optic nerve and tract.
16 separate the proteins by molecular size. The fluorograph of the gel, seen in Fig. 3, provides a quantitative view of the radioactivity contained in each band of radio-labelled protein. Digital quantification of this level of radioactivity was carried out with a scanning laser densitometer and clearly showed the rise and fall of the amounts of radioactive proteins with time. The relative level of radioactivity contained in the proteins at the leading edge of SCb (2-day specimen) are in the same proportion as those at the trailing edge of SCb (10-day specimen). This observation strongly supports the hypothesis that SCb (the soluble enzymes) behaves, in fact, like a single structure rather than a solution of proteins. DISCUSSION In the second century Galen described the nerves as narrow conduits through which cerebral "pneuma" flowed, the result being the actions brought about by the nervous system (Grafstein and Forman 1980). Although incorrect, Galen's idea was the fh'st to indicate an active role for the axon in connecting the cell body with the synapse. In the twentieth century Ramon y Cajal (1928) showed the neuron, in both its normal and its pathologic state, to be a cell with a complex internal network of fibrils (cytoskeleton). Ramon y Cajal suggested that material diffuses down this fibrillar pathway rather than passing through the axon as if it were a simple conduit. In 1948, Weiss and Hiscoe demonstrated that placing a ligature around a peripheral nerve caused a swelling proximal to the area of constriction; If the ligature was then removed, the area of swelling moved down the nerve as a unit at 1-2 mm/day (the rate of SCb). The one large swelling would frequently separate into smaller swellings resembling the peristaltic movement seen in the intestine. These f'mdings led some to believe that the mechanism of transport was a peristaltic motion of the neuron. Experimental techniques using pulse labelling with 3H- or 35S-labelled amino acids (Droz and LeBlond 1963; Lasek 1968) or using specific binding lectins (Borges and Sidman 1981) have added greatly to our understanding of the characteristics of axonal transport. The radioactive wave of protein first observed by Droz and Leblond (1963) to move down the axon at 1-2 mm/day was similar in rate of movement to the blebs seen by Weiss and Hiscoe (1948) and corresponded to the SCb proteins. Using similar techniques, Goldberg and Kotani (1967) as well as Lasek (1967) found a portion of radio-labelled material moving approximately 100 times faster than the wave observed by Droz and Leblond. This set of proteins (FC) corresponded to the material observed two years earlier by Burdwood (1965) using time-lapse photography of living axons. This was the first visual demonstration of the movement of a specific structure corresponding to a rate component of axonal transport. At the present time, it is accepted that there are three major rates of movement of material in the axon, two of which are associated with easily observable cytoplasmic structures. The fast component, moving at 240 ram/day, consists of membrane and membrane-bound proteins, such as those included in the smooth endoplasmic reticulum and neurotransmitter vesicles. This material can be observed and its motion seen in real time using computer-enhanced video light microscopy (Ellisman and Porter 1980; Alien
17 et al. 1982). The slowest rate component (SCa), moving at 0.3 ram/day, consists of the tubulins (the subunits of microtubules) and the three subunits that make up the neurofflaments (Hoffman and Lasek 1975). These structures can be observed with light and electron microscopy in the static state but move too slowly for their movement to be observed directly. Finally, there is SCb, which consists of globular "soluble" enzymes and moves 1-2 mm/day (Black and Lasek 1980). Representative examples of SCb proteins are actin (Black and Lasek 1978), nerve-specific enolase, creatinine phosphokinase (Brady and Lasek 1981), calmodulin (Brady et al. 1981), clathrin (Garner and Lasek 1981), and some polyamines (Kremzner and Ambron 1982). The application of high-voltage electron microscopy and quick-freezing preservation to the neuron and other cells has revealed a level of free structure called the microtrabecular network (Ellisman and Porter 1980; Schnapp and Reese 1982). This morphologically demonstrable matrix probably corresponds at least in part to the biochemically defined complex called SCb. We have demonstrated that following a pulse label, the proteins synthesized by the retinal ganglion cell move down the axon as if they were a part of a definite axonal complex. At the leading and trailing edges of SCb the relative concentration of large proteins to small