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Axonal transport of actin: Slow component b is the principal source of actin for the axon
Brain Research, 171 (1979)401-413 © Elsevier/North-Holland Biomedical Press
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A X O N A L T R A N S P O R T OF ACTIN: SLOW C O M P O N E N T b IS T H E PRINCIPAL SOURCE OF ACTIN FOR T H E AXON
MARK M. BLACK* and RAYMOND J. LASEK Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 (U.S.A.)
(Accepted November 23rd, 1978)
SUMMARY Axonally transported proteins were studied in guinea pig retinal ganglion cells using the standard radioisotopic labeling procedure. Two slowly moving groups of proteins were identified in guinea pig retinal ganglion cells. The more slowly moving group of proteins, designated slow component a (SCa) was transported at 0.2-0.5 mm/day. Five polypeptides contained greater than 75~o of the total radioactivity transported in SCa. Two of these polypeptides correspond to the subunits of tubulin, while the other three correspond to the slow component triplet16,1L The other slowly moving group of proteins, which is designated slow component b (SCb), was transported at approximately 2 ram/day. Twenty labeled polypeptides were identified in SCb. The major labeled polypeptides transported in SCb differ from those transported in SCa. One of the polypeptides transported in SCb co-migrates with skeletal muscle actin in SDS-polyacrylamide slab gels. This polypeptide behaved identically to skeletal muscle actin on DNaseI affinity columns. Since DNaseI is a highly specific affinity ligand for actin 21, we conclude that the labeled SCb polypeptide which comigrates with actin in SDS-gels is actin. Between 1.4 and 5.7 of of the total radioactivity transported in SCb is attributable to actin. Detailed comparison of the distribution of total radioactivity in the optic axons with the distribution of radioactive actin in the optic axons at post-injection times between 6 and 77 days showed that actin was transported specifically in SCb, and not in SCa. Furthermore, analyses of the proteins transported in the fast component of guinea pig retinal ganglion cells by DNaseI affinity chromatography failed to reveal an actin-like moiety. Slow component a, SCb and the fast component are the major corn* Present address: Neuroscience Department, The Children's Hospital Medical Center, 300 Longwood Avenue, Boston, Mass. 02115, U.S.A.
402 ponents of axonal transport in guinea pig retinal ganglion cells. Thus, in these neurons, actin is transported principally and possibly only in SCb. Guinea pig retinal ganglion cell axons project principally to the lateral genicutate nucleus and superior colliculus. The fate of actin axonally transported to the region of the axon terminals was studied by determining the kinetics by which radioactivity associated with actin accumulates and then decays in the superior colliculus. The results of these studies indicate that labeled actin has a half-life in the superior colticulus of approximately 28 days.
INTRODUCTION The presence of actin in axons and axon terminals is well documented4,7, 2a,2~,29-~1. Although much is known about the chemistry2,12,26,29 and morphological organization7,22-25 of neuronal actin, there is little information available regarding the physiological properties of actin in vivo. Yet, such information is essential to fully understand the functional properties of actin in axons and their terminals. In the present study, we have begun to characterize the properties of actin in living axons by studying its transport within axons. Hoffman and Lasek identified a slowly transported polypeptide in rat ventral motor neurons which co-migrated with actin in SDSgels 16,19. This observation raised the possibility that actin was transported slowly in these axons. We have identified a similar slowly transported polypeptide in guinea pig retinal ganglion cell axons, and have provided strong evidence that this polypeptide is actin. Detailed analyses of the transport kinetics of actin indicate that it is transported principally, and possibly only in one of the three major rate components of guinea pig retinal ganglion cells. This work has been published in preliminary form s . MATERIALS AND METHODS
Radioactively labeling axonally transported proteins in guinea pig retinal ganglion celia" We employed the retinal ganglion cell system of guinea pigs (Harttey strain) in our studies. The axons of retinal ganglion cells project to the lateral geniculate nucleus and superior colliculus via the optic nerve and optic tract. In the strain of guinea pigs employed, this projection is completely crossed 1~. Thus, we were able to use both eyes of each animal, and treat each optic nerve and its contralateral optic tract as individual samples. Guinea pigs, 700-900 g, were anesthetized with sodium pentobarbital (30 mg/kg) administered intraperitoneally. Nine microliters of a [all]amino acid solution were injected into the posterior chamber of each eye. The injection system consisted of a 26gauge needle attached to a 10/zl Hamilton microsyringe. The plunger of the microsyringe was driven by a handoperated micrometer screw. In most experiments, [aH]lysine (40-60 Ci/mmol; NEN) was employed. Immediately before use, an aliquot of the stock solution of [aH]lysme was brought to dry-
403 ness by lyophilization and then was dissolved in water at a final concentration of 20 #Ci//zl. For those experiments examining the behavior of axonally transported proteins on DNaseI affinity columns (see below) a 1:1 mixture of [3H]lysine and [ZH]proline (20 #Ci/#l) was used.
The distribution of total radioactivity & the optic axons At several post-injection times, animals were anesthetized with ether and then decapitated. Each optic nerve, together with its contralateral optic tract and contralateral superior colliculus, was carefully dissected from each animal. Since both eyes were injected with [3H]amino acids, the optic chiasm (approximately 2 mm in length) was not included in these analyses. Each optic nerve together with its contralateral optic tract was cut into consecutive 3 mm segments. Each segment and the superior colliculus was homogenized in 0.325 ml of 8 M urea, 1 ~ SDS and 5 ~o 2-mercaptoethanol with a glass microhomogenizer. The homogenates were incubated at 95 °C for 5 rain and then were centrifuged at 27,000 × g for 1 h (Sorvall SS-34 rotor), and the supernates collected (the superior colliculus sample was centrifuged at 200,000 × g for 1 h in the Beckman SW50.1 rotor). Regardless of the centrifugation conditions, greater than 98 ~ of the radioactivity in the tissue homogenate remained in the supernate. The radioactivity in the supernate from each 3 mm segment was determined by scintillation counting (data were corrected for quenching by the method of external standardization). The radioactivity in each segment was then plotted against distance from the eye (see Fig. 1). The remainder of each sample was analyzed by SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis (SDS-page). Electrophoresis was performed in 7.5 ~ SDS-polyacrylamide slab gels in the discontinuous buffer system of Neville 27. Gels were stained with 0.25 ~o Coomassie blue, then the labeled polypeptides in each gel were visualized by fluorographya, zo. Actin prepared from guinea pig skeletal muscle 3a was used as a molecular weight standard. Other molecular weight standards included vitellogenin (mol. wt. 240,000, kindly supplied by L. Gehrke), rabbit skeletal muscle myosin (mol. wt. 200,000), phosphorylase a and bovine serum albumin (tool. wt. 94,000 and 68,000, respectively, from Sigma Chemical Co.). After obtaining the necessary fluorographs from the gels, the amount of radioactivity within selected labeled polypeptides was determined. The fluorographs were used to locate the position of the labeled polypeptides in the gels. The appropriate region of the gel was then cut out and incubated in 0.5 ml of 30 ~ HzO2 at 60 °C for 2 days to solubilize the radioactivity. Five milliliters of a toluene-Triton X100 scintillation cocktail was added to each sample and the amount of radioactivity determined. Data were corrected for quenching by the method of internal standardization (the counting efficiency ranged between 18 and 23 °/oo). D NaseI affinity chromatography DNase[ is a highly specific affinity ligand for actin 21. We used DNaseI affinity columns to determine if actin was axonally transported in guinea pig retinal ganglion
