Growth Cones Contain a DynamicPopulation of Neurofilament Subunits
Walter K.-H. Chan,1 Jason T. Yabe,1 Aurea F. Pimenta,2 Daniela Ortiz,1
and Thomas B. Shea1*
1Center for Cellular Neurobiology and Neurodegeneration Research, Department ofBiological Sciences, University of Massachusetts-Lowell
2Department of Neurobiology, University of Pittsburgh School of Medicine,Pittsburgh, Pennsylvania
Neurofilaments (NFs) are classically considered to transport in a primarily an-terograde direction along axons, and to undergo bulk degradation within thesynapse or growth cone (GC). We compared overall NF protein distribution withthat of newly expressed NF subunits within NB2a/d1 cells by transfection with aconstruct encoding green fluorescent protein (GFP) conjugated NF-M subunits.GCs lacked phosphorylated NF epitopes, and steady-state levels of non-phosphos-phorylated NF subunits within GC were markedly reduced compared to those ofneurite shaft as indicated by conventional immunofluorescence. However, GCscontained significant levels of GFP-tagged subunits in the form of punctate orshort filamentous structures that in some cases exceeded that visualized along theshaft itself, suggesting that GCs contained a relatively higher concentration ofnewly synthesized subunits. GFP-tagged NF subunits within GCs co-localizedwith non-phosphorylated NF immunoreactivity. GFP-tagged subunits were ob-served within GC filopodia in which steady-state levels of NF subunits were toolow to be detected by conventional immunofluorescence. Selective localization offluorescein versus rhodamine fluorescene was observed within GCs followingexpression of NF-M conjugated to DsRed1-E5, which shifts from fluorescein torhodamine fluorescence within hours after expression; axonal shafts contained amore even distribution of fluorescein and rhodamine fluorescence, further indi-cating that GCs contained relatively higher levels of the most-recently expressedsubunits. GFP-tagged structures were rapidly extracted from GCs under condi-tions that preserved axonal structures. These short filamentous and punctatestructures underwent rapid bi-directional movement within GCs. Movement ofGFP-tagged structures within GCs ceased following application of nocodazole,cytochalasin B, and the kinase inhibitor olomoucine, indicating that their motilitywas dependent upon microtubules and actin and, moreover, was due to activetransport rather than simple diffusion. Treatment with the protease inhibitorcalpeptin increased overall NF subunits, but increased those within the GC to agreater extent than those along the shaft, indicating that subunits in the GCundergo more rapid turnover than do those within the shaft. Some GCs containedcoiled aggregates of GFP-tagged NFs that appeared to be contiguous with axonal
The Supplemental Material referred to in this section can be found at:http://www.interscience.wiley.com/jpages/0886-1544/suppmat/index.html
Contract grant sponsor: National Science Foundation.
*Correspondence to: Thomas B. Shea, Center for Cellular Neurobiol-ogy and Neurodegeneration Research, Department of Biological Sci-
ences, University of Massachusetts-Lowell, One University AvenueLowell, MA 01854.E-mail:[email protected]
Received 5 June 2002; Accepted 31 July 2002
Cell Motility and the Cytoskeleton 54:195–207 (2003)
Neurofilaments (NFs), and in particular their phos-pho-isoforms, are thought to provide stability to thematuring axon. This is consistent with their delayedappearance within the axon relative to other major cy-toskeletal polymers such as microtubules (MTs) and fil-amentous actin [for review, see Pant and Veeranna,1995].
The growth cone (GC) is considered the most plas-tic area of developing axons. Unlike the trailing axon,which progressively stabilizes during continued elonga-tion, the growth cone maintains its plasticity and under-goes constant remodeling [Pfenninger, 1986; Small-heiser, 1990; VanHooff et al., 1989]. Consistent with thisrequirement for plasticity, the cytoskeleton of the GCdiffers in several key respects from that of the axon.Actin forms a complex network within the GC, includingassembling into bundles that extend into and support themovements of filopodia and lamellopodia [Challacombeet al., 1996; Letourneau, 1983; Letourneau and Ressler,1983; Lewis and Bridgmann, 1992]. MTs extend fromthe axonal shaft into the central portion of the GC and tothe base of filopodia, but, in contrast to the MTs of theaxonal shaft [Baas and Black, 1990; Lim et al., 1989; Yiand Black, 1995], MTs within GCs are present in apredominantly labile form [Gordon-Weeks, 1991; Gor-don-Weeks and Lang, 1988; Letourneau and Ressler,1983]. Also consistent with ongoing remodeling, studiesindicate that the GC is devoid of NFs even following theaccumulation of phospho-NFs along the axonal shaft[Roots, 1983; Tang and Goldberg, 2000]. NFs are appar-ently restricted from accumulating within the GC byregional calcium-activated proteolysis, since treatmentwith the protease inhibitor leupeptin fostered the appear-ance of NFs within the GC [Roots, 1983].
