Nerve growth cones isolated from fetal rat brain: Subcellular fractionation and characterization

Cell, Vol. 35, 573-584,

December

1983 (Part I), CopyrIght

0092-8674/83/l

0 1983 by MIT

20573-l 2 $02.00/O

Nerve Growth Cones Isolated from Fetal Rat Brain: Subcellular Fractionation and Characterization Karl H. Pfenninger, Leland Ellis,* Marian Linda B. Friedman, and Stefan Somlo Department of Anatomy and Cell Biology Columbia University College of Physicians and Surgeons New York, New York 10032

P. Johnson,

Summary The biochemical and functional characterization of the nerve growth cone is of major interest for studies on mechanisms involved in nervous system development. We describe the isolation from fetal brain of membrane-bound fragments of nerve growth cones by density gradient fractionation. These socalled growth cone particles are highly uniform and identifiable on the basis of their organelle complement. Furthermore, they co-purify in mixing experiments with fragments of radiolabeled and light microscopically identified nerve growth cones from primary cultures. The possibility of isolating growth cone fragments in quantity renders feasible the analysis of molecular mechanisms involved in growth cone function. Introduction After their terminal mitosis, neurons sprout to form long and slender processes, axons and dendrites. The formation of neurites establishes cellular polarity and leads to the highly complex and selective intercellular connectivity that characterizes the mature nervous system. During development, the neurite’s leading edge is formed by a nerve growth cone (Harrison, 1910; Ramon y Cajal, 1960; Tennyson, 1970; Yamada et al., 1971; Bunge, 1973). This specialized structure is capable of finding its path to the appropriate target area and of recognizing appropriate target cells for synapse formation. Thus, the nerve growth cone plays a pivotal role in nervous system development, and further progress in this field depends upon our ability to isolate and study it with modern biochemical methods, Previous studies of subcellular fractions from developing brain (Gonatas et al., 1971; Grove et al., 1973; Kanerva et al., 1978) have been focused on the isolation of immature synapses; the methods used have been based on protocols for the preparation of pinched-off nerve terminals, synaptosomes (Gray and Whittaker, 1962). A procedure specifically designed to isolate nerve growth cones has heretofore not been available. A major problem with the isolation of growth cones from fetal brain is the lack, at present, of known biochemical markers. Therefore, the initial phase of studies on growth cone isolation is bound to rely heavily on ultrastructural criteria. The strategy of the l

Present address: Dept. of Biochemistry University of California at San Francisco,

and Biophysics, MedIcal Center, San Francisco, California 94143.

present study is to prepare subcellular fractions of low buoyant density from fetal rat brain and to characterize them using the well-documented ultrastructural features of the growth cone (del Cerro and Snyder, 1968; Tennyson, 1970; Yamada et al., 1971; Bunge, 1973) as a measure of enrichment. In this first paper, we describe the method of isolation, cytological characteristics, and polypeptide composition of a subcellular fraction that is highly enriched in pinched-off fragments of nerve growth cones. A forthcoming paper (Ellis and Pfenninger, in preparation) will focus on the biochemical characterization of major polypeptides of the growth cone. Preliminary reports of these findings have been presented in abstract form (Ellis et al., 1982; Pfenninger et al., 1982). Results The flow diagram shown in Figure 1 summarizes the fractionation method used for the studies reported here. The low speed supernatant of fetal brain homogenate is loaded onto a discontinuous sucrose density gradient and spun to equilibrium in a vertical rotor. The cytological features of the various resulting fractions are described in the following sections. Ultrastructure of Subcellular Fractions of Fetal Brain Homogenate and Low Speed Subfractions As expected, gently prepared homogenate of fetal brain consists of a wide range of large and small cellular fragments (Figure 2) which can be separated by a brief low speed spin. The low speed pellet is composed of large elements, mainly cell perikarya whose processes have been sheared off, and nuclei. (Note that Figures 2, 3, 5, and 7 are all at the same total magnification). Most of the small elements are retained in the low speed supernatant, as shown in Figures 3 and 4. These small elements fall into two classes: (i) individual organelles such as mitochondria, rough endoplasmic reticulum, and unidentified membrane sacs and (ii) considerably larger, membranebound structures that contain microfilamentous material and various other organelles. Some of these pinched-off cell fragments contain numerous ribosomes whereas others seem to be free of these organelles (Figure 4). Subfractions of the Low Speed Supernatant The denser subfractions B and C of the low speed supernatant (cf. Figure 1) are illustrated in Figures 5 and 6. The B-fraction (Figure 5) is enriched in highly electron-dense, small cell fragments whose appearance suggests hypertonic shrinkage during preparation. Some organelles, especially structures resembling isolated Golgi cisternae, are also evident. Fraction C is considerably more heterogeneous and contains a wide range of highly electron-dense or partially lysed cell fragments and numerous individual organelles. At higher magnification, endoplasmic reticulum with polyribosomes attached, mitochondria, and membrane ghosts can be identified (Figure 6). Furthermore,

