Cell Motility and the Cytoskeleton 17:10&117 (1990)

Transmembrane Cytoskeletal Modulation in Preterminal Growing Axons: I. Arrest of Bulk and Organelle Transport in Goldfish Retinal Ganglion Cell Axons Regenerating In Vitro by Lectins Binding to Sialoglycoconjugates Brian T. Edmonds and Edward Koenig Department of Physiology, University at Buffalo, Buffalo, New York Goldfish retinal ganglion cell (RGC) axons regenerating in vitro exhibit a novel mode of axoplasmic transport that entails a rapid bidirectional bulk redistribution of axoplasm, “packaged” as protruding varicosities and non-protruding phasedense inclusions (Koenig et al.: J . Neurusci. 5:715-729, 1985; Edmonds and Koenig Bruin Res. 406:288-293, 1987). We have used phase-contrast video microscopy to study transmembrane effects of surface-binding lectins on bulk transport and transport of single visible organelles in RGC axons. Our findings show that certain lectins which crosslink sialoglycoconjugates, such as wheat germ agglutinin (WGA) and the more specific sialic acid-binding lectin Limax flavus agglutinin (LFA), induce a rapid inhibition of transport activity. The LFAinduced inhibition of transport can be reversed by appropriate simple sugar haptens, and can also be antagonized by pretreatment with cytochalasin D. One of the consequences of LFA binding is an increase in RITC-conjugated phalloidin fluorescence staining of preterminal axons. The latter observation in conjunction with the antagonistic action of cytochalasin D suggests that one possible explanation for the transmembrane arrest of transport induced by crosslinking of surface sialoglycoconjugates may involve a polymerization and/or reorganization of the actin filament network which hinders translocation of mobile axoplasmic components. Key words: Wheat germ agglutinin, Limnrflavus agglutinin, axonal cytoskeleton, actin, cytochalasin D, axoplasmic transport

INTRODUCTION Goldfish retinal ganglion cell (RGC) axons regenerating in culture have provided an opportunity to document by video microscopy a novel form of axoplasmic transport which may be characterized as “rapid bulk transport” [Koenig et al., 19851. Mobile entities, which provide the structural basis for this form of bulk transport, move bidirectionally at rapid rates and range in size from large protruding varicosities to smaller varicosities and nonprotruding phase-dense inclusions. Larger varicosities can form through a fusion of smaller varicosities or phase-dense inclusions; they can also diminish in size and even disappear by giving off axoplasmic phase0 1990 Wiley-Liss, Inc.

dense inclusions. Thus, the dynamic changes in bulk contents of varicosities, through gain or loss of phasedense inclusions, indicate a significant intrinsic structural plasticity which represents an important characteristic of this form of bulk transport in regenerating RGC axons in vitro [Koenig et al., 1985; Edmonds and Koenig, 1987al. At the electron microscopic level, varicosities contain a tubulo-vesicular smooth endoplasmic

Received March 28, 1990; accepted June 22, 1990 Address reprint requests to Dr. Edward Koenig, State University of New York at Buffalo, 313 Cary Hall, Buffalo, N Y 14214.

Transmembrane Arrest of Transport

reticulum and an actin-based cytomatrix, which are cotransported during movement of aggregate structures [Koenig et al., 19851. Rapid bulk transport is not unique to regenerating RGC axons of the goldfish, as Hollenbeck and Bray [ 19871 have also observed the presence of mobile aggregates in axons of cultured dorsal root and sympathetic ganglion cells of the chick. Mobile varicosities and nonprotruding phase-dense inclusions appear to be aggregates “packaged” for rapid bulk redistribution of axoplasm [Koenig et al., 19851. The higher transport frequency of smaller varicosities and phase-dense inclusions indicates that they represent the preferred mobile bulk forms. A subsequent video microscopic study has indicated that the movements of varicosities seem to be passive, in that they appear to be carried “piggyback” by highly phase-dense particles [Edmonds and Koenig, 1987al. Pharmacological evidence, furthermore, has suggested that the transport of these phase-dense particles is probably mediated by microtubule-dependent mechanisms similar to those described for single organelles in axoplasm [Allen et al., 1985; Brady, 1985; Paschal et al., 1987; Vale et al., 1985a-c; Vallee et al., 19881. The present report focuses on the effects of selected lectins on the rapid transport of phase-dense particles, nonprotruding inclusions, and varicosities that are visible under phase-contrast video microscopy in growing RGC axons in vitro. Our findings show that lectins having a selective affinity for sialic-acid-containing surface glycoconjugates induce a rapid inhibition of visible transport activity intrinsic to these immature axons. The inhibition is reversed or antagonized by either simple carbohydrate haptens or cytochalasin D. The results show that there is a potential for transmembrane modulation, presumably involving an actin-based cytoskeleton, which can affect transport in growing RGC axons of the goldfish. Preliminary reports of some aspects of this work have appeared in abstract form [Edmonds and Koenig, 1987b, 19881.