Transfection with NF sequences conjugated togreen fluorescent protein (GFP) has allowed real-timeintracellular visualization of newly synthesized subunits,the localization of which has yielded images markedlydistinct from those of steady-state subunits in conven-tional immunofluorescent analyses, and has highlightedNF dynamics. Such analyses have allowed visualization
of axonal transport of short filamentous [Roy et al., 2000;Wang et al., 2000; Yabe et al., 1999, 2000] as well asnon-filamentous punctate structures [Yabe et al., 1999,2001a,b; Prahlad et al., 2000] that are analogous to thoseobserved for other intermediate filament species such asvimentin and keratin during cellular remodeling [Martyset al., 1999; Prahlad et al., 1998; Windorffer and Leube,1999; for review, see Chou and Goldman, 2000]. Trans-fection with GFP-tagged NF sequences has also demon-strated the presence of two experimentally separablepopulations of NFs within growing axons that can bedifferentiated by transport rate, phosphorylation state,and extent of NF-NF interactions [Yabe et al., 2001b].
In the present study, we monitored the distributionof GFP-conjugated NF subunits within GCs of growingaxonal neurites in culture. We demonstrate that GCs,despite containing low overall NF subunit levels, areenriched in highly dynamic, newly synthesized NF sub-units that may participate in regional cytoskeletal remod-eling during axonal elongation.
MATERIALS AND METHODS
Cell Culture
NB2a/d1 cells were cultured in DMEM (high glu-cose formulation) containing 10% horse serum on 35mm2 plates in which a 1-mm hole had been drilled in thecenter and a coverslip sealed beneath the hole with par-affin [e.g., Baas and Ahmad, 1993]. Cells were differen-tiated with 1 mM dbcAMP as described previously [Sheaand Beermann, 1994; Shea et al., 1990]. Some cultureswere treated with 330 �M nocodazole or 5 �M cytocha-lasin B in order to perturb MT and actin dynamics,respectively [Ahmad et al., 2000; Shea et al., 1990; Yabeet al., 1999, 2001a]. Additional cultures received 1 �Mcalpeptin [Shea and Beermann, 1994] or 50 �M olo-moucine [Ratner et al., 1998] for the final 2 h beforeobservation.
Transfection
Cells were transfected with 33.2 �g/ml eGFP-NF-M or eGFP without an NF-M insert in 238 �g/ml
Superfect (Qiagen) for 3 h in the presence of 10% serum,after which the medium was replaced [Yabe et al., 1999].Cells were incubated for an additional 18–24 h to allowaccumulation of sufficient eGFP-conjugated NF-M forconsistent visualization [e.g., Yabe et al., 1999]. Trans-fection with the identical conjugate lacking NF-Myielded only diffuse fluorescence [Yabe et al., 1999].
Cells were also transfected using the above method-ology, and viewed 18–24 h later, with the same construct inwhich the sequence coding for eGFP was replaced withDsRed1-E5, a mutated form of the red fluorescent proteinDsRed1 [Matz et al., 1999]. DsRed1-E5 contains two aminoacid substitutions that increase its fluorescence intensity andendow it with a distinct spectral property: as the proteinages, it changes color from bright green to bright red[Terskikh et al., 2000]. Initial fluorescence is, therefore,detectable under fluorescein optics but shifts withinhours such that it is then instead detectable under rhoda-mine optics. While newly synthesized NF subunits canbe distinguished from the overall subunit population fol-lowing expression of GFP-conjugated NF subunits, con-jugation of NF-M to DsRed1-E5 provides a further indexof the relative age of various NF-M subunits within thisnewly expressed population; as with GFP-tagged sub-units, not only have all fluorescent subunits been ex-pressed within the last 18–24 h, but those that fluoresceunder fluorescein optics represent subunits that wereexpressed more recently than those that fluoresce underrhodamine optics.