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Fig. 1. The distribution of total radioactivity within the optic axons at post-injection times of 6, 9, 15 and 25 days. The 3H content of consecutive 3 mm segments is plotted against distance from the eye. Each profile is representative of from 2 to 6 determinations. cells. DNaseI (from bovine pancreas: Sigma Chemical Co.) was bound to Sepharose 4B (Pharmacia). The Sepharose 4B was activated by CNBr treatment as described previously 21. The activated resin was washed on a Buchner funnel with 0.1 M NaHCO3 and resuspended in 0.1 M CaC12. DNaseI, dissolved in 0.1 M NaHCOs, 0.1 mM CaCIz, was incubated with the activated Sepharose overnight. The resulting material was extensively washed with distilled water and then with buffer 1 (0.15 M NaCI, 1.0 mM CaC12, 0.05 M Tris. pH 7.4). Labeled axonally transported proteins were obtained from the optic nerves and tracts of 4 guinea pigs sacrificed 9 days post-injection. The tissue was processed at 0-4 °C. The tissue was homogenized in 1 ml of 0.25 M sucrose. 1 mM CaCI2, 0.05 M Tris. pH 7.4, and centrifuged at 12,000 x g for 20 rain (Sorvall SS-34 rotor). The supernate was saved and the pellet was rehomogenized in 0.25 ml of the homogenization solution and re-centrifuged. The supernate from the second spin was combined with that of the first, and the combined sample was passed over a DNaseI affinity column (packed volume, 1 ml). The subsequent steps were performed at room temperature. The column was washed with a series of 4 buffers; first with 5 ml of buffer 1, followed by 2 ml of buffer 2 (0.5 M sodium acetate, lmM CAC!2,0.05 M Tris, pH 7.4), then with 2 ml of buffer 3 (buffer 2 -- 0.5 M urea) and finally with 5 ml of buffer 4 (8 M urea). One milliliter fractions were collected. Aliquots of each fraction were taken for determination of total radioactivity. Appropriate fractions were pooled and dialyzed against 8 M urea and 1% SDS. After dialysis. 2-mercaptoethanol was added to each sample to a final concentration of 5 %, and the samples were then incubated at 95 °C for 5 min. The stained and labeled polypeptides in each sample were analyzed by SDS-page and fluorography. RESULTS
Two slowly moving groups of axonally transported proteins Two slowly moving groups of proteins are identifiable in guinea pig retinal ganglion cells. Fig. 1 shows the distribution of total radioactivity within the optic nerve and contralateral optic tract at 6, 9, 15 and 25 days post-injection, The more rapidly moving group of proteins was apparent as a wave of: radioactivity within the
405
Fig. 2. The profile of labeled polypeptides conveyed in the two slowly transported groups of proteins in guinea pig retinal ganglion cells. The figure shows a fluorograph depicting the labeled polypeptides located 4-7 mm from the eye at 6 (SCb) and 25 (SCa) days post-injection. SCa and SCb nomenclature is explained in the text. The samples were electrophoresed on different gels. Molecular weights are indicated adjacent to appropriate bands. optic nerve and tract at 6 and 9 days post-injection. The proximo-distal shift in the position of this wave with time corresponds to a transport rate of approximately 2 m m / day. By 15 days post-injection, only the trailing portion of this wave was present within the optic nerve and tract; the rest of the radioactively labeled proteins comprising this wave had entered the terminal region of the optic axons located in the lateral geniculate nucleus and superior colliculus (data not shown). When the distribution of radioactivity within the optic axons was determined at progressively longer post-injection times, the second, more slowly moving group of axonally transported proteins appeared. At 25 days post-injection, this group of proteins appeared as a wave of radioactivity within the optic nerve and tract, the crest of which was located 4-7 mm from the optic nerve head. The proximo-distal shift in the position of this wave between 25 and 77 days post-injection (see Fig. 5) corresponds to a transport rate of 0.2-0.5 mm/day. These two slowly transported groups of proteins correspond to the movement of distinctly different sets of polypeptides within the axon (Fig. 2). The more slowly moving group of proteins is designated slow component a (SCa). Five polypeptides contain greater than 75 °/ooof the radioactivity transported in SCa. These 5 polypeptides correspond to those originally identified in the slow component (SCa) of rat
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Fig. 3. The profile of radioactivity eluting from a DNasel affinity column. The tissue, which was obtained from the optic nerves and tracts of 4 guinea pigs sacrificed 9 days post:injection, was processed as described in the materials and methods section. The column was eluted with a series of 4 buffers (see Materials and Methods). The beginning of buffer washes 2, 3 and 4 are indicated by arrows. These data are representative of two such experiments.
ventral motor neurons16,19. The labeled polypeptides with molecular weights of 53,000 and 57,000 daltons correspond to the subunits of tubulin, the major microtubule protein. The polypeptides with molecular weights of 200,000, 145,000 and 68,000 daltons correspond to the slow component triplet and apparently are the subunits of neurofilaments 16,19. In addition to these 5 major labeled polypeptides, 4 minor labeled polypeptides are also transported in SCa of' guinea pig retinal ganglion cells. Two of these have molecular weights of approximately 240,000 daltons, while the other two have molecular weights of 62,000 and 64,000 daltons. The more rapidly moving group of slowly transported proteins is designated slow component b (SCb). At least 20 polypeptides are transported in SCb. The major labeled polypeptides have molecular weights of > 400,000, 105,000, 85,000, 70,000, 60,000, 48,000, 43,000, 35,000, 27,000 and 25,000 daltons. The pattern of labeled polypeptides comprising SCb differs completely from that of SCa. The axonal transport o f actin in slow component b The labeled polypeptide in SCb with a molecular weight of 43,000 daltons comigrates with skeletal muscle actin in SDS-polyacrylamide slab gels. In order to determine whether or not this labeled SCb polypeptide was actin, we examined its behavior on DNaseI affinity columns. Labeled SCb proteins o f guinea pig retinal ganglion cells were obtained from the optic nerves and tracts of 4 animals sacrificed 9 days postinjection. A soluble fraction was obtained from the tissue (see Materials and Methods) and applied to the DNaseI affinity column. This fraction contained 95 % of the total radioactivity in the tissue. A representative example of the profile of radioactivity eluting from the DNaseI affinity column is illustrated in Fig, 3. Of the radioactivity applied to the column, approximately 90 % passed through in the first buffer wash. The second and third buffer washes eluted 1% and 0.5 %, respectively, of t h e radioactivity
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Fig. 4. The profile of stained (panel A) and labeled (panel B) polypeptides eluting from the DNasel affinity column depicted in Fig. 3. The gel columns contain the following samples: H, the tissue homogenate; S, the supernate from the tissue homogenate (this material was applied to the column); 1, the material passing through the column in buffer 1 ; 2, the material eluting in buffer 2; 4, the material eluting in buffer 4. Columns H, S and 1 in panel B are from a fluorograph developed after 1 week of exposure to the X-ray film, while columns 2 and 4 are from a fluorograph developed after 4 weeks of exposure to the X-ray film. applied to the column. The final buffer wash, which was 8 M urea, eluted 2 - 4 ~ o f the r a d i o a c t i v i t y applied to the column. The labeled m a t e r i a l eluting in the various buffer washes was analyzed by S D S - p a g e and f t u o r o g r a p h y (Fig. 4). The 8 M urea fraction c o n t a i n e d a single m a j o r b a n d (Fig. 4A, well 4). Its e l e c t r o p h o r e t i c m o b i l i t y was identical to t h a t o f skeletal muscle actin. The r e m a i n i n g stained m a t e r i a l m i g r a t e d with the dye front. As seen in