Cell 574

PREPARATION

OF NERVE

GROWTH

CONE PARTICLES

LSP, low speed pellet; LSS, low speed supematant. The fractions from the discontinuous sucrose density gradient are referred to in the text as indicated (A, B, C). For further explanations, see text.

only 1.1% of the points classified fall onto elements whose structure is incompatible with that of an A-particle (see below). Ribosome-containing structures form the largest class of such contaminants (0.6%). The other contaminants (0.5%) include free mitochondria and, rarely, small cell fragments containing lipid droplets. The balance of the fraction (22.7%) is comprised of tangentially cut elements that cannot be identified for lack of ultrastructural detail in the plane of section. This stereologic study has been extended to the low speed supernatant and the homogenate in order to trace back the characteristic A-particles and to assess their enrichment. Seventy-six-fold enrichment of A-particles as a result of the two fractionation steps is evident (Table 1). The relative amounts of protein recovered during preparation of the A-fraction are listed in Table 2. In a representative experiment, one obtains approximately 30 mg of protein in the A-fraction from approximately 6 g (wet weight) of fetal brain. However, as explained below, most of the protein in the A-fraction consists of soluble contaminants from the load (LSS). The actual yield of A-particle protein is approximately 6 mg, which represents less than 2% of homogenate protein. Without biochemical markers for the growth cone, “specific activities” of the subcellular fractions cannot be determined. Therefore, it is not possible at present to determine the actual recovery of Aparticles from fetal brain.

one can sometimes see elongated profiles containing microtubules in parallel array and an occasional dense core vesicle (Figure 6). Such cytological features are typical of neuritic shafts. The A-fraction, collected at the interface between the load and 0.75 M sucrose, has different ultrastructural features (Figure 7): it is highly homogeneous and consists almost exclusively of membrane-bound cell fragments of medium electron density. (A detailed account of the ultrastructure of these “A-particles” is presented in the next section.) Isolated organelles, membrane ghosts, or “myelin figures” are very rare in, or absent from, the A-fraction. In order to assess the purity of the A-fraction, a stereologic analysis has been carried out using the point-counting method to determine the fractional volume of A-particles and contaminants (Weibel and Bolender, 1973). As shown in Table 1, 76.2% of all elements present in the A-fraction have ultrastructural features characteristic of A-particles;

As already pointed out, A-particles are uniformly sized (0.3 to 1.5 pm diameter), membrane-bound cell fragments of medium electron density that are filled with fibrillar material (Figures 7 and 8). The further cytological characterization of these structures can best be done by comparing them with intact growth cones of sprouting neurons (Figures 9 and IO). The nerve growth cone is a terminal enlargement, sometimes several micrometers in diameter, that contains numerous filopodial and veil-like extensions and a characteristic set of cytoplasmic organelles (cf. Tennyson, 1970; Yamada et al., 1971; Bunge, 1973). As shown in Figures 9 and IO, the typical organelles include, in the growth cone periphery, a plethora of microfilaments and clusters of irregularly shaped, large clear vesicles. In the more central portions of the nerve growth cone agranular reticulum, large dense core vesicles, mitochondria, secondary lysosomes, and microtubules are typical. However, ribosomes

I t LSP

t LSS I

discord

Frgure 1. Flow Diagram for the Preparation

Figures 2-6.

Ultrastructure

of Growth

Cone Particles

of Low Speed, B and C Fractions

Ultrastructural Characterization of “A-Particles”

of Fetal Brain

The homogenate, as shown in Figure 2, consists of a mixture of large and small cellular fragments and organelles. Note the elongated profiles (asterisk), putative neurite fragments. Figures 3 and 4 show the ultrastructural characteristics of the low speed supernatant. As can be seen at higher magnification (Figure 4) the low speed supernatant consists of numerous organelles (e.g. rough endoplasmic reticulum, r) and various sheared-off cell fragments and processes. Some of these structures (open arrows) contain filamentous material and various other organelles, including prominent clusters of large, clear vesicles, but no nbosomes. Other cell fragments (*) are denser in texture and contain numerous ribosomes. The B-fraction (Figure 5) is characterized by pleomorphic. highly electron-dense cellular fragments containrng various organelles. In contrast, fraction C (Figure 6) consists mainly of disrupted, lysed cell fragments (f) and many organelles such as mitochondria and rough endoplasmic reticulum (r). In addition, elongated profiles containing parallel arrays of microtubules can regularly be seen. These have the cytological characteristics of partially lysed neuritic shafts (s). Calibration, 2 pm for Figures 2, 3, and 5 (for easier comparison, Figures 2, 3, 5, and 7 are shown at the same magnification): calrbration for Figures 4 and 6, 0.5 pm.