To generate this construct, the cDNA encoding theEGFP was excised from plasmid pEGFP-N3/NF-M[Yabe et al., 1999] and replaced by the cDNA encodingthe DsRed1-E5. pEGFP-N3/NF-M was initially linear-ized at a unique BamH I site present in the linker betweenthe NF-M and the EGFP cDNAs and converted to bluntend. Subsequently, EGFP was excised by restriction en-zyme digestion with Not I. The cDNA encoding theDsRed1-E5 was excised from plasmid pTimer (Clon-tech) by sequential restriction enzyme digestion withXma I (converted to a blunt end) and Not I. The DsRed1-E5cDNA was then subcloned into the BamH I (blunted) andNot I site of the pN3/NF-M plasmid, in the same translationframe of the NF-M, confirmed by nucleotide sequencing.Expression of the NF-M/DsRed1-E5 fusion protein is,therefore, under control of the CMV promoter, with theinitiation codon provided by the NF-M cDNA and thetermination codon by the DsRed1-E5 cDNA.
Immunofluorescence
Cultures were processed for endogenous NF immu-noreactivity using antibodies directed against phosphor-ylated (RT97) and non-phosphorylated (SMI-32) NFepitopes followed by Texas red-conjugated secondaryantibody [Jung et al., 1998], and a polyclonal antibody
(L3) raised in this laboratory against purified NF-L. Thedistribution of MTs within GCs was determined using amonoclonal antibody directed against �-tubulin [Shea,1999] following extraction with saponin in the presenceof 10 �M taxol to deplete free tubulin subunits yet retainassembled MTs [Brown et al., 1992].
Densitometric and Confocal Analyses
Phase-contrast and epifluorescent images of indi-vidual cells were captured via a Dage CCL-72 cameraconnected to a Scion LG-3 frame grabber housed in aMacintosh 7100AV operated by NIH Image software(version 1.62). Images were then stored as TIFF or PICTfiles. In some experiments, images were captured alongthe Z-axis of transfected cells and subjected to deconvo-lution using Open Lab software. For densitometric anal-yses, GCs and axonal neurites in stored images wereencircled using the NIH Image freehand tool and therelative intensity determined. The relative amount ofGFP signal versus endogenous subunit immunoreactivityin GCs and axonal neurite shafts was determined bydividing the mean intensity of the respective signal inGCs by the mean intensity in GCs plus the mean inten-sity within axons. For cells transfected with DsRed1-E5-NF-M, these ratios were independently calculated forvalues obtained under fluorescein and rhodamine optics.For comparison of the influence of nocodazole, cytocha-lasin B and olomoucine on motility of GFP-tagged struc-tures, sequential images of GCs of cells transfected 24 hpreviously were captured prior to and following additionof agents, and the percentage of motile GFP-tagged par-ticles was determined as described [Yabe et al., 1999].For comparison of the effect of calpain on subunit levels,GCs and neurites of 12 calpeptin-treated and 11 non-treated individual cells expressing GFP-M in culturestransfected 24 h previously were encircled using thefreehand tool of NIH Image (1.57) and the density ofGFP fluorescence was recorded. The relative increase inGFP-NF-M in GCs and neurites obtained following cal-peptin treatment was determined by dividing the meandensity of the GFP signal in calpeptin-treated images bythe mean of untreated images. In additional experimentswith cells transfected with DsRed1-E5-NF-M, the meanintensity of fluorescence obtained under fluorescein op-tics was divided by the mean intensity of fluorescenceobtained under rhodamine optics. Statistical analyses wascarried out via Student’s t-test. Real-time analyses oftransfected cells was carried out by repetitive imaging ofcells and conversion of sequential images into a single“stack” with NIH Image, followed by saving as an un-compressed QuickTime movie file [Yabe et al., 1999,2001a]. Images were annotated in Adobe Photoshop.