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Fig. 5. The distribution of total radioactivity (O) and radioactivity associated with acfin (C.) within the optic axons at 6, 9, 15, 25, 35 and 77 days post-injection. The distribution of total radioactivity was determined as described in the materials and methods section. The distribution of radioactivity associated with actin was determined as follows, Guinea pigs were sacrificed atthe indicatedtimes and the optic nerves, together with the contralateral optic tracts, removed. Each optic nerve-optic tract was cut into consecutive 3 mm segments. The labeled polypeptides within each segment was analyzed by SDS-page and fluorography. The radioactivity co-migrating with actin were quantitated as described in Materials and Methods. The amount of radioactivity co-migrating with actin was then graphed against distance from the eye. Each profile is representative of from 2 to 6 determinations. Fig. 4B, well 4, most of the radioactivity (approximately 70 °/o) eluting in 8 M urea is contained within the 43,000 dalton band; of the remaining radioactive material, most migrated with the dye front. The nature of this latter material was not determinedl Actin is not transported in slow component a In order to determine whether actin was transported in both SCb a n d SCa, or only in SCb, we compared the distribution of radioactivity in the optic axons which co-migrated with actin with the distribution of total radioactivity in the optic axons at several post-injection times between 6 and 77 days. (Analyses of the labeled SCb polypeptides by two-dimensional I E F - S D S gels have shown that actin is the most prominent labeled species which co-migrates with actin in one-dimensional SDS gels (Tytell and Lasek, unpublished). The results of this analysis are shown in Fig. 5. At 6 and 9 days post-injection, the distribution of radioactive actin within the optic nerve and contralateral optic tract exhibits a wave-like form. At each of these times, the shape and position of the radioactive actin wave is very similar to that of the radioactive SCb wave. These observations clearly demonstrate that the labeled actin within the optic axons at 6 and 9 days post,injection is transported in SCb. If actin were also transported in SCa, then, at appropriate post-injection times, the distribution of radioactive actin should exhibit a wave-like form that closely resembles the radioactive SCa wave. As seen in Fig. 5, the radioactive actin wave and the radioactive SCa wave are out of phase. In fact, only the trailing portion of the actin wave Overlaps the SCa wave. These observations demonstrate that, in guinea pig retinalganglion cells,
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Fig. 6. The amount of radioactivity in the superior colliculus which co-migrates with actin at several post-injection times between 15 and 100 days. At the indicated times, the labeled polypeptides in the superior colliculus were analyzed by SDS page and fluorography. The amount of radieactivity comigrating with actin was quantitated as described in the materials and metheds section. The mean standard errors are indicated (the values in parentheses indicate n).
actin is not transported coherently with the proteins of SCa. The radioactive actin in the optic axons at post-injection times greater than 25 days was deposited in the optic axons during the transit of the labeled SCb proteins from the cell body to the axon terminal. Actin is not transported in the fast component Labeled fast component proteins were harvested from the superior colliculus and lateral geniculate nucleus at 16 h post-injection and prepared for DNasel affinity chromatography (using the same protocol described in Materials and Methods). Of the 2.5 x 105 cpm in the tissue homogenate, approximately 1% was recovered in the 8 M urea fraction from a DNasel affinity column. When this labeled material was analyzed by SDS-page and fluorography, using an exposure time calculated to detect 20 dpmS, 20, we were unable to detect any labeled polypeptides. Thus, if actin is present in the fast component, it accounts for less than 0.002% of this class of axonally transported materials. Axonal transport o f actin to the axon terminal Actin has been identified in the axon terminal T M . If the actin in the axon terminal is delivered by SCb, then at appropriate post-injection times we should be able to identify labeled actin in the region of the CNS containing the terminals of the optic axons. We examined this by measuring the amount of radioactivity in the superior colliculus (a major termination site for retinal ganglion cell axons) which comigrated with actin in SDS slab gels at several post-injection times between 6 and 100 days. The results of these analyses are shown in Fig. 6. We were unable to detect labeled actin in the superior colliculus at 6 and 9 days post-injection. Subsequent to this time, radioactivity associated with actin appeared in the superior colliculus in concert with the entry of the radioactive SCb wave. The amount of radioactivity in the superior colliculus which co-migrated with actin increased through day 49 post-injection, subsequent to which it decreased in an apparantly exponential manner. The half-life of the radioactivity associated with actin in the superior colliculus was
410 approximately 28 days. The radioactive actin located in the superio~ colliculus between 15 and 100 days post-injection could be located in the synaptic terminals of the optic axons and/or in the preterminal portion of the optic axons.
DISCUSSION
The behavior of the 43,000 dalton polypeptide of SCb on DNaseI affinity columns provides strong evidence that this polypeptide is actin. DNasel is an affnity ligand for actin 21. The binding of actin to DNaseI is so tight that it can only be disrupted by strong denaturants such as 8 M urea or 3 M guanidine HC1. Furthermore, actin is apparently the only protein which DNaseI binds so tightly. Since the 43,000 dalton polypeptide transported in SCb interacts with DNasei in a manner which is characteristic of actin, we conclude that this polypeptide is actin. In support of this conclusion is the observation that the labeled 43,000 dalton SCb polypeptide co-migrates with actin on two-dimensional I E F - S D S gels (Tytell and Lasek, unpublished). Is actin transported in components of axonal transport other than SCb? The observation that the radioactive actin wave is out of phase with the radioactive SCa wave (Fig. 5) indicates that actin is not transported in SCa of guinea pig retinal ganglion cells. Components of axonal transport which are comparable to SCa and SCb in both transport rate and polypeptide composition have been identified in other neuronal systems (guinea pig hypoglossal motor neurons, Black and Lasek, in preparation; rat ventral motor neurons, Hoffman and Lasek, submitted). In these neuronal systems, as in guinea pig retinal ganglion cells, a 43,000 dalton polypeptide (presumably actin) is also transported in SCb, but not SCb. Several observations indicate that actin is not transported in the fast component of axonaltransport. First, we were unable to detect actin in the fast component of guinea pig retinal ganglion cells on the basis of DNaseI affinity chromatography. Second, analyses of the proteins transported in the fast component of frog spinal ganglion cells 35 and guinea pig retinal ganglion cells (Tytell and Lasek, unpublished) by two-dimensional IEF-SDS gels did not reveal an actin-like polypeptide. Since two independent assays failed to detect actin in the fast component, we conclude that actin. if it is present in the fast component, is present in vanishingly small amounts. In addition to SCa, SCb and the fast component, there are groups of proteins transported at rates in-between that of SCb and the fast component 17,1s,'~8. It is not known whether or not actin is a constit uent of any of these intermediate components. Relative to SCb, the intermediate components contribute a small amount of material to the axon. Thus, if actin is present in any of the intermediate components, it can only account for a small percentage o f the total axonally transported actin. On the basis of these considerations, we conclude that in guinea pig retinal ganglion cells, SCb is the principal and possibly only vehicle for the delivery of actin to the axon and its terminals. Quantitative evaluation of the gel profile of labeled material transported in SCb