107

Retinal Explant Preparation Goldfish retinal explants were prepared as described elsewhere [Koenig and Adams, 1982; Koenig et al., 19851 and were used for experimental observations after 3-5 days in culture. Briefly, 2-4 weeks after crushing the optic nerve, the retina was isolated, chopped into squares (0.65 X 0.65 mm), plated out onto polylysine-coated no. 1.5 circular coverslips, and cultured in L-15 (Gibco) medium, supplemented with 10% fetal calf serum (Flow), 0.02 M Hepes, 0.1 mM 5-fluorodeoxyuridine, 0.1 mg/ml gentamycin sulfate, 0.2 mM uridine and 0.6% methyl cellulose. Explants were cultured in humid air atmosphere. Video Microscopy

For viewing, the circular coverslip was either mounted in a Dvorak-Stotler chamber (Nicolson Precision Instruments), or was inverted over a 35 X 50 mm no. 2 coverslip supported by 0.5-1 mm-thick spacers polymerized and trimmed from Silastic medical elastomer (Dow). While both chambers permit the total exchange of bathing medium within 1 minute, the latter one allowed for more uniform Kohler illumination of axonal fields. The standard bathing medium was a modified Cortland physiological fish saline [Koenig and Adams, 19821 composed of (in mM): 132 NaCl, 5 KCl, 1.6 MgCl,, 1.8 Ca:Cl,, 5.5 glucose, 20 Hepes, adjusted to pH 7.2 with Tris. All experiments were conducted at 22-24°C. Axonal fields were viewed under phase-contrast microscopy (Olympus BHS microscope) with a X 100 oil immersion planapochromat objective (Zeiss N.A. = 1.25) combined with an achromat condenser (Olympus, N.A. = 1.4) oiled to the bottom coverslip. The microscope stage was isolated from external vibration by a Vibraplane air-suspension table-top platform (Kinetic Systems, Inc.). The phase image was displayed on a video monitor (Sanyo) by using a DAGE NC-67M video camera with a Newvicon tube (DAGE-MTI, Inc.) mounted on a trinocular head of the microscope. Experiments were recorded in a time-lapse mode with a video MATERIALS AND METHODS recorder (TLC 2001, GYYR, Inc.), where time was compressed by a factor of 12. Still photographs were Biochemicals The following were purchased from E-Y Labora- taken from the monitor screen by using a Polaroid CU-5 tories: purified Triticum vulgaris (wheat germ agglutinin, Land camera type 665 positivehegative Polaroid film, or WGA), succinylated WGA (sWGA), Limax f l a w s ag- directly through the microscope with an attached 35 mm glutinin (LFA), Texas Red-conjugated LFA, and con- photomicrographic system (Olympus PM- 1OAD) on canavalin A (Con A). Unconjugated Con A, N-acetyl Kodak panatomic X film (ASA 32) developed in D-76. D-glucosamine, N-acetyl neuraminic acid (type VI, E. coli), poly-1-lysine, 5-fluorodeoxyuridine, gentamycin Focal Application of Lectins In some experiments, lectins were applied focally sulfate, uridine, methyl cellulose, cytochalasin D, pertussis toxin, and cholera toxin were purchased from to a targeted varicosity of single axons with a micropiSigma; rhodamine isothiocyanate (R1TC)-conjugated pette. For these experiments, retinal explants were grown on no. 1 rectangular coverslips (1 1 X 40 mm) and phalloidin was obtained from Molecular Probes.

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inverted over a glass viewing chamber containing Cortland fish saline. The chamber was placed on the stage of a Zeiss GFL microscope having a X 100 objective (N.A. = 1.25) and an achromatic aplanatic condenser (N.A. = 0.63 with 7 mm focal intercept in air). Glass micropipettes with tip diameters of 1-2 pm, bent at a 45” angle, and attached to a de Fonbrune micromanipulator were filled at the tip with Texas Red-conjugated lectin solution (1 mg/ml). The tip was positioned about 5-10 pm below the targeted varicosity, and the lectin was expelled by pressure onto the varicosity by using a glass syringe, while the preparation was imaged on the video monitor and recorded. After 1-3 minutes of lectin application, the pipette tip was moved out of the field, while transport activity continued to be recorded. After 10 minutes, the field was viewed with fluorescence video microscopy to evaluate distribution of lectin fluorescence. Longer application times resulted in spread of axonal surface labeling outside of the circumscribed target region. Analysis of Organelle Transport Activity An evaluation of changes in organelle transport activity was obtained as follows. During video playback, a line transecting a single axon or fascicle was drawn on the screen of the video monitor. The number of bidirectionally mobile entities (i.e., particles, mitochondria, phase-dense inclusions, and varicosities) crossing the transection line within multiple 1 minute intervals of elapsed time of the video record was counted before and after a given treatment. Generally, distinct organelles within a single axon could be observed to enter the field of view, crossing the transection line, and to exit the field of view on the monitor screen. This continuous viewing of organelle translocations reduces potential counting inaccuracies that could potentially arise from thermal effects of the microscope stage or axon displacement. A “transport change index” (TCI) of organelle transport activity was computed by the following ratio: TCI = frequency of motile organelles after/ frequency of motile organelles before Results were interpreted such that a ratio of 1.O indicated no change, while a ratio < 1 indicated a reduced frequency of movement activity. Tabulated results were expressed as median and inner quartile range (IQR) values, as these were values used in determining significance. Nonparametric statistical analysis of ratios was performed with a microcomputer software package (Number Cruncher Statistical Systems, Kayesville, UT) at a 0.05 level of significance. The two-sided sign test was used to determine if a given treatment had an effect on transport activity; i.e., if the median ratio after/before the treatment was or was not equal to 1.0. In addition,