The form in which NF subunits undergo transport,and whether or not an appreciable population of non-filamentous subunits exists within axonal neurites, re-main controversial [e.g., Yabe et al., 2001b and refer-ences therein]. We, therefore, refer to “NF subunits”and/or “GFP-tagged NF subunits” in some occasionsmerely to avoid arbitrarily suggesting any particular po-lymerization state; this is also not intended to imply thatsuch subunits are not polymerized. Only in cases whereit is clear that certain GFP-tagged NF subunits arepresent in punctate, filamentous, or bundled form do werefer to them as such [e.g., Yabe et al., 1999, 2001a,b].All transfections were carried out with GFP-NF-M un-less specifically indicated to have been carried out withDsRed1-E5-NF-M.
RESULTS
Following transfection of differentiated cells withour GFP-conjugated NF-M subunits, we noted that GCscontained significant levels of GFP that often exceededthat within respective neurite shafts (Fig. 1). NF subunitswithin GCs expressing or not expressing GFP-conju-gated subunits did not react with an antibody (RT97)directed against NF phospho-epitopes, but were reactivewith an antibody (SMI-32) directed against non-phos-phorylated NF epitopes (Fig. 1). The ratio of mean fluores-cence intensity with the GC vs. the shaft was markedlyhigher for GFP than for phospho-NFs (as ascertained by
phospho-dependent antibodiesRT97 and SMI-31) or totalNFs (as ascertained by use of phospho-independent anti-bodies directed against NF-L and NF-M; Fig. 1), indicatinga higher proportion of newly expressed subunits within GCsthan within respective axonal shafts.
Higher magnification revealed that the GFP-taggedNF subunits within GCs were present in the form of shortfilamentous and punctate structures (Fig. 2) that resem-bled those previously demonstrated to undergo transportalong the axonal shaft [Yabe et al., 1999, 2001a,b].Considerable variance was observed in the relativeamount of filamentous and punctate structures amongindividual GCs, as well as the total amount of GFP-labeled structures (e.g., compare Fig. 2A–E). Most GCspresented a mixture of both filamentous and punctatestructures, while some contained apparently exclusivelypunctate (e.g., Fig. 2A) or filamentous (e.g., Fig. 2B)GFP-labeled structures. This degree of variance resem-bled that previously reported for GFP-labeled structureswithin axonal neurites themselves [Yabe et al., 2001a]. Inmany neurites, a GFP-labeled aggregate was observed inthe area of the GC adjacent to the shaft (e.g., Figs. 3,4).Confocal microscopy as well as favorable conven-tional fluorescent images demonstrated this aggregate tobe comprised of coiled GFP-labeled filamentous struc-tures (Fig. 3). Filamentous profiles apparently extendedover time from these aggregates as neurites elongated(Fig. 3B).
Both punctate and short filamentous structureswithin GC exhibited highly dynamic motion, while bun-
Fig. 1. GFP-M accumulates in the GCs of transiently-transfectedcells and co-localizes with nonphosphorylated NF epitopes. Cell 1presents a cell transfected with GFP-NF-M, then immunostained witha monoclonal antibody (RT97) that reacts with phospho-epitopes ofNF-H. Note that GFP-M is present along the entire shaft and GC, andis more intense within the GC than the axonal shaft, while phospho-NFimmunoreactivity is present along the axonal shaft but relativelyabsent from the GC. Cell 2 presents the GC and terminal region of theaxonal shaft of a second transfected cell. Note that GFP-M co-localizeswith non-phospho (SMI-32) NF immunoreactivity. The accompanying
graph presents the relative levels (mean densitometric signal � SEM)of GFP-M, NF-L (L3; as an index of total NFs), NF-M (M2, as anindex of total NFs and to facilitate comparison of endogenous NF-Mwith GFP-tagged NF-M), and phospho-NFs (SMI-31, RT97) withinGCs vs. their respective axonal shafts. Note that the mean density ofGFP-M within GCs markedly exceeds that of respective shafts. Bycontrast, the mean density of overall NFs as determined by NF im-munoreactivity is markedly higher within axonal neurites versus thatwithin respective GCs.