Isolation of Nerve Growth 575

Cones

Cell 576

Figure 7. Low Power Survey Picture of the A-Fraction Note the high degree of homogeneity of this fraction magnification In Figure 8. Calibration, 2 gm.

compared

to that of the previously

are absent from axonal nerve growth cones. All these ultrastructural features can be rediscovered in A-particles, as shown in Figure 8. Microfilaments are abundant, and clusters of large, clear vesicles are seen in most crosssections of A-particles. However, these vesicles often appear ballooned and/or coalesced. Furthermore, mitochondria, agranular reticulum, large dense core vesicles, and secondary lysosomes can always be seen in a crosssection through a group of several A-particles. As in the axonal nerve growth cones, ribosomes are strikingly absent from A-particles, Microtubules have not been seen in A-particles. The relative contribution by different organelles to the total membrane complement of A-particles is of major interest for biochemical and functional studies. The assessment of the relative size of membrane compartments was carried out by stereologic intercept analysis (Weibel and Bolender, 1973) on electron micrographs from two

illustrated

preparations.

The A-particles

are shown

at higher

different A-particle preparations (Table 3). By far the largest compartments are plasmalemma and large clear vesicles, which constitute together over 70% of the membrane area. All other membranous organelles, including the agranular reticulum, form only minor fractions. As can be seen in Table 3, small, uniformly spherical vesicles (approximately 60 nm in diameter) are found in rare instances. They are morphologically identical with synaptic vesicles. Aside from their unusual organelle content, nerve growth cones exhibit characteristic plasmalemmal structure as revealed by freeze-fracture analysis (Figure 11). Growth cone plasmalemma is unusually poor in intramembranous particles (IMPS), especially on mound-like protrusions covering superficial clusters of the large clear vesicles (Pfenninger and Bunge, 1974; Small and Pfenninger, submitted). These almost particle-free plasmalemmal protrusions are surrounded by a series of pits, the openings of invaginations (cf. Yamada et al., 1971; Bunge, 1973; Pfenninger

lsolatron of Nerve Growth 577

Table 1. Enrichment

Cones

of A-Particles

in Subfractions

of Fetal Brain

Volume Fraction” A-Particles

Other Particles

Purification

n/P

Homogenate

0.010 + 0.004

0.990 + 0.004

4/l 763

Low speed supernatant

0.147 + 0.015

0.853 + 0.015

41794

15

A-fractron

0.762 ir 0.008

0.011 T!z 0.004” (0.227 k 0.008’)

41822

76

“Volume density of A-particles as a fraction of all formed elements present, determined stereologrcally. Means + S.E.M. ’ n/p, number of determinations/total number of points classified. ’ Non-A particles: elements whose structure is incompatible with A-parkcle identity (e.g. rrbosome-containing). d Unidentifiable elements that could not be classified because they were cut tangentially, revealing insufficient ultrastructural

Table 2. Protein Recoveries

in Subfractrons Fraction

of Fetal Brain

(?b ? S.E.M.) of:

Protern”

(mg)

Homogenate

Homogenate

680

100.0

Low speed supernatant

300

49.6 + 4.5

A-fractionb

30

5.4 + 0.8

A-particles”

6

1.3 + 0.2

A-Fraction

Number of Determrnations 13 a

100.0 27.0 f 11.7

13 3

a Yield from a typical fractionation of approximately 72 fetal brarns (17 days gestatron, 6 litters, wet weight approximately 1 g/12 brains). ’ Total A-fraction removed from gradient. ’ Peak I from chromatography of A-fraction on column of controlled-pore glass (Figure 16).

and Bunge, 1974). Freeze-fracture electron micrographs of A-particles yield a strikingly similar image (Figure 12). Aparticle plasmalemma is poor in IMPS and frequently forms nearly IMP-free protrusions surrounded by a number of pits. Co-purification of Identified Nerve Growth Cones from Primary Culture If A-particles are indeed fragments of nerve growth cones, as their ultrastructure suggests, then it should be possible to co-purify identified nerve growth cones with the Afraction. To test this hypothesis experimentally, growth cones of neurons radiolabeled in vitro were mixed with fetal brains, the subcellular fractionation was carried out, and the A-fraction was examined for the presence of labeled growth cones. After several days in vitro, explants of dorsal root ganglia or olfactory bulbs produce a broad halo of neurites tipped with nerve growth cones. By the use of appropriate medium (see Experimental Procedures), the outgrowth is essentially free of supporting cells. Such cultures were labeled with either 35S-methionine or a mixture of 14Clabeled amino acids and then chased. The explants and the more central two-thirds of the neuritic outgrowth were then removed by microdissection (cf. Estridge and Bunge, 1978). The most distal neurite segments, containing the light microscopically identified growth cones, were then