the two-sided Mann-Whitney U test (Wilcoxan rank sum test) was used to determine if differences existed between treatment medians. The resulting test statistic T values from the rank sum test were used to calculate z values which were then compared for significance with critical values of the unit normal distribution. The rationale for the use of these specific tests is discussed by Daniel [ 19831. Phalloidin and lmmunofluorescence Staining Retinal explants were rinsed first in Cortland saline and then in modified PHEM buffer [Schliwa and van Blerkom, 19811, which consisted of (in mM) 60 Pipes, 25 Hepes, 15 EGTA, 2 MgCl,, adjusted to pH 6.9 with KOH, and then exposed to a lysis-fixation buffer containing 2% paraformaldehyde, 0.2% glutaraldehyde, 0.5% Triton X-100 in PHEM buffer. After lysis-fixation, coverslips were washed three times for 15 minutes with TM buffer (in mM): 130 NaCl, 10 Tris, 5 KCl, 5 MgCl,, 1 EGTA (pH 7.2) containing sodium borohydride (1.0 mg/ml), and then washed three times for 10 minutes in TM buffer. Incubation with RITC-conjugated phalloidin (0.33 pM) was for 20 minutes. For tubulin immunocytochemistry , coverslips were blocked with 1% BSA in TM buffer for 1 hour before incubation with tubulin antiserum (Polysciences), diluted 1500, in TM buffer. After three 5 minute washes in TM buffer, coverslips were incubated with Texas Red-conjugated goat anti-rabbit IgG (1:2000, EY Labs) for 1 hour. After three 5 minute rinses in TM buffer, coverslips were air dried and mounted with Elvanol (Dow). RESULTS Several classes of mobile aggregate structures and particles of varying sizes and mobilities are visible at the light microscope level within RGC axons [Koenig et al., 1985; Edmonds and Koenig, 1987a1, including the following: i) large varicosities (0.2 p d s e c ) ; ii) smaller varicosities and nonprotruding phase-dense inclusions ( 1-2 p d s e c ) ; iii) mitochondria (- 1 p d s e c ) ; iv) phase-dense (“hyperdense”) particles ranging from small (-1.5 p d sec) to large (- 1.3 p d s e c ) ; and v) low-density particles (-2 p d s e c ) . Mitochondria are easily identified by their elongated morphology and staining with rhodamine 123 (Kodak; unpublished observations). Ultrastructural correlates of mobile hyperdense particles have not been characterized systematically, but probably correspond to several identifiable structures visible in electron micrographs. For example, in addition to uniformly dense and dense-core vesicles [Koenig et al., 19851, which may correspond to small hyperdense particles, there are large electron-dense, multilamellar, lysosomal-like residual bodies (Koenig and Adams, unpublished observations),

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109

TABLE I. Median Transport Change Index Ratios 20 Minutes After Lectin Application* Concentration (wdml) Treatment 1. WGA

P value n 2. LFA P value n 3. LFA vs. WGA T statistic P value

0.98 (.32) 1 .o

5 1.15 (.25) 0.2188 6 24 0.1003

100

150

200

300

0.59 (.24) 0.038 9 0.07 (.23) 0.0156

-

0. (0) 0.156

0.36 (.45) 0.00002 20 0 (0) 0.156

0.16 (.25) 0.00002 22 0 (0) 0. I56

7

7

50

25

0.67 (.33) 0.004 9 0.24 (.51) 0.125 7 19 0.1858

*n = number of transport frequency measurements;( )

=

-

7 -

2 0.0032

7

0 0.0003

-

12

0.0025

inner quartile range; significance at 0.05 level.

which very likely correspond to the large hyperdense particles. Similar structures have also been described in electron micrographs of varicosities in regenerating RGC axons of the goldfish in vivo [Murray, 19761. Both at the electron microscopic (unpublished observations) and video microscopic [Edmonds and Koenig, 1987al levels, one-to-several of the large hyperdense particles may be lodged in a given varicosity. Hyperdense particles are implicated in mediating apparent retrograde ‘‘piggyback” translocation of mobile aggregate structures [Edmonds and Koenig, 1987al. In the present study, the effects of lectin surface binding on transport activity in naked RGC axons were evaluated quantitatively by determining a transport change index (TCI). This entailed comparing the frequency with which visible mobile entities (i.e., hyperdense particles, mitochondria, dense inclusions, and varicosities) in axons crossed a transection line drawn on the monitor screen before with that after lectin treatment (see Methods). Data from single axons and axon fascicles were combined because differences in the TCI ratio between them were not discernable. The smallest, lowdensity particles were excluded from this quantitative analysis because they could not be reliably counted due to their size, although the loss of their movements could be confirmed qualitatively in real-time video microscopy when larger organelle movements were completely arrested. In general, organelle transport frequencies in a single untreated axon were variable with approximately 3-5 mobile species crossing the transection line per minute. WGA-Induced Inhibition of Transport

WGA decreased the frequency of moving mobile species in a dose-dependent manner, as shown in Table I and Figure 1. Frequency measurements were made 20 minutes after application, when a steady state was achieved. As no differences were detected in the frequencies between anterograde or retrograde movements,