Fig. 2. GFP-M is present in GCs in filamentous and punctate forms. Epiflourescent imagesof portions of the axonal shaft and GCs of cells transfected with GFP-NF-M. The transitionfrom the shaft to the GC is indicated. A,D,E: Images were captured at 30-sec intervals;numbers within panels in C indicate times (in minutes) at which images were captured. Notethe dynamic aspect of GFP-M within GCs as indicated by continued movement of thesestructures within the GC. Representative short filamentous structures are indicated within
GCs in A. B: Two images of the same GC, captured at different focal planes; note the overallfilamentous nature of GFP in this GC. Note the continuous movement of punctate andfilamentous profiles within the GC in C, D, and E, while the NF bundle (indicated in the firstimage) exhibits little if any motion. Video sequences of A, C and D are presented inMovie2A, Movie2C and Movie2D, respectively (http://www.interscience.wiley.com/jpages/0886-1544/suppmat/index.html).
dled filamentous structures within the adjacent axonalshaft were relatively motionless (Figs. 2, 4). Individualfilamentous and punctate structures rapidly traversed theaxonal length in a predominantly anterograde direction(e.g., Fig. 4A) [see also Yabe et al., 1999, 2001b]. While
GFP-labeled structures within axonal neurites demon-strated movement either anterograde or retrograde withrespect to the longitudinal axonal axis (e.g., Fig. 4A)[Yabe et al., 1999, 2001a], movement of GFP-labeledstructures within GCs was not always linear with respect
Fig. 3. The aggregates of GFP-M within GCs are com-prised of filamentous structures. Confocal (A) and con-ventional (B) epifluorescent images of two representa-tive neurites that contain aggregations of GFP-M withinthe GC adjacent to the shaft. A: Five deconvolvedimages obtained via a series along the Z-axis of a celltransfected with GFP-NF-M along with the image com-piled from all 5 focal planes. Note the filamentous natureof the aggregate as revealed by individual images. Inset:Higher magnification of the image obtained at level 4indicates a filamentous profile (arrows) within the ag-gregate. B: Sequential, conventional epifluorescent im-ages of a second axonal neurite transfected with GFP-NF-M obtained at 1-min intervals that also highlights thefilamentous nature of the aggregate. Note the progres-sive elongation of the filamentous profile from the ag-gregate into the GC (arrow).
Fig. 4. GFP-labeled structures within GCs are highly dynamic. Sequential images of cellstransfected with GFP-NF-M obtained at the indicated intervals (in minutes) of the distalregion of the axonal shaft and GC of a representative transfected cell. Punctate and shortfilamentous GFP-tagged structures translocate along the axonal shaft (arrowheads along theshaft in A) around the centrally-situated NF bundle; it is difficult to track the movement ofindividual GFP-tagged structures, however, some of these apparently translocate along thelength of the axonal shaft and into the GC. Note the large aggregation of GFP-M at the areaof the GC adjacent to the shaft. Video sequences are presented in Movie4A. B: Confocal
images captured at 15-sec intervals from a single plane along the z-axis of the distal shaft andGC of a transfected cell. Images 1–4 depict punctate structures moving along a filopodia(arrow 1 in image 1) and the apparent formation of a filamentous profile from severalpunctate structures followed by its dissolution (arrow 2 in image 1; insets present this regionat higher magnification). Images 5–12 denote apparent looping of punctate structures at theleading edge of the GC; these areas are encircled in image 5. The motion of a representativepunctate structure is indicated in sequential images (arrows in images 6–12). Note also thatthe bundled NFs do not exhibit any net motion. Video sequences are presented in Movie4B.
to the axonal shaft. Some punctate structures were ob-served within GC filopodia (Fig. 4B) as previously dem-onstrated for short MTs [Dent et al., 1999]. Sequentialconfocal images also demonstrated some punctate struc-tures to loop around at the leading edge of GCs (Fig. 4B),in a manner similar to the “fountains” of non-filamentousGFP-vimentin observed at the periphery of non-neuronalcells [Ho et al., 1988; Martys et al., 1999]. The non-linearmotions observed for some punctate structures withinGCs could be mediated by translocation along MTs that“loop” through GCs (Fig. 5) [see also Dent et al., 1999;Tanaka and Kirschner, 1991] and by those MTs thatextended into filopodia and lamellopodia (Fig. 5). Alter-natively, and/or in addition, subunits may translocatealong actin fibers within GCs. These possibilities weresupported by the rapid cessation of movement of punc-tate and filamentous NF containing structures followingapplication of nocodazole and cytochalasin B (Fig. 6),indicating a requirement of an intact MT and actin cy-toskeleton to support translocation of NF subunits withinGCs. Movement of GFP-tagged structures also ceasedfollowing treatment with the kinase inhibitor olo-moucine, which was previously demonstrated to inhibitaxonal transport [Ratner et al., 1998] (Fig. 6). Cessationof movement following application of each of these threereagents confirms, as previously shown for GFP-taggedstructures in neurites [Yabe et al., 2001a], that the move-ment of GFP-tagged structures within GCs is derivedfrom active transport rather than simple diffusion.