Factor

detail.

scraped from the dishes and homogenized together with unlabeled fetal rat brains for fractionation. It was not possible to assess accurately the specific radioactivity of the growth cone material for the following reasons: (i) the presence in the dishes of residual serum protein from the culture medium precludes accurate assay of neuronal protein and (ii) much of the labeled protein recovered from the cultures is soluble, i.e. derived from explants and neurites damaged or fragmented during microdissection. Thus, a meaningful biochemical balance sheet cannot be generated for this mixing experiment. Therefore, the distribution of labeled growth cones among the various fractions has to be assessed by electron microscopic radioautography and their ultrastructure compared with that of the Aparticles. The homogenate (not illustrated) contains a variety of labeled structures including isolated organelles, unidentifiable fragments, and intact A-particles. The low speed supernatant contains a similar array of labeled elements except that the largest structures were deleted during the low speed spin (not illustrated). During subfractionation, most elongated neurite-like elements, smaller cell fragments, and organelles marked by silver grains band with the B- and C-fractions. At the 0.32/0.75 M interface, an occasional radiolabeled A-particle is found among hundreds of A-particles not marked by silver grains (Figure 13). This result is to be expected because a relatively small number of growth cones from the cultures has been mixed with a large excess of growth cones from fetal brain. Closer examination of the labeled particles in this preparation reveals that they are identical in structure and organelle content with the unlabeled A-particles derived from fetal brain (Figures 14 and 15). Thus, resealed particles derived from identified growth cones of neurons sprouting in culture co-purify with A-particles during subcellular fractionation Polypeptides of A-Particles Because the A-fraction is collected from the interface between the load and the lightest band of the sucrose density gradient, it is heavily contaminated with soluble proteins released during homogenization of the fetal brains. Two strategies can be employed to separate A-particles from soluble protein: centrifugation through sucrose (how-

Cell 578

lsolatron of Nerve Growth 579

Table 3. Membrane

Cones

Compartments

of A-Particles Relative Surface Density”

Organelle

Raw % f S.E.M.

Adjusted

a. Plasmalemma

40.4 + 1.4

45

b. “Growth

33.4 + 1.4

37

cone vesrcles”

c. Agranular

reticulum

d. Mrtochondna e. “Synaptoid” f.

vesrcles

Large dense-core

9. Lysosomal h. Unrdentrfied

vesrcles

structures membranes

6.4 f 1.4

7

4.4 f 1.7

5

2.2 -t 0.2

2

1.8 f 0.4

2

2.1 f 0.6

2

9.4 * 2.2 100.1

Total

%

100

a Relative membrane area contributed by all membranous organelles contained in the A-fraction, determined by stereological intercept analysis. “Adjusted percentage” was calculated from raw percentages, assuming that all elements (items a-g) contribute approximately equally to the unidentified category (h). The analysis was carried out on six groups of four random electron micrographs each. Organelle categories: a, self-explanatory; b, large clear vesicles characteristic of growth cones c, membrane cisternae structurally similar to smooth endoplasmic reticulum; d, because the inner and outer mitochondrial membranes could not be resolved separately at the magnification used for the analysis, the intercept number with these membranes was multiplied by 2; e, clear vesicles of 50-60.nm drameter, similar to synaptic vesicles; f, self-explanatory; g, includes prrmary and secondary lysosomes as well as multivesicular bodies; h, structures that were drstorted or only partially seen so that classification was not possible.

ever, as described in a forthcoming paper, pelleting leads to breakage of A-particles; Ellis and Pfenninger, in preparation) or chromatography of the A-fraction on a column of controlled-pore glass (c-pg). Chromatography of the A-fraction on c-pg (300.nm mean pore diameter) results in the elution of two highly symmetrical peaks as detected by extinction at 280 nm and assay of protein content (Figure 16). Peak I, which contains about 30% of the protein loaded onto the column (see Table 2) elutes with the void volume. Electron microscopic analysis of this peak (Figure 17) reveals A-particles cytologically identical to those described above (cf. Figures 7 and 8) with their plasma membranes essentially intact and without signs of swelling or lysis (A-particles have been found to exclude inulin; Clark and Pfenninger, unpublished observations). Furthermore, the elution of A-particles as a sharp, highly symmetrical peak indicates that they form a homogeneously sized population of elements with

Figures 8-12.