!I 1004

v -

4

0 75

1

1

-

0.25 0501

25

50

100

150

200

300

Concentration Lug/ml)

Fig. 1 . Median Transport Change Index (TCI) ratios from Tables I and I1 plotted as a function of WGA or LFA concentration. Ratios were calculated 20 minutes after addition of the specific lectin concentration. Asterisks (*) indicate a significant difference between the median TCI ratios for the same solute concentration of WGA and LFA (note that lectin concentrations are given on a wiv basis; see text for discussion).

data of bidirectional movements were combined. For each concentration of WGA, there was a measurable reduction in frequency which reached significance at concentrations greater than 50 pg/ml. Usually, a clear reduction was evident within 1 minute after application, and was followed by a progressive reduction which reached a maximum by 20 minutes. The inhibition by WGA at each concentration was quite variable (note large inner quartile range values in Table I) in that some axons were devoid of detectable mobile species, while a small pecentage of axons exhibited little or no reduction. Axon morphology appeared unchanged following WGA treatment and there was no apparent correlation between the size of axons monitored and the magnitude of the TCI ratio. It is possible, however, that residual organelle movements within large fascicles may have been due to axons inaccessible to lectin binding.

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TABLE 11. Median Transport Change Index Ratios for Wheat Germ Aeelutinin and Combined Treatments* ~~~~~~

~~

Treatment

n

Median

1QR

P-value

1 . WGA + GlcNAc (200 Fg/ml) (25 mM) 2. GlcNAc (25 mM) 3. WGA + NeuNAc (100 mM) 4. NeuNAc (100 mM) 5. sWGA before WGA (200 Fgirnl each) 6. sWGA

50

1.12

0.49

0.15

29

1.05

0.26

1 .0

I

0.98

0.28

1.0

14

1.12

0.26

0.02

6

0.58

0.28

0.03

12

1.09

0.53

0.17

*n, number of frequency transport measurements; IQR, inner quartile range.

Generally, the larger motile components were affected first. Thus, the movements of varicosities, phasedense inclusions, larger hyperdense particles, and the largest mitochondria were rapidly arrested. In addition, the typical axial micro-oscillations of stationary varicosities caused by particles entering or leaving [Edmonds and Koenig, 1987al ceased. The smooth and continuous movements of smaller hyperdense particles became saltatory and interrupted, and then became arrested or exhibited axial oscillations in a delimited axon segment. Addition of N-acetyl D-glucosamine (GlcNAc; 25 mM), a hapten of WGA, restored all transport activity to normal levels following WGA treatment, and it antagonized WGA-induced inhibition when added simultaneously with the lectin (TCI = 1.12; IQR = 0.49; Table 11). GlcNAc alone had no effect on frequency of particle movements (TCI = 1.05; IQR = 0.26; Table 11). WGA also binds to surface glycoconjugates containing N-acetyl neuraminic acid (NeuNAc) residues [Kronis and Carver, 1982, 1985a,b; Peters et al., 1979; Wright, 19801. When NeuNAc (100 mM) is added simultaneously with the lectin, the WGA-induced inhibition of transport is blocked. Furthermore, if NeuNAc is added after WGA has already established its maximal reduction in transport frequency, an immediate reversal occurs (TCI = 0.98; IQR = 0.28; Table 11). Thus, both GlcNAc and NeuNAc are each capable of antagonizing WGA-induced inhibition of transport in RGC axons. To discriminate between which of the WGAbinding carbohydrate moieties may be associated with the lectin’s inhibitory effect on transport, a modified form of WGA was tested. Succinylated WGA (sWGA) is specific for GlcNAc and does not bind NeuNAc [Monsigny et al., 19791. When RGC axons were treated with sWGA alone, transport frequencies were unaffected (TCI = 1.09; IQR = 0.53; Table 11). If unmodified WGA was applied after treatment with sWGA, then the

reduction in transport activity was not as marked as when axons were treated with WGA alone (i.e., WGA-induced inhibition was attenuated). Specifically, the TCI ratio for WGA (300 pg/ml) alone was 0.16; IQR = 0.25 (Table I), whereas sWGA (200 pg/ml) followed by WGA (300 pg/ml) yielded a TCI ratio of 0.58; IQR = 0.28 (Table 11). This difference between medians was significant (T = 106; P = 0.025). These observations suggested that it was the binding by WGA to NeuNAc-containing surface glycoconjugates that may have been important for inhibiting transport activity.

