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The role of microtubules in axoplasmic transport in vivo
Brain Research, 369 (1986) 75-82 Elsevier
75
BRE 11550
The Role of Microtubules in Axoplasmic Transport In Vivo SI-DE GAN, MING-MING FAN and GUO-PING HE
Department of Neurobiology, Institute of Basic Medical Sciences, 11 Tai Ping Road, Beijing, 100800 (P. R. China) (Accepted July 30th, 1985)
Key words: axoplasmic transport - - in vivo - - microtubule - - Ca2+ - - Cd2+ - - tubulin - - SH content
To verify whether microtubules are involved in the mechanism of axoplasmic transport in vivo, [3H]leucine was injected into ventral horns of rats, and 3 h later Ca2÷ or other drugs injected into sciatic nerves. The injection of 50-200 mM Ca2÷, raising intra-axoplasmic Ca2÷ levels, blocked transport above the intraneurai injecting site and decreased microtubular density. Conversely, injection of 10 mM EGTA lowering the intra-axoplasmic Ca2+ induced the same changes. By combining the injection of 50 mM colchicine with 25 mM Ca2+ or 5 mM EGTA, the effects were additive in that transport was weakened further or even blocked and microtubules disappeared. Therefore, microtubules seemed to be a mediator between the injected drug and the blockade of transport and Ca2÷ to be a regulator of axoplasmic transport in vivo. Tubulin, a subunit of microtubules, contains SH groups and Cd2+ is a chelate of them. By injection of 50-100 mM Cd 2+, transport was weakened or blocked. The sulfhydryl inhibitor, N-ethylmaleimide increased, but the sulfhydryl donor, dimercaptosuccinate, abolished the effect of Cd2+ on transport. N-ethylmaleimide also amplified the Cd2+ effect on decreasing SH group content of sciatic nerve homogenate. There were 8.7 SH groups per tubulin monomer isolated from rabbit brain. The SH groups of tubulin in vitro and microtubular density in vivo were decreased with the increase of Cd2+ concentration. All these results indicated that microtubules play a role in the mechanism of axoplasmic transport. INTRODUCTION The physiological significance of neurocytoplasmic transport is well known but its mechanism is far from clear 11. The essential p r o b l e m s are to determine the force which drives the t r a n s p o r t a b l e materials and what is (are) the carrier(s) of transportation, if any. Some hypotheses were delivered during the last 10 years, i.e. the S E R hypothesis 7, and hypotheses with a relation to microtubules such as microstream model 13, transport filament model22, 23 and others 11,19. A l t h o u g h some findings support a relation of microtubules to transport, contrary data have been r e p o r t e d recently2,3.6,10,:4. F o r example, under treatment with Ca 2+, colchicine or other drugs, a block of transport might exist with a normal microtubular density6.10, or a n o r m a l transport with a disappearance of microtubules2.3. H o w e v e r , most o f , t h e conclusions were reached from experiments in vitro. On the contrary, the present study was u n d e r t a k e n to observe the effects of Ca 2÷, E G T A , colchicine and
some sulfhydryl reagents on microtubules and cellifugal transport in vivo to identify whether microtubules might be involved in the mechanism of axoplasmic transport. MATERIALS AND METHODS A l b i n o rats (180-200 g) were anaesthetized with urethane and [3H]leucine (Shanghai A t o m i c Nuclear Institute, 53 Ci/mM, 1 mCi/ml, concentrated to 5 mCi/ml) was injected into left and right L 4 - L 5 ventral horns through micropipettes connected with a constant velocity p u m p , 2/A per site injected within 10 min. Three hours later, u n d e r half-dose of the anaesthetic, CaCI2, E G T A or colchicine (Fluke) in 20 /~1, or CdCI 2, sodium dimercaptosuccinate or N-ethylmaleimide (Koch-Light) in 30 g l injected into right sciatic nerve (10/~l/h), equivolume of saline into left as control. The intraneural injecting sites were about 60 m m from which L4 ventral roots left spinal cord. A t the end of injection (30/~1 injected) or 1 h
Correspondence: S.-D. Gan, Department of Neurobiology, Institute of Basic Medical Sciences, 11 Tai Ping Road, Beijing, 100800, People's Republic of China. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
76 !04i
\
'~
---
ContrOl
~0 ~
10 2 .
10 I 0
phate buffer (pH 7.2) containing 6.85% sucrose. The samples were post-fixed with 1% OsO 4, stained with uranyi acetate and lead citrate routinely. Under electronmicroscope J E M 6C, microtubule and other organelles of all axons in every ultrathin section were examined throughout, for easy comparison, only axons in a similar size range are presented in the figures.
mm
Fig. 1. Blocked effect of Ca2+ on axoplasmic transport. Three hours after injection of [3H]ieucine into both sides of L4-L5 ventral horns, 100 mM CaCI2 injected into the right sciatic nerve, 1 h later, the radioactivity in each 5 mm nerve segment showed that the transport was blocked at the site of intraneural injection (arrow). Abscissa: transported distance, 5 rats/group, For details see text.
later (20 #1 injected), animals were sacrificed and every 5 mm segment was taken from both sides of L 4 - L 5 ventral roots, sciatic, peroneal and tibiai nerves, digested with 0.5 N N a O H 0.3 ml at 75 °C, for 1 h. After cooling, 3 ml ethylene glycol monoethyl ether and 5 ml scintillating cocktail were added into each. The radioactivity of every segment was determined with a LKB scintillator. The background of intact nerve segments was 49.42 + 19.44 cpm (X + S.D., n 100). In morphological studies, nerve segments were taken 2 mm beyond the intraneurai injected site, and fixed immediately in 3% glutaraldehyde for two hours, then transferred into 0.2 M phos-
10C
Contrlzl 8C
.
+
RI:
6C
~4c 2O
10 5 2.5 ' ~ ~EGTA mM~ E×p
5
25
50
100 200
Ca2*mM
Fig. 2. Relationship between intra-axoplasmic Ca2+ level and transport, pH]leucine injected into ventral horns and Ca2+ or EGTA into right sciatic nerve as Fig. 1. In a sham experiment, the micropipette was inserted into the nerve but without injection of any drug. Abscissa: assumed changes of intra-axoplasmic Ca2+ concentration due to injection of Ca2+ or EGTA. Ordinate: transported distance of labeled protein. 5 rats/group, ,X + S.E.M. *, P > 0.05; *% P < 0.05; ***. P < 0.01; ****, P < 0.001.