Comparison

of Nerve Growth

Cones Sprouting

a diameter greater than 0.3 pm. These results are consistent with the electron microscopic observations. Analysis of phospholipid phosphorus of the c-pg fractions demonstrates a single phospholipid peak coincident with the eluted A-particle peak (Figure 16). When peak I is analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 5-15% gradient gels, silver stain reveals approximately 75 major polypeptides (Figure 18). While the gel of only one fraction of the peak is shown here, the patterns of fractions collected across peak I are identical, again illustrating the homogeneity of the A-particle preparation. Prominent bands are visible across a wide range of molecular weights: at >260, 150, 97 (doublet), 66, 52, 45, 41, 39 (broad), 38 (broad), 35, 34, 33, 31, 22, and 13 kilodaltons (kd). With the conditions of electrophoresis employed, purified adult rat brain tubulin co-migrates with the 52-kd band (the (Y- and fl-tubulin subunits are not well resolved), and actin (prepared as actomyosin from mouse heart) migrates at 41 kd (see arrowheads, Figure 18). Further characterization of the polypeptides of this fraction is the subject of a forthcoming paper (Ellis and Pfenninger, in preparation). Peak II contains approximately 70% of the protein load (Figure 16). Ultrastructural analysis reveals no identifiable cellular organelles or membrane fragments in this material (not shown). This finding is consistent with the fact that peak II contains very little detectable phospholipid phosphorus. Analysis of peak II on silver-stained SDS gels reveals 70-80 major bands, many of which are shared with peak I (Figure 18). However, there are a number of polypeptides that are present in peak II but absent (or reduced) in peak I: 215, 78, 74, 71, 48, 46, 43, 42, and 19 kd and several bands in the range of 23-30 kd. Conversely, there are a number of polypeptides in peak I that are absent from, or present in much smaller amounts, in peak II: at 45, 39, and 38 (broad bands), and at 34, 24, and 22 kd. Discussion The Identity of A-Particles The A-fraction is an unusually uniform preparation consisting of cell fragments that contain all the organelles characteristic of axonal nerve growth cones: microfilaments, large clear vesicles, dense core vesicles, agranular reticulum, and mitochondria, but no ribosomes (del Cerro and Snyder, 1968; Tennyson, 1970; Yamada et al., 1971;

In Culture (Frgures 9-l 1) with A-Fraction

Partrcles (Figures

8 and 12)

Note the hrgh degree of organellar similarity between the two structures. Of particular importance are: mrcrofilaments, in peripheral regions of the nerve growth cone (‘) including growth cone filopodia (f), and in A-particles; clusters of large, clear, pleomorphic vesicles (arrowheads) rn both structures; large dense core vesrcles (double arrows); agranular reticulum (ar); mitochondria (not labeled). I, lysosomal structures; mt. microtubules. Note that ribosomes are absent from axonal growth cones as well as A-partrcles. Figures 11 and 12 show freeze-fracture images of a nerve growth cone of rat spinal cord sproutrng in culture and of an A-panrcle, respectively. Note the low density of intramembrane panrcles In growth cone plasmalemma (gp, protoplasmic leaflet) and in the plasmalemma of the A-particle (ap, protoplasmrc leaflet). A supporting cell profile in culture (open arrow rn Frgure 11, protoplasmic leaflet) has a considerably higher particle densrty. Of further interest are mound-like protrusions surrounded by small prts (arrowheads), which are frequently seen rn both nerve growth cones and Aparticles. Shadowrng direction, from the lower right (Figure 11) and from the lower lefl (Figure 12). Calibration for all figures, 0.5 Wm.

Cell 580

Figures

13-15.

EM Radroautograms

from Co-purification

Experiment

as Described

in the Text

Figure 13 shows the typical, homogeneous composition of the A-fraction. Note the presence of a single labeled A-particle (open arrow) among many unlabeled elements. Labeled A-particles are shown at higher magnification in Figures 14 and 15. Note that the labeled particles exhibit the same ultrastructural features as the unlabeled A-parkles in this experiment as well as those in other experiments (cf. Figures 7 and 8). The labeled A-particles are derived from the distal outgrowth of an olfactory bulb culture previously incubated with “‘C-amino acids. For further description, see text. Calibration, 1 Mm for Figure 13 and 0.5 pm for Figures 14 and 15.