Inhibition of Transport by a Sialic-Acid-Specific Lectin

Lirnax f l a w s agglutinin (LFA), a lectin which is specific for NeuNAc [Miller, 1982; Miller et al., 19821, dramatically decreased transport frequency. Compared to WGA, lower solute concentrations of LFA resulted in significantly greater reductions of all mobile species resolvable with our system (Fig. 1, Table I). Whereas a progressive reduction in transport frequency occurred with most concentrations of WGA, a gradual reduction was only seen at the two lowest concentrations of LFA tested. In all cases where higher LFA concentrations were used, a virtually complete arrest of transport activity occurred within the first few minutes of exposure. Another difference from that of WGA effects on transport was the lower variability within the LFA-treated population, as indicated by the smaller inner quartile ranges at higher concentrations. While WGA and LFA are compared on a w/v basis, the molecular weights of WGA (36 kD) and LFA (44 kD) are not sufficiently different to affect the overall relationship shown in Figure 1. The hapten sugar also antagonizes the effect of LFA on transport activity. In accordance with the reported carbohydrate specificity of LFA, addition of NeuNAc (25 mM) blocked (TCI = 1.03; IQR = 0.38; Table 111) or immediately reversed (TCI = 1.17; IQR = 0.15; Table 111) the typical inhibitory effects. In contrast to GlcNAc, NeuNAc alone slightly enhanced the frequency of particle transport frequency of untreated axons (TCI = 1.12; IQR = 0.26; P < 0.02; Table 11). Concanavalin A (Con A), a tetravalent lectin that possesses an affinity for terminal mannosyl and glucosyl residues [Lis and Sharon, 19861, had little or no effect on transport frequency up to concentrations of 400 pg/ml (TCI = 0.9; IQR = 0.41, P < 1.0); lectin binding to RGC axons was confirmed by using Texas Redxonjugated Con A (data not shown). This suggests that the property of lectin polyvalency alone is insufficient to explain the results observed with WGA and LFA.

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111

TABLE 111. Median Transport Change Index Ratios for L i m a .flavus Agglutinin and Combined Treatments* Treatment

I . LFA + GlcNAc (100 pg/ml) (25 mM) 2. NeuNAc before LFA (25 mM) 3. NeuNAc after LFA (25mM) (100 pgiml) 4. WGA before LFA (300 pg/ml) 5. CD before LFA (10 FM) 6. CD (10 u.M)

n Median 8 0.28

IQR

P-value

0.19

0.008

.o

12

1.03

0.38

1

7

1.17

0.15

0.125

6

0.04

0.01

0.03

9

0.9

0.22

0.5

6

0.96

0.25

0.2

*n, number of transport frequency measurements; IQR, inner quartile range; CD, cytochalasin D.

Inhibition of Transport by Focal Application of LFA

Cytochemical survey of LFA binding distributions showed that labeling was preferentially localized to varicosities and to a lesser extent to growth cones [Edmonds and Koenig, 1990~1.It was of interest, therefore, to determine if a local blockade of transport could be induced by applying LFA to a single varicosity of an isolated axon. Texas Red-conjugated LFA (1 mg/ml) was applied focally with a micropipette (Fig. 2a) over a period varying from 1 to 3 minutes. Particle traffic through the targeted region was monitored by video microscopy while the lectin was being expelled and was always arrested during the period of application. The blockade of transport was transient, however, as indicated by a gradual return of transport frequency to near control levels after 5 minutes. Nevertheless, LFA caused a significant reduction in particle frequency at the zone of application during the first 5 minutes following cessation of application (TCI = 0.395; IQR = 0.24; P = 0.0002; Table IV). The time required for recovery of particle transport seemed directly related to the duration of the application; i.e., a longer application time resulted in a longer period before particle traffic recovered to normal frequency. LFA binding at the application site was confirmed by viewing the live preparation with fluorescence video microscopy (Fig. 2c). Particle traffic was unaffected in those areas displaying no lectin binding, except in regions immediately contiguous to the application site. Texas Red-conjugated Con A ( 1 mg/ml) was similarly applied to single varicosities of individual axons. While fluorescence due to Con A binding was present after application (data not shown), no effects on particle frequency were observed either during or after application (TCI = 0.78; IQR = 0.45; P = 0.2266; Table IV).

Fig. 2. Focal application of LFA onto a single varicosity. a: Phasecontrast video micrograph taken from the video record showing a single axon, while LFA-conjugated Texas Red ( I mg/ml) is expelled by micropipette onto a single varicosity (open arrow). Asterisks (*) indicate debris on the coverslip. b: Same axon as in a within 1 minute after cessation of LFA application. c: Same as in a,b viewed with fluorescence video microscopy 10 minutes after cessation of LFA application. Note that LFA labeling is confined to the target varicosity, a small de-novo-formed varicosity immediately to the left of the target, and to the debris adjacent to the location of the pipette tip, while the intervening segments and the varicosities in the upper right corner show little or no LFA binding. The bright area in the field is due to non-uniform distribution of light. Bar: 10 pm.

The difference between TCI medians of the LFA and Con A treatments applied in this manner was highly significant (T = 154; P = 0.00001).

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TABLE IV. Median Transport Change Index Ratios for Lectins Focally Applied to a Single Varicosity' Lectin

n

Median

IQR

P value

1. LFA (1 mg/ml) 2. Con A (1 mgiml)

14 11

0.395*

0.24 0.45

0.0002 0.2266

0.78*

'TCI ratios determined from data obtained within 5 minute period after cessation of application. n, number of transport frequency measurements; IQR, inner quartile range. *Significantly different from each other; P < 0.00001; T statistic = 154.

LFA-Induced Increase in Phalloidin Fluorescence in Preterminal Axons

works [Schliwa, 19821, blocked LFA-induced inhibition of transport when axons were pretreated 1 hour before LFA exposure (TCI = 0.9; IQR = 0.22; Table 111). The antagonism of inhibition by CD was not due to an interference with lectin binding, as the distribution of Texas Red-conjugated LFA fluorescence was normal (data not shown). However, an assessment of phalloidin fluorescence patterns was inconclusive when CD treatment in combination with that of LFA was compared with LFA treatment alone because CD produces focal actin aggregates which exhibit strong phalloidin fluorescence staining [Edmonds and Koenig, 1990bl (see Discussion).