proteins is the same. This type of behavior would not be predicted for a protein solution, where diffusion would be the primary determinant of motion and large proteins would be expected to move more slowly than the smaller proteins. Thus our data strongly support the structural theory of axonal transport. The continued investigations of the transport characteristics of the axoplasmic matrix proteins of SCb in a manner similar to that reported here will yield a picture of the organization of these proteins at the molecular level. Since the SCb proteins seem closely tied to the capacity of axons to grow and regenerate (reviewed by Cancalon 1984), this information should be of value in defining procedures and treatments designed to promote neuron survival and recovery subsequent to neurological disease or trauma. REFERENCES Allen, R. D., J. Metuzals, I. Tasaki, S. Brady and S. Gilbert (1982) Fast axonal transport in squid giant axon, Science, 218:1127-1129. Black, M. M. and R.J. Lasek (1979) Axonal transport of actin - - Slow component b is the principal source of actin for the axon, Brain Res., 171: 401-413. Black, M.M. and R.J. Lasek (1980) Slow components of axonal transport - - Two cytoskeletal networks, J. Cell BioL, 86: 616-623. Bonner, W.M. and R.A. Laskey (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels, Europ. J. Biochem., 46: 83-88. Borges, L. F, and R.L. Sidman (1982) Axonal transport of lectins in the peripheral nervous system, J. Neurosci., 2: 647-653. Brady, S.T. and R.J. Lasek (1981) Nerve-specific enolase and creatine phosphokinase in axonal transport - - Soluble proteins and the axoplasmic matrix, Cell, 23: 515-523. Brady, S.T. and R.J. Lasek (1982) Axonal transport - - A cell-biological method for studying proteins that associate with the cytoskeleton, In: L. Wilson (Ed.), Methods in CellBiology, Vol. 25, Part b, Academic Press, New York, pp. 365-398. Brady, S.T., M. Tytell, K. Heriot and R.J. Lasek (1981) Axonal transport of calmodulin - - A physiologic approach to identification of long-term associations between proteins, 3. Cell BioL, 89: 607-614.
18 Burdwood, W.O. (1965) Rapid bidirectional particle movement in neurons, J. Cell Biol., 27:115A. Cancalon, P. (1984)The relationship of slow axonal flow to nerve elongation and degeneration. In: J. S. Elam and P. Cancalon (Eds.), Advances in Neurochemistry, Vol. 6, Plenum Press, New York, pp. 211-241. Droz, B. and C. P. Leblond (1963) Axonal migration of proteins in the central nervous system and peripheral nerves as shown by radioautography, J. Comp. Neurol., 121: 325-337. Ellisman, M. H. and K. R. Porter (1980) Microtrabecular structure of the axoplasmic matrix - - Visualization of cross-linking structures and their distribution, J. Cell Biol., 87: 464-479. Garner, J.A. and R.J. Lasek (1981) Clathrin is axonally transported as part of slow component b - - The microfilament complex, J. Cell Biol., 88: 172-178. Garner, J.A. and R.J. Lasek (1982) Cohesive axonal transport of the slow component b complex of polypeptides, J. Neurosci., 2: 1824-1835. Goldberg, S. and M. Kotani (1967) The projection of optic nerve fibers in the frog Rana catesbeiana as studied by radioautography, Anat. Rec., 158: 325-332. Grafstein, B. and D.S. Forman (1980) Intercellular transport in neurons, Physiol. Rev., 60: 1167-1283. Hoffman, P.N. and R.J. Lasek (1975)The slow component of axonal transport - - Identification of major structural polypeptides of the axon and their generality among mammalian neurons, J. Cell Biol., 66: 351-366. Hoffman, P.N. and R.J. Lasek (1980) Axonal transport of the cytoskeleton in regenerating motor neurons - - Constancy and change, Brain Res., 202: 317-333. Kremzner, L.T. and R.T. Ambron (1982) Metabolism and axonal transport of polyamines in a single identified neuron of Aplysia californica, J. Neurochem., 38: 1719-1727. Lasek, R. (1968) Axoplasmic transport in cat dorsal root ganglion cells - - As studied with [3H]-L-leucine, Brain Res., 7: 360-377. Lasek, R.J., J.A. Garner and S.T. Brady (1984) Axonal transport of the cytoplasmic matrix, J. Cell Biol., 99: 212s-221s. Laskey, R.A. and A.D. Mills (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography, Europ. J. Biochem., 56: 335-341. Ramon y Cajal, S. (1928) Degeneration and Regeneration of the Nervous System (transl. by R. M. May), Oxford University Press, Cambridge. Schnapp, B. J. and T. S. Reese (1982) Cytoplasmic structure in rapid-frozen axons,J. CellBiol., 94: 667-679. Tytell, M., M.M. Black, J.A. Garner, and R.J. Lasek (1981) Axonal transport - - Each major rate component reflects the movement of distinct macromolecular complexes, Science, 214: 179-181. Weiss, P. and H.B. Hiscoe (1948) Experiments on the mechanism of nerve growth, J. Exp. ZooL, 107: 315-395.