Isolation and identification of tubulin Tubulin was isolated from rabbit brain by the Shelanski method31. We used the modified Lowry method to determine protein content 26. By P A G E the isolated tubulin showed a single band and a molecular weight of 57,000 according to relative mobility. It occupied a peak wave area about 87.9% by Shimadzu CS9101 thin layer scanner (590 nm). After incubation and negative staining, the isolated tubulin polymerized into microtubules and intermediate discs under Philipe 400T electronmicroscope, thus its biological activity was demonstrated. Determination of SH group content The SH group content was determined by modified Ellman method 29. One sample was 1 ml supernatant of sciatic nerve homogenate (16 mg wet wt./1 ml saline, x8000 g for 10 min at 4 °C), the other was 0.5 mg/ml tubulin. For identifying the effect of Cd 2+ on SH groups, we added 30/A CdCI 2 into the supernatant or 5/A into tubulin, then mixed with 0.1 M Tris buffer 3 ml (pH 6.8) and 5 mM D T N B 0.1 ml. The SH group content of supernatant was calculated from O.D. reading (412 nm) and standard curve of cysteine, or further divided by 57,000 to get the SH content per tubulin monomer. RESULTS
Effect of Ca2+ on axoplasmic transport in vivo After uptake and incorporation of injected [3H]leucine, the labeled protein was transported cellifugally to peripheral nerves. The radioactivities of ventral root segments decreased gradually in sequence with the increases of distance from spinal cord, but in sciatic, peroneal and tibial nerve segments they fluctuated about a certain level a n d i n appearance of a plateau until the end of transportation. On the control sides the average transported distance
77
Fig. 3. Microtubules (arrow) in axons of intact rat sciatic nerves. Abundant microtubules distributed along the longitudinal axis of a myelinated fiber with a certain length and density (A) and looked like rings in transverse section of unmyelinated fibers (B). Bars in Figs. 3-6 and 8 represent 0.5am.
of labeled p r o t e i n was 87.43 + 0.6 m m (X + S . E . M . , N 143) d u r i n g 6 h. It was a b o u t 30 m m over the nerve site of saline injection. I n t r a n e u r a l injec-
W h e n 5 0 - 2 0 0 m M Ca 2+ was used it b l o c k e d the t r a n s p o r t at the i n j e c t e d site, above which the radioactivities of a d j a c e n t n e r v e s e g m e n t s a c c u m u l a t e d
tion of Ca 2÷ raised i n t r a - a x o p l a s m i c Ca 2+ level 4.
a n d below which radioactivity fell off to b a c k g r o u n d
Fig. 4. Disappearance of microtubules by changes of intra-axoplasmic Ca2÷ concentration induced with transport-blocking-doses of 10 mM EGTA (A, transverse section) or 100 mM CaClz (B, longitudinal section) injected into sciatic nerves. Samples taken from the nerve segments 2 mm beyond the intraneural injected site and at 1 h after the end of injection (20/~1, 10/d/h).
7~ TABLE 1 Effect of colchicine and in combined injection with Ca:~ or EG TA on cellifugal transport ofl labeled protein Total volume 20.ul (10~tl/h) injected into sciatic nerves either in single or in combination (X _+ S.E.M.). For details see Fig. 1. N.S., not significant. Drug Saline Colchicine
EGTA Colchicine EGTA Caz+ Colchicine Ca2+
Dose (mM) 25 50 100 200 5 50 5 25 50 25
n
Transported distance (mm)
40 5 5 5 5 5
88 + 89 + 86 + -64 + 62 + 79 +
0.7 1.8 2.9 2.4 2.5 3.6
P
N.S. N.S. <0.001 <0.00l <0.05
5 5
71 ___4.8 75 _+5.7
<0.01 <0.05
5
65 +_2.7
<0.001
level (Fig. 1). Conversely, injection of 10 mM E G T A , a chelate of Ca 2+ to lower endogenous intraaxoplasmic Ca -'+ level, also blocked transport. 5 m M E G T A or 25 m M Ca 2+ weakened transport only mildly, with no effect seen under 5 rnM Ca 2+ or 2.5 m M E G T A . By merely inserting the micropipette, without containing any drug, into the nerve, no change of the transport was caused (Fig. 2). These results indicated that normal axoplasmic transport in vivo required a certain level of intra-axoplasmic Ca 2+ and Ca 2+ would be a regulator of axoplasmic transport. Effect o f Ca 2+ on microtubules in vivo Microtubules were a b u n d a n t in intact myelinated
Fig. 5. Disappearance of microtubules and blockade of transport of mitochondria with other organelles by 200 mM colchicine injected into the sciatic nerve, transverse section. Sample taken as Fig. 4.
or unmyelinated fibers of rats (Fig. 3). Saline (20 ul) injected into sciatic nerve did not make any observable change in microtubular density (figures not shown). Injection of 25 mM Ca ",+ or 5 mM E G T A induced only a slight change but with the transportblocking doses, 50-200 mM Ca z+ or 10 mM E G T A , microtubules decreased or disappeared (Fig. 4). Thus, for maintaining the normal microtubular density, a certain intra-axoplasmic Ca 2+ was also necessary. Effect o f combined injection o f colchicine with Ca 2+ or E G T A on microtubules and transport Some in vitro experiments d e m o n s t r a t e d that colchicine depolymerized microtubules and/or blocked axoplasmic transport 8. No a p p a r e n t effect on microtubules or transport was observed by injecting 25 or 50 mM colchicine into nerves but with 100 or 200 mM, microtubules decreased or disappeared and transport was blocked (Table I, Fig. 5). If 50 mM colchicine was used in combination with 5 mM E G T A or 25 mM Ca '-+, the effects on microtubules and transport were additive, apparently greater than that by any single drug (Table I, Fig. 6). This synergic effect as well as the fact that associated changes in microtubules and transport by drugs all indicated that microtubules might be mediators affecting the process of axoplasmic transport. Effect o f Cd 2+ and sulfhydryl reagents on axoplasmic transport in vivo It was r e p o r t e d that the polymerization of tubulin into microtubules might be in relation to its SH groups 20 which Cd 2+ could chelate with 9. For further demonstrating the role of microtubules in axoplasmic transport, Cd 2+ was injected into sciatic nerves, 75 or 100 m M blocked the transport, 50 m M w e a k e n e d it and no effect was seen with 12.5 mM or 25 mMi Nethylmaleimide, a sulfhydryl inhibitor injected in a "concentration of 200 m M decreased transport only moderately, but blocked the transport when in a combined injection with 50 mM Cd 2+. On the contrary, as expected, the SH groups donor, sodium dimercaptosuccinate, abolished the blocked effect of 100 m M Cd 2+ completely (Table I l l so that the blocking effect of Cd 2+ might be due to a decrease of SH groups of the nerve tissue.