Probes of Second-Messenger Involvement in Phalloidin labeling of RGC axons is qualitatively Transport Inhibition different from that of actin immunofluorescence [EdIt is possible that inhibition of transport in response monds and Koenig, 1990bl. For example, while both to lectin binding to the membranecould involve a signal phalloidin fluorescence and actin immunofluorescence transduction pathway which would increase one or more are generally strong in growth cones, phalloidin fluores- intracellular second messengers such as calcium or cence is generally very weak in preterminal axons, where CAMP. Lectin-induced inhibition of transport is unafit is represented by infrequent fluorescent puncta or short fected by the presence or absence of extracellular calstreaks (Fig. 3), with streaks observed occasionally only cium. As shown elsewhere [Edmonds and Koenig, in fascicles (Fig. 3d). Actin immunofluorescence, on the 1990a1, however, the use of a calcium ionophore and other hand, is strong, and shows a preferential distribu- extracellar [Ca2'] buffered to 100 p M to increase intration to varicosities and phase-dense inclusions in preter- cellular [Ca"] in RGC axons without lectin treatment minal axons [Koenig et al., 19851. significantly reduces TCI ratios; however, this occurs After LFA treatment there was a notable increase in over a time course of about 20 minutes and is correlated overall RITC-conjugated phalloidin fluorescence stain- with an axonal shrinkage caused by an apparent activaing (Fig. 4). Growth cones generally became involuted tion of calcium-dependent K channels, which leads to and became frozen when LFA took full effect (Fig. 4a); a loss of cellular K + and osmotically obligated water. however, they still exhibited a strong fluorescence sig- Not only is the latency of onset in the decrease in transnal. In preterminal axons, most varicosities showed an port activity an order of magnitude larger, but antagoincreased level of fluorescence, and many also exhibited nists of K' channels, which block shrinkage and inhistrongly fluorescent puncta. On the other hand, the dis- bition of transport [Edmonds and Koenig, 1990a1, also tribution of phalloidin fluorescence following exposure have no effect on the lectin-induced inhibition of transto Con A was not different from that of control (data not port (data not shown). shown), a finding consistent with Con A's lack of effect The use of permeant cAMP analogs, such as dibuon transport activity. Although limitations in resolution tyryl cAMP (20 mM) or 8-bromo cAMP (10 mM), also hindered definitive assessment of microtubule distribu- causes a reduction of organelle transport activity; howtion, there appeared to be little change from that of con- ever, both the latency of the effect and the mechanism trol following LFA treatment as indicated by simulta- appear to be similar to those of increasing intracellular neous lysis-fixation and indirect immunofluorescence of calcium concentration. Thus, the volume decrease intubulin (data not shown). These findings suggested that duced by cAMP analogs appears also to be caused by a LFA appeared to alter the actin-based cytoskeleton in an loss of cellular K + , and the latter effect is dependent on apparently selective manner. the presence of extracellular calcium [Edmonds and Koenig, 1990al. Cytochalasin D Antagonism of the LFA-Induced Experiments probing potential involvement of G Inhibition of Transport proteins in mediating lectin-imediated inhibition of transThe increase in phalloidin signal in response to port by perturbing G protein function failed to yield evLFA binding provided presumptive evidence of an in- idence of any modification. For example, pretreatment crease polymerization and/or reorganization of F-actin. of axonal fields for 18-24 hours prior to lectin exposure Cytochalasin D (10 pM; from an ethanol stock solution), with either pertussis toxin (15 pg/ml) or cholera toxin which perturbs actin filament elongation [Bonder and (15 Fg/ml), established modifiers of G protein function Mooseker, 1986; Cooper, 19871 and actin filament net- [see Stryer and Bourne, 1986; Gilman, 19871, were in+

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Fig. 3. Distribution of phalloidin fluorescence staining in RGC axons. a, c: Phase-contrast and (b, d) fluorescence micrographs of areas labeled with RITC-conjugated phalloidin. a, b: Growth cones showing typical strong fluorescence staining. c, d: A proximal axon field showing fascicles that contain several varicosities. Phalloidin fluorescence staining is typically weak with occasional strongly stained puncta and/or streaks (e.g., note streak associated with the fascicle in d). Bar: 10 p n .