GFP-tagged NF subunits within GCs were rapidlyextracted from GCs under conditions that retained both
the axonal NF bundle (Fig. 7) as well as some of thepunctate structures within neurites and perikarya [seeYabe et al., 1999, 2000a]. The increased solubility ofsubunits within GCs, coupled with the relative lack of NFphospho-epitopes in this region, is consistent with thepurported role of phosphorylation with NF-NF associa-tions that promote Triton-insolubility [e.g., Pant andVeeranna, 1995].
Treatment of intact cells with the protease inhibitorcalpeptin has been shown previously to increase NFsubunit levels, including that of GFP-tagged NF subunits[Shea, 1995; Yabe et al., 2001a]. Herein, we observedthat calpeptin increased GFP-tagged subunit levelswithin the GC approximately 4-fold more than those inthe corresponding neurite shafts (Fig. 8A). Followingtreatment with calpeptin, GFP-labeled structures withinGCs were more resistant to extraction (Fig. 8B). Consis-tent with previous studies [Roots, 1983], calpeptin alsoinduced an increase in filamentous GFP-M within theGCs (Fig. 8C).
Finally, we transfected additional cultures with aconstruct expressing NF-M conjugated to DsRed1-E5, amutated form of the red fluorescent protein DsRed1[Matz et al., 1999], that is initially detectable underfluorescein optics but shifts its fluorescence emissionwithin hours such that it is then instead detectable underrhodamine optics [Terskikh et al., 2000]. DsRed1-E5-NF-M subunits that fluoresce under fluorescein optics,therefore, represent those subunits that have been syn-thesized more recently than those that fluoresce underrhodamine optics. If, as the above data indicate, GCscontain a population of the most recently synthesized NFsubunits, we reasoned that the ratio of fluorescein signalin GCs versus their respective axonal shafts might ex-ceed that of rhodamine. Fluorescence was detectableunder both fluorescein and rhodamine optics 18–24 hafter transfection with DsRed1-E5-NF-M (Fig. 9). How-ever, the fluorescein signal was relatively concentratedwithin perikarya and GCs versus that along the axonalshaft, while the rhodamine signal was more evenly dis-tributed throughout cells. Densitometric analyses of mul-tiple cells confirmed these visual impressions; the meanintensity of fluorescein signal in GCs versus their respec-tive axonal shafts was 2-fold greater than that of rhoda-mine (Fig. 9). These data indicate that GCs contained arelatively higher proportion of the most recently synthe-sized subunits than did their respective axonal shafts.
DISCUSSION
Consistent with prior studies [Roots, 1983; Tangand Goldberg, 2000], GCs of differentiated NB2a/d1cells contained low levels of endogenous NF subunits.However, we document herein that they contained levels
Fig. 5. MTs are present throughout NB2a/d1 Gcs. Two representa-tive GCs extracted and processed under conditions that preserve MTs,then immunostained with a monoclonal antibody directed againstbeta-tubulin as described in Materials and Methods. Note that aspreviously described for other neuronal culture systems (see text), MTprofiles extend into and loop throughout GCs.