79
Fig. 6. Further decrease or disappearance of microtubules by combined injection of 50 mM colchfcine with 5 mM EGTA (A, longitudinal section) or with 25 mM CaCI 2 (B, transverse section). Total injected volume 20pl (10/A/h). Effect o f Cd 2+ on S H group content in supernatant o f
n e r v e h o m o g e n a t e was e q u a l to 2.7 + 0 . 0 6 / ~ m o l cys-
nerve h o m o g e n a t e and tubulin
teine/g w e t wt. It was d e c r e a s e d by 1 2 . 5 - 1 0 0 m M
T h e S H g r o u p c o n t e n t in s u p e r n a t a n t of rat sciatic
Cd 2÷ ( d a t a not s h o w n ) and N - e t h y l m a l e i m i d e a m p l i fied t h e C d 2÷ effect synergically ( T a b l e III). F o r fur-
TABLE II
t h e r clarification, tubulin was isolated f r o m rabbit
Effect of Cd2+ and sulfhydryl reagents on cellifugal transport of labeled protein
brain. T h e r e w e r e 8.7 S H g r o u p s p e r tubulin m o n o m -
Cd 2÷ or other sulfhydryl reagents injected into sciatic nerves either in single or in combination with each other via a doubletubed micropipette. The injected volume was 30 ~1 (10 pl/h) in every case. (,X + S.E.M.). N.S., not significant.
ship with the increase of C d 2+ c o n c e n t r a t i o n (Fig. 7).
Drug Saline Cd 2+
N-ethylmaleimide N-ethylmaleimide Cd2+ Na-dimercaptosuccinate Na-dimercaptosuccinate Cd2+
Dose (rnM) 12.5 25 50 75 100 200 200 50 50 50 100
n
Transported P distance (mm)
45 5 5 5 5 5 5
90 + 90 + 87 + 81 + 63 + 61 + 77 +
0.8 0 4.6 5.8 3.7 1.0 2.5
N.S. <0.05 <0.001 <0.001 <0.001
5 5
62 + 1.2 94 + 1.0
<0.001 N.S.
5
92 + 1.2
N.S.
N.S.
er. T h e d e c r e a s e of S H g r o u p s was in linear r e l a t i o n -
TABLE Ill Effect of Cd2+ and N-ethylmaleimide on SH group content of rat sciatic nerve 50 mM CdCI 2 or/and 50 mM N-ethylmaleimide added into 1 ml supernatant of nerve homogenate. Triple determinations, _+S.D. Drug
Control Cd 2÷ N-ethylmaleimide N-ethylmaleimide Cd 2+
Dose (IA)
30 0.5 0.5 30
n
SH content (pmol cysteine/ g wet wt.)
P
5 5 5
2.70 _+ 0.06 2.43 _+ 0.05 1.94 + 0.06
<0.05 <0.001
5
1.50 -+ 0.10
<0.001
8t)
.
.
.
.
.
.
.
.
.
.
.
.
.
SH groups could cause microtubule depolymerization with blockade of transport as a consequence of microtubule disassembly.
'3rlw OJ
eoiu n
i
DISCUSSION
S 49~ 8 c
s©
8 x ~9 t
12 5
j
2~5
5JO Cd 2. mfvl
75
_ _ _ ~
100
Fig. 7. Decrease of SH groups in tubulin by Cd2+; 5 ~1 CdCI2 added into 1 ml tubulin (0.5 mg/ml) isolated from rabbit brain. SH groups in tubulin control decreased with the increase of Cd2+ concentration. Each group, n = 5; triple determinations, X + S.D.
Effect o f Cd 2+ on microtubules in vivo Microtubules were examined after Cd 2+ injection into sciatic nerves in vivo to compare its effect on SH group content with those seen in vitro. The changes of microtubular density from a decrease to disappearance were observed with an increase of Cd2+ concentration from 50 to 100 m M (Fig. 8, Table IV). Taking the results together, Cd 2÷ inhibition of free
Fig. 8. Decrease of microtubules by Cd 2+ ; 75 mM CdCI 2 30/A
injected into sciatic nerve (10/A/h), only a few microtubules and/or fragments (arrow) remained. Samples taken as Fig. 4 but immediately after the end of injection. Longitudinal section.
The results d e m o n s t r a t e d that a c o m p a r a b l e associated change of microtubule and transport coexists with experimental drugs, i.e. where microtubules decreased or disappeared the transport usually weakened or blocked (Table V). Therefore, the depolymerization of microtubules seems to participate or mediate a block of transport. In o t h e r words, the microtubules must play a role in axoplasmic transport in vivo. Although the d e t e r m i n a t i o n of intra-axoplasmic Ca 2+ will devote a further p r o o f to the conclusion, unfortunately, that the Ca 2+ concentration in mammalian nerve is unknown and no quantitative m e t h o d can be used efficientlyl,25. H o w e v e r , Ochs et al. 4 in an ultrastructural study with X-ray microanalysis showed that the Ca-containing electron-dense particle density in axoplasm and some organelles was related to Ca 2+ level in m e d i u m , and d e m o n s t r a t e d a Ca 2+ effect on transport in d e s h e a t h e d nerve in vitro5,23; the latter has been confirmed by others ~6,~7 also. As calmodulin facilitated the Ca 2+ effect on inhibition of tubulin assembly (unpublished) and on disassembly of polymerized microtubules ~2, one way in which microtubules b e c o m e d e p o l y m e r i z e d and transport blocked by Ca 2+ may be via calmodulin 23. In addition, according to axonal demyelination of cutting nerve incubated in high Ca 2+ m e d i u m , Schlaepfer 27 suggested that Ca z+ may activate protease. But in our cases, except the b r e a k d o w n of microtubules, all other organeiles kept normal morphological features. Thus, it also did not seem that the effect of experimental drugs on microtubules would be due to a change of osmotic pressure of the injected solution, for example, a d i s a p p e a r a n c e of microtubules was observed with 100 m M Ca 2+ or 100 m M C d 2+, which are isotonic. The SH group loss of tubulin delivered an explanation on molecular basis why Cd 2+ could depolymerize microtubules and block transport. Having observed that the rate or degree of microtubule polymerization in vitro was a function of tubulin concentration ~2, we deduced that axoplasmic free tubulin might be in dy-
81 TABLE IV Summary of Cd2÷ effects on tubulin SH group content, microtubules and ceUifugal transport of labeled protein
CdC12injected into rat sciatic nerve 3 h after the injection of [3H]leucineinto ventral horns. The site of intraneural injection was 60 mm from which L4 ventral roots left the spinal cord. Tubulin was isolated from rabbit brain. N.S., not significant. Cd 2+ (triM)
SH content (%)
Microtubules
Transported distance (ram)
Coiatrol 12.5 25 50 75 100
100 66.5 54.4 45.7 39.5 27.3
With a certain density and length No change No change Decreased mildly Decreased apparently Only a few fragments or disappeared
88 + 0.7 90 + 0 87 + 4.6 81 + 5.8 63 + 3.7 61 + 1.0
namic equilibrium with the tubulin polymerized into microtubules in viv0. Cd 2÷ or colchicine could react with the SH groups of free tubulin and change them into disulfide bondsSAS, 20, thus the polymerization of tubulin was inhibited, the equilibrium might shift to tubulin side and microtubules depolymerized. If it is the case, the SH group loss by Cd2÷ in vitro (Table IV), the converse effects o f - S H inhibitor and -SH donor on transport in vivo (Table II) and the microtubules disassembly by Cd2÷ (Fig. 8) all demonstrated a further proof that a correlation exists among SH content, microtubular density and axoplasmi c transport. However, it could not rule out the possibility that Cd 2+ might react with the SH groups of microtubule associated proteins9, zl, dynein protein 14 or ATPase14As,23,30; all of them would activate microtubule assembly. The associated change of microtubule and transport in vivo presented in this work were different from other studies in vitro, in which only either microtubules or transport were seen to suffer changes by the same drug2,3,6A0. In some of those experiments in vitro, both the fewer experimental drugs selected
P N.S.