[I
Elsevier JNS 2591
Slow Axonal Protein Transport and Axoplasmic Organization David J. G o w e r 1'2 and Michael Tytell 2 ~Section on Neurosurgery, Department of Surgery and 2Department of Anatomy, Wake Forest University Medical Center, Bowman Gray School of Medicine, Winston-Salem, NC (U.S.A.)
(Received 20 May, 1985) (Revised, received 5 August, 1985) (Accepted 7 August, 1985)
SUMMARY A recently formulated structural theory of axonal transport suggests that each group of transported material is a distinct class of functionally related structures and that the proteins making up those structures, with few exceptions, are found in only one rate component, thus maintaining a close noncovalent association during transport. If such a relationship exists, one would postulate that the labelled proteins at the leading edge and trailing edge of a rate component following a pulse of radioactive amino acid would be present in similar proportions. After retinal ganglion cell proteins were labelled by intraocular injection of radioactive amino acid in the guinea pig eye, the optic nerve and tract were analyzed at several post-injection intervals. The data thus derived support the existence of a close relationship among the "soluble" proteins of slow component b and are consistent with the structural concept of axonal transport. If diffusion were the mechanism of transport, the larger proteins would be expected to move slower than the smaller proteins. Such insights into the process of axonal transport should help to identify the variables critical to survival of neurons following acute trauma and degenerative diseases.
Key words: A x o n a l t r a n s p o r t - Guinea p i g - Visual system
Correspondence to: David J. Crower, M.D., Section on Neurosurgery,Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem,NC 27103, U.S.A. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (BiomedicalDivision)
12 INTRODUCTION The orderly anterograde movement of proteins within the axon has been observed by a number of investigators (Hoffman and Lasek 1975; Black and Lasek 1980; Hoffman and Lasek 1980; TyteU et al. 1981; Brady and Lasek 1982; Garner and Lasek 1982; Lasek et al. 1984). This protein transport occurs in three groups, or rate components: "fast component" (FC); "slow component b" (SCb); and "slow component a" (SCa). The family of proteins contained within each rate component move at unique speeds and undergo coordinate transport with little mixing. Two of the rate components contain structural elements that may easily be identified with the electron microscope. The membranous organelles and transmitter vesicles constitute part of the FC, and the cytoskeleton, made up of neurofflaments and microtubules, is included in SCa. However, in the case of SCb, the phenomenon of coordinate transport has been difficult to understand since no easily identifiable structure is contained within SCb, and its proteins traditionally have been thought to be "soluble." Although the idea that cytoplasm behaves like a solution of proteins remains popular, recent studies have suggested that SCb (axoplasm) may correspond, in part, to the structural matrix between the dements of the axonal cytoskeleton, or the "axoplasmic ground substance" (Black and Lasek 1980; Schnapp and Reese 1982; Lasek et al. 1984) and maintain a close non-covalent association with the characteristics of a single structure during axonal transport. This report provides additional evidence that the proteins of SCb behave as an organized aggregate rather than a simple solution. MATERIALS AND METHODS A unilateral intraocular injection of 500 #CI of [aSS]methionine (specific activity > 800 Ci/mmol) in 10 #1 of H 2 0 was administered to eighteen 60-day-old male Hartley guinea pigs under ether anesthesia. The animals were allowed to recover and groups of three were killed at 2, 4, 6, 8, 10 and 25 days following the injection. The appropriate optic nerve with tract was removed and divided into seven 3-ram segments (Fig. 1). Each segment was homogenized in 100 #1 of 1 ~ sodium dodecyl sulfate, 8 M urea, and 2% beta-mercaptoethanol to solubilize the proteins. The three segments from equivalent positions in the optic nerves and tracts from each animal were pooled in order to provide
500#Ci
~
~sS]Methionine~ ~
1JTeetum _~~ , / / ~ "
Fig. 1. Opticsystemofthe guineaNg showingintraoeular injectionfollowedby sectionof the visualsystem.