Bunge, 1973). In contrast to their axonal counterpart, dendritic growth cones have an electron-lucent cytoplasm that is sparsely populated by organelles and contains ribosomes (Hinds and Hinds, 1972; Skoff and Hamburger, 1974; Vaughn et al., 1974). Therefore, the ultrastructural features of A-particles are very similar, or identical, to those of axonal growth cones. Both agranular reticulum and, especially, microtubules, which are concentrated in the more central and proximal portions of axonal nerve growth cones, are less prominent in A-particles. This suggests that these particles may be sheared off peripheral regions (i.e. the leading edge) of the growth cone during homogenization. The more proximal neuritic shafts, characterized by bundles of parallel microtubules, virtual absence of microfilaments and paucity of membranous organelles, seem to be disrupted into small fragments that are not

seen in the A-fraction but are frequently observed in the C-fraction. The various ultrastructural observations that point to the identity of A-particles are strongly supported by the results of the mixing experiments. It is important to note that the distal segments of neuritic outgrowth in the explant cultures used for the experiments are (i) highly enriched in nerve growth cones (whose presence can readily be established by light and electron microscopy) and (ii) essentially devoid of supporting cells. Hence, the co-purification experiments demonstrate that fragmentation of identified nerve growth cones by shearing forces (scraping off the dish, homogenization) produces structures that are cytologically identical with, and that co-purify at the same buoyant density as, A-particles, If A-particles are indeed derived from axonal growth

;$ation

of Nerve Growth

Cones

I

II 0.6

. ..-. .- .. .,...._x”

2001 60

70

116m ‘,;, 20

67m

30

40 FRACTION

016

50

60

70

NUMBER

018 Figures

16-18.

Separation

of A-Particles

from Soluble Components

by Chromatography

on a Column of Controlled-Pore

Glass

Figure 16, elution profiles as determined by absorbance at 280 nm, protein assay, and phospholipid (PL) determination (protein and phospholipid data are from two drfferent experiments). Note the symmetry of the first peak (I). It contains essentially intact A-particles, as shown in Frgure 17 (calibration, 1 pm). Frgure 18 shows the polypeptide profiles of A-particles (peak I) and soluble proteins (peak II), as analyzed on an SDS-polyacrylamide gradient (5-i%) gel. Note that these patterns have some bands in common, e.g. the proteins co-migrating with tubulin and actin (upper and lower arrowheads, respectively), while there are numerous bands that are characterrstic of one or the other of the two peaks (see text).

cones, this raises the question of the fates in this fractionation scheme of the growing tips and ruffling edges of non-neuronal cells of the nervous system. In fact, previous studies (e.g. Hinds and Ruffet, 1971) have noted a morphological similarity between axonal growth cones and neuroepithelial endfeet of the developing brain. However, neuroepithelial endfeet have ribosomes scattered in the cytoplasm, are covered with a basal lamina at the external limiting layer, and are interconnected by junctions, especially on the ventricular face of the neuroepithelium (Hinds and Ruffet, 1971; Rakic, 1972; Peters and Feldman, 1973; Henrickson and Vaughn, 1974). However such features have not been observed in the A-fraction. Furthermore,

cellular fragments with an electron-dense cytoplasm and containing numerous rosettes of polysomes are abundant in the low speed supernatant and band in the heavier Band C-fractions. In rare cases, we have observed clusters of small, uniformly sized clear vesicles (reminiscent of synaptic vesicles) in A-particles. This raises the question of the contamination of the A-fraction by synaptosomes. However, neither synaptic junctional complexes nor presynaptic junctional specializations have been seen in our large ultrastructural sample of A-particles. Furthermore, mature synaptosomes band at higher density (1.2 M sucrose) than A-particles (0.75 M sucrose) during isopycnic gradient

Cell 582

centrifugation (e.g. Gray and Whittaker, 1962; Cotman and Taylor, 1972; Cohen et al., 1977). Thus, A-particles containing “synaptoid” vesicles may be at an early transitional stage between growth cone and presynaptic terminal or may be derived from growing axons that have already formed a synaptic contact en passage. Unfortunately, biochemical markers for nerve growth cones are not known at the present time. Therefore, the identification of nerve growth cones (in vivo and in vitro) rests upon ultrastructural criteria. Both the ultrastructural features of A-particles and the co-purification experiments suggest that A-particles are sheared-off fragments of axonal nerve growth cones. They will henceforth be termed “growth cone particles” (GCPs). Evaluation of the Method GCPs can be prepared quite rapidly, using a vertical rotor, and in quantities that make biochemical analyses possible (1 mg protein/g wet weight of fetal brain). With the method reported here, the A-fraction removed from the interface between load and 0.75 M sucrose is heavily contaminated with soluble protein from the homogenate. All attempts to include a gradient step of sucrose of intermediate density to “wash” the A-particles during centrifugation have resulted in the loss of particles at the 0.75 M interface due to shrinkage or breakage. We have attempted to use less osmotically active density reagents (metrizamide, Ficoll, Percoll) and, especially, to employ a flotation scheme to prepare GCPs. However, the A-fraction equivalent is heavily contaminated with ribosome-containing cell fragments after isopycnic centrifugation with reagents other than sucrose, and GCPs lyse during flotation in a density gradient (even under strictly isotonic conditions). Thus, in order to prepare intact GCPs, we have employed the additional step of c-pg chromatography of the A-fraction to remove soluble protein. The polypeptide composition of the intact GCP (peak I from the c-pg column) is complex, as might be expected for a membrane-bound cellular fragment. A forthcoming paper (Ellis and Pfenninger, in preparation) will be focused on the biochemical analysis of polypeptides of GCPs and on the preparation of a membrane subfraction from them. Of particular interest will be the identification of characteristic polypeptides of growth cone membranes that may be useful as biochemical markers. Perspective Several publications document earlier attempts to study subcellular fractions of immature brain (Gonatas et al., 1971; Grove et al., 1973; Kanerva et al., 1978). These fractions, unlike the one presented here, were prepared from the crude mitochondrial pellet, essentially according to synaptosome preparation schemes (cf. Gray and Whittaker, 1962). Thus, they contained mainly elements that could be identified as (immature) synaptosomes, combined with some GCP-like structures and a variety of other, unidentified elements. As a consequence, it was not pos-