of GFP-tagged subunits that often exceed GFP-NF levelsalong the axonal shaft. This higher ratio of GFP-taggedversus endogenous subunits indicates that that overallpopulation of NF subunits within GCs is more recentlyexpressed than the overall population of subunits withinthe axon. Were NF subunits within the GC instead de-rived from regional disassembly of the overall NF pop-ulation, they would be expected instead to contain a ratioof GFP/endogenous NF immunoreactivity that was
equivalent to, or less than, their respective axonal neuriteshafts. These differences were further highlighted by theselective localization of fluorescein versus rhodaminefluorescene within GCs versus axonal shafts followingexpression of NF-M conjugated to DsRed1-E5, whichshifts from fluorescein to rhodamine fluorescence withinhours after expression. The selective concentration ofmore recently expressed NF subunits within GCs ofgrowing axonal neurites is likely to result from the rapid
Fig. 6. Movement of GFP-M-containing structures within GCs ismediated by active transport and is dependent upon MTs and actin.Images of representative GCs of cells transfected with GFP-NF-Mobtained prior to and following application of nocodazole, cytochala-sin B, and the kinase inhibitor olomoucine as indicated. The accom-panying graphs present the percentage of GFP-tagged structures thatdid or did not exhibit net motion in any direction quantified over a
range of 5–15 min prior to application of reagents and for an additional5–15 min following their application; values represent the average ofat least 50 GFP-tagged punctate or filamentous structures within GCsfrom at least 3 different cells. Note that movement of GFP-M-con-taining punctate and filamentous structures within GCs was halted byall three agents.
NF Subunits in Growth Cones 203
transport of some subunits, in either punctate and fila-mentous form, along the axonal length, in contrast to therelatively slow turnover of the prominent bundle of NFsalong the axonal shaft [Yabe et al., 2001b].
The concentration of punctate and filamentous sub-units within the GC may reflect the requirement for rapidelongation of the NF network to provide regional stabi-lization of the axonal cytoskeleton during axonal out-growth. The rapid motion of punctate structures and shortNFs within GCs resembles that demonstrated for MTswithin GCs [Dent et al., 1999]. Interestingly, non-fila-mentous, punctate actin-containing punctate structureshave also been observed at the leading edge and inlamellopodia of cultured non-neuronal cells. These struc-tures were rich in microinjected actin subunits, movedcentripetally towards the cell center following their for-mation, and have been considered to contribute to actinfilament assembly [Cao et al., 1993]. The general simi-larities in remodeling dynamics of GCs and the leadingedge of migratory, non-neuronal cells warrants investi-gation of whether or not similar punctate assemblies ofactin contribute to GC dynamics. The relationship of NF
Fig. 7. GFP-M subunits within GCs are Triton-soluble. Epifluores-cent images of GFP-NF-M-transfected cells before (A,B) and after (B)extraction with 1% Triton X-100. Note that extraction depletes GFP-labeled structures from the GC while the axonal NF bundle is retained.
Fig. 8. Levels of GFP-tagged subunits within GCs are regulated byproteolysis Cultures transfected with GFP-NF-M were treated with theprotease inhibitor calpeptin (1 �M). Densitometric analyses (A) ofGCs and their respective axonal shafts before and after treatment with1 mM calpeptin demonstrated that calpeptin increased GFP-taggedsubunit levels within both the GC and the shaft. However, levelswithin GCs were increased approximately 4-fold more than those inthe corresponding shafts. B: Representative GC following treatment
with calpeptin and extraction with 1% Triton. Note the persistence ofGFP following extraction in contrast to its depletion following extrac-tion of GCs not treated with calpeptin (e.g., compare with Fig. 7).Consistent with previous studies [Roots, 1983], calpeptin also inducedan increase in filamentous GFP-M within the GCs (C); in someinstances, following calpeptin treatment, the axonal NF bundle ex-tended into and along the full length of GCs (C). Inset shows this moreclearly, arrows.
Fig. 9. Growth cones contain a relatively higher proportion of the most recently synthe-sized NF subunits derived from transfection. A,B: Representative cells transfected 24 hpreviously with a construct expressing DsRed1-E5-NF-M. Note that the fluorescein signal inA is more concentrated within perikaryon and GC (arrow) than within the axonal shaft, whilerhodamine fluorescence is more evenly distributed among all 3 regions. B: Higher magni-fication image of the distal portion of the axonal shaft and GC of a second cell. Note that thefluorescein signal is more highly concentrated within the GC than the adjacent shaft, whilethe rhodamine signal is more evenly distributed between the GC and shaft. In both A and B,merged images highlight the differential distribution of these respective signals, areas
containing substantial both are yellow-orange, indicating the presence of significant fluores-cein signal, while the axonal shafts are predominantly red, indicating the relative lack offluorescein signal. C,D: Densitometric analyses derived from 15 individual cells from twoindependent transfections. C presents the ratio (mean � standard error of the mean) of themean fluorescent intensity under fluorescein (green) and rhodamine (red) in GCs vs. theirrespective axonal shafts. D presents the above ratio of fluorescein signal divided by that ofrhodamine. Note that the ratio of fluorescein signal in GCs vs. their respective axonal shaftssignificantly (P � 0.05) exceeds that of rhodamine.