N.S. <0.05 <0.001 <0.001
and the narrow range of doses of the tested drugs would be causes which restrict varied and meaningful observations. Our results did show a strict dose response from none to maximum effects among levels of drugs (Table V), the conclusions therefore were reliable. Except the study of Ca 2÷ on microtubules of heliozoan axopodium 2s, few data on the effects of Ca 2÷, E G T A or Cd 2+ on microtubule and axoplasmic transport in vivo have been reported. We further verified the relationship between microtubule and transport with morphological and biochemical studies and compared the in vitro results2,3,5,6,9,10,16,17 to those in vivo. This present in vitro and in vivo work provides further evidence that Ca 2÷ is a key regulator of axoplasmic transport and microtubules are critically involved in the mechanism of this transport. ACKNOWLEDGEMENTS The authors are indebted to Prof. Chiao Tsai for his kind supporting on this work and thank Miss Wang G u o - H w a for her technical assistance.
TABLE V Summary of effects of drugs on microtubules and cellifugal transport of labeled protein in vivo Effects
Ca2+
EG TA
Colchicine
Cd2+
(mM)
On microtubules
No effect Decreased mildly Decreased apparently Disappeared
5-25 25 50 50-200
2.5 5 10 10
25-50 50 100 100-200
12.5-25 50 75-100 100
On transport
No effect Decreased mildly Blocked
5 25 50-200
2.5 5 10
25-50
12.5-25 50 75-100
100-200
82 REFERENCES 1 Baker, P.F., The regulation of intracellular calcium in giant axons of Loligo and Myxicola, Ann. N Y Acad. Sci.. 3(17 (1978) 250-268. 2 Brady, S.T., Chrothers, S.D., Nosal, C. and McClure, W.O., Fast axonal transport in the presence of high Ca2+: evidence that microtubules are not required, Proc. ,Natl. Acad.Sci. U.S.A.. 77 (1980) 5909-5913. 3 Byers, M.R., Structural correlates of rapid axonal transport: evidence that microtubules may not be directly involved, Brain Research, 75 (1974) 97-113. 4 Chan, S.Y., Ochs, S. and Jersild, R.A., Jr., Localization of calcium in nerve fibers, J. Neurobiol., 15 (1983) 89-108. 5 Chan, S.Y., Ochs, S. and Worth, R.M., The requirement for calcium ions and the effect of other ions on axoplasmic transport in mammalian nerve, J. Physiol. (London), 301 (1980) 477-504. 6 Donoso, J.A., lllanes, J.P. and Samson, F., Dimethylsulfoxide action on fast axoplasmic transport and ultrastructure of vagal axons, Brain Research, 120 (1977) 287-301. 7 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. 8 Dustin, P., Microtubules, Springer-Verlag, Berlin, 1978, pp. 167-225. 9 Edstrom, A. and Mattson, H., Inhibition and stimulation of rapid axonal transport in vitro by sulfhydryl blockers, Brain Research, 108 (1976) 381-395. 1(1 Fernandez, H.L., Hrneeus, F.C. and Davison, P.F., Studies on the mechanism of axoplasmic transport in the crayfish cord, J. Neurobiol., 1 (1970) 395-409. 11 Gan, S.-D., Neurocytoplasmic transport and transmission of molecular information, Physiol. Sci., 2(5) (1982) 1-4; 2(6) (1982) 11-16 (in Chinese). 12 Gan. S.-D., Fan, M.-M. and He, G.-P., The role of calcium and calmodulin in axoplasmic transport in vivo, News Commu. Chinese Pharmacol. Soc., 1(3-4) (1984) 16 (in Chinese). 13 Gross, G.W., The microstream concept of axoplasmic and dendritic transport, Adv. Neurol., 12 (1975) 283-296. 14 Haimo, L.T. and Telzer, B.R., Dynein-microtubule interactions: ATP-sensitive dynein binding and the structural polarity of mitotic microtubules, Cold Spring Harbor Syrup. Qaant. Biol., 46 (1982) 207-217. 15 Hammerschlag, R. and Bobinski, J.A., Ca 2+- or Mg 2+stimulated ATPase activity in bullfrog spinal nerve: relation to Ca 2+ requirements for fast axonal transport, J. Neu-
rochem., 36 (1981) 1114-1121. 16 Hammerschlag, R., David, A.R. and Chiu, A.Y., Mechanism of axonal transport: a proposed role for calcium ions, Science, 188 (1975) 273-275. 17 Kanje, M., Edstr6m, A. and EdstrOm, P., Divalent cations and fast axonal transport in chemically desheathed (Triton X-treated) frog sciatic nerve, Brain Research, 241 (1982) 67-74. 18 Kuriyama, R. and Sakai, H., Role of tubulin in sulfhydryl groups in polymerization to microtubules, J. Biochem., 76 (1974) 651-654. 19 Lasek, R.J. and Brady, S.T., The axon: a prototype for studing expressional cytoplasm. Cold Spring Harbor Syrup. Quant. Biol., 46 (1982) 113-124. 20 Mellon, M.G. and Rebhum, L.I., Sulfhydryls and the in vitro polymerization of tubulin, J. Cell Biol., 70 (1976) 226-278. 21 Murphy, D.W., Valle, R.B. and Borisy, G.G., Identity and polyinerization in stimulatory activity of the non-tubutin protein associated with microtubules, Biochemistry, 16 (1977) 2598-2605. 22 Ochs, S., Characteristics and a model for fast axoplasmic transport in nerve, J. NeurobioL, 2 (1971) 331-345. 23 Ochs, S. and Jersild, R.A., Jr., Calcium localization in nerve fibers in relation to axoplasmic transport, Neurochem. Res., 9 (1984) 823-836. 24 Pollard, T.D., Which organelles are necessary for fast neuronal transport?, Neurosci. Res. Progr. Bull., 20 (1981) 92-97. 25 Scarpa, A. and Carafoli, E., Calcium transport and cell function. Part I. Measurements of calcium, Ann. NYAcad. Sci., 307 (1978) 28-124. 26 Schacterle, G.R., A simplified method for the quantitative assay of small amounts of protein in biological material, Anal. Biochem., 51 (1973) 654-655. 27 Schlaepfer, W.W., Experimental alternations of neurofilaments and neurotubules by calcium and other ions, Exp. CellRes., 67 (1971) 73-80. 28 Schliwa, M., The role of divalent cation in the regulation of microtubules assembly, J. Cell Biol., 70 (1976) 527-540. 29 Sedlak, G. and Lindsay, R.H., Estimation of total, proteinbound and non-protein sulfhydryl groups in tissue with Ellman's reagent, Anal. Biochem., 25 (1968) 192-205. 30 Sharp, G.A., Fitzsimons, J.T.R. and Kerkut, G.A., Microtubular ATPase in Carcinus and Helix nerve, Comp, Biochem. Physiol., 61A (1978) 297-301. 31 Shelanski, M.L., Gaskin, F. and Cantor, C.R., Microtubule assembly in the absence of added nucleotides, Proc. Natl. Acad. Sci. U.S.A.. 70 (1973) 765-786.