411 (see Fig. 5 for example) indicates that approximately 5.7 ~ of this labeled material comigrates with actin. However, not all of this labeled material is attributable to actin, since analyses of the labeled SCb polypeptides by two-dimensional IEF-SDS gels have shown that the labeled material co-migrating with actin in SDS gels includes two or three polypeptides in addition to actin (Tytell and Lasek, unpublished). Thus, actin contains less than 5.7 ~ of the radioactivity in the SCb wave. Another estimate of the amount of radioactivity which actin contributes to the SCb wave can be obtained from the behavior of the labeled SCb proteins on DNaseI affinity columns. Since 2--4 ~ of the radioactivity applied to the colunm eluted in 8 M urea, and approximately 70 ~ of this radioactivity is in actin, 1.4-2.8 ~ of the labeled material applied to the column is actin. However, this value may underestimate the fraction of radioactivity in the SCb wave which is attributable to actin since actin may not have been quantitatively bound by the DNaseI affinity column. On the basis of these considerations, we conclude that between 1.4 and 5.7 °/o of the total radioactivity in the SCb wave is attributable to actin. Four to six nanometer actin-containing microfilaments have been identified in axons and their terminals 7,~3,z4,3°. The actin comprising these microfilaments most likely entered the axon in SCb since SCb is the principal and possibly only vehicle for the delivery of actin to the axon and axon terminal. Thus, the 4-6 nm microfilament is a morphological representation of the actin transported in SCb. It is also likely that other proteins transported in SCb are constituents of microfilaments. In this regard, tropomyosin, a protein component of microfilaments in muscle and non-muscle cells, may also be transported in SCb. The evidence for this is as follows. First, using a protocol for the isolation of brain tropomyosin 11, we have partially purified a labeled protein from SCb of guinea pig retinal ganglion cells. Secondly, a protein has been identified in SCb (group IV according to Willard et al. 3s) of rabbit retinal ganglion cells which has a subunit molecular weight closely resembling that of brain tropomyosin, and which also exhibits a specific affinity for actin filaments in vitro 37. Within axons and axon terminals, actin-containing microfilaments often appear physically associated with membrane structures such as the smooth endoplasmic reticulum, synaptic vesicles and the axonal and synaptic plasma membrane23,24, z0. As discussed previously, the actin comprising these microfilaments enters the axon in SCb. However, the membrane vesicles with which actin microfilaments interact are not transported in SCb. Rather, the available evidence indicates that these vesicular structures, which include transmitter storage vesicles and the smooth endoplasmic reticulum, are transported in the fast component of axonal transportl,8-10,14,15. Since the transport rate of actin is two orders of magnitude slower than that of the membrane structures conveyed in the fast component (2-4 ram/day for SCb; 250-400 ram/day for the fast component28), the interaction between actin microfilaments and these membrane structures inferred from ultrastructural observations must be of a transitory natme. The transient association of actin microfilaments with membrane structures transported in the fast component may be indicative of the participation of actin microfilaments in the convection of these structures within the axon. If so, then actin microfilaments can be viewed as relatively stationary elements against which these membrane structures move.
412 M a n y models which have been proposed for the motile mechanism of a x o n a l t r a n s p o r t are based o n a muscle-like a c t o m y o s i n system6,16,19,23,28, 33. The presence of actin7, 2z a n d m y o s i n 82,36 in axons fulfills a requisite of these models. Since actin enters the axon principally a n d possibly only in SCb, c o m p o n e n t s of axonal t r a n s p o r t which are m o v e d by a n actomyosin-like system must utilize the actin which entered the axon in SCb. F u r t h e r m o r e , if a muscle-like a c t o m y o s i n system generates the m o v e m e n t of c o m p o n e n t s which lack actin, such as the fast c o m p o n e n t , then these c o m p o n e n t s m u s t c o n t a i n a myosin-like p a r t n e r which can interact with the actin in SCb. In this regard, a myosin-like p r o t e i n has been tentatively identified in the fast c o m p o n e n t of r a b b i t retinal g a n g l i o n cells 37. ACKNOWLEDGEMENTS We are very grateful to Ms. Shirley Ricketts for her excellent technical assistance t h r o u g h o u t the course of this work. This work was s u p p o r t e d by N a t i o n a l I n s t i t u t e s of Health G r a n t s NS09299 a n d NS13658 to Dr. Lasek a n d N a t i o n a l Institutes of Health Traineeship HD00020 to Dr. Black.
REFERENCES 1 Bennett, G., Di Giamberardino, L., Koenig, H. L. and Droz, B., Axonal migration of protein and glycoprotein to nerve endings. II. Radioautographic analysis of the renewal of glycoproteins in nerve endings of chick ciliary ganglion after intracerebral injection of [3H]fucose and [ZH]glucosamine, Brain Research, 60 (1973) 129-146. 2 Berl, S. and Puszken, S., Mg-Ca activated adenosinetryphosphatase system isolated from mammalian brain, Biochemistry, 9 (1970) 2058-2067. 3 Black, M. M. and Lasek, R. J., The axonal transport of actin, J. Cell Biol., 75 (1977) 110a. 4 Blitz. A. L. and Fine, R. E., Muscle-like contractile proteins and tubulin in synaptosomes, Proc. nat. Acad. Sci. (Wash.), 71 (19741 4472-4476. 5 Banner, W. M. and Laskey, R. A., A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels, Europ. J. Biochem., 46 (1974) 83-88. 6 Byers, M. R., Structural correlates of rapid axonal transport: evidence that microtubules may not be directly involved, Brain Research, 75 (1974) 97-113. 7 Chang, C. M. and Goldman, R. D., The localization of actin-like fibers in cultured neuroblastoma cells as revealed by heavy meromyosin binding, J. Cell Biol., 57 (1973) 867-874. 8 Di Giamberardino, L., Bennett, G., Koenig, H. L. and Droz, B., Axonal migration of protein and glycoprotein to nerve endings. III. Cell fractionation analysis of chicken ciliary ganglion after intracerebral injection of labeled precursors of proteins and glycoproteins, Brain Research, 60 (1973) 147-159. 9 Droz, B., Koenig, H. L. and Di Giamberardino, L.. Axonal migration of protein and glycoprotein tc nerve endings. I. Radioautographic analyses of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [aH]lysine, Brain Research, 60 (1973) 93-127. 10 Droz, B., Rambourg, A. and Koenig, H. L., The smooth endoplasmic reticulum: structure and role in the renewal of axonal membrane and synaptic vesicles by fast axonal transport, Brain Research, 93 (1975) 1-13. 1t Fine, R. E., Blitz, A. L., Hitchcock, S. E. and Kaminer, B., Tropomyosin in brain and growing neurones, Nature New Biol., 245 (1973) 182-186. 12 Fine, R. E and Bray, D., Actin in growing nerve cells, Nature New Biol., 234 (1971) 115-118. 13 Giolli, R. A. and Creel, D. J., The primary optic projection in pigmented and albino guinea pigs: an experimental degeneration study, Brain Research, 105 (1973) 25-39. 14 Goldman, J. E., Kim, K. S. and Schwartz, J. H., Axonal transport of [aH]serotonin in an identified neuron of Aplysia californica, J. Cell. Biol., 70 (1976) 304-318.