13 adequate material for repeated analysis, and the trichloroacetic acid-precipitable radioactive protein was measured by liquid scintillation counting. The combined 3-mm segments of optic tracts just posterior to the optic chiasm defined the site of the "axonal window" (Fig. 1). A 30-#1 aliquot of each of these samples was loaded onto a 6-19~/o gradient SDS-polyacrylamide gel and electrophoresis was carried out under a constant power of 8 W for 3 h. The gel was then stained with Serva Blue R ® stain and photographed (Fig. 2). The stained gel was processed for fluorographic detection of radioactive proteins (Bonnet and Laskey 1974; Laskey and Mills 1975), dried and placed in contact with pre-flashed high-speed X-ray film at - 7 0 ° for 2 weeks. The fluorographs were developed as are standard X-ray trims (Fig. 3) and scanned with a laser densitometer to provide a quantitative measure of band density (Fig. 4).
SDS-PA 6-19% Fig. 2. SDS-polyacrylamide gel electrophoresis of 3 mm of optic tract 6-19~ gradient gel concentration: Lane 1 pooled 2 days post-injection, Lane 2 pooled 4 days post-injection, Lane 3 pooled 6 days postinjection, Lane 4 pooled 8 days post-injection, Lane 5 pooled 10 days post-injection, Lane 6 pooled 25 days post-injection.
14
Fig. 3. Fluorograph of gel in Fig. 2.
RESULTS
The graph in Fig. 5 represents the passage of a radioactive wave of labelled proteins from the site ofintraocul~ injection to the superior colliculus over I0 days. The rate of movement of this wave of proteins is consistent with the movement of SCb previously reported (Black and Lasek 1979). A second wave of radioactive proteins may be appreciated at the optic chiasm at 25 days and corresponds to the proteins of SCa. Since scintillation counting gives no information about specific protein species that are undergoing transport, polyacrylamide gel electrophoresis was performed to
15
2 DAY
4 DAY
DAY
8 DAY
10 DAY
25 DAY
Fig. 4. Scanninglaser densitometryoffluorographinFig. 3. These data indicate that the proportion oflarge to small proteins is the same in the leading edge (Day 2) and the trailing edge (Day 10) of slow component b.
Radioactivity 4O.3O
27.57" 1 14.83~
2.1o-1< Retina
,oo
~1~ ~ 1 0 . 0 0 Position in Optic Nerve
Pest.Injecton (Days)
2.00 Superior Colliculus
Fig. 5. Graphic representation of movement of radioactive material associated with slow component b along the optic nerve and tract.