75
BRE 11550
The Role of Microtubules in Axoplasmic Transport In Vivo SI-DE GAN, MING-MING FAN and GUO-PING HE
Department of Neurobiology, Institute of Basic Medical Sciences, 11 Tai Ping Road, Beijing, 100800 (P. R. China) (Accepted July 30th, 1985)
Key words: axoplasmic transport - - in vivo - - microtubule - - Ca2+ - - Cd2+ - - tubulin - - SH content
To verify whether microtubules are involved in the mechanism of axoplasmic transport in vivo, [3H]leucine was injected into ventral horns of rats, and 3 h later Ca2÷ or other drugs injected into sciatic nerves. The injection of 50-200 mM Ca2÷, raising intra-axoplasmic Ca2÷ levels, blocked transport above the intraneurai injecting site and decreased microtubular density. Conversely, injection of 10 mM EGTA lowering the intra-axoplasmic Ca2+ induced the same changes. By combining the injection of 50 mM colchicine with 25 mM Ca2+ or 5 mM EGTA, the effects were additive in that transport was weakened further or even blocked and microtubules disappeared. Therefore, microtubules seemed to be a mediator between the injected drug and the blockade of transport and Ca2÷ to be a regulator of axoplasmic transport in vivo. Tubulin, a subunit of microtubules, contains SH groups and Cd2+ is a chelate of them. By injection of 50-100 mM Cd 2+, transport was weakened or blocked. The sulfhydryl inhibitor, N-ethylmaleimide increased, but the sulfhydryl donor, dimercaptosuccinate, abolished the effect of Cd2+ on transport. N-ethylmaleimide also amplified the Cd2+ effect on decreasing SH group content of sciatic nerve homogenate. There were 8.7 SH groups per tubulin monomer isolated from rabbit brain. The SH groups of tubulin in vitro and microtubular density in vivo were decreased with the increase of Cd2+ concentration. All these results indicated that microtubules play a role in the mechanism of axoplasmic transport. INTRODUCTION The physiological significance of neurocytoplasmic transport is well known but its mechanism is far from clear 11. The essential p r o b l e m s are to determine the force which drives the t r a n s p o r t a b l e materials and what is (are) the carrier(s) of transportation, if any. Some hypotheses were delivered during the last 10 years, i.e. the S E R hypothesis 7, and hypotheses with a relation to microtubules such as microstream model 13, transport filament model22, 23 and others 11,19. A l t h o u g h some findings support a relation of microtubules to transport, contrary data have been r e p o r t e d recently2,3.6,10,:4. F o r example, under treatment with Ca 2+, colchicine or other drugs, a block of transport might exist with a normal microtubular density6.10, or a n o r m a l transport with a disappearance of microtubules2.3. H o w e v e r , most o f , t h e conclusions were reached from experiments in vitro. On the contrary, the present study was u n d e r t a k e n to observe the effects of Ca 2÷, E G T A , colchicine and
some sulfhydryl reagents on microtubules and cellifugal transport in vivo to identify whether microtubules might be involved in the mechanism of axoplasmic transport. MATERIALS AND METHODS A l b i n o rats (180-200 g) were anaesthetized with urethane and [3H]leucine (Shanghai A t o m i c Nuclear Institute, 53 Ci/mM, 1 mCi/ml, concentrated to 5 mCi/ml) was injected into left and right L 4 - L 5 ventral horns through micropipettes connected with a constant velocity p u m p , 2/A per site injected within 10 min. Three hours later, u n d e r half-dose of the anaesthetic, CaCI2, E G T A or colchicine (Fluke) in 20 /~1, or CdCI 2, sodium dimercaptosuccinate or N-ethylmaleimide (Koch-Light) in 30 g l injected into right sciatic nerve (10/~l/h), equivolume of saline into left as control. The intraneural injecting sites were about 60 m m from which L4 ventral roots left spinal cord. A t the end of injection (30/~1 injected) or 1 h
Correspondence: S.-D. Gan, Department of Neurobiology, Institute of Basic Medical Sciences, 11 Tai Ping Road, Beijing, 100800, People's Republic of China. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
76 !04i
\
'~
---
ContrOl
~0 ~
10 2 .
10 I 0
phate buffer (pH 7.2) containing 6.85% sucrose. The samples were post-fixed with 1% OsO 4, stained with uranyi acetate and lead citrate routinely. Under electronmicroscope J E M 6C, microtubule and other organelles of all axons in every ultrathin section were examined throughout, for easy comparison, only axons in a similar size range are presented in the figures.
mm
Fig. 1. Blocked effect of Ca2+ on axoplasmic transport. Three hours after injection of [3H]ieucine into both sides of L4-L5 ventral horns, 100 mM CaCI2 injected into the right sciatic nerve, 1 h later, the radioactivity in each 5 mm nerve segment showed that the transport was blocked at the site of intraneural injection (arrow). Abscissa: transported distance, 5 rats/group, For details see text.
later (20 #1 injected), animals were sacrificed and every 5 mm segment was taken from both sides of L 4 - L 5 ventral roots, sciatic, peroneal and tibiai nerves, digested with 0.5 N N a O H 0.3 ml at 75 °C, for 1 h. After cooling, 3 ml ethylene glycol monoethyl ether and 5 ml scintillating cocktail were added into each. The radioactivity of every segment was determined with a LKB scintillator. The background of intact nerve segments was 49.42 + 19.44 cpm (X + S.D., n 100). In morphological studies, nerve segments were taken 2 mm beyond the intraneurai injected site, and fixed immediately in 3% glutaraldehyde for two hours, then transferred into 0.2 M phos-
10C
Contrlzl 8C
.