413 15 Goldman, J. E. and Schwartz, J. H., Cellular specificity of serotonin storage and axonal transport in identified neurones of Aplysia ealiforniea, J. Physiol. (Lond.), 242 (1974) 61-76. 16 H offman, P. N. and Lasek, R. J., The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons, J. Cell Biol., 66 (1975) 351 366. 17 Karlsson, J. O. and Sj6strand, J., Synthesis, migration and turnover of protein in retinal ganglion cells, J. Neurochem., 18 (1971) 749 767. 18 Lasek, R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289 324. 19 Lasek, R. J. and Hoffman, P. N., The neuronal cytoskeleton, axonal transport and axonal growth. In R. Goldman, T. Pollard and J. Rosenbaum, (Eds.), Cell Motility, Cold Spr. Harb. Conf Cell ProliJeration, Vol. 3, 1976, pp. 1021 1049. 20 Laskey, R. A. and Mills, A. D., Quantitative film detection of [all] and [a4c] in polyacrylamide gels by fluorography, Europ. J. Biochem., 56 (1975) 335 341. 2l Lazarides, E. and Lindberg, U., Actin is the naturally occurring inhibition of deoryribonuclease 1, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 4742-4746. 22 LeBeux, Y. J., An ultrastructural study of the synaptic densities, neoatosomes, neurotubular neurofilaments and of a further three-dimensional filamentous network as disclosed by the E-PTA staining procedure, Z. Zellforsch., 143 (1973) 239-272. 23 LeBeux, Y. J. and Willemot, J., An ultrastructural study of the microfilaments in rat brain by means of heavy meromyosin labeling. I. The peukaryon, the dendrites and the axon, Cell Tiss. Res., 160 (1975) 1 36. 24 LeBeux, Y. J. and Willemot, J., An ultrastructural study of the microfilaments in rat brain by means of E-PTA staining and heavy meromyosin labeling, Cell Tiss. Res., 160 (1975) 37-68. 25 Metuzals, J. and Muchynski, W. E., Election microscope and experimental investigations of the neurofilamentous network in Deiters' neurons: relationship with the cell surface and nuclear pores, J. Cell Biol., 61 (1974) 701 702. 26 Moring, S., Nuscha, M., Cooke, P. and Samson, F., Isolation and polymerization of brain actin, J. Neurobiol., 6 (1975) 245-255. 27 Neville, D. M., Molecular weight determination of protein dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system, J. biol. Chem., 246 (1971) 6328 6334. 28 Ochs, S., Fast transport of materials in mammalian nerve fibers, Science, 176 (1972) 252 260. 29 Puszken, S. and Kochwa, S., Regulation of neurotransmitter release by a complex of actin with relaxing protein isolated from rat brain synaptosomes, J. biol. Chem., 249 (1974) 7711-7714. 30 Puszken, S., Kochwa, S. and Rosefield, R. E., Ultrastructural evidence of association of contractile proteins in synaptosomes. In R. Goldman, T. Pollard and J. Rosenbaum (Eds.), Cell Motility, Cold Spr. Harb. CoJl[i Cell Proliferation, Vol. 3, 1976, pp. 665 670. 31 Puszken, S., Nicklas, W. J. and Berl, S., Actomyosin-like protein in brain: subcellular distribution, J. Neurochem., 19 (1972) 1319-1333. 32 Roisen, F., lnezedy-Marcsek, M., Hus, L. and Yorke, W., Myosin: immunofluorescent localization in neuronal and glial cultures, Science, 199 (1978) 1445 1448. 33 Schmitt, F. O., Fibrous proteins, neuronal organelles, Proc. nat. Aead. Sci. (Wash.), 60 (1968) 1092-1101. 34 Spudich, J. A. and Watts, S., The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin, J. biol. Chem., 246 (1971) 4866 4871. 35 Stone, G. C., Wilson, D. L. and Hall, M. E., Two-dimensional gel electrophoreses of proteins in rapid axoplasmic transport, Brain Research, 144 (1978) 287-302. 36 Unsicker, K., Drenckhahn, D. and Gr6schel-Stewart, U., Further immunofluorescence-microscopic evidence for myosin in various peripheral nerves, Cell Tiss. Res., 188 (1978) 341 344. 37 Willard, M., The identification of two intra-axonally transported polypeptides resembling myosin in some respects in the rabbit visual system, J. Cell Biol., 75 (1977) 1-11. 38 Willard, M., Cowan, W. M. and Vagelos, P. R., The polypeptide composition of intra-axonally transported proteins: evidence for four transport velocities, Proc. nat. Aead. Sci. (Wash.), 71 (1974) 2183-2187.
40l
A X O N A L T R A N S P O R T OF ACTIN: SLOW C O M P O N E N T b IS T H E PRINCIPAL SOURCE OF ACTIN FOR T H E AXON
MARK M. BLACK* and RAYMOND J. LASEK Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 (U.S.A.)
(Accepted November 23rd, 1978)
SUMMARY Axonally transported proteins were studied in guinea pig retinal ganglion cells using the standard radioisotopic labeling procedure. Two slowly moving groups of proteins were identified in guinea pig retinal ganglion cells. The more slowly moving group of proteins, designated slow component a (SCa) was transported at 0.2-0.5 mm/day. Five polypeptides contained greater than 75~o of the total radioactivity transported in SCa. Two of these polypeptides correspond to the subunits of tubulin, while the other three correspond to the slow component triplet16,1L The other slowly moving group of proteins, which is designated slow component b (SCb), was transported at approximately 2 ram/day. Twenty labeled polypeptides were identified in SCb. The major labeled polypeptides transported in SCb differ from those transported in SCa. One of the polypeptides transported in SCb co-migrates with skeletal muscle actin in SDS-polyacrylamide slab gels. This polypeptide behaved identically to skeletal muscle actin on DNaseI affinity columns. Since DNaseI is a highly specific affinity ligand for actin 21, we conclude that the labeled SCb polypeptide which comigrates with actin in SDS-gels is actin. Between 1.4 and 5.7 of of the total radioactivity transported in SCb is attributable to actin. Detailed comparison of the distribution of total radioactivity in the optic axons with the distribution of radioactive actin in the optic axons at post-injection times between 6 and 77 days showed that actin was transported specifically in SCb, and not in SCa. Furthermore, analyses of the proteins transported in the fast component of guinea pig retinal ganglion cells by DNaseI affinity chromatography failed to reveal an actin-like moiety. Slow component a, SCb and the fast component are the major corn* Present address: Neuroscience Department, The Children's Hospital Medical Center, 300 Longwood Avenue, Boston, Mass. 02115, U.S.A.
402 ponents of axonal transport in guinea pig retinal ganglion cells. Thus, in these neurons, actin is transported principally and possibly only in SCb. Guinea pig retinal ganglion cell axons project principally to the lateral genicutate nucleus and superior colliculus. The fate of actin axonally transported to the region of the axon terminals was studied by determining the kinetics by which radioactivity associated with actin accumulates and then decays in the superior colliculus. The results of these studies indicate that labeled actin has a half-life in the superior colticulus of approximately 28 days.
INTRODUCTION The presence of actin in axons and axon terminals is well documented4,7, 2a,2~,29-~1. Although much is known about the chemistry2,12,26,29 and morphological organization7,22-25 of neuronal actin, there is little information available regarding the physiological properties of actin in vivo. Yet, such information is essential to fully understand the functional properties of actin in axons and their terminals. In the present study, we have begun to characterize the properties of actin in living axons by studying its transport within axons. Hoffman and Lasek identified a slowly transported polypeptide in rat ventral motor neurons which co-migrated with actin in SDSgels 16,19. This observation raised the possibility that actin was transported slowly in these axons. We have identified a similar slowly transported polypeptide in guinea pig retinal ganglion cell axons, and have provided strong evidence that this polypeptide is actin. Detailed analyses of the transport kinetics of actin indicate that it is transported principally, and possibly only in one of the three major rate components of guinea pig retinal ganglion cells. This work has been published in preliminary form s . MATERIALS AND METHODS
Radioactively labeling axonally transported proteins in guinea pig retinal ganglion celia" We employed the retinal ganglion cell system of guinea pigs (Harttey strain) in our studies. The axons of retinal ganglion cells project to the lateral geniculate nucleus and superior colliculus via the optic nerve and optic tract. In the strain of guinea pigs employed, this projection is completely crossed 1~. Thus, we were able to use both eyes of each animal, and treat each optic nerve and its contralateral optic tract as individual samples. Guinea pigs, 700-900 g, were anesthetized with sodium pentobarbital (30 mg/kg) administered intraperitoneally. Nine microliters of a [all]amino acid solution were injected into the posterior chamber of each eye. The injection system consisted of a 26gauge needle attached to a 10/zl Hamilton microsyringe. The plunger of the microsyringe was driven by a handoperated micrometer screw. In most experiments, [aH]lysine (40-60 Ci/mmol; NEN) was employed. Immediately before use, an aliquot of the stock solution of [aH]lysme was brought to dry-
403 ness by lyophilization and then was dissolved in water at a final concentration of 20 #Ci//zl. For those experiments examining the behavior of axonally transported proteins on DNaseI affinity columns (see below) a 1:1 mixture of [3H]lysine and [ZH]proline (20 #Ci/#l) was used.