subunit–containing punctate structures to filamentousstructures has not been fully resolved [e.g., see Wang andBrown, 2001; Wang et al., 2000; Yabe et al., 1999].However, prior studies indicate that punctate structuresare not derived from over-expression of GFP, and can beformed from endogenous subunits in non-transfectedcells. In addition, they undergo axonal transport at thesame rate, and within the same axonal neurites, as fila-mentous subunits, and the balance of punctate versusfilamentous structures shifts during axonal outgrowthand following treatment with agents that promote NFformation [Yabe et al., 2001a]. In this regard, punctateNF-containing structures may be analogous to the vimen-tin-containing punctate structures that contribute to theestablishment of vimentin filaments during remodeling innon-neuronal cells [Ho et al., 1998; Martys et al., 1999;Prahlad et al., 1998]. The observation of a preponderanceof punctate structures and short NFs within GCs, even incells in which most NF subunits were present almostentirely in filamentous form along the axonal shaft, isconsistent with such subunits participating in regionalremodeling of the axonal cytoskeleton.
In many cells, the base of GCs contained an aggre-gate of GFP-tagged NFs that was apparently contiguouswith the axonal NF bundle. This aggregate was moreprominently labeled with relatively more GFP than wereNFs along the axonal shaft, implying regional incorpo-ration of newly transported subunits into pre-existingNFs and/or fusion of short NFs/punctate structures on thedistal end of axonal NFs [e.g., Okabe et al., 1993].Filamentous profiles apparently extended from aggre-gates during axonal elongation. It is unclear at presentwhether this was accomplished by the uncoiling of NFsfrom the aggregate, essentially as has been shown forsimilarly-coiled MTs [Dent et al., 1999] and/or by thefusion of subunits/polymers with axonal NFs at the baseof the GC, or instead merely by chance translocation ofshort filamentous structures across aggregates duringcapturing of sequential images. Punctate NF-containingstructures and short NFs within the GC provide a poten-tial source for such addition. The overall increase inGFP-tagged NF subunits within GCs following treatmentwith a protease inhibitor supports the notion of subunit/polymer fusion with existing NFs. In either event, thepresence of an intensely labeled NF aggregate at thedistal-most portion of the shaft suggests that NF subunitpolymerization and/or incorporation of NF subunits intothe axonal cytoskeletal matrix need not occur exclusivelyat the perikaryal-most end of pre-existing NFs, but in-stead supports the prior suggestion that such events mayoccur at multiple sites along the axonal shaft [Okabe etal., 1993; Takeda et al., 1994]. These findings are alsoreminiscent of the demonstration that the most proximaland most distal portions of the axon contain the highest
levels of newly assembled MTs, suggesting that theyrepresent the most active sites of MT assembly [Brown etal., 1992].
While our findings indicate that GCs contain apopulation of dynamic NF subunits, it should perhaps beemphasized that this population must be relatively smallwith respect to axonal NFs levels, since GCs lackedsubstantial NF immunoreactivity. However, a small buthighly dynamic population of NF subunits may stillcontribute to regional remodeling of the NF cytoskeletonduring axonal elongation. The findings of the presentstudy remain fully consistent with prevention of accu-mulation of NFs within GCs by regional proteolysis[Roots, 1983]; indeed, the increase in GFP-tagged sub-units in GCs following treatment with a protease inhib-itor confirms proteolytic regulation of GFP-tagged sub-unit levels. However, these findings extend these earlierobservations by highlighting that NFs also undergo re-gional dynamics within GCs and may contribute to re-gional remodeling of the axonal cytoskeleton.
CONCLUSION
The findings of the present study indicate thatgrowth cones contain a population of newly synthesizedNF subunits that can participate in regional cytoskeletalremodeling. Concentration of these subunits within thedistal-most portion of axonal neurites challenges the no-tion that NF assembly is confined to the perikaryon orproximal neurite.
ACKNOWLEDGMENTS
We are grateful to Ron Liem and the members ofhis laboratory for their generous sharing of NF-M cDNA.
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