+
RI:
6C
~4c 2O
10 5 2.5 ' ~ ~EGTA mM~ E×p
5
25
50
100 200
Ca2*mM
Fig. 2. Relationship between intra-axoplasmic Ca2+ level and transport, pH]leucine injected into ventral horns and Ca2+ or EGTA into right sciatic nerve as Fig. 1. In a sham experiment, the micropipette was inserted into the nerve but without injection of any drug. Abscissa: assumed changes of intra-axoplasmic Ca2+ concentration due to injection of Ca2+ or EGTA. Ordinate: transported distance of labeled protein. 5 rats/group, ,X + S.E.M. *, P > 0.05; *% P < 0.05; ***. P < 0.01; ****, P < 0.001.
Isolation and identification of tubulin Tubulin was isolated from rabbit brain by the Shelanski method31. We used the modified Lowry method to determine protein content 26. By P A G E the isolated tubulin showed a single band and a molecular weight of 57,000 according to relative mobility. It occupied a peak wave area about 87.9% by Shimadzu CS9101 thin layer scanner (590 nm). After incubation and negative staining, the isolated tubulin polymerized into microtubules and intermediate discs under Philipe 400T electronmicroscope, thus its biological activity was demonstrated. Determination of SH group content The SH group content was determined by modified Ellman method 29. One sample was 1 ml supernatant of sciatic nerve homogenate (16 mg wet wt./1 ml saline, x8000 g for 10 min at 4 °C), the other was 0.5 mg/ml tubulin. For identifying the effect of Cd 2+ on SH groups, we added 30/A CdCI 2 into the supernatant or 5/A into tubulin, then mixed with 0.1 M Tris buffer 3 ml (pH 6.8) and 5 mM D T N B 0.1 ml. The SH group content of supernatant was calculated from O.D. reading (412 nm) and standard curve of cysteine, or further divided by 57,000 to get the SH content per tubulin monomer. RESULTS
Effect of Ca2+ on axoplasmic transport in vivo After uptake and incorporation of injected [3H]leucine, the labeled protein was transported cellifugally to peripheral nerves. The radioactivities of ventral root segments decreased gradually in sequence with the increases of distance from spinal cord, but in sciatic, peroneal and tibial nerve segments they fluctuated about a certain level a n d i n appearance of a plateau until the end of transportation. On the control sides the average transported distance
77
Fig. 3. Microtubules (arrow) in axons of intact rat sciatic nerves. Abundant microtubules distributed along the longitudinal axis of a myelinated fiber with a certain length and density (A) and looked like rings in transverse section of unmyelinated fibers (B). Bars in Figs. 3-6 and 8 represent 0.5am.
of labeled p r o t e i n was 87.43 + 0.6 m m (X + S . E . M . , N 143) d u r i n g 6 h. It was a b o u t 30 m m over the nerve site of saline injection. I n t r a n e u r a l injec-
W h e n 5 0 - 2 0 0 m M Ca 2+ was used it b l o c k e d the t r a n s p o r t at the i n j e c t e d site, above which the radioactivities of a d j a c e n t n e r v e s e g m e n t s a c c u m u l a t e d
tion of Ca 2÷ raised i n t r a - a x o p l a s m i c Ca 2+ level 4.
a n d below which radioactivity fell off to b a c k g r o u n d
Fig. 4. Disappearance of microtubules by changes of intra-axoplasmic Ca2÷ concentration induced with transport-blocking-doses of 10 mM EGTA (A, transverse section) or 100 mM CaClz (B, longitudinal section) injected into sciatic nerves. Samples taken from the nerve segments 2 mm beyond the intraneural injected site and at 1 h after the end of injection (20/~1, 10/d/h).
7~ TABLE 1 Effect of colchicine and in combined injection with Ca:~ or EG TA on cellifugal transport ofl labeled protein Total volume 20.ul (10~tl/h) injected into sciatic nerves either in single or in combination (X _+ S.E.M.). For details see Fig. 1. N.S., not significant. Drug Saline Colchicine
EGTA Colchicine EGTA Caz+ Colchicine Ca2+
Dose (mM) 25 50 100 200 5 50 5 25 50 25
n
Transported distance (mm)
40 5 5 5 5 5
88 + 89 + 86 + -64 + 62 + 79 +
0.7 1.8 2.9 2.4 2.5 3.6
P
N.S. N.S. <0.001 <0.00l <0.05
5 5
71 ___4.8 75 _+5.7
<0.01 <0.05
5
65 +_2.7
<0.001
level (Fig. 1). Conversely, injection of 10 mM E G T A , a chelate of Ca 2+ to lower endogenous intraaxoplasmic Ca -'+ level, also blocked transport. 5 m M E G T A or 25 m M Ca 2+ weakened transport only mildly, with no effect seen under 5 rnM Ca 2+ or 2.5 m M E G T A . By merely inserting the micropipette, without containing any drug, into the nerve, no change of the transport was caused (Fig. 2). These results indicated that normal axoplasmic transport in vivo required a certain level of intra-axoplasmic Ca 2+ and Ca 2+ would be a regulator of axoplasmic transport. Effect o f Ca 2+ on microtubules in vivo Microtubules were a b u n d a n t in intact myelinated
Fig. 5. Disappearance of microtubules and blockade of transport of mitochondria with other organelles by 200 mM colchicine injected into the sciatic nerve, transverse section. Sample taken as Fig. 4.