The distribution of total radioactivity & the optic axons At several post-injection times, animals were anesthetized with ether and then decapitated. Each optic nerve, together with its contralateral optic tract and contralateral superior colliculus, was carefully dissected from each animal. Since both eyes were injected with [3H]amino acids, the optic chiasm (approximately 2 mm in length) was not included in these analyses. Each optic nerve together with its contralateral optic tract was cut into consecutive 3 mm segments. Each segment and the superior colliculus was homogenized in 0.325 ml of 8 M urea, 1 ~ SDS and 5 ~o 2-mercaptoethanol with a glass microhomogenizer. The homogenates were incubated at 95 °C for 5 rain and then were centrifuged at 27,000 × g for 1 h (Sorvall SS-34 rotor), and the supernates collected (the superior colliculus sample was centrifuged at 200,000 × g for 1 h in the Beckman SW50.1 rotor). Regardless of the centrifugation conditions, greater than 98 ~ of the radioactivity in the tissue homogenate remained in the supernate. The radioactivity in the supernate from each 3 mm segment was determined by scintillation counting (data were corrected for quenching by the method of external standardization). The radioactivity in each segment was then plotted against distance from the eye (see Fig. 1). The remainder of each sample was analyzed by SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis (SDS-page). Electrophoresis was performed in 7.5 ~ SDS-polyacrylamide slab gels in the discontinuous buffer system of Neville 27. Gels were stained with 0.25 ~o Coomassie blue, then the labeled polypeptides in each gel were visualized by fluorographya, zo. Actin prepared from guinea pig skeletal muscle 3a was used as a molecular weight standard. Other molecular weight standards included vitellogenin (mol. wt. 240,000, kindly supplied by L. Gehrke), rabbit skeletal muscle myosin (mol. wt. 200,000), phosphorylase a and bovine serum albumin (tool. wt. 94,000 and 68,000, respectively, from Sigma Chemical Co.). After obtaining the necessary fluorographs from the gels, the amount of radioactivity within selected labeled polypeptides was determined. The fluorographs were used to locate the position of the labeled polypeptides in the gels. The appropriate region of the gel was then cut out and incubated in 0.5 ml of 30 ~ HzO2 at 60 °C for 2 days to solubilize the radioactivity. Five milliliters of a toluene-Triton X100 scintillation cocktail was added to each sample and the amount of radioactivity determined. Data were corrected for quenching by the method of internal standardization (the counting efficiency ranged between 18 and 23 °/oo). D NaseI affinity chromatography DNase[ is a highly specific affinity ligand for actin 21. We used DNaseI affinity columns to determine if actin was axonally transported in guinea pig retinal ganglion
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Fig. 1. The distribution of total radioactivity within the optic axons at post-injection times of 6, 9, 15 and 25 days. The 3H content of consecutive 3 mm segments is plotted against distance from the eye. Each profile is representative of from 2 to 6 determinations. cells. DNaseI (from bovine pancreas: Sigma Chemical Co.) was bound to Sepharose 4B (Pharmacia). The Sepharose 4B was activated by CNBr treatment as described previously 21. The activated resin was washed on a Buchner funnel with 0.1 M NaHCO3 and resuspended in 0.1 M CaC12. DNaseI, dissolved in 0.1 M NaHCOs, 0.1 mM CaCIz, was incubated with the activated Sepharose overnight. The resulting material was extensively washed with distilled water and then with buffer 1 (0.15 M NaCI, 1.0 mM CaC12, 0.05 M Tris. pH 7.4). Labeled axonally transported proteins were obtained from the optic nerves and tracts of 4 guinea pigs sacrificed 9 days post-injection. The tissue was processed at 0-4 °C. The tissue was homogenized in 1 ml of 0.25 M sucrose. 1 mM CaCI2, 0.05 M Tris. pH 7.4, and centrifuged at 12,000 x g for 20 rain (Sorvall SS-34 rotor). The supernate was saved and the pellet was rehomogenized in 0.25 ml of the homogenization solution and re-centrifuged. The supernate from the second spin was combined with that of the first, and the combined sample was passed over a DNaseI affinity column (packed volume, 1 ml). The subsequent steps were performed at room temperature. The column was washed with a series of 4 buffers; first with 5 ml of buffer 1, followed by 2 ml of buffer 2 (0.5 M sodium acetate, lmM CAC!2,0.05 M Tris, pH 7.4), then with 2 ml of buffer 3 (buffer 2 -- 0.5 M urea) and finally with 5 ml of buffer 4 (8 M urea). One milliliter fractions were collected. Aliquots of each fraction were taken for determination of total radioactivity. Appropriate fractions were pooled and dialyzed against 8 M urea and 1% SDS. After dialysis. 2-mercaptoethanol was added to each sample to a final concentration of 5 %, and the samples were then incubated at 95 °C for 5 min. The stained and labeled polypeptides in each sample were analyzed by SDS-page and fluorography. RESULTS
Two slowly moving groups of axonally transported proteins Two slowly moving groups of proteins are identifiable in guinea pig retinal ganglion cells. Fig. 1 shows the distribution of total radioactivity within the optic nerve and contralateral optic tract at 6, 9, 15 and 25 days post-injection, The more rapidly moving group of proteins was apparent as a wave of: radioactivity within the
405
Fig. 2. The profile of labeled polypeptides conveyed in the two slowly transported groups of proteins in guinea pig retinal ganglion cells. The figure shows a fluorograph depicting the labeled polypeptides located 4-7 mm from the eye at 6 (SCb) and 25 (SCa) days post-injection. SCa and SCb nomenclature is explained in the text. The samples were electrophoresed on different gels. Molecular weights are indicated adjacent to appropriate bands. optic nerve and tract at 6 and 9 days post-injection. The proximo-distal shift in the position of this wave with time corresponds to a transport rate of approximately 2 m m / day. By 15 days post-injection, only the trailing portion of this wave was present within the optic nerve and tract; the rest of the radioactively labeled proteins comprising this wave had entered the terminal region of the optic axons located in the lateral geniculate nucleus and superior colliculus (data not shown). When the distribution of radioactivity within the optic axons was determined at progressively longer post-injection times, the second, more slowly moving group of axonally transported proteins appeared. At 25 days post-injection, this group of proteins appeared as a wave of radioactivity within the optic nerve and tract, the crest of which was located 4-7 mm from the optic nerve head. The proximo-distal shift in the position of this wave between 25 and 77 days post-injection (see Fig. 5) corresponds to a transport rate of 0.2-0.5 mm/day. These two slowly transported groups of proteins correspond to the movement of distinctly different sets of polypeptides within the axon (Fig. 2). The more slowly moving group of proteins is designated slow component a (SCa). Five polypeptides contain greater than 75 °/ooof the radioactivity transported in SCa. These 5 polypeptides correspond to those originally identified in the slow component (SCa) of rat
406
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Fig. 3. The profile of radioactivity eluting from a DNasel affinity column. The tissue, which was obtained from the optic nerves and tracts of 4 guinea pigs sacrificed 9 days post:injection, was processed as described in the materials and methods section. The column was eluted with a series of 4 buffers (see Materials and Methods). The beginning of buffer washes 2, 3 and 4 are indicated by arrows. These data are representative of two such experiments.