Rapidly moving hyperdense particles are prevalent in growing RGC axons and appear to play an important role in mediating redistribution of varicosities and phasedense inclusions by a “piggyback” mode [Edmonds and DISCUSSION Koenig, 1987al. Exposure of RGC axons to WGA caused a signifSurface-binding lectins were utilized in the present study to probe for transmembrane modulation of or- icant inhibition in the bidirectional transport frequency of ganelle and bulk axoplasmic transport activity in grow- visible mobile entities, while exposure to Con A had no ing RGC axons. Bulk axoplasmic transport is a form of such effect (Table I, Fig. 1). Mobility of the larger structransport that appears to be associated with immature, tures, such as varicosities, phase-dense inclusions, migrowing axons [Koenig et al., 1985; Hollenbeck and tochondria, and large hyperdense particles, was preferBray, 19871. It entails a rapid, bidirectional redistribu- entially blocked by the WGA treatment. While the tion of axoplasm in a bulk aggregate form, appearing as incomplete transport blockade induced by WGA was remobile varicosities and nonprotruding phase-dense inclu- versed after adding the hapten sugar GlcNAc, sWGA, sions [Koenig et al., 19851. Axonal varicosities contain which binds GlcNAc residues exclusively [Monsigny et a tubulo-vesicular SER embedded in an actin-based cy- al., 19791, had no effect on transport (Table 11). As tomatrix, and from an immunocytochemical standpoint, WGA binds GlcNAc and NeuNAc [Peters et al., 1979; they also contain spectrin, calmodulin [Koenig et al., Wright, 1980; Kronis and Carver, 1982, 1985a,b; Lis 19851, and myosin [Edmonds and Koenig, 1990b,c]. and Sharon, 19861, NeuNac also reversed the inhibition

effective in altering the typical inhibition of transport activity by LFA.

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Fig. 4. a, c: Phase-contrast and (b, d) fluorescence micrographs, showing distribution of RITCconjugated phalloidin in RGC axonal fields after treatment with LFA (100 kg/ml). b, d: Pretenninal axons, including varicosities, exhibit an overall enhanced fluorescence staining (cf. Fig. 3d).

(Table II), albeit at a higher concentration (see below). LFA, which is specific for sialic acid residues [Miller, 1982; Miller et al., 19821, was more potent than WGA in inhibiting transport activity, both in the rapidity of onset and in the extent of inhibition (Table I, Fig. 1). Obviously, these findings may or may not necessarily apply to mobile particles below the level of resolution of phasecontrast video microscopy; nevertheless, they suggest that one or more sialoglycoconjugates may be involved in mediating the visible transmembrane inhibitory action of the lectins. Given the carbohydrate specificity, the rapidity of the onset of inhibition, the rapid reversal of inhibition by the hapten, and the antagonism of the inhibition by CD, it is unlikely that the transport perturbation is simply caused by a nonspecific irreversible loss of viability of affected axons. While each of the two sugar haptens reversed WGA-induced inhibition, GlcNAc was four times more p e n t compared to NeuNAc (Table II), and is in agreement with other reports [Peters et al., 19791. The stereochemical similarities and chemical differences between

GlcNAc and NeuNAc account for differences in relative affinities by WGA for the two sugars [Bhavanandan and Katlic, 19791. On the other hand, the measured affinities of WGA to whole cells or to carbohydrate oligosaccharides common to membranes are higher than those for free sugars [Kronis, and Carver, 1982, 1985a,b) due to multiple interactions with the four available sugarbinding sites per WGA dimer. In any case, the differences in efficacy for hapten-mediated reversal appear to parallel the relative affinities for the free sugars. Since both lectins bind NeuNAc, it is unclear why WGA-induced inhibition failed to exceed -SO%, while that of LFA produced 100% (Fig. 1). An explanation for the difference is a matter of conjecture because the nature of the membrane signal that triggers the inhibition of transport in response to lectin binding is unknown. One possibility may relate to the broader carbohydrate specificity of WGA. For example, the heterogeneity of WGA surface-binding reactions may attenuate the membrane signal and cause a suboptimal activation of the specific pathways responsible for inhibiting transport activity.