or unmyelinated fibers of rats (Fig. 3). Saline (20 ul) injected into sciatic nerve did not make any observable change in microtubular density (figures not shown). Injection of 25 mM Ca ",+ or 5 mM E G T A induced only a slight change but with the transportblocking doses, 50-200 mM Ca z+ or 10 mM E G T A , microtubules decreased or disappeared (Fig. 4). Thus, for maintaining the normal microtubular density, a certain intra-axoplasmic Ca 2+ was also necessary. Effect o f combined injection o f colchicine with Ca 2+ or E G T A on microtubules and transport Some in vitro experiments d e m o n s t r a t e d that colchicine depolymerized microtubules and/or blocked axoplasmic transport 8. No a p p a r e n t effect on microtubules or transport was observed by injecting 25 or 50 mM colchicine into nerves but with 100 or 200 mM, microtubules decreased or disappeared and transport was blocked (Table I, Fig. 5). If 50 mM colchicine was used in combination with 5 mM E G T A or 25 mM Ca '-+, the effects on microtubules and transport were additive, apparently greater than that by any single drug (Table I, Fig. 6). This synergic effect as well as the fact that associated changes in microtubules and transport by drugs all indicated that microtubules might be mediators affecting the process of axoplasmic transport. Effect o f Cd 2+ and sulfhydryl reagents on axoplasmic transport in vivo It was r e p o r t e d that the polymerization of tubulin into microtubules might be in relation to its SH groups 20 which Cd 2+ could chelate with 9. For further demonstrating the role of microtubules in axoplasmic transport, Cd 2+ was injected into sciatic nerves, 75 or 100 m M blocked the transport, 50 m M w e a k e n e d it and no effect was seen with 12.5 mM or 25 mMi Nethylmaleimide, a sulfhydryl inhibitor injected in a "concentration of 200 m M decreased transport only moderately, but blocked the transport when in a combined injection with 50 mM Cd 2+. On the contrary, as expected, the SH groups donor, sodium dimercaptosuccinate, abolished the blocked effect of 100 m M Cd 2+ completely (Table I l l so that the blocking effect of Cd 2+ might be due to a decrease of SH groups of the nerve tissue.
79
Fig. 6. Further decrease or disappearance of microtubules by combined injection of 50 mM colchfcine with 5 mM EGTA (A, longitudinal section) or with 25 mM CaCI 2 (B, transverse section). Total injected volume 20pl (10/A/h). Effect o f Cd 2+ on S H group content in supernatant o f
n e r v e h o m o g e n a t e was e q u a l to 2.7 + 0 . 0 6 / ~ m o l cys-
nerve h o m o g e n a t e and tubulin
teine/g w e t wt. It was d e c r e a s e d by 1 2 . 5 - 1 0 0 m M
T h e S H g r o u p c o n t e n t in s u p e r n a t a n t of rat sciatic
Cd 2÷ ( d a t a not s h o w n ) and N - e t h y l m a l e i m i d e a m p l i fied t h e C d 2÷ effect synergically ( T a b l e III). F o r fur-
TABLE II
t h e r clarification, tubulin was isolated f r o m rabbit
Effect of Cd2+ and sulfhydryl reagents on cellifugal transport of labeled protein
brain. T h e r e w e r e 8.7 S H g r o u p s p e r tubulin m o n o m -
Cd 2÷ or other sulfhydryl reagents injected into sciatic nerves either in single or in combination with each other via a doubletubed micropipette. The injected volume was 30 ~1 (10 pl/h) in every case. (,X + S.E.M.). N.S., not significant.
ship with the increase of C d 2+ c o n c e n t r a t i o n (Fig. 7).
Drug Saline Cd 2+
N-ethylmaleimide N-ethylmaleimide Cd2+ Na-dimercaptosuccinate Na-dimercaptosuccinate Cd2+
Dose (rnM) 12.5 25 50 75 100 200 200 50 50 50 100
n
Transported P distance (mm)
45 5 5 5 5 5 5
90 + 90 + 87 + 81 + 63 + 61 + 77 +
0.8 0 4.6 5.8 3.7 1.0 2.5
N.S. <0.05 <0.001 <0.001 <0.001
5 5
62 + 1.2 94 + 1.0
<0.001 N.S.
5
92 + 1.2
N.S.
N.S.
er. T h e d e c r e a s e of S H g r o u p s was in linear r e l a t i o n -
TABLE Ill Effect of Cd2+ and N-ethylmaleimide on SH group content of rat sciatic nerve 50 mM CdCI 2 or/and 50 mM N-ethylmaleimide added into 1 ml supernatant of nerve homogenate. Triple determinations, _+S.D. Drug
Control Cd 2÷ N-ethylmaleimide N-ethylmaleimide Cd 2+
Dose (IA)
30 0.5 0.5 30
n
SH content (pmol cysteine/ g wet wt.)
P
5 5 5
2.70 _+ 0.06 2.43 _+ 0.05 1.94 + 0.06
<0.05 <0.001
5
1.50 -+ 0.10
<0.001
8t)
.
.
.
.
.
.
.
.
.
.
.
.
.
SH groups could cause microtubule depolymerization with blockade of transport as a consequence of microtubule disassembly.
'3rlw OJ
eoiu n
i
DISCUSSION
S 49~ 8 c
s©
8 x ~9 t
12 5
j
2~5
5JO Cd 2. mfvl
75
_ _ _ ~
100
Fig. 7. Decrease of SH groups in tubulin by Cd2+; 5 ~1 CdCI2 added into 1 ml tubulin (0.5 mg/ml) isolated from rabbit brain. SH groups in tubulin control decreased with the increase of Cd2+ concentration. Each group, n = 5; triple determinations, X + S.D.
Effect o f Cd 2+ on microtubules in vivo Microtubules were examined after Cd 2+ injection into sciatic nerves in vivo to compare its effect on SH group content with those seen in vitro. The changes of microtubular density from a decrease to disappearance were observed with an increase of Cd2+ concentration from 50 to 100 m M (Fig. 8, Table IV). Taking the results together, Cd 2÷ inhibition of free
Fig. 8. Decrease of microtubules by Cd 2+ ; 75 mM CdCI 2 30/A
injected into sciatic nerve (10/A/h), only a few microtubules and/or fragments (arrow) remained. Samples taken as Fig. 4 but immediately after the end of injection. Longitudinal section.