ventral motor neurons16,19. The labeled polypeptides with molecular weights of 53,000 and 57,000 daltons correspond to the subunits of tubulin, the major microtubule protein. The polypeptides with molecular weights of 200,000, 145,000 and 68,000 daltons correspond to the slow component triplet and apparently are the subunits of neurofilaments 16,19. In addition to these 5 major labeled polypeptides, 4 minor labeled polypeptides are also transported in SCa of' guinea pig retinal ganglion cells. Two of these have molecular weights of approximately 240,000 daltons, while the other two have molecular weights of 62,000 and 64,000 daltons. The more rapidly moving group of slowly transported proteins is designated slow component b (SCb). At least 20 polypeptides are transported in SCb. The major labeled polypeptides have molecular weights of > 400,000, 105,000, 85,000, 70,000, 60,000, 48,000, 43,000, 35,000, 27,000 and 25,000 daltons. The pattern of labeled polypeptides comprising SCb differs completely from that of SCa. The axonal transport o f actin in slow component b The labeled polypeptide in SCb with a molecular weight of 43,000 daltons comigrates with skeletal muscle actin in SDS-polyacrylamide slab gels. In order to determine whether or not this labeled SCb polypeptide was actin, we examined its behavior on DNaseI affinity columns. Labeled SCb proteins o f guinea pig retinal ganglion cells were obtained from the optic nerves and tracts of 4 animals sacrificed 9 days postinjection. A soluble fraction was obtained from the tissue (see Materials and Methods) and applied to the DNaseI affinity column. This fraction contained 95 % of the total radioactivity in the tissue. A representative example of the profile of radioactivity eluting from the DNaseI affinity column is illustrated in Fig, 3. Of the radioactivity applied to the column, approximately 90 % passed through in the first buffer wash. The second and third buffer washes eluted 1% and 0.5 %, respectively, of t h e radioactivity
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Fig. 4. The profile of stained (panel A) and labeled (panel B) polypeptides eluting from the DNasel affinity column depicted in Fig. 3. The gel columns contain the following samples: H, the tissue homogenate; S, the supernate from the tissue homogenate (this material was applied to the column); 1, the material passing through the column in buffer 1 ; 2, the material eluting in buffer 2; 4, the material eluting in buffer 4. Columns H, S and 1 in panel B are from a fluorograph developed after 1 week of exposure to the X-ray film, while columns 2 and 4 are from a fluorograph developed after 4 weeks of exposure to the X-ray film. applied to the column. The final buffer wash, which was 8 M urea, eluted 2 - 4 ~ o f the r a d i o a c t i v i t y applied to the column. The labeled m a t e r i a l eluting in the various buffer washes was analyzed by S D S - p a g e and f t u o r o g r a p h y (Fig. 4). The 8 M urea fraction c o n t a i n e d a single m a j o r b a n d (Fig. 4A, well 4). Its e l e c t r o p h o r e t i c m o b i l i t y was identical to t h a t o f skeletal muscle actin. The r e m a i n i n g stained m a t e r i a l m i g r a t e d with the dye front. As seen in
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Fig. 5. The distribution of total radioactivity (O) and radioactivity associated with acfin (C.) within the optic axons at 6, 9, 15, 25, 35 and 77 days post-injection. The distribution of total radioactivity was determined as described in the materials and methods section. The distribution of radioactivity associated with actin was determined as follows, Guinea pigs were sacrificed atthe indicatedtimes and the optic nerves, together with the contralateral optic tracts, removed. Each optic nerve-optic tract was cut into consecutive 3 mm segments. The labeled polypeptides within each segment was analyzed by SDS-page and fluorography. The radioactivity co-migrating with actin were quantitated as described in Materials and Methods. The amount of radioactivity co-migrating with actin was then graphed against distance from the eye. Each profile is representative of from 2 to 6 determinations. Fig. 4B, well 4, most of the radioactivity (approximately 70 °/o) eluting in 8 M urea is contained within the 43,000 dalton band; of the remaining radioactive material, most migrated with the dye front. The nature of this latter material was not determinedl Actin is not transported in slow component a In order to determine whether actin was transported in both SCb a n d SCa, or only in SCb, we compared the distribution of radioactivity in the optic axons which co-migrated with actin with the distribution of total radioactivity in the optic axons at several post-injection times between 6 and 77 days. (Analyses of the labeled SCb polypeptides by two-dimensional I E F - S D S gels have shown that actin is the most prominent labeled species which co-migrates with actin in one-dimensional SDS gels (Tytell and Lasek, unpublished). The results of this analysis are shown in Fig. 5. At 6 and 9 days post-injection, the distribution of radioactive actin within the optic nerve and contralateral optic tract exhibits a wave-like form. At each of these times, the shape and position of the radioactive actin wave is very similar to that of the radioactive SCb wave. These observations clearly demonstrate that the labeled actin within the optic axons at 6 and 9 days post,injection is transported in SCb. If actin were also transported in SCa, then, at appropriate post-injection times, the distribution of radioactive actin should exhibit a wave-like form that closely resembles the radioactive SCa wave. As seen in Fig. 5, the radioactive actin wave and the radioactive SCa wave are out of phase. In fact, only the trailing portion of the actin wave Overlaps the SCa wave. These observations demonstrate that, in guinea pig retinalganglion cells,
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Fig. 6. The amount of radioactivity in the superior colliculus which co-migrates with actin at several post-injection times between 15 and 100 days. At the indicated times, the labeled polypeptides in the superior colliculus were analyzed by SDS page and fluorography. The amount of radieactivity comigrating with actin was quantitated as described in the materials and metheds section. The mean standard errors are indicated (the values in parentheses indicate n).
actin is not transported coherently with the proteins of SCa. The radioactive actin in the optic axons at post-injection times greater than 25 days was deposited in the optic axons during the transit of the labeled SCb proteins from the cell body to the axon terminal. Actin is not transported in the fast component Labeled fast component proteins were harvested from the superior colliculus and lateral geniculate nucleus at 16 h post-injection and prepared for DNasel affinity chromatography (using the same protocol described in Materials and Methods). Of the 2.5 x 105 cpm in the tissue homogenate, approximately 1% was recovered in the 8 M urea fraction from a DNasel affinity column. When this labeled material was analyzed by SDS-page and fluorography, using an exposure time calculated to detect 20 dpmS, 20, we were unable to detect any labeled polypeptides. Thus, if actin is present in the fast component, it accounts for less than 0.002% of this class of axonally transported materials. Axonal transport o f actin to the axon terminal Actin has been identified in the axon terminal T M . If the actin in the axon terminal is delivered by SCb, then at appropriate post-injection times we should be able to identify labeled actin in the region of the CNS containing the terminals of the optic axons. We examined this by measuring the amount of radioactivity in the superior colliculus (a major termination site for retinal ganglion cell axons) which comigrated with actin in SDS slab gels at several post-injection times between 6 and 100 days. The results of these analyses are shown in Fig. 6. We were unable to detect labeled actin in the superior colliculus at 6 and 9 days post-injection. Subsequent to this time, radioactivity associated with actin appeared in the superior colliculus in concert with the entry of the radioactive SCb wave. The amount of radioactivity in the superior colliculus which co-migrated with actin increased through day 49 post-injection, subsequent to which it decreased in an apparantly exponential manner. The half-life of the radioactivity associated with actin in the superior colliculus was
410 approximately 28 days. The radioactive actin located in the superio~ colliculus between 15 and 100 days post-injection could be located in the synaptic terminals of the optic axons and/or in the preterminal portion of the optic axons.