sible to study the nerve growth cone with biochemical methods and to compare it with its mature counterpart, the presynaptic nerve terminal. In contrast, the GCP fraction presented here is the result of an entirely different fractionation strategy. It is highly homogeneous and makes such biochemical studies possible. Overall, the GCP fraction opens various new avenues for the biochemical investigation of growth cone function during nervous system development, i.e. for the analysis of molecular mechanisms involved in neuritic growth and network formation. Experimental

Procedures

Materials Controlled-pore glass (No. CPG03000, 60/120 mesh, 300.nm mean pore diameter, lot No. 11 All) was from Electra-Nucleonics, Inc. (Fairfield, NJ). Ultrapure sucrose was from Schwarz/Mann. N-Tris(hydroxymethyl)methyi2-aminoethanesulfonic acid (TES) and tris(hydroxymethyl)aminomethane (Tris, as Trizma base) were from Sigma. Protein standards for SDS-PAGE were from Phamacia (14.4-94 kd) and from Bio-Rad (14.4-200 kd). Sodium dodecyl sulfate (SDS) was from British Drug House. All chemicals for SDSPAGE were electrophoresis grade. All other chemicals were reagent grade. Fractionation Protocols The brains of fetal Sprague-Dawley rats of 16 to 16 days (ideally 17 days) gestation were gently homogenized with five strokes in a Teflon-glass homogenizer (clearance, 0.15-0.23 mm; A. H. Thomas Co.), in 6 volumes (wet weight:volume) of 0.32 M sucrose containing 1 mM MgCI? and 1 mM TES-NaOH pH 7.3. The homogenate was spun at 1,660 x gmax for 15 min. The low speed supernatant was loaded onto a discontinuous sucrose density gradrent, as shown in Figure 1. The gradients were spun to equilrbrrum, for 40 min at 242,000 X gmar usrng a Beckman VTi50 vertical rotor. Thereafter, the fractions were removed from the various interfaces for further analysis. All these procedures, including the dissection of the uterus and the fetus, were carried out on ice or at 4°C. A-panrcles were separated from the bulk of soluble proteins contained rn the A-fractron (sucrose concentration, approximately 0.65 M) on a column of controlled-pore glass (c-pg; Nagy et al., 1976; Carlson et al., 1976). The c-pg column (60 x 1 cm) was equrlibrated with 0.65 M sucrose containing 1 mM MgCl? and 1 mM TES-NaOH, pH 7.3, and run at a flow rate of approximately 1.7 ml/min at room temperature, under light pressure. Elution was monitored continuously by recording optical density at 260 nm. The volume of the collected fractions was 0.75-I .O ml. Electron Microscopic Procedures Unfixed A-particles tend to break during pelletting (Ellis and Pfenninger, in preparatron). Therefore, the following protocol was used. Aliquots of the various fractions were mixed with gradually increasing amounts of 1.5% glutaraldehyde in 0.2 M phosphate buffer, pH 7.3, with 120 mM glucose and 0.4 mM calcium chloride added. After IO to 15 min, the fixed material was pelleted into the tip of a conical embedding capsule (BEEM No. 5406). The pellets were exposed to the glutaraldehyde fixative for at least another 30 mm at room temperature, then washed with arsenate buffer, osmicated, block-stained with magnesium uranyl acetate, dehydrated, and embedded as described elsewhere (Pfenninger and Maylie-Pfenninger, 1981) except that the incubation times in the various solutions were increased 3. to 4. fold compared to the published protocol in order to allow for permeation of the fixing reagents into the pellet. Thin sections prepared from these pellets, cut at various levels, were examined with a JEOL 1OOC electron microscope. Fixation in suspension leads to the formation of aggregates (combining elements regardless of size and density) that can be pelleted at relatively low speed. The use of small aliquots in pointed embedding capsules leads to little, if any, stratification of the pellet and, therefore, to a representative vrew of the fraction’s contents. Radioautograms of the sections were prepared usrng the flat substrate method of Salpeter and Bachmann (1972) and exposed for a period of 5 months. Nerve tissue cultures were processed for thin section electron