16 separate the proteins by molecular size. The fluorograph of the gel, seen in Fig. 3, provides a quantitative view of the radioactivity contained in each band of radio-labelled protein. Digital quantification of this level of radioactivity was carried out with a scanning laser densitometer and clearly showed the rise and fall of the amounts of radioactive proteins with time. The relative level of radioactivity contained in the proteins at the leading edge of SCb (2-day specimen) are in the same proportion as those at the trailing edge of SCb (10-day specimen). This observation strongly supports the hypothesis that SCb (the soluble enzymes) behaves, in fact, like a single structure rather than a solution of proteins. DISCUSSION In the second century Galen described the nerves as narrow conduits through which cerebral "pneuma" flowed, the result being the actions brought about by the nervous system (Grafstein and Forman 1980). Although incorrect, Galen's idea was the fh'st to indicate an active role for the axon in connecting the cell body with the synapse. In the twentieth century Ramon y Cajal (1928) showed the neuron, in both its normal and its pathologic state, to be a cell with a complex internal network of fibrils (cytoskeleton). Ramon y Cajal suggested that material diffuses down this fibrillar pathway rather than passing through the axon as if it were a simple conduit. In 1948, Weiss and Hiscoe demonstrated that placing a ligature around a peripheral nerve caused a swelling proximal to the area of constriction; If the ligature was then removed, the area of swelling moved down the nerve as a unit at 1-2 mm/day (the rate of SCb). The one large swelling would frequently separate into smaller swellings resembling the peristaltic movement seen in the intestine. These f'mdings led some to believe that the mechanism of transport was a peristaltic motion of the neuron. Experimental techniques using pulse labelling with 3H- or 35S-labelled amino acids (Droz and LeBlond 1963; Lasek 1968) or using specific binding lectins (Borges and Sidman 1981) have added greatly to our understanding of the characteristics of axonal transport. The radioactive wave of protein first observed by Droz and Leblond (1963) to move down the axon at 1-2 mm/day was similar in rate of movement to the blebs seen by Weiss and Hiscoe (1948) and corresponded to the SCb proteins. Using similar techniques, Goldberg and Kotani (1967) as well as Lasek (1967) found a portion of radio-labelled material moving approximately 100 times faster than the wave observed by Droz and Leblond. This set of proteins (FC) corresponded to the material observed two years earlier by Burdwood (1965) using time-lapse photography of living axons. This was the first visual demonstration of the movement of a specific structure corresponding to a rate component of axonal transport. At the present time, it is accepted that there are three major rates of movement of material in the axon, two of which are associated with easily observable cytoplasmic structures. The fast component, moving at 240 ram/day, consists of membrane and membrane-bound proteins, such as those included in the smooth endoplasmic reticulum and neurotransmitter vesicles. This material can be observed and its motion seen in real time using computer-enhanced video light microscopy (Ellisman and Porter 1980; Alien
17 et al. 1982). The slowest rate component (SCa), moving at 0.3 ram/day, consists of the tubulins (the subunits of microtubules) and the three subunits that make up the neurofflaments (Hoffman and Lasek 1975). These structures can be observed with light and electron microscopy in the static state but move too slowly for their movement to be observed directly. Finally, there is SCb, which consists of globular "soluble" enzymes and moves 1-2 mm/day (Black and Lasek 1980). Representative examples of SCb proteins are actin (Black and Lasek 1978), nerve-specific enolase, creatinine phosphokinase (Brady and Lasek 1981), calmodulin (Brady et al. 1981), clathrin (Garner and Lasek 1981), and some polyamines (Kremzner and Ambron 1982). The application of high-voltage electron microscopy and quick-freezing preservation to the neuron and other cells has revealed a level of free structure called the microtrabecular network (Ellisman and Porter 1980; Schnapp and Reese 1982). This morphologically demonstrable matrix probably corresponds at least in part to the biochemically defined complex called SCb. We have demonstrated that following a pulse label, the proteins synthesized by the retinal ganglion cell move down the axon as if they were a part of a definite axonal complex. At the leading and trailing edges of SCb the relative concentration of large proteins to small proteins is the same. This type of behavior would not be predicted for a protein solution, where diffusion would be the primary determinant of motion and large proteins would be expected to move more slowly than the smaller proteins. Thus our data strongly support the structural theory of axonal transport. The continued investigations of the transport characteristics of the axoplasmic matrix proteins of SCb in a manner similar to that reported here will yield a picture of the organization of these proteins at the molecular level. Since the SCb proteins seem closely tied to the capacity of axons to grow and regenerate (reviewed by Cancalon 1984), this information should be of value in defining procedures and treatments designed to promote neuron survival and recovery subsequent to neurological disease or trauma. REFERENCES Allen, R. D., J. Metuzals, I. Tasaki, S. Brady and S. Gilbert (1982) Fast axonal transport in squid giant axon, Science, 218:1127-1129. Black, M. M. and R.J. Lasek (1979) Axonal transport of actin - - Slow component b is the principal source of actin for the axon, Brain Res., 171: 401-413. Black, M.M. and R.J. Lasek (1980) Slow components of axonal transport - - Two cytoskeletal networks, J. Cell BioL, 86: 616-623. Bonner, W.M. and R.A. Laskey (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels, Europ. J. Biochem., 46: 83-88. Borges, L. F, and R.L. Sidman (1982) Axonal transport of lectins in the peripheral nervous system, J. Neurosci., 2: 647-653. Brady, S.T. and R.J. Lasek (1981) Nerve-specific enolase and creatine phosphokinase in axonal transport - - Soluble proteins and the axoplasmic matrix, Cell, 23: 515-523. Brady, S.T. and R.J. Lasek (1982) Axonal transport - - A cell-biological method for studying proteins that associate with the cytoskeleton, In: L. Wilson (Ed.), Methods in CellBiology, Vol. 25, Part b, Academic Press, New York, pp. 365-398. Brady, S.T., M. Tytell, K. Heriot and R.J. Lasek (1981) Axonal transport of calmodulin - - A physiologic approach to identification of long-term associations between proteins, 3. Cell BioL, 89: 607-614.