The results d e m o n s t r a t e d that a c o m p a r a b l e associated change of microtubule and transport coexists with experimental drugs, i.e. where microtubules decreased or disappeared the transport usually weakened or blocked (Table V). Therefore, the depolymerization of microtubules seems to participate or mediate a block of transport. In o t h e r words, the microtubules must play a role in axoplasmic transport in vivo. Although the d e t e r m i n a t i o n of intra-axoplasmic Ca 2+ will devote a further p r o o f to the conclusion, unfortunately, that the Ca 2+ concentration in mammalian nerve is unknown and no quantitative m e t h o d can be used efficientlyl,25. H o w e v e r , Ochs et al. 4 in an ultrastructural study with X-ray microanalysis showed that the Ca-containing electron-dense particle density in axoplasm and some organelles was related to Ca 2+ level in m e d i u m , and d e m o n s t r a t e d a Ca 2+ effect on transport in d e s h e a t h e d nerve in vitro5,23; the latter has been confirmed by others ~6,~7 also. As calmodulin facilitated the Ca 2+ effect on inhibition of tubulin assembly (unpublished) and on disassembly of polymerized microtubules ~2, one way in which microtubules b e c o m e d e p o l y m e r i z e d and transport blocked by Ca 2+ may be via calmodulin 23. In addition, according to axonal demyelination of cutting nerve incubated in high Ca 2+ m e d i u m , Schlaepfer 27 suggested that Ca z+ may activate protease. But in our cases, except the b r e a k d o w n of microtubules, all other organeiles kept normal morphological features. Thus, it also did not seem that the effect of experimental drugs on microtubules would be due to a change of osmotic pressure of the injected solution, for example, a d i s a p p e a r a n c e of microtubules was observed with 100 m M Ca 2+ or 100 m M C d 2+, which are isotonic. The SH group loss of tubulin delivered an explanation on molecular basis why Cd 2+ could depolymerize microtubules and block transport. Having observed that the rate or degree of microtubule polymerization in vitro was a function of tubulin concentration ~2, we deduced that axoplasmic free tubulin might be in dy-
81 TABLE IV Summary of Cd2÷ effects on tubulin SH group content, microtubules and ceUifugal transport of labeled protein
CdC12injected into rat sciatic nerve 3 h after the injection of [3H]leucineinto ventral horns. The site of intraneural injection was 60 mm from which L4 ventral roots left the spinal cord. Tubulin was isolated from rabbit brain. N.S., not significant. Cd 2+ (triM)
SH content (%)
Microtubules
Transported distance (ram)
Coiatrol 12.5 25 50 75 100
100 66.5 54.4 45.7 39.5 27.3
With a certain density and length No change No change Decreased mildly Decreased apparently Only a few fragments or disappeared
88 + 0.7 90 + 0 87 + 4.6 81 + 5.8 63 + 3.7 61 + 1.0
namic equilibrium with the tubulin polymerized into microtubules in viv0. Cd 2÷ or colchicine could react with the SH groups of free tubulin and change them into disulfide bondsSAS, 20, thus the polymerization of tubulin was inhibited, the equilibrium might shift to tubulin side and microtubules depolymerized. If it is the case, the SH group loss by Cd2÷ in vitro (Table IV), the converse effects o f - S H inhibitor and -SH donor on transport in vivo (Table II) and the microtubules disassembly by Cd2÷ (Fig. 8) all demonstrated a further proof that a correlation exists among SH content, microtubular density and axoplasmi c transport. However, it could not rule out the possibility that Cd 2+ might react with the SH groups of microtubule associated proteins9, zl, dynein protein 14 or ATPase14As,23,30; all of them would activate microtubule assembly. The associated change of microtubule and transport in vivo presented in this work were different from other studies in vitro, in which only either microtubules or transport were seen to suffer changes by the same drug2,3,6A0. In some of those experiments in vitro, both the fewer experimental drugs selected
P N.S.
N.S. <0.05 <0.001 <0.001
and the narrow range of doses of the tested drugs would be causes which restrict varied and meaningful observations. Our results did show a strict dose response from none to maximum effects among levels of drugs (Table V), the conclusions therefore were reliable. Except the study of Ca 2÷ on microtubules of heliozoan axopodium 2s, few data on the effects of Ca 2÷, E G T A or Cd 2+ on microtubule and axoplasmic transport in vivo have been reported. We further verified the relationship between microtubule and transport with morphological and biochemical studies and compared the in vitro results2,3,5,6,9,10,16,17 to those in vivo. This present in vitro and in vivo work provides further evidence that Ca 2÷ is a key regulator of axoplasmic transport and microtubules are critically involved in the mechanism of this transport. ACKNOWLEDGEMENTS The authors are indebted to Prof. Chiao Tsai for his kind supporting on this work and thank Miss Wang G u o - H w a for her technical assistance.
TABLE V Summary of effects of drugs on microtubules and cellifugal transport of labeled protein in vivo Effects
Ca2+
EG TA
Colchicine
Cd2+
(mM)
On microtubules
No effect Decreased mildly Decreased apparently Disappeared
5-25 25 50 50-200
2.5 5 10 10
25-50 50 100 100-200
12.5-25 50 75-100 100
On transport
No effect Decreased mildly Blocked
5 25 50-200
2.5 5 10
25-50
12.5-25 50 75-100
100-200
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rochem., 36 (1981) 1114-1121. 16 Hammerschlag, R., David, A.R. and Chiu, A.Y., Mechanism of axonal transport: a proposed role for calcium ions, Science, 188 (1975) 273-275. 17 Kanje, M., Edstr6m, A. and EdstrOm, P., Divalent cations and fast axonal transport in chemically desheathed (Triton X-treated) frog sciatic nerve, Brain Research, 241 (1982) 67-74. 18 Kuriyama, R. and Sakai, H., Role of tubulin in sulfhydryl groups in polymerization to microtubules, J. Biochem., 76 (1974) 651-654. 19 Lasek, R.J. and Brady, S.T., The axon: a prototype for studing expressional cytoplasm. Cold Spring Harbor Syrup. Quant. Biol., 46 (1982) 113-124. 20 Mellon, M.G. and Rebhum, L.I., Sulfhydryls and the in vitro polymerization of tubulin, J. Cell Biol., 70 (1976) 226-278. 21 Murphy, D.W., Valle, R.B. and Borisy, G.G., Identity and polyinerization in stimulatory activity of the non-tubutin protein associated with microtubules, Biochemistry, 16 (1977) 2598-2605. 22 Ochs, S., Characteristics and a model for fast axoplasmic transport in nerve, J. NeurobioL, 2 (1971) 331-345. 23 Ochs, S. and Jersild, R.A., Jr., Calcium localization in nerve fibers in relation to axoplasmic transport, Neurochem. Res., 9 (1984) 823-836. 24 Pollard, T.D., Which organelles are necessary for fast neuronal transport?, Neurosci. Res. Progr. Bull., 20 (1981) 92-97. 25 Scarpa, A. and Carafoli, E., Calcium transport and cell function. Part I. Measurements of calcium, Ann. NYAcad. Sci., 307 (1978) 28-124. 26 Schacterle, G.R., A simplified method for the quantitative assay of small amounts of protein in biological material, Anal. Biochem., 51 (1973) 654-655. 27 Schlaepfer, W.W., Experimental alternations of neurofilaments and neurotubules by calcium and other ions, Exp. CellRes., 67 (1971) 73-80. 28 Schliwa, M., The role of divalent cation in the regulation of microtubules assembly, J. Cell Biol., 70 (1976) 527-540. 29 Sedlak, G. and Lindsay, R.H., Estimation of total, proteinbound and non-protein sulfhydryl groups in tissue with Ellman's reagent, Anal. Biochem., 25 (1968) 192-205. 30 Sharp, G.A., Fitzsimons, J.T.R. and Kerkut, G.A., Microtubular ATPase in Carcinus and Helix nerve, Comp, Biochem. Physiol., 61A (1978) 297-301. 31 Shelanski, M.L., Gaskin, F. and Cantor, C.R., Microtubule assembly in the absence of added nucleotides, Proc. Natl. Acad. Sci. U.S.A.. 70 (1973) 765-786.