Isolation of Nerve Growth Cones 583

microscopy as described elsewhere (Pfennrnger and Maylie-Pfenninger, 1981). For freeze-fracturrng. A-particles were fixed in suspension as descrrbed above, pelleted, and then impregnated with increasing concentrations of glycerol to reach 30%. Fragments of the pellet were fractured in a Balzers BAF300 apparatus according to standard procedures. Nerve tissue cultures were fractured with the same equipment but using a sandwich technique described elsewhere (Pfennrnger and Rinderer, 1975).

References

Morphometric Analysis In order to determine the volume density of GCPs in the various fractions, electron micrographs (12 for LSS and the A-fraction; 20 for the homogenate) were taken at random from thrn sections of each fraction, at a primary magnification of X 6.600. The micrographs were enlarged 2.6 times and then overlard with a grid containing 108 points. According to standard stereologic methods (e.g. Weibel and Bolender, 1973) we counted those points that fell either on GCPs or other cellular or subcellular elements to determine relative volumes. To determine the relative membrane composition of GCPs. we took six groups of four random pictures each from two drfferent A-fractions (primary magnification, x 8,300). The 2.6 times-enlarged micrographs were overlaid with a grid containing 108 lines, each 2 cm long. Line intercepts with the various membrane types were counted and tabulated, and means and standard errors of the mean were determined. The total volume of samples used for morphometric analysis was approximately 0.5-l pi of pelleted maternal, for each fraction.

Bradford, M. M. (1976). A rapid and sensrtive method for the quantitatron of microgram quantities of proteins utilizing the principle of protein dye bindrng. Anal. Biochem. 72, 248-254.

Tissue Culture Techniques For the mrxrng experiments, we prepared explant cultures of dorsal root ganglia (DRG) or of olfactory bulb (OB), dissected from 14.day or 18.day gestation rat fetus, respectively. DRG or OB explants were grown in polylysine-coated (Varon, 1979) 35mm plastic dishes for 7 days, In a medium modified after that of Varon (1979) or Bottenstein and Sato (1980) respectively. For each experiment, we used approximately 250 explants. When they had produced sufficient amounts of neuritic outgrowth, the cultures were pulsed with either 200 pCi/ml L-?+methronine (995 Ci/mmol; New England Nuclear) overnight (DRGs) or wrth 33 &i/ml of a mixture of 16 Y-amino acids (>50 mCi/mg atom of carbon; Amersham) for 3 hr (OBs). and then chased for 1 hr. The central two-thirds of the neuritic outgrowth as well as the explants were cut out and removed under a dissecting microscope. The remaining distal outgrowth was scraped off the dish and the resulting suspensron placed into a Teflon-glass homogenizer. Approximately one dozen fresh fetal rat brains were added and the mixture was processed for the generation of the A-fraction. Biochemical Procedures Protein in the various fractions was assayed by the method of Bradford (1976: Bio-Rad reagent), using bovine plasma r-globulin as standard. Phospholipid phosphorus was determined in chloroform-methanol extracts according to a modification of the method of Ames and Dubin (1960). Analysis of proteins by SDS-PAGE was by the method of Laemmli (1970). Gels consrsted of a 5-15% acrylamide gradient (Piccioni et al., t982), wrth the drmensions 140 X 240 X 0.75 mm, and a PO-mm stacking gel of 3% acrylamide. Gels were run at a constant current of 15 mA for a drstance of 200 mm in the resolving gel. They were stained with ammoniacal silver according to Wray et al. (1981). Acknowledgments The authors wish to thank Dr. Kathryn Howell, now at the European Molecular Biology Laboratory In Heidelberg, for her helpful suggestrons regarding the fractionation procedures. The authors are also grateful to Kathy Srlberman for her help with the completion of this manuscript. This research was supported by NSF grants BNS 76-18513 and BNS 79-14071, an I. T. Hirsch1 Career Scientist Award to K. H. P., an NIH National Research Servrce Award to L. E., and a summer research fellowship to S. S. The costs of publication of this article were defrayed in part by the payment of page charges, This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

June 24, 1983; revised

September

7, 1983

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