18 Burdwood, W.O. (1965) Rapid bidirectional particle movement in neurons, J. Cell Biol., 27:115A. Cancalon, P. (1984)The relationship of slow axonal flow to nerve elongation and degeneration. In: J. S. Elam and P. Cancalon (Eds.), Advances in Neurochemistry, Vol. 6, Plenum Press, New York, pp. 211-241. Droz, B. and C. P. Leblond (1963) Axonal migration of proteins in the central nervous system and peripheral nerves as shown by radioautography, J. Comp. Neurol., 121: 325-337. Ellisman, M. H. and K. R. Porter (1980) Microtrabecular structure of the axoplasmic matrix - - Visualization of cross-linking structures and their distribution, J. Cell Biol., 87: 464-479. Garner, J.A. and R.J. Lasek (1981) Clathrin is axonally transported as part of slow component b - - The microfilament complex, J. Cell Biol., 88: 172-178. Garner, J.A. and R.J. Lasek (1982) Cohesive axonal transport of the slow component b complex of polypeptides, J. Neurosci., 2: 1824-1835. Goldberg, S. and M. Kotani (1967) The projection of optic nerve fibers in the frog Rana catesbeiana as studied by radioautography, Anat. Rec., 158: 325-332. Grafstein, B. and D.S. Forman (1980) Intercellular transport in neurons, Physiol. Rev., 60: 1167-1283. Hoffman, P.N. and R.J. Lasek (1975)The slow component of axonal transport - - Identification of major structural polypeptides of the axon and their generality among mammalian neurons, J. Cell Biol., 66: 351-366. Hoffman, P.N. and R.J. Lasek (1980) Axonal transport of the cytoskeleton in regenerating motor neurons - - Constancy and change, Brain Res., 202: 317-333. Kremzner, L.T. and R.T. Ambron (1982) Metabolism and axonal transport of polyamines in a single identified neuron of Aplysia californica, J. Neurochem., 38: 1719-1727. Lasek, R. (1968) Axoplasmic transport in cat dorsal root ganglion cells - - As studied with [3H]-L-leucine, Brain Res., 7: 360-377. Lasek, R.J., J.A. Garner and S.T. Brady (1984) Axonal transport of the cytoplasmic matrix, J. Cell Biol., 99: 212s-221s. Laskey, R.A. and A.D. Mills (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography, Europ. J. Biochem., 56: 335-341. Ramon y Cajal, S. (1928) Degeneration and Regeneration of the Nervous System (transl. by R. M. May), Oxford University Press, Cambridge. Schnapp, B. J. and T. S. Reese (1982) Cytoplasmic structure in rapid-frozen axons,J. CellBiol., 94: 667-679. Tytell, M., M.M. Black, J.A. Garner, and R.J. Lasek (1981) Axonal transport - - Each major rate component reflects the movement of distinct macromolecular complexes, Science, 214: 179-181. Weiss, P. and H.B. Hiscoe (1948) Experiments on the mechanism of nerve growth, J. Exp. ZooL, 107: 315-395.