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Alternatively, multiple surface-binding reactions of dependent on size in situ, they become independent of WGA may activate at least two distinct transmembrane size when undergoing transport on isolated transport filpathways, the first of which may induce arrest of trans- aments (i.e., microtubules). Based on the foregoing observations, we favor a port, and a second of which may incompletely antagonize the first. In this context, it is of interest that pre- mechanism in which an increased hindrance to axial treatment of RGC axons with sWGA, which is specific translocations causes a blockade of transport. The infor GlcNAc [Monsigny et al., 19791, causes an attenu- crease in phalloidin fluorescence in preterminal axonal ation of response to subsequent WGA exposure (Table fields after LFA (Fig. 4) is consistent with an increased 11) and an increase in the time required for arrest ordi- density of F-actin. This could signify an altered organinarily to occur after LFA treatment (data not shown). zational state of the cytomatrix which could hinder orSuch observations are more consistent with the second ganelle transport in the microtubule domain. It is noteworthy in this context that WGA has been shown to explanation and suggest areas for further investigation. The preferential localization of LFA-binding sites increase the amount of polymerized actin in lymphocytes on varicosities [Edmonds and Koenig, 1990~1prompted [Rao, 19841. Consistent also with such an interpretation experiments aimed at determining the effects of focal is the antagonism of LFA-induced inhibition by CD application of LFA to a single varicosity of a single (Table 111). Cytochalasins act primarily by capping the axon. While LFA inhibited transport through the targeted actively growing ends of actin filaments [Cooper, 19871, varicosity during the application, the inhibition was tem- modifying filament elongation [Bonder and Mooseker, porary. The transient nature of the inhibition was not 19861 and inhibiting actin polymerization [Goddette and simply due to the action of a polyvalent surface-binding Frieden, 19861. Thus, CD may block LFA-induced Fligand because a similar application of Con A had no actin formation and/or reorganization. Cytochalasins are also capable of disrupting actin effect on transport. The efficacy of cytochalasin D to antagonize LFA-induced blockade of transport (Table networks independent of their inhibition of actin poly111) implicates an actin-based cytoskeleton in the inhibi- merization [Schliwa, 19821. In RGC axons [Edmonds tory process (see below). Thus, the observed reversibil- and Koenig, 1990b1, as well as in other cell types [Godity may relate to a greater potential for reversible changes man et al., 1980; Britch and Allen, 1981; Palevitz, in the organizational state of the varicosity’s cytoskele- 19881, CD treatment results in the formation of discrete ton when only a delimited axonal region is affected by cytoskeletal focal aggregates which contain actin and exlectin binding. The spontaneous reversibility notwith- hibit strong phalloidin staining. Focal actin aggregates standing, local changes intrinsic to a single varicosity are especially prevalent in varicosities, and the strong appear to be sufficient to account for the inhibition of phalloidin staining is in contrast to weak phalloidin staining in untreated varicosities (Fig. 3). The formation of transport. Mechanisms underlying the perturbation of trans- focal actin aggregate [Edmonds and Koenig, 1990bl preport are not readily apparent, as current evidence sug- sumably represents a “collapse” of a low-density actingests that transport of single organelles involves ATP- based meshwork, in which a diffuse, weak phalloidinand microtubule-dependent soluble translocators [Brady , staining distribution is transformed into a strong 1985; Paschal et al., 1987; Vale et al., 1985a-c; Vallee phalloidin-staining aggregated distribution. This suget al., 19881. While transport function could be inhibited gests a possible mechanism for CD-mediated antagonism by perturbation of processes directly involved in trans- of LFA-induced inhibition of transport; i.e., CD-induced port and cannot be ruled out at present, an indirect effect “collapse” of the actin-based cytomatrix may remove or seems more likely. There was a size-related order of deplete cytoskeletal impediments in the microtubular doinhibition of mobile entities, with the largest being af- main that might otherwise form in response to lectin fected first. Even in the case of smaller organelles which binding. Although our results suggest that LFA may cause a were not arrested following treatment with WGA, there was a transition from smooth to irregular saltations. superfluity of polymerized actin in the microtubular doVideo microscopic studies of organelle movements indi- main which could hinder transport, there are several recate that interactions between organelles and axoplasm, ports in the literature which indicate that a normal actin particularly of larger particles, interfere with smooth lin- organization is an important ancillary factor in supportear motion [Koles et al., 1982; Vale et al., 1 9 8 5 ~ ; ing axonal transport. For example, DNase I, which comKoenig, 19861, which imply transient attachments and/or plexes G-actin, or gelsolin, a calcium-dependent, F-actin collisions with elements of the cytomatrix. The latter severing and capping protein, each inhibit rapid transport interpretation is consistent with observations by Vale et [Isenberg et al., 1980; Goldberg, 1982; Nemhauser and al. [1985c], who showed that while transport velocities Goldberg, 1985; Brady et al., 19841. In the case of of organelles in the axoplasm of the squid giant axon are DNase I, microinjection into the large axon of the R2

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neuron in Aplysia causes clustering of microvesicles, significant loss of microtubules, and some apparent misorientation of neurofilaments, indicating that polymerized actin may normally play important structuring and stabilizing roles for axonal microtubules in the mature axon [Nemhauser and Goldberg, 19851. On the other hand, dihydrocytochalasin B does not affect transport in the R2 neuron [Goldberg, 19821. In immature axons, disruption of the actin cytoskeleton induced by the cytochalasins may actually faciliate transport in certain instances. For example, cytochalasin E treatment of immature chick dorsal root axons increases the number of retrogradely moving varicosities [Hollenbeck and Bray, 19871. We have also observed that CD treatment causes an increase in the TCI ratio (i.e., > 1.0) in RGC axons; however, the apparent facilitation of transport occurs only in axons that are more mature than those used in the present study (unpublished observations). The basis for the latter agerelated phenomenon is unknown, although it could be explained by an accretion of neurofilaments with maturation [Hoffman et al., 19841, whose normal distribution within the axon may affect transport activity and which may also be influenced by the organization of actin. In summary, lectin cross-linking of one or more surface sialoglycoconjugates of immature RGC axons in vitro causes a significant inhibition of transport of mobile entities visible at the light microscope level. A potential mechanism consistent with available data is that the lectin binding induces a polymerization and/or reorganization of the actin-based cytoskeleton which hinders organelle translocation. Such a mechanism would suggest that perturbation of microtubular-based transport function is secondary to a transmembrane modulation of the state of actin polymerization. ACKNOWLEDGMENTS

We thank Mr. Douglas Currant-Everett for his assistance with the TCI ratio statistical analysis and Mrs. Sarah Finnegan Sloan for her technical assistance. This research was supported in part by BRSG SO7 RR0540026 awarded by the Biomedical Research Support and Grant Program, Division of Research Resources, NIH. REFERENCES Allen, R.D., Weiss, D.G., Hayden, J.H., Brown, D.T., Fugiwake, H . , and Simpson, M. (1985): Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport. J . Cell Biol. 100:1736-1752. Elhavanandan, V., and Katlic, A.W. (1979): The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid. J . Biol. Chem. 254:4000-4008.

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