DISCUSSION
The behavior of the 43,000 dalton polypeptide of SCb on DNaseI affinity columns provides strong evidence that this polypeptide is actin. DNasel is an affnity ligand for actin 21. The binding of actin to DNaseI is so tight that it can only be disrupted by strong denaturants such as 8 M urea or 3 M guanidine HC1. Furthermore, actin is apparently the only protein which DNaseI binds so tightly. Since the 43,000 dalton polypeptide transported in SCb interacts with DNasei in a manner which is characteristic of actin, we conclude that this polypeptide is actin. In support of this conclusion is the observation that the labeled 43,000 dalton SCb polypeptide co-migrates with actin on two-dimensional I E F - S D S gels (Tytell and Lasek, unpublished). Is actin transported in components of axonal transport other than SCb? The observation that the radioactive actin wave is out of phase with the radioactive SCa wave (Fig. 5) indicates that actin is not transported in SCa of guinea pig retinal ganglion cells. Components of axonal transport which are comparable to SCa and SCb in both transport rate and polypeptide composition have been identified in other neuronal systems (guinea pig hypoglossal motor neurons, Black and Lasek, in preparation; rat ventral motor neurons, Hoffman and Lasek, submitted). In these neuronal systems, as in guinea pig retinal ganglion cells, a 43,000 dalton polypeptide (presumably actin) is also transported in SCb, but not SCb. Several observations indicate that actin is not transported in the fast component of axonaltransport. First, we were unable to detect actin in the fast component of guinea pig retinal ganglion cells on the basis of DNaseI affinity chromatography. Second, analyses of the proteins transported in the fast component of frog spinal ganglion cells 35 and guinea pig retinal ganglion cells (Tytell and Lasek, unpublished) by two-dimensional IEF-SDS gels did not reveal an actin-like polypeptide. Since two independent assays failed to detect actin in the fast component, we conclude that actin. if it is present in the fast component, is present in vanishingly small amounts. In addition to SCa, SCb and the fast component, there are groups of proteins transported at rates in-between that of SCb and the fast component 17,1s,'~8. It is not known whether or not actin is a constit uent of any of these intermediate components. Relative to SCb, the intermediate components contribute a small amount of material to the axon. Thus, if actin is present in any of the intermediate components, it can only account for a small percentage o f the total axonally transported actin. On the basis of these considerations, we conclude that in guinea pig retinal ganglion cells, SCb is the principal and possibly only vehicle for the delivery of actin to the axon and its terminals. Quantitative evaluation of the gel profile of labeled material transported in SCb
411 (see Fig. 5 for example) indicates that approximately 5.7 ~ of this labeled material comigrates with actin. However, not all of this labeled material is attributable to actin, since analyses of the labeled SCb polypeptides by two-dimensional IEF-SDS gels have shown that the labeled material co-migrating with actin in SDS gels includes two or three polypeptides in addition to actin (Tytell and Lasek, unpublished). Thus, actin contains less than 5.7 ~ of the radioactivity in the SCb wave. Another estimate of the amount of radioactivity which actin contributes to the SCb wave can be obtained from the behavior of the labeled SCb proteins on DNaseI affinity columns. Since 2--4 ~ of the radioactivity applied to the colunm eluted in 8 M urea, and approximately 70 ~ of this radioactivity is in actin, 1.4-2.8 ~ of the labeled material applied to the column is actin. However, this value may underestimate the fraction of radioactivity in the SCb wave which is attributable to actin since actin may not have been quantitatively bound by the DNaseI affinity column. On the basis of these considerations, we conclude that between 1.4 and 5.7 °/o of the total radioactivity in the SCb wave is attributable to actin. Four to six nanometer actin-containing microfilaments have been identified in axons and their terminals 7,~3,z4,3°. The actin comprising these microfilaments most likely entered the axon in SCb since SCb is the principal and possibly only vehicle for the delivery of actin to the axon and axon terminal. Thus, the 4-6 nm microfilament is a morphological representation of the actin transported in SCb. It is also likely that other proteins transported in SCb are constituents of microfilaments. In this regard, tropomyosin, a protein component of microfilaments in muscle and non-muscle cells, may also be transported in SCb. The evidence for this is as follows. First, using a protocol for the isolation of brain tropomyosin 11, we have partially purified a labeled protein from SCb of guinea pig retinal ganglion cells. Secondly, a protein has been identified in SCb (group IV according to Willard et al. 3s) of rabbit retinal ganglion cells which has a subunit molecular weight closely resembling that of brain tropomyosin, and which also exhibits a specific affinity for actin filaments in vitro 37. Within axons and axon terminals, actin-containing microfilaments often appear physically associated with membrane structures such as the smooth endoplasmic reticulum, synaptic vesicles and the axonal and synaptic plasma membrane23,24, z0. As discussed previously, the actin comprising these microfilaments enters the axon in SCb. However, the membrane vesicles with which actin microfilaments interact are not transported in SCb. Rather, the available evidence indicates that these vesicular structures, which include transmitter storage vesicles and the smooth endoplasmic reticulum, are transported in the fast component of axonal transportl,8-10,14,15. Since the transport rate of actin is two orders of magnitude slower than that of the membrane structures conveyed in the fast component (2-4 ram/day for SCb; 250-400 ram/day for the fast component28), the interaction between actin microfilaments and these membrane structures inferred from ultrastructural observations must be of a transitory natme. The transient association of actin microfilaments with membrane structures transported in the fast component may be indicative of the participation of actin microfilaments in the convection of these structures within the axon. If so, then actin microfilaments can be viewed as relatively stationary elements against which these membrane structures move.
412 M a n y models which have been proposed for the motile mechanism of a x o n a l t r a n s p o r t are based o n a muscle-like a c t o m y o s i n system6,16,19,23,28, 33. The presence of actin7, 2z a n d m y o s i n 82,36 in axons fulfills a requisite of these models. Since actin enters the axon principally a n d possibly only in SCb, c o m p o n e n t s of axonal t r a n s p o r t which are m o v e d by a n actomyosin-like system must utilize the actin which entered the axon in SCb. F u r t h e r m o r e , if a muscle-like a c t o m y o s i n system generates the m o v e m e n t of c o m p o n e n t s which lack actin, such as the fast c o m p o n e n t , then these c o m p o n e n t s m u s t c o n t a i n a myosin-like p a r t n e r which can interact with the actin in SCb. In this regard, a myosin-like p r o t e i n has been tentatively identified in the fast c o m p o n e n t of r a b b i t retinal g a n g l i o n cells 37. ACKNOWLEDGEMENTS We are very grateful to Ms. Shirley Ricketts for her excellent technical assistance t h r o u g h o u t the course of this work. This work was s u p p o r t e d by N a t i o n a l I n s t i t u t e s of Health G r a n t s NS09299 a n d NS13658 to Dr. Lasek a n d N a t i o n a l Institutes of Health Traineeship HD00020 to Dr. Black.
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